July–September 2015

Perspective 1

MG Steve Jones

Baseline Susceptibility to Pyrethroid and Organophosphate Insecticides 3

in Two Old World Sand Fly Species (Diptera: Psychodidae)

Andrew Y. Li, PhD; Adalberto A Perez de Leon, DVM, PhD; Kenneth J. Linthicum, PhD;

Seth C. Britch, PhD; MAJ Joshua D. Bast; Mustapha Debboun, PhD

Effi cacy of Permethrin Treated Bed Nets Against Leishmania major 10

Infected Sand Flies

Tobin Rowland; MAJ Silas A. Davidson; Kevin Kobylinski, PhD;

Claudio Menses; Edgar Rowton, PhD

Controlled Human Malaria Infection at the Walter Reed Army Institute of Research: 16

The Past, Present, and Future From an Entomological Perspective

Lindsey S. Garver, PhD; Megan Dowler; MAJ Silas A. Davidson

Mosquito Fauna of Lao People’s Democratic Republic, with Special Emphasis 25

on the Adult and Larval Surveillance at Nakai District, Khammuane Province

Leopoldo M. Rueda, PhD; Khamsing Vongphayloth, MD; James E. Pecor;

LCDR Ian W. Sutherland, USN; Jeffrey Hii, PhD; Mustapha Debboun, PhD; Paul T. Brey, PhD

Records and Distribution of New World Phlebotomine Sand Flies 33

(Psychodidae, Diptera), With Special Emphasis on Primary Types and Species Diversity

Leopoldo M. Rueda, PhD; Desmond H. Foley, PhD; David Pecor; Matthew Wolkoff

Development of Air Force Aerial Spray Night Operations: 47

High Altitude Swath Characterization

Lt Col Karl A. Haagsma, USAFR; Lt Col Mark S. Breidenbaugh, USAFR;

Kenneth J. Linthicum, PhD; Robert L. Aldridge; Seth C. Britch, PhD

Toxicity Testing in the 21st Century: Refi ning the Army’s Toolbox 60

LTC Erica Eggers Carroll; Mark S. Johnson, PhD, DABT

The New US Military Role in the European Union’s Import Program: 64

Strategic Implications Ensuring Safe Food for the European Theater

MAJ Michael McCown; Jacob L. Hall, II; Megan McCormick, DVM, MPH;

Lt Col Henry H. Triplett, III, USAF

Evaluation of Postdeployment Cancers Among Active Duty Military Personnel 68

Jessica M. Sharkey, MPH; Joseph H. Abraham, ScD

A Preliminary Analysis of Noise Exposure and Medical Outcomes 76

for Department of Defense Military Musicians

Cindy Smith; Sharon Beamer, AuD; Shane Hall, MS; Thomas Helfer, PhD;

Timothy A. Kluchinsky, Jr, PhD

The Benefi ts of Deploying Health Physics Specialists to Joint Operation Areas 83

LTC Scott Mower; MAJ Joshua D. Bast; MAJ Margaret Myers

Forensic and Ethical Issues in Military Behavioral Health

A New Volume in the Borden Institute Textbook of Military Medicine Series 89

 Reviewed by LTC Daniel E. Banks and COL (Ret) Edward Lindeke

JOURNAL

THE UNITED STATES ARMY

MEDICAL DEPARTMENT

FORCE HEALTH PROTECTION: EVOLVING CHALLENGES AND SOLUTIONS
JOURNAL

A Professional Publication

of the AMEDD Community THE UNITED STATES ARMY

MEDICAL DEPARTMENT

Online issues of the AMEDD Journal are available at http://www.cs.amedd.army.mil/amedd_journal.aspx

July – September 2015 US Army Medical Department Center & School PB 8-15-7/8/9

The Army Medical Department Journal [ISSN 1524-0436] is published quarterly 

for The Surgeon General by the AMEDD Journal Office, USAMEDDC&S, AHS CDD

3630 Stanley RD STE B0204, JBSA Fort Sam Houston, TX 78234-6100.

Articles published in The Army Medical Department Journal are listed and 

indexed in MEDLINE, the National Library of Medicine’s premier bibliographic 

database of life sciences and biomedical information. As such, the Journal’s 

articles are readily accessible to researchers and scholars throughout the 

global scientific and academic communities.

CORRESPONDENCE: Manuscripts, photographs, official unit requests to 

receive copies, and unit address changes or deletions should be sent via 

email to usarmy.jbsa.medcom-ameddcs.list.amedd-journal@mail.mil, or by 

regular mail to the above address. Telephone: (210) 221-6301, DSN 471-6301

DISCLAIMER: The AMEDD Journal presents clinical and nonclinical 

professional information to expand knowledge of domestic & international 

military medical issues and technological advances; promote collaborative 

partnerships among Services, components, Corps, and specialties; convey 

clinical and health service support information; and provide a peer-reviewed, 

high quality, print medium to encourage dialogue concerning healthcare 

initiatives.

Appearance or use of a commercial product name in an article published 

in the AMEDD Journal does not imply endorsement by the US Government.

Views expressed are those of the author(s) and do not necessarily reflect 

official policies or positions of the Department of the Army, Department of 

the Navy, Department of the Air Force, Department of Defense, nor any other 

agency of the US Government. The content does not change or supersede 

information in other US Army Publications. The AMEDD Journal reserves the 

right to edit all material submitted for publication (see inside back cover).

CONTENT: Content of this publication is not copyright protected. Reprinted 

ma-terial must contain acknowledgement to the original author(s) and the 

AMEDD Journal.

OFFICIAL DISTRIBUTION: This publication is targeted to US Army Medical 

Department units and organizations, other US military medical organizations, 

and members of the worldwide professional medical community.

By Order of the Secretary of the Army:

Official:

GERALD B. O’KEEFE

Administrative Assistant to the 

Secretary of the Army

Raymond T. Odierno

General, United States Army

Chief of Staff

DISTRIBUTION: Special 1503606

LTG Patricia D Horoho

The Surgeon General

Commander, US Army Medical Command

MG Steve Jones

Commander

US Army Medical Department Center and School
July – September 2015 1

When the Army Medical Department was organized on 

July 27, 1775, it was formed as a team. The act of Congress establishing a hospital for an Army of twenty thousand appointed the following offi cers and attendants: 

one director general and chief physician, four surgeons, 

one apothecary, twenty surgeon’s mates, one clerk, two 

storekeepers and one nurse for every ten sick. Army 

Medicine had been serving our Soldiers for almost a 

year when our nation declared independence—we are 

the country’s fi rst, and arguably best healthcare team. 

Our accomplishments over the past 240 years have been 

signifi cant and have been the result of a team effort. Major Walter Reed discovered the mosquito was the vector 

that carried Yellow Fever, but it was Colonel William 

Gorgas who led the campaign to control mosquitoes 

that allowed construction of the Panama Canal. Today, 

casualty survival rates are at historic levels because of 

the teamwork between AMEDD staff, operational commanders, Soldiers, and Families.

AMEDD doctrine has traditionally described ten medical battlefi eld operating systems; the major healthcare 

functions that are employed to provide care in theater. 

This was a useful concept for planners and helped ensure critical functions were not forgotten. It was aligned 

with Army doctrine which described battlefi eld operating systems as the elements of combat power, but created an artifi cial distinction between care in theater and 

care in garrison. It does not portray the fact that casualty 

care occurs every day in our fi xed hospitals, and that 

every member of the AMEDD makes important contributions to the effort. It also does not adequately refl ect 

recent changes in how we provide healthcare while deployed and at home.

The February 27, 2008, edition of Field Manual 3-0: 

Operations was a fundamental departure from prior 

versions. Just as the 1976 edition of Field Manual 100-

5: Operations brought the Army from the rice paddies 

of Vietnam to the battlefi elds of Western Europe, the 

2008 edition took the Army into 21st century confl icts 

among people. It replaced the older battlefi eld operating 

systems with six warfi ghting functions as the elements 

of combat power:

Mission Command Fires

Movement and Maneuver Sustainment

Intelligence Protection

A seventh warfi ghting function, Engagement, has since 

been added. Warfi ghting functions are defi ned as a 

group of tasks and systems (people, organizations, information, and processes) united by a common purpose 

that commanders use to accomplish missions. It is a useful concept that reinforces the ideas of mutual support 

and fi ghting as combined arms teams.

Field Manual 4-02: Army Health System lists the ten 

medical functions:

Medical Mission Command

Medical Treatment

Hospitalization

Medical Evacuation

Dental Services

Preventive Medicine Services

Combat and Operational Stress Control

Veterinary Services

Medical Logistics

Medical Laboratory Services

It defi nes two missions for the AMEDD: Health Service 

Support and Force Health Protection. The Health Service Support Mission comprising casualty care, medical 

evacuation, and medical logistics falls under the Sustainment Warfi ghting Function. Force Health Protection 

comprising preventive medicine, veterinary services, 

preventive aspects of combat and operational stress control, dental and services, and laboratory services falls 

under the Protection Warfi ghting Function.

The full breadth of AMEDD support to Unifi ed Land 

Operations and the continuum of casualty care are far 

greater than what is described in AMEDD doctrine. 

Efforts to keep Soldiers healthy and fi t allow them to 

perform better while deployed and recover more quickly 

if injured. Medical treatment facilities at home station 

TIME FOR A REVOLUTION IN AMEDD DOCTRINE

Perspective

COMMANDER’S INTRODUCTION

MG Steve Jones
2 http://www.cs.amedd.army.mil/amedd_journal.aspx

provide specialty care through reach-back support and 

telemedicine. Casualty care does not stop after evacuation to the continental United States, but continues 

with defi nitive care and rehabilitation of the wounded, 

the operation of Warrior Transition Units, and the support provided to families. Our approach to healthcare 

has changed signifi cantly since Vietnam. Primary 

healthcare is now provided in Soldier-Centered Medical 

Homes who partner with line unit staff and commanders. The Brigade Healthcare Team now includes a physical therapist and behavioral health provider and nurse 

in addition to medics, physician assistants, dentists, and 

surgeons. The Combat Lifesaver and 68W (Health Care 

Specialist) programs, tactical combat casualty care, 

damage control resuscitation, and damage control surgery have signifi cantly improved prehospital care. Critical care fl ight paramedics and nurses on tactical medevac aircraft have increased survival rates of critically 

injured patients. These patients no longer spend weeks 

in theater hospitals but are evacuated to large medical 

centers within days, with care continuing in hospitals 

along the way. Patient outcomes are monitored closely 

and data collected by the Joint Theater Trauma System 

forms the basis of a robust performance improvement 

system. Regular after action conferences with returning 

units and research teams also provide information that is 

used to improve care. This approach to care and the hard 

work of the entire AMEDD team has raised the survival 

rate of casualties wounded on the battlefi eld from 76% 

in Vietnam to 92% today.

Updating our doctrine will allow the AMEDD to better 

communicate the many ways it supports Unifi ed Land 

Operations every day. It will lead to a better understanding of new concepts in casualty care, the contributions 

of all members of the team, and of how units and leaders 

combine capabilities across medical functions to accomplish their mission. It should start with the premise that 

casualty care begins with the maintenance of a healthy 

and fi t force, extends from the point of injury through 

defi nitive care and rehabilitation, and includes the operation of Warrior Transition Units. After action reviews 

from our fi xed hospitals should be regularly conducted 

and included in lessons learned programs. Gaps in the 

provision of defi nitive care and rehabilitation should be 

identifi ed and included in the requirements process to 

drive development of new capabilities. Medical functions should be updated to refl ect how we provide care 

rather than listing capabilities, and should include:

Medical Mission Command

Primary Care

Prehospital Care

Evacuation and En Route Care

Hospital Care

Defi nite Care and Rehabilitation

Force Health Protection

Medical Sustainment

Medical Engagement

Medical Information

Research and Innovation

Several of these functions are new. The addition of Medical Engagement as a function recognizes the signifi cant 

support the AMEDD provides Combatant Commanders 

in shaping the environment. Including Research and Innovation recognizes the important contributions of these 

members of the team. Medical Information includes not 

only health threats but capabilities, treatment considerations, military, political, economic, and cultural considerations that affect healthcare operations. It includes 

the information needed to develop and sustain a high 

degree of situational understanding while operating in 

complex environments against determined, adaptive enemy organizations and emerging health threats.

Like confl ict, healthcare is a complex, risk-fi lled human 

endeavor. Army healthcare is more complex, has greater 

risk, and is fi lled with even more uncertainty and emotion. We often deliver care under austere and extreme 

conditions, sometimes under fi re, and always under the 

scrutiny of Congress and the media. We understand we 

are part of a bigger team with a bigger mission because 

we serve our nation. Our mission is to keep Soldiers 

healthy and ready to fi ght, giving them confi dence with 

our presence on the battlefi eld and comfort knowing we 

are caring for their families back home. We should refl ect this reality in our doctrine.

PERSPECTIVE
July – September 2015 3

Phlebotomine sand fl ies are vectors of Leishmania species protozoan parasites that cause various forms of 

leishmaniasis and the sand fl y fever viruses (Phlebovirus; Bunyaviridae) in humans and other animals in 

tropical and subtropical regions of the world.1 Leishmania parasites are transmitted by the bite of infected female sand fl ies.2

 An estimated 1.3 million new human 

cases of leishmaniasis and 20,000 to 30,000 deaths occur worldwide annually, which represents a major public 

health problem in affected regions including the Mediterranean basin, Central Asia, Southwest Asia, Southeast Asia, East Africa, Afro-Eurasia, and the Americas.3

This sand fl y-borne disease with no approved vaccines 

and lengthy and costly treatment causes signifi cant 

social and economic burden to civilian populations in 

endemic regions. Phlebotomine sand fl ies also pose a 

signifi cant threat to US military personnel who are deployed to those regions, particularly the Middle East, 

Southwest Asia, and Africa where sand fl y Leishmania 

vectors are endemic.4

 During Operation Iraqi Freedom, 

the US military reported over 1,000 confi rmed cases 

of cutaneous leishmaniasis among deployed personnel 

between May 2003 and November 2004.5

 Various insecticides and application technologies were tested at a 

US airbase in southern Iraq during that period with only 

limited success in reducing sand fl y populations.6

 The 

reasons for failure in completely eliminating or substantially reducing sand fl y populations by frequent insecticide applications remain poorly understood.6

Chemical insecticides have been used for indoor and 

outdoor residual applications and bed net treatments to 

reduce sand fl y populations in endemic regions.1,7-10 The 

insecticide dichlorodiphenyltrichloroethane (DDT) has 

been used to control sand fl ies after World War II.11,12

DDT resistance was reported in Phlebotomus papatasi

(Scopoli) populations in Iran in a fi eld survey conducted 

during 1985-1988, although the use of DDT had been 

discontinued since 1969.13 DDT resistance was similarly 

reported in both P papatasi and P argentipes (Annandale and Brunetti) in India, Nepal, and Sri Lanka.10,14-17

Populations of P papatasi were shown to be susceptible 

to pyrethroid and organophosphate insecticides in laboratory studies of fi eld-collected sand fl y samples from 

North Africa and the Middle East.18,19 However, multiple 

resistances to DDT, organophosphate, and pyrethroid 

insecticides were detected in India and more recently in 

Sudan.14,20 One P papatasi population collected from the 

Surogia village in Sudan demonstrated signifi cant resistance to malathion and propoxur in laboratory bioassays, 

and the observation was also supported by reduced inhibition of acetylcholinesterase by these organophosphate 

Baseline Susceptibility to Pyrethroid

 and Organophosphate Insecticides in

 Two Old World Sand Fly Species

 (Diptera: Psychodidae)

 Andrew Y. Li, PhD Seth C. Britch, PhD

 Adalberto A. Pérez de León, DVM, PhD, MS MAJ Joshua D. Bast, MS, USA

 Kenneth J. Linthicum, PhD Mustapha Debboun, PhD

ABSTRACT

Phlebotomine sand fl ies are blood-feeding insects that transmit Leishmania parasites that cause various forms 

of cutaneous and visceral leishmaniasis and sand fl y fever viruses (Phlebovirus; Bunyaviridae) in humans. 

Sand fl ies pose a signifi cant threat to US military personnel deployed to Leishmania-endemic and sand fl y 

fever endemic regions which include Europe, the Mediterranean basin, Middle East, Central Asia, Southwest 

Asia, and Africa. A research project supported by the Department of Defense Deployed Warfi ghter Protection 

Program was initiated to evaluate the susceptibility of 2 Old World sand fl y species, Phlebotomus papatasi

and P duboscqi, to a number of commonly used pyrethroid and organophosphate insecticides. A new glass vial 

bioassay technique based on the CDC bottle assay was developed for this study. The exposure time-mortality 

relationship at a given insecticide concentration was determined for each insecticide, and their relative toxicity 

against the 2 sand fl y species was ranked based on bioassay results. This study validated the new bioassay 

technique and also generated baseline insecticide susceptibility data to inform future insecticide resistance 

monitoring work.
4 http://www.cs.amedd.army.mil/amedd_journal.aspx

insecticides.20 Compared to mosquitoes and other vector species, there is a general lack of understanding of 

prevalence of insecticide resistance in sand fl ies.1,21 Only 

a few studies have been reported in the literature on possible biochemical or molecular mechanisms of insecticide resistance in sand fl ies.21-23

Previous sand fl y insecticide susceptibility/resistance 

tests were carried out following a World Health Organization (WHO) standard procedure that was originally 

designed for mosquitoes in which insects were exposed 

to an insecticide-impregnated fi lter paper 24,25, or the 

Centers for Disease Control and Prevention (CDC) bottle assay 3,26 that exposes adult sand fl ies to an insecticide-treated surface inside the glass bottle. The WHO 

assay requires sand fl ies to be exposed to insecticide for 

one hour and subsequent transfer of sand fl ies to clean 

containers for incubation under appropriate temperature 

and humidity conditions for 24 hours before mortality 

can be assessed. The CDC bottle assay is more rapid and 

the time-mortality relationship of an insecticide concentration can be established by checking mortality every 

15 minutes during a 2-hour exposure period.3,21 In most 

sand fl y studies, one or two diagnostic concentrations 

were tested for resistance detection.18,19,27,28 Diffi culties 

in collecting suffi cient numbers of live sand fl ies for 

bioassays and the lack of a standard sand fl y bioassay 

technique may have impeded progress in insecticide resistance studies in sand fl ies.

In this study we developed a simplifi ed glass vial bioassay technique based on the CDC bottle assay to test 

exposure time–mortality relationships at selected insecticide concentrations. This test used fewer sand fl ies and 

allowed rapid determination of sand fl y susceptibility. 

The objective of this study was to evaluate this new bioassay technique and determine baseline susceptibility 

to a number of commonly used pyrethroid and organophosphate insecticides in 2 sand fl y species, P papatasi

and P duboscqi (Neveu-Lemaire), which are relevant to 

US military operations in the Middle East and Africa.

MATERIALS AND METHODS

Sand Flies. Adult male P papatasi and P duboscqi were 

used in this study. Adult P papatasi were from a laboratory colony maintained at the US Department of Agriculture Agricultural Research Service Knipling-Bushland 

US Livestock Insects Research Laboratory, Kerrville, 

TX. The colony was established using pupae from a 

long-established Israeli strain of P papatasi maintained 

at the Division of Entomology, Walter Reed Army Institute of Research, Silver Spring, MD. Adult females 

were blood-fed using an in vitro membrane feeding system (Figure 1A). Larvae were fed with a sand fl y larval 

diet consisting of a composted mixture of rabbit feces 

and rabbit food.29 Male and female sand fl ies in the cage 

were fed daily with 30% sucrose water after emergence 

and maintained at 26°±2°C and a relative humidity of 

85%±2% in an environmental chamber. Adult P duboscqi were from a laboratory colony maintained at the 

US Army Medical Research Unit-Kenya (USAMRU-K) 

fi eld station located at the Kenya Agricultural Research 

Institute Marigat Field Station.

Insecticides. Technical-grade permethrin (92.2% active 

ingredient) and coumaphos (97.4% active ingredient) 

were obtained from FMC (Philadelphia, PA) and Bay Vet 

(Shawnee, KS), respectively. All other technical-grade 

insecticides were obtained from Chem Service (West 

Chester, PA). Formulated etofenprox (Zenivex E4 RTU, 

4%) was a product of Wellmark International (Schaumburg, IL). Dilutions of technical insecticide in acetone 

were made to generate test concentrations ranging from 

0.0001% to 0.1% (Figure 1B). To treat the inside surface 

of glass vials with insecticide, a volume of 0.2 mL of 

the test solution was added to a 20 mL glass scintillation vial (Fisher Scientifi c, Pittsburgh, PA), which was 

placed on a hotdog roller (Figure 1C) for 1 hour to allow 

evaporation of solvent and uniform insecticide coating 

of the glass surface. The treated vials were recapped and 

stored at room temperature after drying and used within 

2 days for study of P papatasi or 4 to 7 days for study of 

P duboscqi to allow for travel to the Kenya fi eld location. 

Dilution of formulated etofenprox was made using bottled water at the fi eld station in Kenya. Etofenprox-treated vials were set at ambient room temperature (≈25°C to 

30°C) with frequent rolling by hand for 3 hours to allow 

solvent to evaporate. Three vials were prepared for each 

test concentration.

Bioassays. All bioassays with P papatasi were done using adult males of mixed ages (3 to 10 days) for reasons 

noted below under normal laboratory conditions (Temperature 23°±2°C). Five to 7 treated vials, each representing one concentration, and the control vial that was 

treated with acetone only were placed on the counter 

(Figure 1D, 1E). Adult male sand fl ies were directly aspirated from the holding cage using a mouth aspirator 

and briefl y knocked down with CO2 before being placed 

in the vials (10 sand fl ies/vial). Flies woke up in about 30 

seconds. Fly mortality in each vial was checked every 

10 minutes, according to the order in which fl ies were 

added to each vial, for up to 3 hours, or until all fl ies in 

each vial were dead (Figure 1F). Each experiment was 

repeated 3 times so each test concentration had a total of 

3 replicates. Bioassays with P duboscqi were conducted 

following the same protocol using adult males of mixed 

ages (≈3 to 10 days) kept at room temperature in the 

BASELINE SUSCEPTIBILITY TO PYRETHROID AND ORGANOPHOSPHATE INSECTICIDES

IN TWO OLD WORLD SAND FLY SPECIES (DIPTERA: PSYCHODIDAE)
July – September 2015 5

THE ARMY MEDICAL DEPARTMENT JOURNAL

USAMRU-K fi eld station in Kenya (Figure 2). Room 

temperature in the laboratory ranged between 25°C and 

30°C. All glass vials were pretreated with insecticides 

at the USDA laboratory in Texas, except for etofenprox 

which was prepared on site.

Data Analysis. Probit analysis of time-mortality data for each insecticide and concentration tested were conducted using POLO PLUS 

software.30 The LT50 and LT90 (exposure time at 

which 50% and 90% fl ies died) were generated 

and used to compare the relative toxicity of insecticides tested in this study.

RESULTS AND DISCUSSION

Results with P papatasi. Adult males began to 

die after 30 to 40 minutes of exposure to the 

lowest concentration (0.00001% or 0.5 ng/cm2) 

of pyrethroid insecticides (Figure 3), and it 

took over 120 minutes to reach 100% mortality. Both the time at which sand fl ies started 

to die and the time when 100% mortality was 

reached decreased with increasing insecticide 

concentrations (0.0001, 0.001, and 0.01%). The 

time (minutes) it took to kill 50% of the treated fl ies (LT50) for each of 3 concentrations of 

6 pyrethroid insecticides tested are listed in 

Table 1. Based on LT50 data, prallethrin and 

λ-cyhalothrin were most toxic to sand fl ies, 

followed in order by deltamethrin, cyfl uthrin, 

cypermethrin, and permethrin. Compared to 

pyrethroid insecticides, organophosphate insecticides were generally less toxic (Figure 4). 

At the lowest concentration (0.00001%) tested, 

organophosphate insecticides were slow in killing sand fl ies. Sand fl ies started to die only after 

over 60-minute exposure (diazinon) or even after 120 minutes (chlorpyrifos). Similarly, higher 

concentrations of organophosphate insecticide 

caused fl ies to die more rapidly. Based on LT50

data listed in Table 2, the order of relative toxicity of the 4 organophosphate insecticides 

was diazinon ≥ chlorpyrifos > coumaphos > 

dichlorvos.

Results with P duboscqi. Toxicity bioassay results for 6 pyrethroid and 3 organophosphate 

insecticides against adult males are listed in 

Table 2. Prallethrin was again the most toxic 

pyrethroid insecticide. Susceptibility of P duboscqi sand fl ies to permethrin was similar to 

that of P papatasi. The order of relative toxicity 

was: prallethrin > λ-cyhalothrin > deltamethrin 

> cypermethrin > permethrin > etofenprox. A 

limited data set was obtained for the 3 organophosphate 

insecticides tested against P duboscqi sand fl ies. Chlorpyrifos appeared to be more toxic than malathion and 

carbaryl (Table 2).

B

C D

F

A

E

Figure 1: (A) Membrane feeding of adult sand fl ies; (B,C) treatment of glass 

vials with insecticide; (D,E,F) determination of sand fl y mortality.

Figure 2. Sand fl y bioassays were conducted at the US Army Medical Research Unit-Kenya fi eld station in Marigat, Kenya.
6 http://www.cs.amedd.army.mil/amedd_journal.aspx

The bioassay technique involving a glass vial to measure 

exposure time-mortality relationships has been used 

previously to assess sand fl y susceptibility to a number 

of insecticides.28 Based on data obtained in this study, 

we verifi ed that our modifi ed version of the CDC bottle 

bioassay technique was sensitive and reliable. We were 

able to demonstrate a time-mortality relationship at different concentrations of the same insecticide, as well as 

compare sand fl y susceptibility to different insecticides 

at particular test concentrations. Based on LT50 data, we 

were able to rank relative toxicity of pyrethroid and organophosphate insecticides to both P papatasi and P duboscqi sand fl y species. The susceptibility data obtained 

from this study will help military entomologists in the 

fi eld with the selection of insecticides for sand fl y control. The results from this study could also be used as 

baseline susceptibility data for comparative purposes in 

resistance monitoring of fi eld-collected sand fl y populations. Additionally, having reference susceptibility data 

to several conventional pyrethroid and organophosphate 

insecticides facilitates future work involving the screening of new insecticides, including essential oils and other natural products31 for sand fl y control.

Like mosquitoes, only female sand fl ies are blood feeders. Many insecticide studies use females as test subjects. 

Because we were at the early stage of establishing the P 

papatasi colony, females were reserved for maintaining 

and propagating the sand fl y colony. Therefore, 

we used adult males for toxicity bioassays. Male 

sand fl ies are slightly smaller than females, and 

are known to be more sensitive than females to 

insecticides.32 However, our results indicate that 

relative toxicity of insecticides against sand fl ies 

could be assessed using males in bioassays. Depending on the collection technique employed 

in the fi eld, sand fl y samples can include adult 

males and females. Therefore, future laboratory studies will involve experiments to ascertain if males are as susceptible to insecticides 

as females.

Although P duboscqi is one of the major sand fl y 

species transmitting leishmaniasis in Kenya and 

has been the target of control efforts,33,34 little 

is known about insecticide susceptibility in this 

sand fl y species. To the best of our knowledge, 

this is the fi rst report of laboratory testing of P 

duboscqi susceptibility to commonly used pyrethroid and organophosphate insecticides.

Due to the diffi culty in collecting large numbers 

of live sand fl ies in the fi eld, having enough fl ies 

to run a dose-response bioassay, which requires 

a large number of sand fl ies, is impractical. 

While the standard CDC bottle assay uses 250 

ml Wheaton glass bottles, the 10 ml glass vials we used in this study were smaller, required 

fewer sand fl ies to run a test, and are amenable 

for fi eld deployment. Treatment of glass vials with technical insecticides in a resourceconstrained environment may be diffi cult. Pretreatment of glass vials at a standard laboratory 

equipped with a simple roller and similar devices would allow the use of pretreated glass vials 

in fi eld locations at least for 7 days posttreatment, as demonstrated in this study. Diagnostic 

concentrations are recommended for a number 

BASELINE SUSCEPTIBILITY TO PYRETHROID AND ORGANOPHOSPHATE INSECTICIDES

IN TWO OLD WORLD SAND FLY SPECIES (DIPTERA: PSYCHODIDAE)

20 40 60 80 100 120 140 160

20

0

40

60

80

100

0.00001%

0.0001%

0.001%

0.01%

% Mortality (SE)

Time (minutes)

Permethrin

Time (minutes)

0.00001%

0.0001%

0.001%

0.01%

20 40 60 80 100 120

20

0

40

60

80

100

Cyfluthrin

% Mortality (SE)

Figure 3. Exposure time-mortality responses of male P papatasi to 

different concentrations of representative pyrethroid (permethrin, cyfl uthrin) insecticides.
July – September 2015 7

THE ARMY MEDICAL DEPARTMENT JOURNAL

of insecticides against mosquitoes.25

Although several concentrations have 

been tested in susceptibility studies, no 

standard diagnostic insecticide concentrations are available for most insecticides to detect resistance in sand fl y species.19,28 Collaboration among sand fl y 

researchers is necessary to coordinate 

efforts to develop standards for insecticide resistance monitoring in sand fl ies. 

This study provides baseline susceptibility data that can inform future insecticide resistance monitoring in sand fl ies.

ACKNOWLEDGMENTS

We thank Darci Burchers, DeEsta Hyatt 

(USDA Agricultural Research Service 

Knipling-Bushland US Livestock Insects 

Research Laboratory), Francis Ngere, 

Daniel Ngonga, and Clifford Chepchieng 

(USAMRU-Kenya) for technical assistance during this study. This work was 

funded by US Department of Agriculture 

appropriated funds (USDA-ARS CRIS 

project 6205-32000-033000D) and Deployed Warfi ghter Protection Research 

Program (Project number 6201-32000-

033017R) of the US Department of Defense through the Armed Forces Pest 

Management Board.

REFERENCES

1. Alexander B, Maroli M. Control of 

phlebotomine sandfl ies. Med Vet Entomol. 2003;17(1):1-18.

2. Killick-Kendrick R. The biology and 

control of phlebotomine sand fl ies. 

Clinic Dermatol. 1999;17:279-289.

3. World Health Organization. Leishmaniasis Fact Sheet No. 375 [internet]. February 2015. Available at: 

http://www.who.int/mediacentre/factsheets/fs375/

en/. Accessed April 15, 2015.

4. Cope SE, Strickman DA, White GB. The Deployed 

Warfi ghter Protection Research Program: fi nding 

new methods to vanquish old foes. US Army Med 

Dept J. October-December 2012:9-20.

5. Lay JC. Leishmaniasis among US armed forces, 

January 2003-November 2004. MSMR. 2004;10:2-5.

6. Coleman RE, Burkett DA, Sherwood V, et al. Impact of Phlebotomine sand fl ies on United States 

military operations at Tallil Air Base, Iraq: 6. Evaluation of insecticides for the control of sand fl ies. J 

Med Entomol. 2011;48(3):584-599.

7. Maroli M, Majori G. Permethrin-impregnated 

curtains against phlebotomine sandfl ies (Diptera: 

Psychodidae): laboratory and fi eld studies. Parassitologia. 1991;33(suppl):399-404.

8. Morsy TA, Aboul-Ela RG, El-Gozamy BM, Salama MM, Ragheb DA. Residual effects of four insecticides applied for indoor control of Phlebotomus papatasi (Scopoli). J Egyptian Soc Parasitol.

1993;23(2):485-492.

9. Kasili S, Kutima H, Mwandawiro C, Ngumbi PM, 

Anjili CO. Laboratory and semi-fi eld evaluation of 

long-lasting insecticidal nets against leishmaniasis vector, Phlebotomus duboscqi in Kenya. J Vec 

Borne Dis. 2010;47(1):1-10.

Table 1. Results of glass vial bioassays of pyrethroid and organophosphate insecticides against the Israeli strain of P papatasi maintained at USDA-ARS-KBUSLIRL 

in Kerrville, Texas.

Solution

Concentration

(%)

Surface

Concentration

Slope χ2 (n) LT50 (95% CI)

Pyrethroids

Cypermethrin 0.0001 5 ng/cm2 6.5 13.9 (14) 30.0 (26.9-32.9)

0.001 50 ng/cm2 4.4 17.4 (10) 21.1 (16.1-26.2)

0.01 0.5 μg/cm2 / / <20

Deltamethrin 0.0001 5 ng/cm2 2.6 19.6 (21) 29.9 (24.8-35.0)

0.001 50 ng/cm2 / / <20

0.01 0.5 μg/cm2 n/t

Cyfluthrin 0.0001 5 ng/cm2 6.1 7.4 (17) 32.1 (28.9-35.2)

0.001 50 ng/cm2 4.9 4.9 (9) 16.5 (14.0-19.)

0.01 0.5 μg/cm2 n/t

λ-cyhalothrin 0.0001 5 ng/cm2 2.4 6.5 (14) 12.8 (8.1-16.5)

0.001 50 ng/cm2 / / <10

0.01 0.5 μg/cm2 n/t

Permethrin 0.0001 5 ng/cm2 5.0 11.6 (45) 85.9 (80.1-91.8)

0.001 50 ng/cm2 5.1 13.1 (17) 34.2 (30.5-37.8)

0.01 0.5 μg/cm2 4.4 6.9 (8) 14.2 (11.6-16.7)

Prallethrin 0.0001 5 ng/cm2 4.9 4.1 (8) 13.8 (11.4-16.0)

0.001 50 ng/cm2 / / <10

0.01 0.5 μg/cm2 n/t

Organophosphates

Chlopyrifos 0.001 50 ng/cm2 8.7 26.1 (21) 53.1 (49.1-57.2)

0.01 0.5 μg/cm2 7.5 5.5 (14) 28.3 (25.6-30.9)

0.1 5 μg/cm2 n/t

Coumaphos 0.001 50 ng/cm2 12.2 16.3 (28) 77.6 (74.3-81.2)

0.01 0.5 μg/cm2 5.4 6.5 (17) 34.7 (31.2-38.2)

0.1 5 μg/cm2 n/t

Diazinon 0.001 50 ng/cm2 12.1 13.7 (20) 53.4 (50.5-56.2)

0.01 0.5 μg/cm2 10.1 9.6 (10) 25.5 (23.4-27.6)

0.1 5 μg/cm2 n/t

Dichlovos 0.001 50 ng/cm2 3.3 10.2 (58) 122.0 (112.4-254.8)

0.01 0.5 μg/cm2 6.6 9.1 (32) 58.4 (54.3-62.4)

0.1 5 μg/cm2 n/t

LT50 indicates exposure time (minutes) at which 50% of the flies had died.

n/t indicates not tested.
8 http://www.cs.amedd.army.mil/amedd_journal.aspx

10. Chowdhury R, Akhter S, Huda MM, et 

al. The Indian and Nepalese programmes 

of indoor residual spraying for the elimination of visceral leishmaniasis: performance 

and effectiveness. Ann Trop Med Parasitol. 

2011;105(1):31-45.

11. Jacusiel F. Sandfl y control with DDT residual 

spray-fi eld experiments in Palestine. Bull Entomol Res. 1947;38(3):479-488.

12. Wilamowski A, Pener H. Effi cacy of microencapsulated insecticides against the sandfl y, 

Phlebotomus papatasi Scopoli. J Vector Ecol.

2003;28:229-233.

13. Rashti MA, Panah HY, Mohamadi HS, Jedari M. Susceptibility of Phlebotomus papatasi (Diptera: Psychodidae) to DDT in some 

foci of cutaneous leishmaniasis in Iran. J Am 

Mosq Control Assoc. 1992;8(1):99-100.

14. Amalraj DD, Sivagnaname N, Srinivasan R. 

Susceptibility of Phlebotomus argentipes and 

P papatasi (Diptera: Psychodidae) to insecticides. J Comm Dis. 1999;31(3):177-180.

15. Kishore K, Kumar V, Kesari S, Bhattacharya SK, Das P. Susceptibility of Phlebotomus argentipes against DDT in endemic 

districts of North Bihar, India. J Comm Dis.

2004;36(1):41-44.

16. Singh RK, Mittal PK, Dhiman RC. Insecticide susceptibility status of Phlebotomus argentipes, a vector of visceral leishmaniasis in 

different foci in three states of India. J Vec 

Borne Dis. 2012;49(4):254-257.

17. Dinesh DS, Das P, Das ML, et al. Insecticide 

susceptibility of Phlebotomus argentipes

in visceral leishmaniasis endemic districts 

in India and Nepal. PLoS Negl Trop Dis. 

2010;4(10):e859.

18. Tetreault GE, Zayed AE-BB, Hanafi HA, 

Beavers GM, Zeichner BC. Susceptibility of 

sand fl ies to selected insecticides in North 

Africa and the Middle East. J Am Mosq Control Assoc. 2001;17(1):23-27.

19. Faraj C, Ouahabi S, Adlaoui EB, EL Elkohli M, 

Lakraa L, El Rhazi M, Ameur B. Insecticide susceptibility status of Phlebotomus (Paraphlebotomus) sergenti and Phlebotomus (Phlebotomus) papatasi in endemic foci of cutaneous leishmaniasis 

in Morocco. Parasit Vectors. 2012;5(1):51.

20. Hassan MM, Widaa SO, Osman OM, Numiary 

MSM, Ibrahim MA, Abushama HM. Insecticide 

resistance in the sand fl y, Phlebotomus papatasi

from Khartoum State, Sudan. Parasit Vectors.

2012;5(1):46.

21. Brogdon WG, McAllister JC. Insecticide resistance and vector control. Emerging Infect Dis.

1998;4(4):605-613.

22. Surendran SN, Karunaratne SHPP, Adams Z, 

Hemingway J, Hawkes NJ. Molecular and biochemical characterization of a sand fl y population 

from Sri Lanka: Evidence for insecticide resistance 

due to altered esterases and insensitive acetylcholinesterase. Bull Entomol Res. 2005:95 (4):371-380.

23. Temeyer KB, Brake DK, Tuckow AP, Li AY, Pérez 

de León AA. Acetylcholinesterase of the sand fl y, 

Phlebotomus papatasi (Scopoli): cDNA sequence, 

baculovirus expression, and biochemical properties. Parasit Vectors. 2013;6:31.

BASELINE SUSCEPTIBILITY TO PYRETHROID AND ORGANOPHOSPHATE INSECTICIDES

IN TWO OLD WORLD SAND FLY SPECIES (DIPTERA: PSYCHODIDAE)

Figure 4. Exposure time-mortality responses of male P papatasi to 

different concentrations of representative organophosphate (diazinon, 

chlorpyrifos) insecticides.

20 40 60 80 100 120 140 160 180 200

20

0

40

60

80

100

Time (minutes)

Diazinon

0.00001%

0.0001%

0.001%

0.01%

% Mortality (SE)

Time (minutes)

% Mortality (SE)

20 40 60 80 100 120 140 160 180 200 220

20

0

40

60

80

100

0.00001%

0.0001%

0.001%

0.01%

Chlorpyrifos
July – September 2015 9

THE ARMY MEDICAL DEPARTMENT JOURNAL

24. World Health Organization. Instructions for determining the susceptibility or resistance of adult black 

fl ies, sand fl ies and biting midges to 

insecticides. Geneva, Switzerland: 

World Health Organization; 1981. 

WHO/VBC/81.810.

25. World Health Organization. Vector 

resistance to pesticide. . Geneva, 

Switzerland: World Health Organization; 1992. Tech Rep. Ser 818.

26. Centers for Disease Control and 

Prevention. Guideline for evaluating insecticide resistance in vectors using the CDC bottle bioassay 

[internet]. Available at: http://www.

cdc.gov/malaria/resources/pdf/fsp/

ir_manual/ir_cdc_bioassay_en.pdf. 

Accessed March 3, 2015.

27. Aboul Ela RG, Morsy TA, el-Gozamy BM, Ragheb DA. The susceptibility of the Egyptian Phlebotomus 

papatasi to fi ve insecticides. J Egyp 

Soc Parasitol. 1993;23(1):69-94.

28. Maroli M, Cianchi T, Bianchi R, 

Khoury C. Testing insecticide susceptibility of Phlebotomus perniciosus and P papatasi (Diptera: 

Psychodidae) in Italy. Ann 1st Super 

Sanita. 2002;38(4):419-423.

29. Young, DG, Perkins P, Endris RG. A 

larval diet for rearing phlebotomine 

sand fl ies (Diptera, Psychodidae). J 

Med Entomol. 1981;18(5):446-446.

30. Robertson JL, Russell RM, Savin 

NE. PoloPlus Probit and Logit 

Analysis User’s Guide. Berkeley, 

CA: LeOra Software;2007.

31. Dinesh DS, Kumari S, Kumar V, 

Das P. The potentiality of botanicals and their products as an alternative to chemical insecticides to 

sandfl ies (Diptera: Psychodidae): a review. J Vector Borne Dis. 2014;51(1):1-7.

32. Alexander B, Barros VC, de Souza SF, et al. Susceptibility to chemical insecticides of two Brazilian populations of the visceral leishmaniasis vector Lutzomyia longipalpis (Diptera: Psychodidae). 

Trop Med Int Health. 2009;10;1272-1277.

33. Beach R, Hendricks L, Oster C, Kiilu G, Leeuwenburg J. Cutaneous leishmaniasis in Kenya: transmission of Leishmania major to man by the bite of 

a naturally infected Phlebotomus duboscqi. Trans 

Royal Soc Trop Med Hyg. 1984;78(6):747-751.

34. Britch SC, Linthicum KJ, Walker TW, et al. Evaluation of ULV applications against Old World sand 

fl y (Diptera: Psychodidae) species in equatorial 

Kenya. J Med Entomol. 2011;48(6):1145-1159.

AUTHORS

Dr Li is a Research Entomologist, USDA-ARS Invasive Insect Biocontrol and Behavior Laboratory, Beltsville, MD.

Dr Pérez de León is Director and Research Leader, USDAARS Knipling-Bushland US Livestock Insects Research 

Laboratory, Kerrville, TX.

Dr Linthicum is Director, USDA-ARS Center for Medical, 

Agricultural, and Veterinary Entomology, Gainesville, FL.

When this article was written, MAJ Bast was assigned to 

the US Army Medical Research Unit, Nairobi, Kenya.

Dr Britch is a Research Entomologist, USDA-ARS Center for Medical, Agricultural, and Veterinary Entomology, 

Gainesville, FL.

Dr Debboun is Director, Mosquito Control Division, 

Harris County Public Health & Environmental Services, 

Houston, TX.

Table 2. Results of glass vial bioassays of pyrethroid and organophosphate insecticides against the Israeli strain of P duboscqi strain maintained at USAMRU-K fi eld 

station in Marigat, Kenya.

Solution

Concentration

(%)

Surface

Concentration

Slope χ2 (n) LT50 (95% CI)

Pyrethroids

Cypermethrin 0.0001 5 ng/cm2 2.8 34.3 (19) 109.2 (74.7-503.2)

0.001 50 ng/cm2 6.0 8.3 (13) 24.1 (21.0-27.0)

0.01 0.5 μg/cm2 / / <10

Deltamethrin 0.0001 5 ng/cm2 4.0 58.2 (19) 57.1 (43.6-77.8)

0.001 50 ng/cm2 10.5 6.8 (10) 20.2 (17.8-22.0)

0.01 0.5 μg/cm2 / / <10

Etofenprox 0.0001 5 ng/cm2 5.8 34.6 (13) 25.0 (18.9-30.7)

0.001 50 ng/cm2 3.4 36.8 (22) 32.5 (23.5-40.8)

0.01 0.5 μg/cm2 5.6 5.0 (6) 17.9 (14.3-21.2)

λ-cyhalothrin 0.0001 5 ng/cm2 2.0 8.1 (22) 65.3 (52.9-91.5)

0.001 50 ng/cm2 / / <20

0.01 0.5 μg/cm2 / / <5

Permethrin 0.0001 5 ng/cm2 3.1 11.2 (25) 80.8 (69.8-101.4)

0.001 50 ng/cm2 4.9 19.4 (23) 33.1 (29.4-36.5)

0.01 0.5 μg/cm2 / / <20

Prallethrin 0.0001 5 ng/cm2 2.4 10.5 (13) 22.4 (17.8-27.0)

0.001 50 ng/cm2 / / <10

0.01 0.5 μg/cm2 / / <5

Organophosphates

Carbaryl 0.001 50 ng/cm2 8.1 141.1 (19) 32.2 (21.8-40.3)

0.01 0.5 μg/cm2 7.5 270.4 (13) /

0.1 5 μg/cm2 11.4 3.9 (10) 26.8 (24.8-28.8)

Chlopyrifos 0.001 50 ng/cm2 6.2 33.1 (13) 21.4 (16.6-25.5)

0.01 0.5 μg/cm2 / / <10

0.1 5 μg/cm2 / / <10

Malathion 0.001 50 ng/cm2 n/t

0.01 0.5 μg/cm2 6.3 8.6 (7) 13.1 (10.6-15.5)

0.1 5 μg/cm2 n/t

LT50 indicates exposure time (minutes) at which 50% of the flies had died.

n/t indicates not tested.
10 http://www.cs.amedd.army.mil/amedd_journal.aspx

Leishmaniasis is caused by parasitic protozoa in the genus Leishmania that are vectored by Phlebotomine sand 

fl ies. The disease manifests in cutaneous, mucocutaneous, or visceral forms. There are an estimated 12 million people in tropical and subtropical regions currently 

infected with some form of this disease.1

 The cutaneous form of the disease was frequently observed among 

US service members deployed in Iraq and Afghanistan 

with 1,670 confi rmed cases reported from 2003 to 2014.2

However, the actual number of cases was probably 

much larger. Initial efforts by the US military to control 

sand fl ies relied on insecticides and proved largely unsuccessful due to harsh environmental conditions and 

the cryptic nature of sand fl ies.3

 Current guidelines for 

managing sand fl ies endorse an integrated approach and 

stress the importance of personal protective measures 

that include using topical repellents on the skin, treating 

uniforms with permethrin, and sleeping under insecticide treated nets (ITNs).4

Insecticide treated nets are recognized by the global 

health community as an important tool to help control 

sand fl ies and prevent Leishmaniasis.5,6 They have been 

most frequently used and are most well known for their 

use in malaria control programs where there is a current 

emphasis to switch to long lasting insecticide nets that 

do not require periodic retreatment with an insecticide.7

Sand fl ies cannot be adequately controlled with standard 

mosquito nets because most species are small enough to 

pass through the mesh, and decreasing the mesh size restricts air fl ow and makes the nets uncomfortable to use 

in hot environments. Therefore, protection is only observed after the addition of an insecticide.8

 Insecticide 

treated nets have been compared to baited traps where 

sand fl ies are attracted to host odors emanating from 

inside the net and then contact the insecticide treated 

fabric while trying to enter.5

 Even if sand fl ies are able to 

pass through ITNs, the nets are capable of killing sand 

fl ies or changing their feeding behavior.9,10

One important question that has not been investigated 

is whether ITNs provide the same level of protection 

against Leishmania infected sand fl ies as they do for 

noninfected sand fl ies. It is well documented that Leishmania infection causes many changes to sand fl y feeding behavior and host seeking.11-13 During normal feeding, noninfected sand fl ies will land on a host, search 

for a suitable location, begin probing, and then usually 

obtain a full blood meal within 2 to 3 minutes. In contrast, infected sand fl ies will often probe multiple times 

and for several minutes and never successfully obtain a 

blood meal. They are also more persistent and are more 

Efficacy of Permethrin Treated

 Bed Nets Against Leishmania major

 Infected Sand Flies

 Tobin Rowland

 MAJ Silas A. Davidson, MS, USA

 Kevin Kobylinski, PhD

 Claudio Menses

 Edgar Rowton, PhD

ABSTRACT

Insecticide treated nets (ITNs) are a potential tool to help control sand fl ies and prevent Leishmaniasis. However, little is currently known about the response of Leishmania infected sand fl ies to ITNs. In this study, Phlebotomus duboscqi sand fl ies were infected with the parasite Leishmania major. Infected and noninfected sand 

fl ies were then evaluated against permethrin treated and untreated bed nets in a laboratory assay that required 

sand fl ies to pass through suspended netting material to feed on a mouse serving as an attractive host. The 

number of sand fl ies passing through the nets and blood feeding was recorded. There was not a signifi cant difference in the ability of infected or noninfected sand fl ies to move through treated or untreated nets. Fewer sand 

fl ies entered the permethrin treated nets compared to the untreated nets, indicating that permethrin creates an 

effective barrier. The results show that in addition to reducing the nuisance bites of noninfected sand fl ies, ITNs 

also protect against Leishmania infected sand fl ies and therefore can play in key role in reducing the rates of 

Leishmaniasis. This study is important to the Department of Defense as it continues to develop and fi eld new 

bed nets to protect service members.
July – September 2015 11

likely to return and feed if interrupted.13 This behavior 

likely enhances transmission of Leishmania parasites, 

but it is not known if it makes sand fl ies more likely to 

enter ITNs and seek a blood meal.

In this laboratory study, the ability of Leishmania major

infected Phlebotomus duboscqi to pass through a permethrin treated net and take a blood meal was compared 

to noninfected fl ies. Permethrin was selected because 

it is the insecticide used by the military to treat bed 

nets and belongs to the pyrethroid class of insecticides 

which is preferred by most global health organizations.14

The sand fl y P duboscqi is a major vector of cutaneous 

leishmaniasis in Africa. The parasite L major is found 

in Africa and the Middle East and was the leading cause 

of cutaneous Leishmaniasis among service members in 

Iraq.4

MATERIALS AND METHODS

Sand Flies

Phlebotomus duboscqi from Mali were obtained from 

the National Institute of Health and reared in the insectary at the Walter Reed Army Institute of Research by 

the methods described in Modi and Rowton.15 The sand 

fl ies were maintained at 26°C and 80% relative humidity.

Infection with Leishmania

Leishmania major RYN strain parasites were used for 

infections. A membrane feeding apparatus and water 

bath circulator were used to infect sand fl ies.16 Defi brinated rabbit blood was spiked with L major promastigotes and placed in the feeding apparatus. The feeding 

apparatus had a chicken skin membrane attached to 

the lower opening and was placed on a screened carton holding sand fl ies. The sand fl ies were allowed to 

blood feed to repletion. Dissections on day 13 postinfection and immediately after experimental manipulation revealed 90% to 100% infection rates. Noninfected 

control sand fl ies were blood fed in the same way, but L 

major promastigotes were not added to the blood meal.

Insecticide Treated Nets

Untreated white, 196-mesh polyester netting material 

was obtained from Vestergaad Frandsen, Inc (Lausanne, Switzerland). This material was selected because 

it was known that sand fl ies could pass through. The 

fabric was cut into 232.4 cm2

 squares before treatment. 

A wooden stand was used to hold an aerosol can of Repel Permanon (0.5% permethrin) (United Industries 

Corp, Middleton, WI) with the nozzle 20 cm from the 

center of the material. The fabric was then sprayed for 

5 seconds on each side. This method of permethrin application was selected because it simulates how bed nets 

are currently treated by the US military.14 The treated 

material was allowed to dry for 48 hours in a chemical 

fume hood and then stored individually in plastic bags at 

room temperature until use. Untreated netting material 

was cut in similar squares and hung in a separate chemical hood for 48 hours.

Assays

Assays were conducted using a Grieco module.17 The 

module consists of 2 Plexiglass cylinders each 15.9 cm 

long and 10.2 cm in diameter (Figure 1). A Tefl on linking section (4.4 cm thick, 10.2 cm diameter) connects 

the 2 cylinders and contains a butterfl y valve (5.5 cm 

diameter) that allows movement between the 2 modules. 

Netting material was stretched tightly between the 2 

clear chambers with the butterfl y door closed. With the 

door open, the only way for sand fl ies to move between 

the cylinders is through the netting material.

The assays were performed in a glove box with the temperature maintained between 20°C and 24°C and 70% to 

80% relative humidity. The sand fl ies used in the assays 

were approximately 15 to 17 days postemergence and 

had been water and sugar starved for 12 hours prior to 

use. A single ICR (Institute for Cancer Research) mouse 

(Charles River Laboratory, Frederick, MD) was anesthetized and placed in one chamber to serve as a host 

for sand fl ies. Twenty female P duboscqi were placed 

in the opposite chamber and were given 3 minutes to 

acclimate. The lights were turned off making the room 

completely dark and the butterfl y door was then opened 

allowing the sand fl ies to move about undisturbed. After 

20 minutes, the sand fl ies from each side of the module 

Figure 1. Grieco module used to conduct laboratory assays 

with insecticide treated nets.
12 http://www.cs.amedd.army.mil/amedd_journal.aspx

were counted. The sand fl ies were then dissected to verify blood feeding with any trace of blood considered a 

blood fed sand fl y. The midguts were also checked for 

metacyclic promastigotes to confi rm infection.

Study Design

There were 4 experimental groups based on the combination of netting material (permethrin treated, untreated) and sand fl ies (infected, noninfected). Each combination was replicated 6 times. Data was collected on the 

number of sand fl ies passing through the nets and blood 

feeding for all combinations. The data were analyzed by Fisher exact tests, which were conducted 

using a 2-sided alpha of 0.05.

RESULTS

There was not a signifi cant difference in the movement of Leishmania infected or noninfected sand 

fl ies through treated or untreated nets as shown by 

Figure 2. For the treated netting material, 27 of 73 

(37%) of the infected sand fl ies passed through and 

23 of 77 (30%) of the noninfected sand fl ies passed 

through (P=.6245, χ2

=0.4267). Results for the untreated material were similarly not signifi cant with 

42 of 78 (54%) of infected sand fl ies passing through 

and 49 of 81 (60%) of the noninfected sand fl ies 

passing through (P=.394, χ2=0.988).

There was a signifi cant difference when comparing the 2 types of netting material. Fewer sand fl ies 

passed through the permethrin treated net compared 

to the untreated net for both infected (P=.0369, 

χ2

=4.9784) and noninfected sand fl ies (P=.0002, 

χ2

=14.6701). This indicates that the treated netting 

material was more effective than the untreated net 

at preventing sand fl ies from moving through the 

mesh. It was noted for the permethrin treated net 

that most of the sand fl ies that passed through the 

netting material were knocked down or dead after 20 

minutes. All of the sand fl ies exposed to the untreated 

net were still active.

There were no blood-fed sand fl ies in modules containing the permethrin treated net. When the untreated netting material was used, 7% of the infected sand fl ies 

took a blood meal and 25% of the noninfected sand fl ies 

took a blood meal as shown in the Table.

COMMENT

This is the fi rst study to assess the ability of Leishmania

infected sand fl ies to pass through ITNs. Both infected 

and noninfected sand fl ies passed through permethrin 

treated netting material at similar rates. Infections with 

very high levels of parasites did not lead to changes in 

behavior,11-13 and thus allow infected sand fl ies to bypass 

the protection of ITNs. The results suggest that ITNs 

likely play an important role in reducing the transmission of Leishmaniasis in addition to reducing the nuisance bites of sand fl ies.

Permethrin treated nets reduced vector host interactions 

for both infected and noninfected sand fl ies. This shows 

that permethrin is effective in lowering the movement 

of sand fl ies into bed nets. However, a small percentage 

of sand fl ies were still able to pass through treated nets. 

This may have been an artifact of the testing module 

since it was small and enclosed, and, without the ability to escape, some sand fl ies may have found their way 

through. 

It is worth noting that in this study even when sand fl ies 

did pass through the permethrin treated net, none of 

them took a blood meal and most were knocked down. 

This corresponds to other studies showing that exposure to permethrin alters sand fl y feeding behavior even 

if it does not kill them directly.9,10 There have been a 

Blood feeding results for sand fl ies that were able to pass 

through the netting material to host side of the module.

Permethrin

Treated Net

Untreated

Net

Leishmania infected sand flies 0/27 (0%) 3/42 (7%)

NonInfected sand flies 0/23 (0%) 12/49 (24%)

Proportion of P duboscqi on Host Side of Net

P=.0002

P=.0369

Treated 

Net

Treated 

Net

Untreated

Net

Untreated

Net

0.8

0.0

0.6

0.4

0.2

Uninfected P duboscqi

L major infected P duboscqi

Figure 2. Proportion of Phlebotomus duboscqi that passed through 

treated or untreated bed net material.

EFFICACY OF PERMETHRIN TREATED BED NETS AGAINST LEISHMANIA MAJOR INFECTED SAND FLIES
July – September 2015 13

THE ARMY MEDICAL DEPARTMENT JOURNAL

few unpublished studies at the Walter Reed Army Institute of Research (WRAIR) where blood feeding was 

observed after sand fl ies contacted permethrin treated 

nets, and contact with permethrin may not always prevent blood feeding. In this study, sand fl y probing behavior could not be observed because the assays were 

conducted in the dark. It is possible that some sand fl ies 

moved through the net and probed before being killed or 

knocked down. 

The results showed that infected sand fl ies were less effective at taking blood meals than the noninfected sand 

fl ies. This corresponds to other studies showing that 

Leishmania infection inhibits blood feeding.11,12 Overall, the blood feeding rates of sand fl ies in this study 

were lower than previous studies conducted at WRAIR. 

Those unpublished studies used noninfected fl ies that 

never received a blood meal and feeding rates were 60% 

to 80%. The lower rates in this study were likely a result 

of the sand fl ies receiving a blood meal during the infection process and being gravid at the time of the assay. It 

was not possible to have nongravid females in this study, 

since a blood meal was required to become infected and 

females die in the laboratory immediately after ovipositing their eggs. 

Many large scale studies have evaluated the effectiveness of ITNs against sand fl ies and most have shown 

that they are benefi cial. In a study in Brazil, ITNs were 

associated with reduced indoor human landing rates and 

high sand fl y mortality.18 Visceral leishmaniasis rates in 

Sudan were reduced following the widespread distribution of ITNs.19 In Syria, the rates of cutaneous leishmaniasis dropped by 85% after the distribution of ITNs.20 A 

large World Health Organization effort to eliminate visceral leishmaniasis in India, Bangladesh, and Nepal has 

shown both positive and negative results. Communitywide distribution of ITNs reduced the indoor density of 

sand fl ies by 25% in India and Nepal,21 and the use of 

ITNs reduced indoor sand fl y densities by 60% to 85% 

in Bangladesh.22,23 However, in one large study in India, 

ITNs did not lead to lowered sand fl y densities.24 In another study in India and Bangladesh, the mass distribution of ITNs only slightly lowered sand fl y biting rates 

based on serological data among communities.25

The most important result of this study is that it justifi es 

using noninfected sand fl ies to evaluate the effectiveness of new ITNs. There have been many unpublished 

evaluations of ITNs at WRAIR using noninfected sand 

fl ies, and other published studies have also used noninfected sand fl ies.10 This study indicates that results from 

noninfected sand fl ies are most likely similar and applicable for Leishmania infected sand fl ies. It is much 

easier to use noninfected sand fl ies in laboratory assays since they can be produced in greater numbers, at 

Figure 4. Pop-up style bed net (NSN 3740-01-516-4415). This 

net is factory treated with permethrin and the mesh size is small 

enough to physically exclude sand fl ies. Female sand fl y included 

for size comparison.

Figure 3. Standard mosquito net (NSN 7210-00-266-9736/

9740). This net is not treated with an insecticide and the mesh 

is too large to physically exclude sand fl ies. Female sand fl y 

included for size comparison.
14 http://www.cs.amedd.army.mil/amedd_journal.aspx

cheaper costs and are safer to handle. Research with 

Leishmania infected sand fl ies must be performed in a 

Biosafety level 2 laboratory.26

The Department of Defense will continue to rely upon 

ITNs to protect service members from sand fl y bites and 

diseases. There are two bed nets currently available. The 

standard mosquito net (NSN 7210-00-266-9736/9740), 

shown in Figure 3, is untreated and its mesh size is too 

large to physically exclude sand fl ies. The mesh becomes more permissive to sand fl ies as the nets become 

older and receive more wear.4

 It is important to treat 

these nets with permethrin if they are to provide protection against sand fl ies. The recommended method is to 

spray with an aerosol can as described in this study.14

Pop-up style bed nets (Figure 4) that are now available 

(NSN 3740-01-516-4415) have a mesh size small enough 

to exclude sand fl ies and are issued factory treated with 

permethrin.4

 These nets are the preferred option for the 

prevention of sand fl y bites. The results from this study 

will be useful as the Department of Defense continues 

to develop and evaluate new ITNs and ensure that they 

provide protection from both infected and noninfected 

sand fl ies.

ACKNOWLEDGMENTS

Thanks to Mr John Paul Benante for providing pictures 

of bed nets. This research was funded by a grant from 

the USDA Deployed War-Fighter Protection program. 

Research was conducted in compliance with the Animal 

Welfare Act, other federal statutes and regulations relating to animals, and in accordance with principles stated 

in the Guide for the Care and Use of Laboratory Animals, 

MRC Publication, 1996 edition.

REFERENCES

1. Desjeux P. Leishmaniasis: current situation and 

new perspectives. Comp Immunol Microbiol Infect 

Dis. 2004;27:305-318.

2. Armed Forces Health Surveillance Center. Leishmaniasis. MSMR. 2014;21(9):19.

3. Coleman RE, Burkett DA, Sherwood V, et al. Impact of phlebotomine sand fl ies on United States 

military operations at Tallil Air Base, Iraq: 6. Evaluation of insecticides for the control of sand fl ies. J 

Med Entomol. 2011;48(3):584-599.

4. Technical Guide No. 49. Sand Flies (Diptera: Psychodidae: Phlebotominae): Signifi cance, Surveillance, and Control in Contingency Operations. Silver Spring, MD: Armed Forces Pest Management 

Board; 2015. Available at: http://www.afpmb.org/

sites/default/fi les/pubs/techguides/TG49/TG49.pdf. 

Accessed June 2, 2015.

5. Alexander B, Maroli M. Control of phlebotomine 

sand fl ies. Med Vet Entomol. 2003;17:1-18.

6. Warburg A, Faiman R. Research priorities for the 

control of phlebotomine sand fl ies. J Vector Ecol. 

2011;36(suppl 1):S10-S16.

7. World Health Organization. Long Lasting Insecticide Nets for Malaria Prevention: A Manual for 

Malaria Programme Managers [Trial Edition]. 

Geneva, Switzerland: World Health Organization; 

2013. Available at: http://www.who.int/manage

ment/programme/LongLastingInsecticidalNets

Malaria.pdf. Accessed June 2, 2015.

8. Ostyn B, Vanlerberghe V, Picado A, et al. Vector control by insecticide-treated nets in the fi ght 

against visceral leishmaniasis in the Indian subcontinent, what is the evidence?. Trop Med Int 

Health. 2008;13:1073-1085.

9. Maroli M, Marjori G. Permethrin-impregnated 

curtains against phlebotomine sandfl ies (Diptera: 

Psychodidae): laboratory and fi eld studies. Parassitologia. 1991;33(suppl):399-404.

10. Kasili S, Kutima H, Mwandawiro C, Ngumbi 

PM, Anjili CO, Enayati AA. Laboratory and semifi eld evaluation of long-lasting insecticidal nets 

against leishmaniasis vector, Phlebotomus (Phlebotomus) duboscqi in Kenya. J Vector Borne Dis. 

2010;47:1-10.

11. Beach R, Kiilu G, Leeuwenburg J. Modifi cation of 

sand fl y biting behavior by Leishmania leads to increased parasite transmission. Am J Trop Med Hyg. 

1985;34:278-282.

12. Vaidyanathan RR. Leishmania parasites (Kinetoplastida: Trypanosomatidae) reversibly inhibit 

visceral muscle contractions in hemimetabolous 

and holometabolous insects. J Invertebr Pathol. 

2004;87:123-128.

13. Rogers ME, Bates PA. Leishmania manipulation 

of sand fl y feeding behavior results in enhanced 

transmission. PLoS Pathog. 2007;3:e91.

14. Technical Guide No. 36. Personal Protective Measures Against Insects and Other Arthropods of 

Military Signifi cance. Silver Spring, MD: Armed 

Forces Pest Management Board; 2009. Available 

at: http://www.afpmb.org/sites/default/fi les/pubs/

techguides/tg36.pdf. Accessed June 2, 2015.

15. Modi GB, Rowton ED. Laboratory maintenance of 

phlebotomine sand fl ies. In: Maramorosch K, Mahmood F, eds. Maintenance of Human, Animal, and 

Plant Pathogen Vectors. Enfi eld, NH: Science Pub 

Inc; 1999:109-121.

16. Rowton ED, Dorsey KM, Armstrong KL. Comparison of in vitro (chicken-skin membrane) versus in vivo (live hamster) blood-feeding methods for maintenance of colonized Phlebotomus 

papatasi (Diptera: Psychodidae). J Med Entomol. 

2008;45:9-13.

EFFICACY OF PERMETHRIN TREATED BED NETS AGAINST LEISHMANIA MAJOR INFECTED SAND FLIES
July – September 2015 15

THE ARMY MEDICAL DEPARTMENT JOURNAL

17. Grieco JE, Achee NL, Sardelis MR, Chauhan KR, 

Roberts DR. A novel high-throughput screening 

system to evaluate the behavioral response of adult 

mosquitoes to chemicals. J Am Mosq Control Assoc. 2005;21:404-411.

18. Courtenay O, Gillingwater K, Gomes PAF, Garcez 

LM, Davies CR. Deltamethrin-impregnated bednets reduce human landing rates of sandfl y vector Lutzomyia longipalpis in Amazon households. 

Med Vet Entomol. 2007;21:168-176.

19. Ritmeijer K, Davies C, van Zorge R, Wang SJ, 

Schorscher J, Dongu’du SI, Davidson RN. Evaluation of a mass distribution program for fi nemesh impregnated bednets against visceral leishmaniasis in eastern Sudan. Trop Med Int Health. 

2007;12:404-414.

20. Jalouk L, Al Ahmed M, Gradoni L, Maroli M. Insecticide-treated bednets to prevent anthroponotic 

cutaneous leishmaniasis in Aleppo Governorate, 

Syria: results from two trials. Trans R Soc Trop 

Med Hyg. 2007;101:360-367.

21. Picado A, Das ML, Kumar V, et al. Effect of village-wide use of long-lasting insecticidal nets on 

visceral leishmaniasis vectors in India and Nepal: 

a cluster randomized trial. PLoS Negl Trop Dis. 

2010;4:e587.

22. Mondal D, Chowdhury R, Huda MM, et al. Insecticide-treated bed nets in rural Bangladesh: 

their potential role in the visceral leishmaniasis 

elimination programme. Trop Med Int Health. 

2010;15:1382-1389.

AUTHORS

Mr Rowland is the Sand Fly Lab Manager, Entomology 

Division, Walter Reed Army Institute of Research, Silver 

Spring, Maryland.

MAJ Davidson is the Chief of Vector & Parasite Biology, Entomology Division, Walter Reed Army Institute 

of Research, Silver Spring, Maryland.

Dr Kobylinski is a National Research Council postdoctoral fellow, Entomology Department, AFRIMS, Bangkok, Thailand.

Mr Menenses is a Research Assistant in the Vector Molecular Biology Unit, Laboratory of Malaria and Vector 

Research, National Institute for Allergy and Infectious 

Disease, Rockville, Maryland.

Dr Rowton was formerly a Senior Scientist, Entomology 

Division, Walter Reed Army Institute of Research, Silver 

Spring, Maryland.
16 http://www.cs.amedd.army.mil/amedd_journal.aspx

Malaria remains one of the greatest infectious disease burdens worldwide, with ≈200 million cases and 

≈600,000 deaths reported in 2013.1

 Though not endemic 

to the United States, malaria incidence is reported in 97 

countries, indicating this threat to global health is also 

a threat to travelers and deployed military personnel. 

Resistance to drugs that kill Plasmodium parasites (the 

etiological agent of malaria) is common and spreads rapidly upon introduction. There is no currently available 

vaccine. Therefore, development and testing of antimalarial vaccines and new drugs has been a top priority 

for infectious disease research within the Department of 

Defense for decades.

Plasmodium parasites are delivered to humans by the 

bite of an Anopheles mosquito; as a female mosquito 

takes blood from a human host, she deposits the sporozoite stage of the parasite into the host’s skin along 

with her saliva. Sporozoites navigate to the liver where 

they invade hepatic cells, shift to a new form called the 

merozoite, and multiply, eventually being released into 

circulation where they can continue an invade-multiplyrelease cycle, now dependent on erythrocytes. Since the 

mosquito only deposits about 10 to 100 sporozoites per 

bite2-4 and the ensuing life cycle involves exponential 

multiplication, the pre-erythrocytic sporozoite stage 

represents a bottleneck in the parasite population that is 

vulnerable to vaccine and drug activity.

Interventions that specifi cally target the pre-erythrocytic stage have been in the pipeline since it was fi rst shown 

that sporozoites elicit an immune response capable of 

preventing subsequent infection.5,6 However, as these 

vaccine and drug candidates were showing effi cacy in 

animal models, it became apparent to medical entomologists that clinical testing of such interventions would 

require a method of mimicking the natural acquisition 

of sporozoites by humans via mosquito bite. Previous 

methods used human gametocyte donors to infect mosquitoes intended to deliver sporozoites to vaccines,7

 but 

this method was unpredictable and dependent on the 

availability of people naturally infected with malaria 

as gametocyte sources. A more controlled, reproducible method was needed; thus, an experimental human 

malaria infection, later coined as controlled human malaria infection (CHMI),8

 was developed at the Walter 

Reed Army Institute of Research (WRAIR). The CHMI 

method encompasses the entirety of a purposeful human malaria infection, from mosquito bite to parasite 

detection in the blood, to resolution by drug administration. The entomological part of CHMI is considered 

the “challenge”: the transmission of malaria parasites 

as mosquitoes bite a human volunteer in a safe, reliable, 

and reproducible way.

IDENTIFYING PARASITES AND VECTORS CAPABLE OF

INFECTING HUMANS

To develop a controlled challenge model, entomologists 

at WRAIR tested the feasibility of artifi cially infecting lab-reared Anopheles mosquitoes with lab-cultured 

Plasmodium. A reliable culture method for growing P 

falciparum in vitro was fi nally published in 1976.9

 This 

system became critical for the development of a malaria challenge since it enabled manufacture of parasite 

Controlled Human Malaria Infection at the

 Walter Reed Army Institute of Research:

 The Past, Present, and Future From an

 Entomological Perspective

 Lindsey S. Garver, PhD

 Megan Dowler

 MAJ Silas A. Davidson, MS, USA

ABSTRACT

Thirty years ago, the Entomology Branch at the Walter Reed Army Institute of Research (WRAIR) performed the fi rst 

controlled human malaria infection, in which lab-reared mosquitoes were infected with lab-cultured malaria parasites 

and allowed to feed on human volunteers. The development of this model was a turning point for pre-erythrocytic 

malaria vaccine research and, through decades of refi nement, has supported 30 years of effi cacy testing of a suite of 

antimalarial vaccines and drugs. In this article, we present a historical overview of the research that enabled the fi rst 

challenge to occur and the modifi cations made to the challenge over time, a summary of the 104 challenges performed 

by WRAIR from the fi rst into 2015, and a prospective look at what the next generation of challenges might entail.
July – September 2015 17

lines with known origin and drug sensitivity, followed a 

somewhat consistent schedule, and used blood and sera 

of known type that could be tested for pathogens. The 

downfall of in vitro parasite growth was (and still is) that 

most parasite lines adapted to grow well asexually in vitro infect mosquitoes poorly, if at all. WRAIR and the 

Naval Medical Research Institute (NMRI), predecessor 

to the Naval Medical Research Center, collaboratively 

tweaked the Trager-Jensen method to grow the best 

lines for infecting mosquitoes.10 Foreseeing the need for 

a compatible parasite-vector pair on which to base the 

malaria challenge, WRAIR entomologists exposed various anopheline species to multiple P falciparum parasite 

lines, both lab-adapted and patient-derived, to assay for 

successful mosquito infection. Although the screening 

was exhaustive, infection rates were often disappointing, 

sometimes yielding months of no infectiousness to mosquitoes. By 1983, the 7G8 strain (chloroquine resistant) 

was cloned from a Brazilian patient sample and, in regular production at WRAIR, showed low numbers of oocysts and sporozoites but with greater consistency than 

any other strain. In 1985, WRAIR received the Africanderived, chloroquine sensitive NF54 P falciparum strain 

from NMRI which had received it from collaborators in 

Nijmegen, The Netherlands.11 This quickly became the 

primary culture in production. In 1987, WRAIR subsequently received 3D7, a strain cloned from NF54 by NIH 

researchers,12 from NMRI. Based on the mosquito infectivity studies done at WRAIR, NMRI, and elsewhere, 

7G8, NF54, and 3D7 would become the worldwide standards for cultured parasites suitable for infecting mosquitoes and nearly the only strains of P falciparum used 

in malaria challenges as of 2015.

For vector selection, the breadth of available parasite 

strains were fed to a variety of potential vectors, including An stephensi, An freeborni, An balabacensis, 

An albimanus, An quadrimaculatus, and others. The 

studies showing 7G8, NF54, and 3D7 were infectious 

to mosquitoes also showed that An stephensi was a robust mosquito, amenable to mass rearing with hearty 

feeding propensity, widely used by other mosquito biologists and displayed excellent susceptibility to both P 

falciparum and P berghei, a pre-clinical rodent model 

for infection. Therefore, it is not surprising that An stephensi is the primary colony supported within WRAIR 

and, to date, 3 challenges used An freeborni but the rest 

have used An stephensi.

INITIAL DEVELOPMENT OF THE WRAIR CHALLENGE

MODEL

During vector-parasite compatibility experiments in 

1982, exposure of a laboratory worker to an escaped 

infectious mosquito resulted in accidental transmission 

of cultured 7G8 P falciparum by lab-reared An freeborni

to a human.13 While this study highlighted the acute 

need for a safety regimen to safeguard workers’ health, 

it also showed for the fi rst time that parasites grown in 

culture and capable of infecting a mosquito could also 

retain infectiousness to humans, inadvertently paving 

the way for CHMI.

The fi rst CHMI was performed in 1985 as a proof-ofconcept trial to assess whether 6 volunteers would develop malaria after being bitten by 5 mosquitoes infected 

with NF54.14 Collectively, WRAIR, NMRI, and NIH 

contributed An freeborni and An stephensi that were given a blood meal containing cultured parasites in donor 

blood; only blood-fed (and therefore potentially infected) 

mosquitoes were retained for possible use. At appropriate times, subpopulations were dissected and numbers of 

oocysts and sporozoites were quantifi ed in midgut and 

salivary glands, respectively. Mosquitoes determined to 

likely be infectious were sorted into cups of 5 and allowed access to a volunteer’s arm for 5 minutes. Mosquitoes were then checked for the presence of a blood meal 

(confi rmed they fed on the volunteer) and the presence of 

sporozoites on a 0 to 4 quantifi cation scale (rating of 2 or 

greater confi rmed infectiousness) and, if fewer than all 

5 satisfi ed those criteria, the volunteer was exposed to 

more mosquitoes until 5 infectious bites were confi rmed. 

This process was performed on a rolling basis—volunteers were called when mosquitoes were ready and not 

all on the same day. All 6 of the volunteers came down 

with malaria. This method was independently repeated 

at the University of Maryland15 with success (4 of 4 volunteers infected) and the fundamentals of the process 

are largely how challenges are performed today.

Questions were raised about the validity of using 5 mosquito bites for a challenge. In nature, people are typically infected by the bite of one mosquito; could vaccinederived immunity be overwhelmed by a 5-bite dosage? 

And, if so, would a vaccine that would be effi cacious 

against a natural 1 or 2 bite dose be erroneously perceived as ineffective in a 5-bite challenge? Also, how 

does sporozoite load affect dosage? Compared to natural conditions, laboratory conditions can load mosquito 

salivary glands with a much heavier burden of sporozoites16; however, the number of sporozoites successfully deposited in the skin is orders of magnitudes lower 

than in the salivary glands and highly variable.3,17 Direct 

enumeration of sporozoites put into each volunteer is 

ethically impossible, so 2 challenges were performed by 

WRAIR for the Navy to assess the feasibility of a 1- or 

2-bite challenge. Three out of 5 volunteers receiving a 
18 http://www.cs.amedd.army.mil/amedd_journal.aspx

single bite became malaria positive, while 2 of 5 receiving 2 bites became positive.18 A third 2-bite challenge 

was performed by WRAIR for Johns Hopkins University with only 1 of 3 volunteers becoming malaria positive.19 Therefore, a 5-bite challenge has been standard 

since about 1990. Later studies show that 3 bites from 

aseptically reared An stephensi can result in 100% infectivity,20,21 but the consistency and the theoretical advantages of this model have yet to be demonstrated, so 

WRAIR continued to provide a 5-bite challenge. In 2012, 

a series of meetings were held to generate a consensus 

of all CHMI-capable centers, ultimately agreeing on the 

WRAIR challenge model of 5 bites from An stephensi

using a 0 to 4 rating scale as the global standard.8

After 24 years, a second parasite species was introduced 

to the 5-bite challenge. In 2009, the Armed Forces 

Research Institute of Medical Sciences (AFRIMS) in 

Bangkok, Thailand, sent 2 challenges using P vivax

in An dirus to WRAIR for infectivity studies. Since P 

vivax cannot be easily cultured in vitro, the lab-reared 

mosquitoes were infected with blood from a human 

gametocytemic patient in Thailand, then shipped to 

WRAIR for challenge administration. All 12 volunteers 

from these studies became infected, demonstrating that 

the challenge model has a measure of fl exibility.

VARIATIONS ON THE TRADITIONAL CHALLENGE

Off-site Challenges

Shipping or hand-carrying infected mosquitoes to perform a challenge overseas was initially tested for feasibility in 2000. A batch of prepared mosquitoes was 

fl own from Washington, DC, to London as a mock challenge test of transport and mosquito viability in anticipation of challenges performed by WRAIR personnel 

for collaborators from Oxford University. This validated 

the feasibility of a “traveling” challenge that, with slight 

variations that defer to site-specifi c clinical trial centers, 

is performed similarly to in-house challenges. This includes not just the supply of infectious mosquitoes but 

of dissectors, entomologists, quality assurance/quality 

control, standard operating procedures, and challenge 

day methodology that has produced success in the past. 

This still requires the receiving facility to have minimal insectary infrastructure for mosquito storage but 

requires no entomological experience, parasite culture, 

or mosquito rearing on the part of the receiver.

Mosquito Bites as Vaccines

Soon after the debut of CHMI, a second mosquito-biting-humans method was developed, in which volunteers 

were exposed to hundreds or even thousands of bites 

from mosquitoes infected with attenuated parasites. At 

fi rst, this was radiation-attenuated sporozoites as a natural progression from the animal studies and few human 

studies that already demonstrated this produced a protective immune response.22 These studies, performed 

on a rolling basis over several years, would collectively 

use 23,279 mosquitoes. Later, sporozoites would also be 

genetically attenuated,23 but the role of entomology remained the same, differing from traditional challenges 

in that many more mosquitoes were required and realtime dissections were not necessary. These trials culminated with a traditional challenge to test the effi cacy of 

the mosquito-delivered “vaccine” and/or investigate the 

immune response generated. Eventually, production of 

mosquitoes that functioned as a vaccine would be considered by the Federal Drug Administration (FDA) to be 

a manufacturing process (reviewed later in this article), 

instituting a sum of regulatory requirements that would 

impose the greatest modifi cation of the challenge process since inception.

Challenge in a Bottle

Not surprisingly, challenges can be expensive, timeconsuming, and require specialized facilities and entomological expertise. Innovations in sporozoite cryopreservation by Sanaria, Inc (Rockville, MD)24 initiated 

an effort to overcome these limitations by vialing aseptic, cryopreserved sporozoites into an FDA-regulated 

product called PfSPZ Challenge, colloquially referred 

to as “challenge in a bottle.” This mosquito-free challenge delivers sporozoites by needle inoculation and is 

capable of reasonable infectivity rates at a dose of 3,500 

sporozoites per vial. This type of challenge is most useful in fi eld settings or locations where facilities cannot 

support insect maintenance; however, in bypassing the 

skin, it does not fully mimic the natural route of sporozoite inoculation by mosquito.25 This means it also 

bypasses immune responses elicited by skin-deposited 

parasites in the dermis and draining lymph nodes, and 

may affect the degree of protection observed.26,27

MEETING THE INCREASING NEEDS OF THE CHALLENGE

By 1989, demand for infected mosquitoes, stemming 

from both clinical and preclinical vaccine research, shifted Entomology into a production role. Every aspect of 

producing infected mosquitoes, from obtaining enough 

blood and serum to rearing enough mosquitoes to having the tools and infrastructure to safely handle so many 

infectious mosquitoes, was reexamined and retooled to 

meet the needs of CHMI. General rearing rooms were 

outfi tted with specialized equipment to improve insect 

production and increase effi ciency, while smaller equipment such as aspirators to transfer mosquitoes, water 

CONTROLLED HUMAN MALARIA INFECTION AT THE WALTER REED ARMY INSTITUTE OF RESEARCH:

THE PAST, PRESENT, AND FUTURE FROM AN ENTOMOLOGICAL PERSPECTIVE
July – September 2015 19

THE ARMY MEDICAL DEPARTMENT JOURNAL

jacketed membrane feeders, and mosquito containment 

devices underwent multiple rounds of innovation to comply with increased demand and increased safety precautions. Mosquito rearing conditions and parasite culture 

methods were optimized and Entomology personnel began to routinely record data on prevalence and intensity 

of mosquito infection, no longer as basic research but as 

indicators of mosquito quality for use in CHMI. 

The biggest physical innovation in mosquito production occurred in the late 1990s as WRAIR moved from 

downtown Washington, DC, to the Forest Glen Annex 

in Silver Spring, MD. The insectary facilities in that 

building were specifi cally designed to meet the needs 

of the challenge. The challenge suite exists separate 

from general insect rearing and consists of (1) an empty 

vestibule to discourage accidental mosquito release as 

doors are opened, (2) a main room where volunteers 

and noninsectary personnel are stationed on challenge 

day, (3) an adjacent room that houses both walk-in and 

reach-in incubators for infected mosquitoes, and (4) a 

separate adjacent room for real-time dissection of mosquito salivary glands. Doors with screens allow personnel to communicate with one another but also contain 

any escaped mosquitoes in work areas away from the 

main challenge room where visitors are permitted. Incubator set-up facilitates scale production depending on 

the sizes and numbers of clinical trials in progress and 

enable segregation of mosquitoes infected with different parasite lines. The dissection room is designed for 

the comfort, safety, and effi ciency of up to 5 dissectors. 

A person must pass through 5 doors and a downward 

air current to get from infected mosquito housing to the 

main corridor, ensuring the safety of all who work in the 

building. Two distinct labs specifi c for parasite culture 

exist separately from the insectary and other lab space, 

isolating challenge-specifi c cultures from general lab 

work while simultaneously enabling segregation of different P falciparum strains destined for challenges.

The 1990s also ushered in an extensive suite of methodological innovations, transitioning the orientation 

of challenge preparation from academic to production. 

Extensive screens for fail-proof stocks of NF54, 3D7, 

and 7G8 were undertaken, mass sporozoite harvesting 

methods were adopted, and individualized fi ne-tuning 

of each round of parasite culture/mosquito infection was 

abandoned in favor of a scheduled, standardized culture/

rearing/infection regimen used for every round of production (Figure 1). This was also highly infl uenced by 

the advent of new regulatory requirements as discussed 

in the next section.

REGULATORY INFLUENCE ON THE CHALLENGE

Until 1993, challenges were performed with mosquitoes 

infected as they would be for routine laboratory experiments. At that time, the FDA became interested in the 

challenge as a systemized and monitored part of a clinical trial and introduced a wave of new regulatory requirements, exponentially increasing the labor and planning required to carry out each successful trial. A batch 

master fi le was created in the fall of 1994 and, within 

one year, entire cell banks comprised of 140, 110, and 75 

vials of NF54, 3D7, and 7G8, respectively, were manufactured under good manufacturing practices (GMP) 

conditions at the Pilot Bioproduction Facility also located on the Forest Glen Annex. These cell banks, derived 

from blood collected from clinical trial volunteers, have 

provided the seed parasites for every WRAIR challenge 

through 2015, though a new bank was created for 3D7 

in 2014 as the original lot dwindled. Every cell bank creation was preceded by months of methodical selection of 

line isolates that gave robust infection in An stephensi

mosquitoes. 

The use of infected mosquito bites as a vaccine (reviewed in previous section) precipitated the need to treat 

infected mosquitoes as an investigational product and to 

treat anything related to culture, husbandry, and feeding 

Figure 1. Workfl ow diagram describing the regimen for producing infected mosquitoes for CMHI.

Strain 3D7 : 9 Week Production Run

Strain NF54 : 11 Week Production Run

Spz

Counts

1 Week

Oocyst

Counts

1 Week

Infectious

Feeds

1 Week

Thaw and

Recover

3-10 Days

Acclimate to

Culture Conditions

2 Weeks

Culture Expansion

2-3 Weeks

Rear Mosquitoes

for Infection

1.5-2 Weeks

Challenge

Generate

Eggs

1 Week
20 http://www.cs.amedd.army.mil/amedd_journal.aspx

as a manufacturing process. By 2009, production of 

infected mosquitoes was performed as close to GMP 

standards as possible for a population of live insects: a 

library of standard operating procedures were written; 

the batch master fi le for parasite production was improved; raw materials and equipment were tracked and 

certifi ed; forms were added; and each step of the process was documented, reviewed by quality assurance/

quality control (QA/QC) personnel, and fi led. These 

methods were extended to traditional challenges and 

have become standard.

SUMMARY OF CHALLENGES PERFORMED

One hundred and four challenges or immunizations 

by mosquito bite have been performed or are planned 

through the end of 2015, resulting in over 2200 volunteer mosquito exposures (VME) (some volunteers are 

counted more than once due to rechallenges or cumulative immunizations on the same person). About half of 

all challenges have been with the 3D7 strain of P falciparum and another 20.5% were with NF54. 7G8, P 

vivax, and genetically attenuated parasites with an NF54 

background comprise the remainder (Figure 2). All data 

is summarized from recordkeeping within the Mosquito 

Biology/Vector and Parasite Biology department within 

the Entomology Branch at WRAIR.

While the number of challenges performed by year did 

not remarkably increase until about 2009, the number of 

VME per year displays a growth trend throughout the 

30 year time line. The average number of VME per year 

for the 1980s is 9.8, for the 1990s is 43.6, for the 2000s 

is 64.8, and for 2010 to 2015 is 183, with particularly 

active years in 2014 and 2015 (recorded and projected) 

(Figure 2). Increase in demand for challenges refl ects, 

fi rst, advancement of vaccine and drug interventions to 

clinical trials and, second, tentative success of several 

vaccine candidates leading to follow-up trials to refi ne 

dosage, schedule, and durability. This escalation in activity parallels the advent of many organizations with 

the mission of controlling malaria, such as the Roll Back 

Malaria Partnership in 1998, the PATH-Malaria Vaccine Initiative in 1999, The Global Fund in 2002, and 

the President’s Malaria Initiative in 2005. Funding from 

Figure 2. Volunteer mosquito exposures involved in malaria challenges performed by the WRAIR Entomology Department 

from 1985 through 2015† and distribution of strains of parasites used for those challenges.

*Genetically attenuated parasites.

†Actual and projected volunteer mosquito exposures shown for 2015. Volunteer Mosquito Exposures

7G8

P vivax

NF54

3D7

GAP*

1985

1986

1987

1988

1989

1990

1991

1992

1993

1994

1995

1996

1997

1998

1999

2000

2001

2002

2003

2004

2005

2006

2007

2008

2009

2010

2011

2012

2013

2014

†2015

350

300

250

200

150

100

50

0

CONTROLLED HUMAN MALARIA INFECTION AT THE WALTER REED ARMY INSTITUTE OF RESEARCH:

THE PAST, PRESENT, AND FUTURE FROM AN ENTOMOLOGICAL PERSPECTIVE
July – September 2015 21

THE ARMY MEDICAL DEPARTMENT JOURNAL

PATH-MVI in particular has directly infl uenced the increased demand for the WRAIR challenge model to test 

their sponsored vaccines.

Vaccines as a malaria control intervention has been by 

far the most common use for the WRAIR malaria challenge model with 61% of all challenges administered 

for that reason. Another 14% have used the immunization-by-mosquito-bite, primarily by repeated exposure 

to radiation attenuated parasites. Verifi cation that the 

model (or modifi cation) is infectious comprised 10% of 

all challenges, a prudent step before new parasites or 

changes are made to the model for testing interventions 

or immunity. As shown in Figure 3, 9% of challenges 

have been used for research into experimental therapeutics, and several challenges either served a purpose 

unknown or unique (eg, test of transport and viability 

overseas).

To date, WRAIR has performed 28 off-site challenges 

(27%), both domestically and overseas. The remaining 

73% were performed in the WRAIR insectary suite as 

described in previous sections. Exclusive of challenges 

performed for Oxford University, nearly 65,000 mosquitoes have been used in the CHMIs summarized here 

(through February 2015). Over 23,000 were used for 

the irradiated sporozoite vaccinations in the 1990s and 

over 27,000 were used in irradiated sporozoite vaccination studies in 2014. These numbers denote the numbers 

of mosquitoes actually exposed to human volunteers. 

Exponentially greater numbers of mosquitoes are prepared for QA/QC and to ensure mosquito availability 

is not a limiting factor for CHMI success. Furthermore, 

up to 10,000 mosquitoes are produced weekly by the 

WRAIR insectary to support clinical and preclinical 

malaria research.

CURRENT AND FUTURE DIRECTIONS OF THE WRAIR 

CHALLENGE MODEL

Although the core of the challenge model has not 

changed much since the 1980s, the model has improved 

signifi cantly with new scientifi c knowledge, applications, technology, and varying needs of users. The next 

generation of WRAIR challenges anticipates the following variations:

Heterologous Challenge

As vaccine candidates display effi cacy against homologous parasites that meets or exceeds the levels called for 

by the target product profi le, demand for heterologous 

challenges is increasing. NF54 and its derivative, 3D7 

are of African origin and serve as the template typically 

used when designing vaccines. 7G8, a Brazilian isolate, 

displays a high degree of polymorphism compared to 

NF54 and 3D728 and is, therefore, an excellent heterologous parasite. However, as observed by labs from multiple institutions, 7G8 is unreliably infectious to mosquitoes. An intradepartmental effort at WRAIR to develop new heterologous strains has evaluated a plethora 

of fi eld-isolated parasite strains for in vitro cultivation 

and mosquito infection and, to date, has found none to 

be dually suitable. Ideally, Entomology would possess 

a library of heterologous strains from around the world 

such that parasites with different genetic backgrounds 

could be tested against vaccines, and those with different drug susceptibility profi les could be tested against 

candidate therapeutics.

Additional species

Concurrently, the need for challenges using non-falciparum Plasmodium species is rising. Entomology anticipates at least one challenge using P vivax within the 

next 2 years and more to follow. This encompasses not 

only late-stage testing of the breadth of protection offered by P falciparum vaccines, but also P vivax-specific vaccines currently in research and development. Despite extensive efforts, P vivax in vitro culture is nearly 

impossible and existing workarounds (such as constant 

addition of purifi ed reticulocytes29) are incompatible 

with the challenge model. P vivax-infected mosquitoes 

can be sourced from AFRIMS, but, as this process uses 

gametocyte donors, it is not nearly as fl exible as what 

exists for P falciparum. Nonhuman primate challenges 

Vaccine

61%

Infectivity

10%

Drug

9%

Unknown

4%

Other

4%

Immunization

14%

Figure 3. Proportions of challenges performed for indicated purposes.
22 http://www.cs.amedd.army.mil/amedd_journal.aspx

(NHP) have also been provided by WRAIR using P 

knowlesi-infected mosquitoes sourced from partners.30

In the future, full in-house NHP challenges with P cynomolgi cycled from NHP to mosquito and back are expected at WRAIR. No plans for P ovale or P malariae

challenges are in place. Each new tweak to a challenge 

model requires an investigation into which mosquito 

species (and strain) is the best to vector the target parasite and how well sporozoites can be recovered, both in 

terms of prevalence and intensity of infection.

Transmission Blocking Interventions

Vaccines and drugs with transmission-blocking potential should be investigated for effi cacy at the clinical level, inciting a need for an inverse challenge: a controlled 

human-to-mosquito malaria transmission that elevates 

the standard membrane feeding assay to natural transmission dynamics. Arms of volunteers who received 

a transmission blocking intervention (TBI) or placebo 

and who acquire malaria would be offered to mosquitoes for feeding and the effi cacy of transmission blocking assessed by Plasmodium prevalence and intensity in 

those mosquitoes. Currently, most TBIs are still in development, but at least one is moving on to Phase I trials 

employing this type of methodology.

Dengue Human Infection Model (and Others)

Just as entomologists in the 1980s foresaw the need for 

a way to test candidate malaria vaccines against natural routes of transmission, it is now obvious that virologists will soon need such a way to test candidate dengue 

vaccines. CHMI has the distinct advantage of using a 

pathogen that is susceptible to available drugs and can 

be completely cured by a simple dosing regimen. This 

is not a characteristic of other vector-borne diseases that 

need a CHMI-like challenge to properly test vaccine 

candidates.

A controlled challenge for dengue is the most pressing 

need, but it would present ethical considerations (ie, if 

the vaccine is not protective, you can only provide supportive care, not cure, to a volunteer). From the entomological point of view, CHMI presents an excellent 

template in which to substitute other mosquito-borne 

pathogens but with careful consideration of where the 

processes differ biologically. Dengue human infection 

model (DHIM) requires a different mosquito species, 

Aedes aegypti, which displays high variability in vector competence across strains that is often dependent on 

the specifi c virus strain used.31,32 A suitable Ae aegypti

strain would have to be validated for every viral strain 

desired in challenges. Dengue virus prevalence and intensity cannot be determined in real-time similar to the 

confi rmation of malaria sporozoites via light microscopy, so DHIM would rely on either pre- or postscreening of mosquitoes for positive infection. Additionally, 

the number of bites optimal for guaranteeing dengue 

transmission while avoiding overwhelming the immune 

response would require investigation. The feasibility of 

such a challenge and some theoretical design elements 

were reviewed by Mores et al.33

SUMMARY

Controlled human malaria infection is a powerful tool in 

antimalarial testing that requires or benefi ts from mimicking the natural route of infection. All of the leading 

pre-erythrocytic vaccines have been tested using this 

model, and even after 30 years its utility is still increasing. Preparing infected mosquitoes for a challenge is a 

task that forces a complex and tenuous biological interaction into a manufacturing-style operation of precision 

and predictability. The challenge portion of CHMI as 

it exists at WRAIR today is the result of decades of research and refi nement. Such cumulative effort is refl ected not only in how well the challenge has performed historically but also in the ways that it can adapt to answer 

new questions about malaria and vector-borne disease.

ACKNOWLEDGMENTS

We thank Dr Imogene Schneider, Dr Jack L. Williams, 

Dr Claudia Golenda, and MAJ Jittawadee Murphy for 

their leadership in the Mosquito Biology/Vector and Parasite Biology Department during the development and 

execution of the WRAIR malaria challenge model. We 

are very grateful to the dozens of Entomology researchers and staff members who have carried out the 30 years 

of work described here. We also thank Dr. Frank Klotz 

for fruitful and engaging discussion about past CHMIs.

REFERENCES

1. World Health Organization. World Malaria Report 

2014. Geneva, Switzerland: World Health Organization; December 2014. Available at: http://www.

who.int/malaria/publications/world_malaria_re

port_2014/en/. Accessed May 11, 2015.

2. Ponnudurai T, Lensen AH, van Gemert GJ, Bolmer 

MG, Meuwissen JH. Feeding behavior and sporozoite ejection by infected Anopheles stephensi. 

Trans R Soc Trop Med Hyg. 1991;85(2):175-180.

3. Medica DL, Sinnis P. Quantitative dynamics of 

Plasmodium yoelii sporozoite transmission by 

infected anopheline mosquitoes. Infect Immun.

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4. Jin Y, Kebaier C, Vanderberg J. Direct microscopic 

quantifi cation of dynamics of Plasmodium berghei

sporozoite transmission from mosquitoes to mice. 

Infect Immun. 2007;75(11):5532-5539.

CONTROLLED HUMAN MALARIA INFECTION AT THE WALTER REED ARMY INSTITUTE OF RESEARCH:

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THE ARMY MEDICAL DEPARTMENT JOURNAL

5. Mulligan HW, Russell PF, Mohan BN. Specifi c 

agglutinogenic properties of inactivated sporozoites of Plasmodium gallinaceum. J Malar Inst Ind.

1941;4:25.

6. Nussenzweig RS, Vanderberg J, Most H, Orton C. 

Protective immunity produced by the injection of 

X-irradiated sporozoites of Plasmodium berghei. 

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7. Clyde DF, Most H, McCarthy VC, Vanderberg J. Immunization of man against sporozoite-induced falciparum malaria. Am J Med Sci. 

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8. Laurens MB, Duncan CJ, Epstein JE, et al. A 

consultation on the optimization of controlled human malaria infection by mosquito bite for evaluation of candidate malaria vaccines. Vaccine. 

2012;30(36):5302-5304.

9. Trager W, Jensen JB. Human malaria parasites in continuous culture. Science. 1976;193(4254):673-675.

10. Burkot TR, Williams JL, Schneider I., Infectivity 

to mosquitoes of Plasmodium falciparum clones 

grown in vitro from the same isolate. Trans R Soc 

Trop Med Hyg. 1984;78:339-341.

11. Ponnudurai T, Leeuwenberg AD, Meuwissen JH. 

Chloroquine sensitivity of isolates of Plasmodium 

falciparum adapted to in vitro culture. Trop Geogr 

Med. 1981;33(1):50-54.

12. Walliker D, Quakyi IA, Wellems TE, et al. Genetic 

analysis of the human malaria parasite Plasmodium falciparum. Science. 1987;236(4809):1661-1666.

13. Williams JL, Innis BT, Burkot TR, Hayes DE, 

Schneider I. Falciparum malaria: accidental transmission to man by mosquitoes after infection with 

culture-derived gametocytes. Am J Trop Med Hyg. 

1983;32(4):657-659.

14. Chulay JD, Schneider I, Cosgriff TM, et al. Malaria transmitted to humans by mosquitoes infected 

from cultured Plasmodium falciparum. Am J Trop 

Med Hyg. 1986;35(1):66-68.

15. Herrington DA, Clyde DF, Murphy JR, et al. A 

model for Plasmodium falciparum sporozoite challenge and very early therapy of parasitaemia for effi cacy studies of sporozoite vaccines. Trop Geogr 

Med. 1988;40(2):124-127.

16. Davis JR, Murphy JR, Clyde DF, et al. Estimate 

of Plasmodium falciparum sporozoite content of 

Anopheles stephensi used to challenge human volunteers. Am J Trop Med Hyg. 1988;40(2):128-130.

17. Menard R. The journey of the malaria sporozoite 

through its hosts: two parasite proteins lead the 

way. Microbes Infect. 2000;2:633-642.

18. Rickman LS, Jones TR, Long GW, et al. Plasmodium falciparum-infected Anopheles stephensi inconsistently transmit malaria to humans. Am J Trop 

Med Hyg. 1990;43(5):441-445.

19. Fries LF, Gordon DM, Schneider I, et al. Safety, 

immunogenicity, and effi cacy of a Plasmodium 

falciparum vaccine comprising a circumsporozoite protein repeat region peptide conjugated to 

Pseudomonas aeruginosa Toxin A. Infect Immun. 

1992;60(5):1834-1839.

20. Lyke KE, Laurens M, Adams M, et al. Plasmodium falciparum malaria challenge by the bite 

of aseptic Anopheles stephensi mosquitoes: results of a randomized infectivity trial. PLoS One. 

2010;5(10):e13490.

21. Laurens M, Billingsley P, Richman A, et al. Successful human infection with P falciparum using 

three Anopheles stephensi mosquitoes: a new model for controlled human malaria infection. PLoS 

One. 2013;8(7):e68969.

22. Hoffman SL, Goh LML, Luke TC, et al. Protection 

of humans against malaria by immunization with 

radiation-attenuated Plasmodium falciparum sporozoites. J Infect Dis. 2002;185(8):1155-1164.

23. Spring M, Murphy J, Nielsen R, et al. First-in-human evaluation of genetically attenuated Plasmodium falciparum sporozoites administered by bite of 

Anopheles mosquitoes to adult volunteers. Vaccine. 

201331(43):4975-83.

24. Roestenberg M, Bijker EM, Sim BK, et al. Controlled human malaria infections by intradermal 

injection of cryopreserved Plasmodium falciparum

sporozoites. Am J Trop Med Hyg. 2013;88(1):5-13.

25. Vaughan JA, Scheller LF, Wirtz RA, Azad AF. Infectivity of Plasmodium berghei sporozoites delivered by intravenous inoculation versus mosquito 

bite: implications for sporozoite vaccine trials. Infect Immun. 1999;67(8):4285-4289.

26. Sinnis P, Zavala F. The skin: where malaria infection and the host immune response begin. Semin 

Immunopathol. 2012;34(6):787-792.

27. Sack BK, Miller JL, Vaughn AM, et al. Model for 

in vivo assessment of humoral protection against 

malaria sporozoite challenge by passive transfer of 

monoclonal antibodies and immune serum. Infect 

Immun. 2014;82(2):808-817.

28. Jiang H, Yi M, Mu J, et al. Detection of genome 

wide polymorphisms in the AT rich Plasmodium 

falciparum genome using a high density microarray. BMC Genomics. 2008;9:398.

29. Golenda CF, Li J, Rosenberg R. Continuous in vitro propagation of the malaria parasite Plasmodium 

vivax. Proc Natl Acad Sci USA. 1997;94:6786-6791.

30. Murphy JR, Weiss WR, Fryauff D, et al. Using 

infective mosquitoes to challenge monkeys with 

Plasmodium knowlesi in malaria vaccine studies. 

Malar J. 2014;13:215.
24 http://www.cs.amedd.army.mil/amedd_journal.aspx

31. Bennett KE, Olson KE, de Lourdes Munoz M, et 

al. Variation in vector competence for dengue 2 

virus among 24 collections of Aedes aegypti from 

Mexico and the United States. Am J Trop Med Hyg. 

2002;67(1): 85-92.

32. Sim S, Jupatanakul N, Ramirez JL, et al. Transcriptomic profi ling of diverse Aedes aegypti strains reveals increased basal-level immune activation in 

dengue virus-refractory populations and identifi es 

novel virus-vector interactions. PLoS Negl Trop 

Dis. 2013;7(7):e2295. 

33. Mores CN, Christofferson RC, Davidson SA. The 

role of the mosquito in a dengue human infection 

model. J Infect Dis. 2014;209(suppl 2):S71-S78.

AUTHORS

Dr Garver is a Malariologist in the Vector and Parasite 

Biology Department, Entomology Branch, Walter Reed 

Army Institute of Research, Silver Spring, Maryland.

Ms Dowler is a Biologist in the Vector and Parasite Biology Department, Entomology Branch, Walter Reed 

Army Institute of Research, Silver Spring, Maryland.

MAJ Davidson is Chief, Vector and Parasite Biology Department, Entomology Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland.

CONTROLLED HUMAN MALARIA INFECTION AT THE WALTER REED ARMY INSTITUTE OF RESEARCH:

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July – September 2015 25

Laos People’s Democratic Republic (Lao PDR) (18° 00ʹ

N; 105°

 00ʹ E; area 236,800 km2) is a landlocked Southeastern Asian country, surrounded by 5 countries: Burma, Cambodia, China, Thailand, and Vietnam.1

 These 

5 countries, together with the Lao PDR, formed the 

Greater Mekong Subregion (GMS), which have a combined population of 92 million. Vector-borne diseases 

have a signifi cant effect on morbidity in these countries, 

and of these diseases, malaria causes more deaths in remote and border areas.2,3 In addition to malaria and high 

heterogeneity in Plasmodium falciparum (Welch) risk,4

dengue, scrub typhus, Japanese encephalitis,5

 and fi lariasis6

 are common insect-borne diseases in the GMS. 

However, their effects on human populations are poorly 

characterized and the taxonomic identities of most vectors should be studied and clarifi ed.

The mosquito fauna of the Lao PDR are not well known, 

except for several scattered reports .7-14 In this study, we 

updated the records and checklist of mosquito species 

from the Lao PDR based on the literature, specimens 

deposited at the US National Mosquito Collections (USNMC), National Museum of Natural History (NMNH), 

Smithsonian Institution, Washington, DC, and our latest specimen collections from Khammuane Province, 

particularly at the Phou Hin Poun National Biodiversity 

Conservation Area (PHP NBCA). This area, which has 

a human population of approximately 30,000, is located 

in a limestone tower karst region of the Annamite Range 

in Khammuane Province. It is composed mainly of rugged caves, porous karst terrain, and dry evergreen forest 

and scrubland. It is also the home to a number of rare or 

newly discovered species of animals.15-17 We are in the 

process of confi rming the identifi cation of several species of mosquitoes and sand fl ies, and possibly describing new species from our recent collections in the area.

MATERIALS AND METHODS

Mosquito Field Collection, Museum Specimens and 

Identification

Specimen collections were conducted from May 1 to 

May 31, 2012, and from February 21 to March 10, 2014, 

from various areas in the PHP NBCA (17.99524° N, 

104.82108° E), Ban Natan, Nakai District, Khammuane 

Province (Figure 1). Adults were collected using modifi ed Centers for Disease Control and Prevention traps 

(Figure 2A, B) with light attractants, and were suspended about 1.3 m above ground level on selected sites and 

inside the caves. Larvae were collected using a standard larval dipper (350 ml, 13 cm diameter: BioQuip, 

Rancho Dominguez, CA) (Figure 2C, D) from various 

habitats including water pockets along edges of rivers, 

rock holes, temporary pools in between rocks, caves, etc 

(Figures 2, 3, and 4). They were individually link-reared 

to adult stage, as morphological voucher specimens 

for this work. Emergent adults were pinned on paper 

points, each given a unique collection number, properly 

labeled, and identifi ed using diagnostic morphological 

characters.18-23 Voucher specimens were deposited at the 

USNMC NMNH, Smithsonian Institution, Washington, 

DC, USA, and at the Entomology Laboratory, Institut 

Pasteur du Laos, Vientiane, Lao PDR. In addition, old 

mosquito specimens at the NMNH repository were examined, and their collection data were recorded.

Mosquito Fauna of Lao People’s Democratic

 Republic, With Special Emphasis on the

 Adult and Larval Surveillance at Nakai

 District, Khammuane Province

 Leopoldo M. Rueda, PhD Jeffrey Hii, PhD

 Khamsing Vongphayloth, MD Mustapha Debboun, PhD

 James E. Pecor, BS Paul T. Brey, PhD

 LCDR Ian W. Sutherland, USN

ABSTRACT

This article includes the distribution records and updated checklist of mosquitoes (Culicidae, Diptera) from the 

Lao People’s Democratic Republic (PDR), based on the literature, specimens deposited at the US National Museum of Natural History mosquito collections, and our recent fi eld collections from the Nakai District, Khammuane Province. Ten of 101 species in the updated checklist of mosquitoes are new records for the Lao PDR.
26 http://www.cs.amedd.army.mil/amedd_journal.aspx

RESULTS

The summary of mosquito collections from the PHP 

NBCA, Khammuane Province is presented in Table 1. 

Figure 1 shows the map of the Lao PDR, with 10 of 16 

provinces, Vientiane (capital city) and PHP NBCA (all 

with asterisks as shown in the map) where adult and larval mosquitoes were collected or reported in the literature. In the PHP NBCA, mosquito habitats included water pockets along edges of rivers, rock holes, temporary 

pools along the edges of rivers, in between rocks, and 

in caves (Figures 2-4). A total of 43 mosquito taxa were 

collected from PHP NBCA in 9 genera (Aedes, Anopheles, Culex, Heizmannia, Mansonia, Orthopodomyia, 

Topomyia, Toxorhynchites, Tripteroides). Among the 3 

genera examined, Aedes (19 species) had the greatest 

number of species, followed by Culex (8 species) and 

Anopheles (7 species). Only 18 species out of 43 (42%) 

were morphologically identifi ed, while the rest (25 species; 58%) need further analyses (including molecular 

techniques) to clarify their taxonomic identities. Known 

or potential vectors of human infectious diseases were 

also collected from PHP NBCA, including Aedes vexans (Meigan), Ae albopictus (Skuse), and several unconfi rmed species of Anopheles (Anopheles), An (Cellia), 

Culex (Culex), and Mansonia.

An updated checklist of mosquitoes in the Lao PDR 

(Table 2) includes a total of 101 species. They are in 16 

genera, namely Aedes (22 species), Anopheles (33), Armigeres (14), Coquillettidia (2), Culex (12), Ficalbia (1), 

Heizmannia (1), Hodgesia (1), Mansonia (4), Mimomyia

(2), Orthopodomyia (1), Topomyia (1), Toxorhynchites

(2), Tripteroides (2), Uronotanea (2), Verrallina (1). 

About 80 of 101 species were reported in the Walter 

Reed Biosystematics Unit (WRBU) catalog,23 2 species found from the Smithsonian/NMNH collections, 17 

species from current PHP NBCA collections, and the 

remaining species from the literature. About 10 species 

of mosquitoes are new records for the Lao PDR. They 

include 9 species under 7 subgenera of the genus Aedes 

and one species in the genus Orthopodomyia (Table 2).

COMMENT

The Lao PDR, like other countries comprising the GMS, 

has a high biodiversity of vector species, a great number 

of mosquito species complexes, enormous spatial heterogeneity in distribution patterns, and extensive behavioral plasticity both between and within species2

.

In 19347

 and 1938,8 Anopheles mosquitoes were reported in the Laos PDR (Table 2). In December 1999, 

malaria vector surveys were carried out by Vythilingam 

et al11 in 7 provinces, namely Borikhamxay, Champasak, Luangprabang, Saravane, Savannakhet, Xayaboury, 

and Sekong, and in the capital city of Vientiane in the 

Lao PDR. Using bare leg collections from indoors and 

outdoors from 6 PM to 5 PM, a total of 438 Anopheles

mosquitoes belonging to 19 species were obtained. Of 

these, only 3 species were found infected with oocysts, 

namely An maculatus Theobald, An dirus Peyton and 

Harrison, and An minimus Theobald. Anopheles aconitus Doenitz was the predominant species in the 1999 

collection, but its vectorial status was unknown. The 

prevalence of Anopheles and epidemiology of malaria 

were also reported in the provinces of Xekong12 and 

Attapeu.13,14 In 2014, Hii and Rueda2

 listed 3 species 

in Anopheles (Anopheles) and 20 species in Anopheles 

(Cellia) in the Lao PDR, including known and potential 

MOSQUITO FAUNA OF LAO PEOPLE’S DEMOCRATIC REPUBLIC, WITH SPECIAL EMPHASIS ON

THE ADULT AND LARVAL SURVEILLANCE AT NAKAI DISTRICT, KHAMMUANE PROVINCE

Figure 1. Map of Lao PDR showing 16 provinces and the 

national capital Vientiane.

Key to Provinces:

AU=Attapu OU=Oudomxai

BO=Bokeo PH=Phongsali

BL=Borikhamxay SA=Saravane

CH=Champasak SV=Savannakhet

HO=Houaphan VI=Viangchan

KH=Khammuane XA=Xayaboury

LO=Louangnamtha XE=Sekong

LU=Luangprabang XI=Xiangkhouang

VT indicates national capital city area of Vientiane.

P indicates Phou Hin Poun National Biodiversity Conservation Area, Nakai District where recent collections were conducted.

Provinces or localities where mosquitoes were collected or 

reported in the literature.

BO*

LO

PH

OU

XA*

LU*

HO

VI

XI

BL*

KH*

P*

SV*

SA*

XE*

AU*

CH*

VT*
July – September 2015 27

THE ARMY MEDICAL DEPARTMENT JOURNAL

malaria vectors in countries of the Mekong Subregion. While there are numerous examples of An 

dirus mostly feeding outdoors and much earlier 

in the evening,24,25 Vythilingam et al13 reported 

an unusual stereotypical nocturnal indoor and 

late feeding behavior in Attapeu province, Laos 

PDR. In 2002, Tsuda et al10 conducted an ecological survey of Aedes dengue vectors in the central 

part of the Lao PDR. A new hydroelectric project, 

Nam Theun 2, created ideal conditions for Aedes 

aegypti (Linnaeus) breeding in water storage jars 

and tires, and Ae albopictus was abundant.26

The present study indicates the species diversity 

of mosquitoes in the Lao PDR. The diffi culty in 

doing morphological comparisons among species warrants further molecular analysis to ascertain taxonomic identities and to clarify hierarchic 

classifi cations. With the diversity of the habitats, 

particularly the caves and surrounding areas, we 

expect that more unknown species will be collected and described in the near future. Deforestation, water resources and management,27,28

conventional agricultural practices, and unregulated destruction of many habitats are major human activities that may adversely affect the fl oral 

and animal fauna of the Lao PDR, including the 

creation or elimination of suitable breeding sites 

of mosquitoes and other arthropods. While habitats in some government protected areas are not 

hugely damaged yet, continuous inventories of 

arthropod fauna, particularly those groups (mosquitoes, sand fl ies, ticks, mites, etc) with known 

disease vectors, should be conducted to accumulate much needed data for developing strategies 

to manage and control infectious human diseases. 

Proper vector surveillance, including ecological surveys, should be performed in areas where 

human diseases (malaria, dengue, tick-borne viruses, fi lariasis, etc) are common and severely 

affect the local human populations. The updated 

checklist of mosquitoes in this article (including 

several vector species) may help health personnel in mapping out some risk areas for infectious 

diseases in the Lao PDR.

ACKNOWLEDGMENT

We express sincere gratitude and appreciation to 

Lea Thutkhin for collecting, processing, mounting 

and pinning specimens, and staff of the Institut Pasteur du Laos for assistance; to Sonexay Ounekham 

of the Entomology Unit, Center for Malaria, Parasitology, and Entomology, Vientiane, for help in collecting specimens in Khammuane Province. We are 

Table 1. Summary of collected mosquito adults and larvae in Phou Hin 

Poun NBCA, Ban Natan, Nakai District, Khammuane Province, Lao 

PDR (17.99524° N, 104.82108° E), from May 1 thru May 31, 2012, and 

February 21 thru March 10, 2014.

Species Sex* Collection no.

Aedes (Aedimorphus) alboscutellatus

(Theobald)

3F LN-048, 050, 060

Aedes (Aedimorphus) sp 1F LN-012

Aedes (Aedimorphus) vexans (Meigen) 1F LN-047

Aedes (Bothealla) eldridgei Reinert 3F† LN-002, 041, 068

Aedes (Bothealla) sp 3F, 1M LN-018, 022, 023, 069

Aedes (Collessius) sp 1F LN-013

Aedes (Downsiomyia) ganapathi Colless 1F LN-024

Aedes (Downsiomyia) harinasutai Knight 1F LN-001

Aedes (Downsiomyia) sp 1M LN-008

Aedes (Fredwardsius) vittatus (Bigot) 2F, 1M LN-015, 065, 066

Aedes (Hulecoeteomyia) chrysolineatus

(Theobald)

1F LN-035

Aedes (Hulecoeteomyia) formosensis Yamada 1F, 1M LN-036, 037

Aedes (Hulecoeteomyia) sp

(near reinerti or formosensis) 1F, 1M LN-031, 046

Aedes (Kenknightia) dissimilis (Leicester) 1F LN-063

Aedes (Kenknightia) sp 1F LN-044

Aedes (Stegomyia) albopictus (Skuse) 1F LN-043

Aedes (Stegomyia) pseudoscutellaris

(Theobald)

1F LN-039

Aedes (Tewarius) pseudonummatus Reinert 2F LN-003

Aedes sp 4F, 2M LN-011, 013, 019, 020, 026, 042

Anopheles (Anopheles) sp (Barbirostris Group) 1F LN-011

Anopheles (Anopheles) sp (Asiaticus Group) 1F LN-049

Anopheles (Anopheles) sp 1F LN-046

Anopheles (Anopheles) sp (Culiciformis Group) 1F LN-062

Anopheles (Cellia) pseudowillmori Theobald 1F LN-045

Anopheles (Cellia) sp (Leucosphyrus Group) 1F LN-005

Anopheles (Cellia) sp 2F LN-009, 010

Coquillettidia (Coquillettidia) ochracea

(Theobald)

1F LN-004

Culex (Culex) sp (Vishnui Complex) 1F LN-052

Culex (Culex) sp (Sitiens Group) 1F LN-054

Culex (Culex) sp 3F LN-028, 055, 071

Culex (Culex) tritaeniorhynchus Giles 1F LN-053

Culex (Culiciomyia) nigropunctatus Edwards 2F, 1M LN-064, 074,075

Culex (Culiciomyia) sp 1F, 1M LN-067, 070

Culex (Eumelanomyia) sp

(Temipalpus Complex)

1F, 2M LN-017, 072, 073

Culex (Lophoceraomyia) sp 1F, 1M LN-007, 016

Heizmannia sp 3F LN-025, 038, 059

Mansonia (Mansonioides) uniformes

(Theobald) 1F LN-057

Mansonia sp 1F LN-051

Orthopodomyia albipes Leicester 1M LN-033

Orthopodomyia sp 1F LN-032

Topomyia sp 1F LN-030

Toxorhynchites sp 3M† LN-076, 077, 078

Tripteroides sp 2F, 1M LN-021, 040, 058

*F indicates female adult, M indicates male adult.

†Larvae collected using plastic larval dipper.
28 http://www.cs.amedd.army.mil/amedd_journal.aspx

MOSQUITO FAUNA OF LAO PEOPLE’S DEMOCRATIC REPUBLIC, WITH SPECIAL EMPHASIS ON

THE ADULT AND LARVAL SURVEILLANCE AT NAKAI DISTRICT, KHAMMUANE PROVINCE

Table 2A. Updated checklist of mosquito species from Lao PDR.

Species Referencea

Aedes (Aedimorphus) alboscutellatus (Theobald) 14, 23, X

Aedes (Aedimorphus) pipersalatus (Giles) 14, 23

Aedes (Aedimorphus) vexans (Meigen) 14, 23, X

Aedes (Bothaella) eldridgei Reinertb X

Aedes (Collessius) macfarlanei (Edwards) 23

Aedes (Diceromyia) iyengari Edwards 14, 23

Aedes (Downsiomyia) ganapathi Collessb X

Aedes (Downsiomyia) harinasutai Knightb X

Aedes (Downsiomyia) niveus (Ludlow) 23

Aedes (Fredwardsius) vittatus (Bigot) 10, X

Aedes (Hulecoeteomyia) chrysolineatus

(Theobald)

14, 23, X

Aedes (Hulecoeteomyia) formosensis Yamadab X

Aedes (Hulecoeteomyia) reinerti Rattanarithikul 

and Harrisonb

X

Aedes (Kenknightia) dissimilis (Leicester)b X

Aedes (Neomelaniconion) lineatopennis (Ludlow) 19

Aedes (Paraedes) ostentatio (Leicester) 19

Aedes (Phagomyia) prominens (Barraud)b X

Aedes (Stegomyia) albopictus (Skuse) 14, X

Aedes (Stegomyia) aegypti (Linnaeus) 10, 23, M

Aedes (Stegomyia) pseudalbopictus Borel 14, 23

Aedes (Stegomyia) pseudoscutellaris (Theobald)b X

Aedes (Tewarius) pseudonummatus Reinertb X

Anopheles (Anopheles) albotaeniatus (Theobald) 11

Anopheles (Anopheles) argyropus

(Swellengrebel)

2

Anopheles (Anopheles) baileyi Edwards 23

Anopheles (Anopheles) barbirostris Van der Wulp 2, 8, 11, 14, 23

Anopheles (Anopheles) donaldi Reid 2, 14, 23

Anopheles (Anopheles) sinensis Wiedemann 3, 8

Anopheles (Anopheles) umbrosus (Theobald) 12

Anopheles (Cellia) aconitus Doenitz 2, 7, 11, 14, 23

Anopheles (Cellia) annularis Van der Wulp 2

Anopheles (Cellia) culicifacies Giles 2, 7, 23

Anopheles (Cellia) dirus Peyton and Harrison 2, 10, 11, 12, 23

Anopheles (Cellia) dravidicus Christophers 2, 14, 23

Anopheles (Cellia) harrisoni Harbach and 

Manguin

23

Anopheles (Cellia) indefi nitus (Ludlow) 2, 23

Anopheles (Cellia) jamesii Theobald 2, 23

Anopheles (Cellia) jeyporiensis James 2, 7, 8, 12, 23

Anopheles (Cellia) karwari (James) 2, 11, 12, 23

Anopheles (Cellia) kochi Donitz 2, 8, 11, 14, 23

Anopheles (Cellia) maculatus Theobald 2, 7, 8, 11, 12, 

14, 23

Anopheles (Cellia) minimus Theobald 2, 8, 11, 12, 

14, 23

Anopheles (Cellia) nivipes (Theobald) 11, 12, 14, 23

Anopheles (Cellia) notanandai Rattanarithikul 

and Green

2, 14, 23

Anopheles (Cellia) pallidus Theobald 11, 12

a X indicates observed, fi eld collection; M indicates observed, Smithsonian/National Museum of Natural History museum collection.

b New record for Lao PDR.

Table 2B. Updated checklist of mosquito species from Lao PDR 

(continued).

Species Referencea

Anopheles (Cellia) pampanai Buttiker and Beales 11, 14, 23

Anopheles (Cellia) philippinensis Ludlow 2, 8, 11, 12, 14, 

23

Anopheles (Cellia) pseudowillmori Theobald 2, 14, 23, X

Anopheles (Cellia) rampae Harbach and 

Somboon

29

Anopheles (Cellia) sawadwongporni 

Rattanarithikul and Green

14, 23

Anopheles (Cellia) splendidus Koidzumi 11, 12, 14, 23

Anopheles (Cellia) subpictus Grassi 2, 11

Anopheles (Cellia) sundaicus (Rodenwaldt) 2

Anopheles (Cellia) tessellatus Theobald 2, 11, 14, 23

Anopheles (Cellia) vagus Donitz 2, 7, 8, 11, 12, 

14

Anopheles (Cellia) varuna Iyengar 2, 11, 12, 14, 23

Armigeres (Armigeres) aureolineatus (Leicester) 23

Armigeres (Armigeres) durhami (Edwards) 23

Armigeres (Armigeres) kuchingensis Edwards 23

Armigeres (Armigeres) laoensis Toma and 

Miyagic 23, M

Armigeres (Armigeres) moultoni Edwards 23

Armigeres (Armigeres) setifer Delfi nado 14, 23

Armigeres (Armigeres) subalbatus (Coquillett) 14, 23

Armigeres (Armigeres) theobaldi Barraud 14, 23

Armigeres (Leicesteria) annulitarsis (Leicester) 23

Armigeres (Leicesteria) dolichocephalus

(Leicester)

23

Armigeres (Leicesteria) fl avus (Leicester) 23, M

Armigeres (Leicesteria) longipalpis (Leicester) 23

Armigeres (Leicesteria) magnus (Theobald) 23

Armigeres (Leicesteria) pectinatus (Edwards) 23

Coquillettidia (Coquillettidia) crassipes

(Van der Wulp)

14, 23

Coquillettidia (Coquillettidia) ochracea

(Theobald)

23, X

Culex (Culex) fuscocephala Theobald 14, 23

Culex (Culex) gelidus Theobald 23

Culex (Culex) hutchinsoni Barraud 14, 23

Culex (Culex) pseudovishnui Colless 14, 23

Culex (Culex) quinquefasciatus Say 14, 23, M

Culex (Culex) tritaeniorhynchus Giles 14, 23, X

Culex (Culex) vishnui Theobald 14, 23

Culex (Culex) whitmorei (Giles) 14, 23

Culex (Culiciomyia) nigropunctatus Edwards 14, 23, X

Culex (Oculeomyia) bitaeniorhynchus Giles 14, 23

Culex (Oculeomyia) pseudosinensis Colless 14, 23

Culex (Oculeomyia) sinensis Theobald 14, 23

Ficalbia minima (Theobald) 23

Heizmannia (Heizmannia) complex (Theobald) 23

Hodgesia malayi Leicester 23

a X indicates observed, fi eld collection; M indicates observed, Smithsonian/National Museum of Natural History museum collection.

c Holotype male, 1 paratype female, 4 females, 3 whole larvae, and 3

larval exuviae, deposited in the Smithsonian/National Museum of 

Natural History museum collection.
July – September 2015 29

THE ARMY MEDICAL DEPARTMENT JOURNAL

Figure 2. Nakai District cave showing mosquito adult wall resting areas (A, D) and larval habitats inside the cave (B, C). A modifi ed light trap hung from the cave wall (D at arrow). Samples were obtained from the cave water pocket (C at arrow) using a larval 

dipper (C inset).

A B

C D

Table 2C. Updated checklist of mosquito species from Lao PDR (continued).

Species Referencea Species Referencea

Mansonia (Mansonioides) annulifera (Theobald) 14, 23 Toxorhynchites (Toxorhynchites) albipes (Edwards) 23

Mansonia (Mansonioides) dives (Schiner) 14 Toxorhynchites (Toxorhynchites) kempi (Edwards) 23

Mansonia (Mansonioides) indiana Edwards 14, 23 Tripteroides (Rachionotomyia) aranoides (Theobald) 23

Mansonia (Mansonioides) uniformes (Theobald) 14, 23, X Tripteroides (Rachionotomyia) ponmeki Miyagi and Toma 9, 23

Mimomyia (Mimomyia) chamberlaini Ludlow 23 Uranotaenia (Pseudofi calbia) nivipleura Leicester 23, M

Mimomyia (Mimomyia) hybrida (Leicester) 23 Uranotaenia (Pseudofi calbia) novobscura Barraud 23, M

Orthopodomyia albipes Leicesterb X Verrallina (Verrallina) dux (Dyar and Shannon) 23

Topomyia (Topomyia) gracilis Leicester 23

a X indicates observed, fi eld collection; M indicates observed, Smithsonian/National Museum of Natural History museum collection.

b New record for Lao PDR.

grateful to Yiau-Min Huang, Jeffrey Clark, and Belen P. 

Rueda for reviewing this manuscript and for their valuable comments. Special thanks go to the staff of the Public Health Offi ce, Nakai District, Khammuane Province 

for cooperation and fi eld assistance.

Partial funding was provided by the US Naval Medical 

Research Center-Asia through the Global Emerging Infections Surveillance and Response System, a Division of 

the US Armed Forces Health Surveillance Center, Silver 

Spring, MD. Institut Pasteur du Laos and the Lao Ministry of Health also contributed to support this project.

This research was performed under a Memorandum of 

Understanding between the Walter Reed Army Institute 

of Research and the Smithsonian Institution, with institutional support provided by both organizations.
30 http://www.cs.amedd.army.mil/amedd_journal.aspx

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Chindavongsa K, Michael E, Aratchige P. A survey 

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ijid.2010.02.2100. Accessed April 27, 2015.

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8. Lefebvre M. Recherches sur la faune Anophelinae au Laos. Bull Soc Pathol Exot Filiales 

1938;31:381-386.

9. Miyagi I, Toma T. Tripteroides (Rachionotomyia) 

ponmeki (Diptera: Culicidae): a new species from 

Khammouane Province, Lao PDR. Med Entomol 

Zool. 2001;52(4):269-277.

MOSQUITO FAUNA OF LAO PEOPLE’S DEMOCRATIC REPUBLIC, WITH SPECIAL EMPHASIS ON

THE ADULT AND LARVAL SURVEILLANCE AT NAKAI DISTRICT, KHAMMUANE PROVINCE

Figure 3. Nakai District river showing mosquito larval habitats along river edge (A), and in rock holes (B, C, D).

B

C D

A
July – September 2015 31

THE ARMY MEDICAL DEPARTMENT JOURNAL

10. Tsuda Y, Kobayashi J, Nambanya S, Miyagi I, Toma 

T, Phompida S, Manivang K. An ecological survey 

of dengue vectors in Central Lao PCDR. Southeast 

Asian J Trop Med Public Health. 2002;33(1):63-67.

11. Vythilingam I, Keokenchanh K, Phommakot S, 

Nambanya S, Inthakone S. Preliminary studies of 

Anopheles mosquitoes in eight provinces in Lao 

PDR. Southeast Asian J Trop Med Public Health. 

2001;32(1):83-87.

12. Vythilingam I, Phetsouvanh R, Keokenchanh K, 

et al. The prevalence of Anopheles (Diptera: Culicidae) mosquitoes in Sekong Province, Lao PDR 

in relation to malaria transmission. Trop Med Int 

Health. 2003;8(6):525-635.

13. Vythilingam I, Sidavong B, Chan ST, et al. Epidemiology of malaria in Attapeu Province, Lao PDR 

in relation to entomological parameters. Trans R 

Soc Trop Med Hyg. 2005;99(11):833-839.

14. Vythilingam I, Sidavong B, Thim CS, Phonemixay T, Phompida S, Jeffrey J. Species composition 

of mosquitoes of Attapeu Province, Lao’s People 

Democratic Republic. J Am Mosq Control Assoc.

2006;22:140-143.

15. Wikipedia [online]. Phou Hin Poun National Biodiversity Conservation Area. Available at: http://

en.wikipedia.25es of Thailand. II. Genera Culex

and Lutzia. Southeast Asian J Trop Med Public 

Health. 2005;36(suppl 2):1-97.

16. Lao National Tourism Administration. Phou Hin 

Poun NBCA. Ecotourism Laos web site. Available 

at: http://www.ecotourismlaos.com/directory/pro

tected_areas/phouhinpoun.htm. Accessed January 

15, 2015.

17. Musser GG, Smith AL, Robinson MF, Lunde DP. 

Description of a new genus and species of rodent 

(Murinae, Muridae, Rodentia) from the Khammouan Limestone National Biodiversity Conservation Area in Lao PDR. American Museum Novitates. 2005;3497:1-31. Available at: http://digi

tallibrary.amnh.org/dspace/handle/2246/5666. Accessed April 28, 2015.

Figure 4. Nakai District river and tributaries showing typical mosquito larval habitats including river edge with fl oating grasses (A, 

B), water pocket (C inset), and temporary water pool (D).

A

C

B

D
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18. Rattanarithikul R, Harbach RE, Harrison BA, Panthusiri P, Jones JW, Coleman RE. Illustrated key to 

the mosquitoes of Thailand. II. Genera Culex and 

Lutzia. Southeast Asian J Trop Med Public Health.

2005;36(suppl 2):1-97.

19. Rattanarithikul R, Harbach RE, Panthusiri P, Peyton EL, Coleman RE. Illustrated key to the mosquitoes of Thailand. III. Genera Aedeomyia, Ficalbia, 

Mimomya, Hodgesia, Coquillettidia, Mansonia

and Uranotaenia. Southeast Asian J Trop Med 

Public Health. 2006;37(suppl 1):1-85.

20. Rattanarithikul R, Harrison BA, Harbach RE, Panthusiri P, Coleman RE. Illustrated key to the mosquitoes of Thailand. IV. Anopheles. Southeast Asian 

J Trop Med Public Health. 2006;37(suppl 2):1-128.

21. Rattanarithikul R, Harbach RE, Harrison BA, 

Panthusiri P, Coleman RE. Illustrated key to the 

mosquitoes of Thailand. V. Genus Orthopodomyia, 

Kimia, Malaya, Topomyia, Tripteroides, and Toxorhynchites. Southeast Asian J Trop Med Public 

Health. 2007;38(suppl 2):1-65.

22. Rattanarithikul R, Harbach RE, Harrison BA, Panthusiri P, Coleman RE, Richardson JH. Illustrated 

key to the mosquitoes of Thailand. VI. Tribe Aedini. Southeast Asian J Trop Med Public Health. 

2010;41(suppl 1):1-225.

23. Walter Reed Biosystematics Unit. Keys to the 

Medically Important Mosquito Species [database 

online]. Suitland, MD: Walter Reed Biosystematics Unit, Smithsonian Institution; 2015. Available 

at: http://www.wrbu.org/command_aors_MQkeys.

html. Accessed January 15, 2015.

24. Obsomer V, Defourny P, Coosemans M. The 

Anopheles dirus complex; spatial distribution and 

environmental factors. Malaria J. 2007;6:26.

25. Killeen GF. Characterizing, controlling and eliminating residual malaria transmission. Malaria J. 

2014;13:330.

26. Hiscox A, Kaye A, Vongphayloth K, et al. Risk 

factors for the presence of Aedes aegypti and Aedes albopictus in domestic water-holding containers in areas impacted by the Nam Theun 2 

hydroelectric project, Laos. Am J Trop Med Hyg. 

2013;88:1070-1078.

27. Erlanger TE, Sayasone S, Krieger GR, et al. Baseline health situation of communities affected by 

the Nam Theun 2 hydroelectric project in central 

Lao PDR and indicators for monitoring. Int J Env 

Health Res. 2008;18:223-242.

28. Ziegler AD, Petney TN, Grundy-Warr C, et al. 

Dams and disease triggers on the lower Mekong 

river. PLoS Negl Trop Dis. 2013;7:e2166.

AUTHORS

Dr Rueda is a Research Entomologist, Principal Investigator, and former Chief of the Walter Reed Biosystematics Unit, Entomology Branch, Walter Reed Army Institute of Research located at the Smithsonian Institution, 

Museum Support Center, Suitland, Maryland.

Dr Vongphayloth is a Medical Doctor and Entomologist, 

Institut Pasteur du Laos, Vientiane, Laos PDR

Mr Pecor is a Museum Specialist at the Walter Reed 

Biosystematics Unit, Entomology Branch, Walter Reed 

Army Institute of Research located at the Smithsonian 

Institution, Museum Support Center, Suitland, Maryland.

LCDR Sutherland is the Chief of Entomological Sciences, US Naval Medical Research Center – Asia located 

at the U.S. Navy Region Center, Sembawang, Singapore.

Dr Hii, formerly a WHO Malaria Scientist, is an Adjunct 

Principal Research Fellow in the School of Public Health, 

Tropical Medicine and Rehabilitation Sciences, James 

Cook University based in Bangkok, Thailand.

Dr Debboun is the Director, Mosquito Control Division, 

Harris County Public Health & Environmental Services, 

Houston, TX.

Dr Brey is a Research Entomologist and Director of the 

Institut Pasteur du Laos, Vientiane, Laos PDR.

MOSQUITO FAUNA OF LAO PEOPLE’S DEMOCRATIC REPUBLIC, WITH SPECIAL EMPHASIS ON

THE ADULT AND LARVAL SURVEILLANCE AT NAKAI DISTRICT, KHAMMUANE PROVINCE

Erratum

In the article “A Heart Gripping Case: Carcinoid Heart Disease” published on pages 93-96 of the 

January-March 2015 issue of the AMEDD Journal, the byline entry “Capt John P. Magulik” is incorrect. The correct byline entry is “Capt John P. Magulick.”

The article “Performance Differences Between Male and Female Marines on Standardized Physical Fitness Tests and Combat Proxy Tasks: Identifying the Gap” appearing on pages 12-21 in the 

print edition of the April-June 2015 issue of the AMEDD Journal has been retracted by the authors. 

The article does not appear in the online digital version of that issue, nor does data describing the 

article appear in the PubMed MEDLINE record database.
July - September 2015 33

Phlebotomine sand fl ies (Subfamily Phlebotominae, 

Family Psychodidae, Order Diptera) are of major health 

importance because they are capable of transmitting 

pathogens, including protozoans (Leishmania), bacteria 

(Bartonella), and viruses (Phleboviruses, sand fl y fever).1

 Like mosquitoes, only female sand fl ies, particularly species of Phlebotomus and Lutzomyia, suck blood, 

including humans. Species of Sergentomyia species 

primarily feed on reptiles, and rarely bite man.2

 Of approximately 900 sand fl y species, only about 70 species 

are capable of transmitting protozoan Leishmania parasites that cause visceral leishmaniasis (kala-azar) and 

various forms of cutaneous leishmaniasis (oriental sore, 

espundia, etc.) in man.3,4 A few sand fl y species have 

been associated with Phlebovirus and other viruses,3-6

and only one, Lutzomyia verrucarum (Townsend) sensu 

lato, can transmit the bacterium Bartonella bacilliformis (Strong, Tyzzer, Brues, Sellards and Gastiaburu) 

causing bartonellosis (Oroya fever, Carrion’s disease) 

in the Andean Region of South America.7,8 Ready9

 reviewed the biology of Phlebotomine sand fl ies as vectors 

of disease agents, including the transmission cycles of 

human leishmaniasis both in the Old and New Worlds, 

mostly in rural communities. Additional Phlebotomine 

reviews also focused on sand fl y biology,10 and emphasis 

on leishmaniasis control.11

Leishmaniasis has a great impact on military operations, particularly those of the United States.12 Since 

World War II, more than 1,000 US service personnel 

were infected with cutaneous leishmaniasis.13 In Afghanistan (Operation Enduring Freedom, OEF) and 

Iraq (Operation Iraqi Freedom, OIF), more US soldiers 

have been exposed to signifi cant leishmaniasis risk than 

any time since World War II. During the disease surveillance period from 2001-2006, there were 1,287 incident diagnoses/reports of leishmaniasis, both cutaneous 

(1,283 cases) and visceral (4 cases) forms, among OEF/

OIF deployers.13 Furthermore, in an effort to establish 

the Leishmaniasis Control Program (LCP) during OIF, 

US military entomologists conducted comprehensive 

phlebotomine sand fl y surveillance at Tallil Air Base 

(TAB), Iraq from April 2003-November 2004. They determined the biology and temporal distribution of sand 

fl ies at TAB, and noted the impact of sand fl y vectors on 

military operations, including the leishmanial threat to 

deployed troops in Iraq.14-16

The phlebotomine sand fl ies are found between 50°N 

and 40°S, with the majority distributed in the tropics 

and subtropics, and none reported on Pacifi c Islands or 

in New Zealand. In the Old World, the anthropophilic 

Phlebotomus sand fl ies (and principally Leishmaniasis 

transmission) are confi ned in the subtropics (particularly in dry, semiarid areas), with a few human biting 

species in Africa south of the Sahara and none in Southeast Asia (although Phlebotomus species are found). In 

the New World (Nearctic and Neotropical Regions), the 

Records and Distribution of New World

 Phlebotomine Sand Flies (Psychodidae,

 Diptera), With Special Emphasis on Primary

 Types and Species Diversity

 Leopoldo M. Rueda, PhD

 Desmond H. Foley, PhD

 David Pecor, BS

 Matthew Wolkoff, BA

ABSTRACT

This article includes the records and distribution of Phlebotomine sand fl ies (Psychodidae, Diptera) in the New 

World based on the specimen collections housed in 2 repositories, the US National Museum of Natural History 

and the Museum of Entomology, Florida State Collection of Arthropods. Approximately 128 species have primary types housed in the 2 repositories, including holotypes (47 species, 3 subspecies), “types” (7 species), allotypes (52 species, 6 subspecies), lectotypes (4 species), paratypes (93 species, 10 subspecies), and neoallotype 

(1 species), mounted on slides, with a total of 1,107 type slides. For species diversity, collection data from 24 

countries in the sand fl y database were analyzed according to the number of species present, specimen records, 

decade of collections, and countries where collections were conducted.
34 http://www.cs.amedd.army.mil/amedd_journal.aspx

transmission of leishmaniasis is mainly in the tropics 

(particularly in the forests and savanna areas) of South 

America.2

In this article, we examine the types and related specimens of New World Phlebotomine sand fl ies housed in 

the US National Museum of Natural History (USNMNH), 

and those borrowed from the Museum of Entomology, 

Florida State Collection of Arthropods (MEFSCA). We 

record the collection data of sand fl ies, including their 

geographical distribution, past and present taxonomic 

arrangement and related information. The species occurrence and diversity of these sand fl ies, according to 

the number of collections for each country over certain 

periods, were analyzed and reported. Other collection or 

occurrence data of sand fl y specimens (including nontypes, from the Nearctic and Neotropical Regions) from 

the 2 repositories (USNMNH and MEFSCA) were also 

examined and recorded, and will be posted later to the 

Walter Reed Biosystematics Unit (WRBU)/VectorMap 

RECORDS AND DISTRIBUTION OF NEW WORLD PHLEBOTOMINE SAND FLIES (PSYCHODIDAE, DIPTERA), 

WITH SPECIAL EMPHASIS ON PRIMARY TYPES AND SPECIES DIVERSITY

Figure 1. Phlebotomine sand fl y collection sites on the New World, based on specimens deposited in the USNMNH 

and MEFSCA.
July - September 2015 35

THE ARMY MEDICAL DEPARTMENT JOURNAL

website (www.vectormap.org).17 They may be helpful in 

developing world sand fl y taxonomic catalogs, and in 

creating sand fl y vector risk maps and prediction distribution models for WRBU/VectorMap. In addition to 

increasing the knowledge of sand fl y distribution, the 

collection holdings in these repositories, particularly the 

primary types, will assist future phlebotomine researchers in their taxonomic and related studies.

MATERIALS AND METHODS

Species Types and Related Specimens

New World Phlebotomine sand fl y specimens used in 

this study are either housed in the USNMNH repository in Suitland, MD, or were borrowed from the MEFSCA in Gainesville, FL. The slide mounted specimens 

(about 10,000 slides in more than 300 slide boxes) were 

examined and their collection data were recorded. All 

collection data from both repositories were entered into 

the USNMNH/MEFSCA database. They were processed and used for analyses in this article. The primary types (holotypes, allotypes, neoallotype, paratypes, 

metatypes) of sand fl ies were examined for collection 

records and related information.

Other sand fl y slides (more than 3,000 slides, mainly 

from Afrotropical and Palearctic Regions) housed in 5 

other repositories were also examined and their collection or occurrence data were also recorded in separate 

databases. Those sand fl y repositories included Institut 

de Reserche pour le Developpement, Montpellier, France; Institut Pasteur, Paris, France; 

Museum National d’Historie Naturelle, Paris, 

France; and Royal Museum for Central Africa, Tervuren, Belgium. However, data from 

the above 5 repositories were not included in 

this article, but may be processed for another 

report.

Species Diversity

The number of sand fl y species according to 

each country in the New World was compiled 

in MS Excel and maps were constructed in 

ArcMap 10.1 (ESRI, Redlands, CA). Georeferences for individual specimens were 

determined and uncertainty calculated using 

the point-radius method.18-20 Label data from 

each specimen was recorded verbatim and 

entered into an Excel spreadsheet. These text 

descriptions were then assigned coordinates 

using a web-based gazetteer.21 For named 

places, the geographic center of the locality 

was used as the latitude and longitude anchor. Once the coordinates were established, 

a measurement of uncertainty was calculated for each 

point. This measurement is defi ned as the radius of a 

circle surrounding the coordinate anchor, indicating that 

the collection site is within this circle. The uncertainty 

measurement takes into consideration errors involving 

the extent of the named place, the geographic datum, 

map scale, and imprecision of collectors’ location descriptions. All information including the verbatim locality description, gazetteer results and geo-referencing 

calculations were recorded and will be available for user 

review via VectorMap.17 The sand fl y data from our 

database (USNMNH and MEFSCA) were sorted and 

ranked according to: (a) number of species per country, (b) number of collection records per species, and (c) 

number of records by decade of collections.

RESULTS

Species Types

The list of New World Phlebotomine species with type 

specimens housed in the USNMNH and MEFSCA is 

shown in Table 1, using the new taxonomic arrangements.22,23 The number of slides for each type and species and the country of type origin are also included in 

Table 1 at the end of this article. A comparison of the 

new22,23 and old4

 generic and subgeneric classifi cations 

of types at both repositories is shown in Table 2 at the 

end of this article. About 139 species have primary types 

housed in those 2 repositories, including holotypes (49 

species, 3 subspecies), “types” (8 species) , allotypes (51 

Figure 2. Map of the New Word showing the number of New World Phlebotomine sand fl y species in the USNMNH and MEFSCA sand fl y database 

according to country of collection.

Number of Species

20-44

45-167

2

3

4

5-7

8-10

11-12

13-15

16-19
36 http://www.cs.amedd.army.mil/amedd_journal.aspx

species, 6 subspecies), paratypes (93 species, 10 subspecies), lectotypes (4 species), neoallotype (1 species), and 

metatype (1 species), mounted on slides, with a total of 

1,113 type slides. The number of paratypes (103) ranged 

from 1 to 53/species or subspecies, with a total of 917. 

Those specimens on slides labeled “Type” could be considered as holotypes, but proper designations should be 

done later. Attempts to check for any additional primary 

types housed in the MEFSCA are still ongoing, and they 

will be listed in separate reports, if any types are found 

later. Species, without type specimens, will be posted 

later on the VectorMap website.17

Species Diversity

The total number of specimen slides considered in this 

database numbered 2,743. Depending on the amount of 

location detail, georeference uncertainty ranged from 

highest (country only information recorded) through 

to lowest (country, province and village information recorded), with an average of 234,678 m (n=2,573). Twenty-four countries of the New World were represented 

in our database of species collection record. The maps 

showing the collections sites in the New World countries, based on the specimens housed in the USNMNH 

and MEFSCA, are presented in Figures 1 and 2. They 

include (from smallest to largest number of species 

occurring in the collection database): Belize= Cuba=

Haiti =Jamaica = Puerto Rico (2 species) < Dominican Republic=El Salvador (3) <French Guyana=Paraguay (4) <Nicaragua=Argentina=Honduras=Venezuela

(5) <Mexico (10) <Bolivia=United States (12) < Guatemala (15) <Ecuador =Trinidad and Tobago (17) <Costa 

Rica (19) < Peru (22) < Colombia (44) < Panama (77) <

Brazil (167). These New World countries, with the top 4 

countries (Brazil, Panama, Colombia, Peru) in the ranks 

according to the number of sand fl y species present in 

the USNMNH/MEFSCA database, are shown in Figure 

3. When the sand fl y collection data were further analyzed according to the number of records present in the 

database, Psychodopygus geniculatus (Mangabeira) was 

the dominant species, followed by Nyssomyia ylephiletor (Fairchild and Hertig) and Lutzomyia panamensis 

(Shannon) (Figure 4). The number of New World sand 

fl y records in the database according to the decade of collections is shown in Figure 5. Most collections were done 

during the 1950s followed by the 1970s then the 1940s. 

The earliest collection was done during 1906 and no collections in the database occurred beyond 1986. Based on 

the number of sand fl y species in the database according 

to the country of collections, Brazil has the greatest number of species (167), followed by Panama (77), Colombia 

(44), and Peru (22) (Figure 3). Mapping species numbers 

by countries reveals that the greatest diversity appears to 

occur around equatorial regions (Figures 1 and 2). When 

the primary types were considered, according to the 

country of occurrence, Panama has the greatest number 

of primary types (21 species with holotypes), followed 

by Brazil (19 spp) and Colombia (7 spp) (Table 1).

COMMENT

The Phlebotomine sand fl y collections, including primary types and voucher specimens, are essential for vector identifi cations, surveillance, and control efforts. The 

USNMNH and MEFSCA have voucher specimens of 

7 (of 8) major species incriminated as vectors9

 of various Leishmania species involved in the transmission of 

human leishmaniasis in the New World. 

Twenty of 27 suspected vector species9

 of 

Leishmania in the New World have types 

and/or voucher specimens deposited in 

the 2 repositories. For example, the holotypes or “types” of the suspected vectors, 

namely Nyssomyia anduzei (Rozeboom), 

Psychodopygus panamensis (Shannon), 

and Lutzomyia diabolica (Hall), are found 

in the USNMNH. Considering the distribution records (mostly from 1940 to 1970) 

at our database (USNMNH/MEFSCA), 

there is an urgent need for additional collections of New World sand fl ies to obtain 

fresh voucher specimens for both molecular and morphological studies, and for safe 

deposits in the USNMNH, MEFSCA, or 

local Neotropical country repositories.

Specimen collections from USNMNH 

and MEFSCA were used in developing 

RECORDS AND DISTRIBUTION OF NEW WORLD PHLEBOTOMINE SAND FLIES (PSYCHODIDAE, DIPTERA), 

WITH SPECIAL EMPHASIS ON PRIMARY TYPES AND SPECIES DIVERSITY

0 5 30 10 15 20 25

20

0

40

60

80

100

120

140

160

180

Colombia (44)

Panama (77)

Brazil (167)

Peru (22)

Country Rank

Number of Species

Figure 3. New World countries ranked according to the number of Phlebotomine 

sand fl y species present in the USNMNH and MEFSCA sand fl y database. The top 

4 countries (and number of species) in the database are identifi ed.
July - September 2015 37

THE ARMY MEDICAL DEPARTMENT JOURNAL

the LUCID24 interactive keys for the New 

World Neotropical Phlebotomine sand 

fl ies, particularly Neotropical Region 

(South and Central America, Southern 

Command, SOUTHCOM). Twenty-four 

morphological keys for males and females of the Neotropical Region (South 

and Central America), were created by 

L. M. Rueda with assistance from the 

WRBU staff, particularly J. Stoffer for 

the Automontage images which are now 

posted at the WRBU website,25 namely:

 Phlebotomine Sand Fly Genera, Neotropical (SOUTHCOM): Females, Males

 Phlebotomine Sand Flies, Subgenera, 

Neotropical (SOUTHCOM): Females, 

Males

 Subgenus Dampfomyia Sand Flies, 

Adults, Neotropical (SOUTHCOM): Females, Males

 Subgenus Evandromyia Sand Flies, 

Adults, Neotropical (SOUTHCOM): Females, Males

 Subgenus Helcocyrtomyia Sand Flies, 

Adults, Neotropical (SOUTHCOM): Females, Males

 Subgenus Lutzomyia Sand Flies, Adults, 

Neotropical (SOUTHCOM): Females, 

Males

 Subgenus Nyssomyia Sand Flies, Adults, 

Neotropical (SOUTHCOM): Females, 

Males

 Subgenus Pintomyia Sand Flies, Adults, 

Neotropical (SOUTHCOM): Females, 

Males

 Subgenus Psathyromyia Sand Flies, 

Adults, Neotropical (SOUTHCOM): 

Males

 Subgenus Psychodopygus Sand Flies, 

Adults, Neotropical (SOUTHCOM): Females, Males

 Subgenus Sciopemyia Sand Flies, Adults, 

Neotropical (SOUTHCOM): Females, 

Males

 Species Grp. Verrucarum Sand Flies, 

Adults, Neotropical (SOUTHCOM): Females, Males

 Subgenus Trichophoromyia Sand Flies, 

Adults, Neotropical (SOUTHCOM): 

Males

The old arrangement of sand fl y taxa by Young and Duncan4

 was followed in the above keys. Other interactive 

keys for Africa and Central, Eastern, and Southwest 

Asia are still in preparation by the WRBU staff. 

Concerning species diversity, a latitudinal biodiversity 

gradient was observed for mosquitoes, with species richness increasing toward the equator.26 For mosquitoes, the 

total number of species increases with geographic area, 

according to a linear log-log relationship, and island 

Figure 4. New World Phlebotomine sand fl y species ranked according to the number of records present in the USNMNH and MEFSCA sand fl y database. The top 

3 most common species (and number of records) in the database are identifi ed.

Psychodopygus geniculatus (122)

Nyssomyia ylephiletor (116)

Lutzomyia panamensis (111)

Species Rank

Number of Records

0 50 100 200 300 150 250

20

0

40

60

80

100

120

140

First Year of Decade

Number of Records

0

100

200

300

400

500

600

700

800

900

1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000

1 1 1 0 14 10

551

97

252

648

831

Figure 5. Number of New World Phlebotomine sand fl y specimen records in the 

USNMNH and MEFSCA sand fl y database according to decade of collection.
38 http://www.cs.amedd.army.mil/amedd_journal.aspx

countries are more species-rich and have a higher number of endemic species than do mainland countries. With 

17 sand fl y taxa in the USNMNH/MEFSCA sand fl y database, Trinidad-Tobago is the most species-rich. Foley et 

al26 also found that this country is species-rich for mosquitoes, even in comparison with other island nations. 

As in mosquitoes, there appears to be little relationship 

(ie, no shared species) between the sand fl y fauna in the 

database for Trinidad-Tobago and Venezuela (the closest continental area), despite these countries having been 

joined during the Pleistocene.27 Separating the effects of 

sampling effort, taxonomic output and species richness 

may be diffi cult, as a species-rich or endemic area will 

initially result in higher numbers of new species per sampling effort, and may attract the greatest sampling effort. 

According to Foley et al,26 Brazil, Panama, French Guiana, and Costa Rica had the highest number of mosquito 

species, including endemic species. With the exception 

of French Guiana, this pattern is also seen for sand fl ies 

from the USNMNH/MEFSCA database. The list of New 

World countries with above average species-level mosquito taxonomic output (type locations, taxonomic publications) included El Salvador, Venezuela, Brazil, Ecuador, Guatemala, Costa Rica, Panama, French Guiana, 

Belize, and Trinidad-Tobago, while Haiti and Uruguay 

were below average.26 The numbers of sand fl y species in 

the USNMNH/MEFSCA database from Haiti and Uruguay were also low, possibly refl ecting a similar lack of 

taxonomic output. A number of assumptions and limitations are inherent in the present study. For example, Hijmans et al28 identifi ed 4 types of bias that could apply in 

the present case, namely species bias (eg, oversampling 

species of sand fl ies due to greater abundance); speciesarea bias (eg, oversampling island endemics compared 

with mainland species); hotspot bias (eg, oversampling 

areas where previous studies indicated a high species 

richness); and infrastructure bias (eg, oversampling near 

roads and towns).

Recently, about 12,000 slides of Phlebotomine sand 

fl ies were donated by retired COL Philip Lawyer to the 

USNMNH for safekeeping. These slides were temporarily mounted using Hoyer’s medium, and should be 

remounted permanently. Their locality data from slide 

labels and collection sheets will be retrieved and recorded. Additional collection data from other regions of the 

world (including Old World countries) will be loaded 

into VectorMap to enable further analysis of species diversity, and to create sand fl y vector distribution models 

that will be useful for leishmaniasis risk assessments.

ACKNOWLEDGMENTS

We are very grateful to Richard Wilkerson and Thomas 

Gaffi gan for help in loaning and retrieving sand fl y slides 

from the MEFSCA; to Gary J. Steck, Curator of Diptera, 

MEFSCA, for facilitating the loan of Phlebotomine sand 

fl y specimens to WRBU; to David Levin; Tracy Brown; 

Victoria Adeboye; Lori Makauskas; and WRBU interns, 

students, and volunteers for help in retrieving collection 

data from sand fl y slides; and to James Pecor for maintaining the sand fl y collections.

This research was performed under a Memorandum of 

Understanding between the Walter Reed Army Institute 

of Research and the Smithsonian Institution, with institutional support provided by the Armed Forces Health 

Surveillance Center’s Global Emerging Infections Surveillance and Response System Operations Division and 

the Military Infectious Diseases Research Program.

REFERENCES

1. Bayer HealthCare AG. Sand Fly-Borne Diseases. 

Companion Vector-borne Diseases Web site. Available at: http://www.cvbd.org/en/sand-fl y-bornediseases/. Accessed March 24, 2015. 

2. Lane R. Sandfl ies (Phlebotominae). In: Lane RP, 

Crosskey RW, eds, Medical Insects and Arachnids.

London, UK: Chapman & Hall; 1993:78-119.

3. Seccombe AK, Ready PD, Huddleston LM. A catalogue of Old World Phlebotomine sandfl ies (Diptera: Psychodidae, Phlebotominae). The Natural 

History Museum (London) Occasional Papers on 

Systematic Entomology. 1993;8:1-58.

4. Young DG, Duncan MA. Guide to the Identifi cation and Geographic Distribution of Lutzomyia 

Sand Flies in Mexico, the West Indies, Central and 

South America (Diptera: Psychodidae). Gainesville, FL: American Entomological Institute. 1994. 

Available at: http://www.amentinst.org/memoirs.

php. Accessed May 1, 2015.

5. Depaquit J, Grandadam M, Fouque F, Andry PE, 

Peyrefitte C. Arthropod-borne viruses transmitted 

by phlebotomine sandfl ies in Europe: a review. Eurosurveillance. 2010;15(10):1-8.

6. Tesh RB. The genus Phlebovirus and its vectors. 

Annu Rev Entomol. 1998;33:169-181.

7. Cohnstaedt LW, Beati L, Caceres AG, Ferro C, 

Munstermann LE. Phylogenetics of the phlebotomine sand fly group Verrucarum (Diptera: 

Psychodidae: Lutzomyia). Am J Trop Med Hyg.

2011;84:913-922.

8. Hambuch TM, Handley SA, Ellis B, Chamberlin J, 

Romero S, Regnery R. Population genetic analysis 

of Bartonella bacilliformis isolates from areas of 

Peru where Carrion’s disease is endemic and epidemic. J Clin Microbiol. 2004;42:3675-3680.

9. Ready PD. Biology of Phlebotomine sand fl ies 

as vectors of disease agents. Annu Rev Entomol.

2013;58:227-250. 

RECORDS AND DISTRIBUTION OF NEW WORLD PHLEBOTOMINE SAND FLIES (PSYCHODIDAE, DIPTERA), 

WITH SPECIAL EMPHASIS ON PRIMARY TYPES AND SPECIES DIVERSITY
July - September 2015 39

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10. Maroli M, Feliciangeli MD, Bichaud L, Charrel RN, 

Gradoni L. Phlebotomine sandfl ies and the spreading of leishmaniases and other diseases of public 

health concern. Med Vet Entomol. 2013;27:123-147.

11. Bates, PA, Depaquit J, Galati EAB, et al. Recent advances in phlebotomine sand fl y research related to 

leishmaniasis control. Parasit Vectors. 2015;8:131.

12. Claborn DM. The biology and control of leishmaniasis vectors. J Glob Infect Dis. 2010;2(2):127-134.

13. Aronson NE. Leishmaniasis in relation to service 

in Iraq/Afghanistan, US Armed Forces, 2001-2006. 

MSMR. 2007;14:2-5.

14. Coleman RE, Burkett DA, Putnam JL, et al. Impact of phlebotomine sand fl ies on US military 

operations at Tallil Air Base, Iraq: 1. Background, 

military situation, and development of a “Leishmaniasis Control Program”. J Med Entomol.

2006;43(4):647-642.

15. Coleman RE, Burkett DA, Sherwood V, et al. Impact of phlebotomine sand fl ies on US military operations at Tallil Air Base, Iraq: 2. Temporal and 

geographic distribution of sand fl ies. J Med Entomol. 2007;44(1):29-41.

16. Coleman RE, Hochberg LP, Swanson KI, et al. Impact of phlebotomine sand fl ies on US military operations at Tallil Air Base, Iraq: 4. Detection and 

identifi cation of leishmania parasites in sand fl ies. 

J Med Entomol. 2009;46(3):649-663.

17. Walter Reed Biosystematics Unit. VectorMap Website. Available at: http://www.vectormap.org/. Accessed March 25, 2015.

18. Wieczorek J, Guo Q, Hijmans R. The point-radius 

method for geo-referencing locality descriptions 

and calculating associated uncertainty. Int J Geogr 

Inf Sci. 2004;18(8):745-767.

19. Wieczorek J, Bloom D. Georeferencing calculator 

[internet]. Berkeley, CA: University of California; 

2008. Available at: http://manisnet.org/gci2.html. 

Accessed March 23, 2015.

20. Foley DH, Wilkerson RC, Rueda LM. Importance of 

the “what,” “when,” and “where” of mosquito collection events. J Med Entomol. 2009;46(4):717-722

21. Wick M. GeoNames [internet]. Available at: http://

www.geonames.org/. Accessed March 23, 2015.

22. Walter Reed Biosystematics Unit. Catalog of subfamily Phlebotominae (Diptera: Psychodidae) [internet]. Available at: http://www.sandfl ycatalog.

org//. Accessed March 25, 2015.

23. Galati EAB. 2. Morfologia e taxonomia, 2.1. Classifi cacao de Phlebotominae, 2.2. Morfologia, terminologia de adultos e identifi cacao dos taxons da 

America. In: Rangel EF, Lainson R, eds. Flebotomineos do Brazil. Rio de Janeiro, Brazil: Editora 

Fiocruz; 2003:23-175.

24. LUCID. Tools for identifi cation and diagnosis (LUCID v. 3.5 software). Brisbane, Australia: University of Queensland. Available at: http://www.lucid

central.com/. Accessed March 25, 2015.

25. Walter Reed Biosystematics Unit. Keys to medically important species of sand fl ies, SOUTHCOM 

[internet]. Available at: http://www.wrbu.org/south

com_SFkeys.html. Accessed March 11, 2015.

26. Foley D, Rueda LM, Wilkerson RC. Insight into 

global mosquito biogeography from country species records. J Med Entomol. 2007;44(4):554-567.

27. Koopman KF. Land bridges and ecology in bat distribution on islands off the northern coast of South 

America. Evolution. 1958;12:429-439.

28. Hijmans RJ, Garrett KA, Huaman Z, et al. Assessing the geographic representativeness of gene bank 

collections: the case of Bolivian wild potatoes. 

Conserv Biol. 2000;14:1755-1765.

AUTHORS

Dr Rueda is a Research Entomologist, Principal Investigator and former Chief of the Walter Reed Biosystematics Unit, Entomology Branch, Walter Reed Army Institute of Research, located at the Smithsonian Institution, 

Museum Support Center, Suitland, Maryland.

Dr Foley is a Research Entomologist of the Walter Reed 

Biosystematics Unit, Entomology Branch, Walter Reed 

Army Institute of Research, located at the Smithsonian 

Institution, Museum Support Center, Suitland, Maryland.

Mr Pecor is a VectorMap technical assistant of the Walter 

Reed Biosystematics Unit, Entomology Branch, Walter 

Reed Army Institute of Research, located at the Smithsonian Institution, Museum Support Center, Suitland, 

Maryland.

Mr Wolkoff is an intern of the College Student Leadership Program, Walter Reed Biosystematics Unit, Entomology Branch, Walter Reed Army Institute of Research, 

located at the Smithsonian Institution, Museum Support 

Center, Suitland, Maryland.
40 http://www.cs.amedd.army.mil/amedd_journal.aspx

RECORDS AND DISTRIBUTION OF NEW WORLD PHLEBOTOMINE SAND FLIES (PSYCHODIDAE, DIPTERA), 

WITH SPECIAL EMPHASIS ON PRIMARY TYPES AND SPECIES DIVERSITY

Table 1A. Types of New World sand fl ies (Phlebotominae, Psychodidae) deposited in the USNMNH and MEFSCA, including country of type origin (continued through 1B, 1C, 1D).

Species Repository Type*

(No. of Slides)

Country of Type

Origin†

Bichromomyia olmeca olmeca (Vargas and Najera 1959) F: P(1) Brazil

Bichromomyia olmeca bicolor (Fairchild and Theodor 1971) F: H, A, P(44) Panama

Bichromomyia olmeca nociva (Young and Arias 1982) F: A, P(14); U: P(10) Brazil

Brumptomyia galindoi (Fairchild and Hertig 1947) F: H Panama

Brumptomyia hamata (Fairchild and Hertig 1947) F: H, A, P(1) Panama

Brumptomyia leopoldoi (Rodriguez 1953) F: P(3) Panama

Dampfomyia (Coromyia) steatopyga (Fairchild and Hertig 1958) F: P(2); U: P(1) Mexico

Dampfomyia (Coromyia) vesicifera (Fairchild and Hertig 1947) F: H, A, P(8); U: P(2) Panama

Dampfomyia (Coromyia) vespertilionis (Fairchild and Hertig 1947) F: H, A, P(24) Panama

Dampfomyia (Coromyia) viriosa (Fairchild and Hertig 1958) F: P(1); U: P(1) Costa Rica

Dampfomyia (Coromyia) zeledoni Young and Murillo 1984 F: H, P(1) Costa Rica

Dampfomyia (Dampfomyia) anthophora (Addis 1945) U: P(1) United States

Dampfomyia (Dampfomyia) rosabali (Fairchild and Hertig 1956) F: P(2) Panama

Dampfomyia (Incertae sedis) caminoi (Young and Duncan 1994) F: H,A,P(1) Mexico

Evandromyia (Aldamyia) sericea (Floch and Abonnenc 1944) U: H Brazil

Evandromyia (Aldamyia) williamsi (Damasceno, Causey and Arouck 1945) U: H Brazil

Evandromyia (Evandromyia) begonae (Ortiz and Torres 1975) U: P(1) Brazil

Evandromyia (Evandromyia) inpai (Young and Arias 1977) F: P(11) Brazil

Evandromyia (Evandromyia) wilsoni (Damasceno and Causey 1945) U: H Brazil

Hertigia hertigi Fairchild 1949 F: A Costa Rica

Lutzomyia (Helcocyrtomyia) botella (Fairchild and Hertig 1961) F: H, P(6) Panama

Lutzomyia (Helcocyrtomyia) cirrita Young and Porter 1974 F: A, P(5) Colombia

Lutzomyia (Helcocyrtomyia) hartmanni (Fairchild and Hertig 1957) F: H, P(10) Panama

Lutzomyia (Helcocyrtomyia) imperatrix (Alexander 1944) U: H Peru

Lutzomyia (Helcocyrtomyia) noguchii (Shannon 1929) U: T Peru

Lutzomyia (Helcocyrtomyia) peruensis (Shannon 1929) U: T Peru

Lutzomyia (Helcocyrtomyia) pescei (Hertig 1943) U: L Peru

Lutzomyia (Helcocyrtomyia) strictivilla Young 1979 F: A, P(6) Colombia

Lutzomyia (Helcocyrtomyia) tortura Young and Rogers 1984 F: A Ecuador

Lutzomyia (Incertae sedis) tanyopsis Young and Perkins 1984 F: P(1) United States

Lutzomyia (Lutzomyia) battistinii (Hertig 1978) U: L, P(2) Peru

Lutzomyia (Lutzomyia) lichyi (Floch and Abonnenc 1950) F: A, P(6) Panama

Lutzomyia (Tricholateralis) carvalhoi (Damasceno, Causey and Arouck 1945) U: H Brazil

Lutzomyia (Tricholateralis) cruciata (Coquillett 1907) U: T Guatemala

Lutzomyia (Tricholateralis) diabolica (Hall 1936) U: T United States

Lutzomyia (Tricholateralis) falcata Young, Morales and Ferro 1994 F: P(7) Brazil

Lutzomyia (Tricholateralis) marinkellei Young 1979 F: A, P(3) Colombia

Martinsmyia gasparviannai Martins, Godoy and Silva 1962 F: P(1) Brazil

Martinsmyia waltoni Arias, Freitas and Barrett 1984 F: P(2); U: P(2) Brazil

Micropygomyia (Coquillettimyia ) apache (Young and Perkins 1984) F: A, P(2) United States

Micropygomyia (Coquillettimyia) stewarti (Mangabeira and Galindo 1944) F: H; U: P(1) United States

Micropygomyia (Coquillettimyia) vexator (Coquillett 1907) U: H United States

Micropygomyia (Micropygomyia) cayennensis cayennensis (Floch and Abonnenc 1941) F: P(3) Guatemala

Micropygomyia (Micropygomyia) cayennensis hispaniolae (Fairchild and Trapido 1950) U: P(2) Dominican Republic

Micropygomyia (Micropygomyia) cayennensis jamaicensis (Fairchild and Trapido 1950) F: H, A, P(1) Jamaica

Micropygomyia (Micropygomyia) cayennensis maciasi (Fairchild and Hertig 1948) F: (P1); U: (P1) Mexico

*Key to Repository Type:

H=holotype (1 specimen) A=allotype (1 specimen)

F=MEFSCA P=paratype (1 or more specimens)

N=neoallotype (1 specimen) T=Type (1 specimen)

U=USNMNH

†Based on types and repositories, as listed in adjacent column.
July - September 2015 41

THE ARMY MEDICAL DEPARTMENT JOURNAL

Table 1B. Types of New World sand fl ies (Phlebotominae, Psychodidae) deposited in the USNMNH and MEFSCA, including country of type origin (continued).

Species Repository Type*

(No. of Slides)

Country of Type

Origin†

Micropygomyia (Micropygomyia) cayennensis puertoriciensis

(Fairchild and Hertig 1948)

F: A, P(5); U: P(1) Puerto Rico

Micropygomyia (Micropygomyia) cayennensis viequesensis

(Fairchild and Hertig 1948)

F: H, A, P(4);

U: P(2)

Puerto Rico: H, A, P(4);

Panama: P(2)

Micropygomyia (Micropygomyia) cubensis (Fairchild and Trapido 1950) F: H, A, P(5);

U: P(1)

Cuba

Micropygomyia (Micropygomyia) duppyorum (Fairchild and Trapido 1950) F: A, P(6); U: P(2) Jamaica

Micropygomyia (Micropygomyia) hispaniolae (Fairchild and Trapido 1950) F: A, P(8) Dominican Republic: A, P(5);

Haiti: P(3)

Micropygomyia (Micropygomyia) pilosa (Damasceno and Causey 1944) U: H Brazil

Micropygomyia (Micropygomyia) xerophila (Young, Brener, and Wargo 1983) F: A, P(10); U: P(1) United States

Micropygomyia (Sauromyia) atroclavata (Knab 1913) U: P(1) Trinidad and Tobago

Micropygomyia (Sauromyia) ferreirana

(Barretto, Martins, and Pellegrino 1956)

U: H Brazil

Micropygomyia (Sauromyia) quechua (Martins, Llanos, and Silva 1975) F: A, P(1) Peru

Micropygomyia (Sauromyia) quinquefer (Dyar 1929) U: T, A Argentina

Migonemyia (Blancasmyia) cerqueirai (Causey and Damasceno 1945) U: H Brazil

Migonemyia (Blancasmyia) gorbitzi (Blancas 1959) F: A, P(50) Panama

Nyssomyia anduzei (Rozeboom 1942) U: H Venezuela

Nyssomyia trapidoi (Fairchild and Hertig 1952) F: H, A, P(29);

U: P(2)

Panama

Nyssomyia ylephiletor (Fairchild and Hertig 1952) F: H, P(36) Panama

Nyssomyia yuilli Young & Porter 1972 U: H, A; F: P(20) Colombia

Oligodontomyia oligodonta (Young, Pérez, and Romero 1985) F: A, P(9) Peru

Pintomyia (Pifanomyia) andina Osorno, Osorno-Mesa, and Morales 1972 U: P(1) Colombia

Pintomyia (Pifanomyia) boliviana (Velasco and Trapido 1974) U: H Bolivia

Pintomyia (Pintomyia) christenseni Young and Duncan 1994 F: H, A, P(34) Panama: H, A , P(20);

Colombia: P(12); Brazil (P2)

Pintomyia (Pifanomyia) christophei (Fairchild and Trapido 1950) F: H, A, P(3) Dominican Republic

Pintomyia (Pifanomyia) gruta Ryan 1986 F: P(1) Brazil

Pintomyia (Pifanomyia) moralesi Young 1979 F: P(4) Colombia

Pintomyia (Pifanomyia) odax (Fairchild and Hertig 1961) F: A, P(17) Panama

Pintomyia (Pifanomyia) oresbia (Fairchild and Hertig 1961) F: A, P(2) Panama

Pintomyia (Pifanomyia) orestes (Fairchild and Trapido 1950) F: H, P(1) Cuba

Pintomyia (Pifanomyia) pia (Fairchild and Hertig 1961) F: H, A, P(10) Panama

Pintomyia (Pifanomyia) torvida Young, Morales, and Ferro 1994 F: A, P(1) Colombia

Pintomyia (Pifanomyia) youngi Feliciangeli and Murillo 1985 F: P(2) Venezuela

Pressatia camposi (Rodriguez 1952) F: A, P(31) Panama

Pressatia dysponeta (Fairchild and Hertig 1952) F: A, P(53) Panama

Pressatia trispinosa (Mangabeira 1942) F: H, A, P(7) Colombia

Psathyromyia (Incertae sedis) ignacioi (Young 1972) F: P(1) Venezuela

Psathyromyia (Forattiniella) barrettoi barrettoi (Mangabeira 1942) U: P(1) Panama

Psathyromyia (Forattiniella) barrettoi majuscula (Young 1979) F: A, P(15); U: P(2) Panama: A, P(11); Colombia: P(3); 

Costa Rica: P(1); Ecuador (P=1); 

Nicaragua: P(1)

Psathyromyia (Forattiniella) carpenteri (Fairchild and Hertig 1953) F: H, A, P(36) Panama

Psathyromyia (Forattiniella) runoides (Fairchild and Hertig 1953) F: H, A, P(28) Panama

Psathyromyia (Forattiniella) texana (Dampf 1938) U: T United States

*Key to Repository Type:

H=holotype (1 specimen) A=allotype (1 specimen)

F=MEFSCA P=paratype (1 or more specimens)

N=neoallotype (1 specimen) T=Type (1 specimen)

U=USNMNH

†Based on types and repositories, as listed in adjacent column.
42 http://www.cs.amedd.army.mil/amedd_journal.aspx

RECORDS AND DISTRIBUTION OF NEW WORLD PHLEBOTOMINE SAND FLIES (PSYCHODIDAE, DIPTERA), 

WITH SPECIAL EMPHASIS ON PRIMARY TYPES AND SPECIES DIVERSITY

Table 1C. Types of New World sand fl ies (Phlebotominae, Psychodidae) deposited in the USNMNH and MEFSCA, including country of type origin (continued).

Species Repository Type*

(No. of Slides)

Country of Type

Origin†

Psathyromyia (Psathyromyia) campbelli (Damasceno, Causey, and Arouck 1945) U: H Brazil

Psathyromyia (Psathyromyia) cratifer (Fairchild and Hertig 1961) F: H, P(1) Mexico

Psathyromyia (Psathyromyia) dasymera (Fairchild and Hertig 1961) F: H, A, P(45) Panama: H, A, P(42); Mexico: P(1);

Nicaragua: P(2)

Psathyromyia (Psathyromyia) guatemalensis Porter and Young 1986 F: A, P(1) Guatemala

Psathyromyia (Psathyromyia) shannoni (Dyar 1929) U: L, P(1); F: P(2) Argentina: H; Panama: L, P(2);

Peru (P1)

Psathyromyia (Psathyromyia) soccula (Fairchild and Hertig 1961) F: P(2) Panama

Psathyromyia (Psathyromyia) souzacastroi (Damasceno and Causey 1944) U: H Brazil

Psathyromyia (Psathyromyia) undulata (Fairchild and Hertig 1950) U: P(1) Guatemala

Psathyromyia (Psathyromyia) volcanensis (Fairchild and Hertig 1950) F: N, P(3) Panama

Psathyromyia (Xiphomyia) aclydifera (Fairchild and Hertig 1952) F: A Panama

Psychodopygus amazonensis (Root 1934) U: (L); F: P(1) Peru: L; French Guyana: P(1)

Psychodopygus ayrozai (Barretto and Coutinho 1940) F: P(2) Panama

Psychodopygus bispinosus (Fairchild and Hertig 1951) F: H, A, P(4) Panama

Psychodopygus carrerai carrerai (Barretto 1946) F: P(1) Panama

Psychodopygus carrerai thula (Young 1979) F: A, P(27) Panama: A, P(17); Colombia: P(10)

Psychodopygus davisi (Root 1934) U: P(1) Brazil

Psychodopygus fairchildi Barretto 1966 F: H,A, P(4) Colombia

Psychodopygus fairtigi (Martins 1970) F: H, P(1) Colombia

Psychodopygus nocticolus (Young 1973) F: A, P(7) Colombia

Psychodopygus panamensis (Shannon 1926) U: T Panama

Psychodopygus recurvus (Young 1973) F: A, P(12) Colombia

Sciopemyia nematoducta Young and Arias 1984 F: A, P(23); U: P(8) Brazil

Sciopemyia pennyi Arias and Freitas 1981 F: P(2); U: P(1) Brazil

Sciopemyia preclara Young and Arias 1984 F: P(1) Peru

Sciopemyia servulolimai (Damasceno and Causey 1945) U: H Brazil

Sciopemyia sordellii (Shannon and Del Ponte 1927) U: L Argentina

Trichophoromyia castanheirai (Damasceno, Causey, and Arouck 1945) U: H Brazil

Trichophoromyia dunhami (Causey and Damasceno 1945) U: H Brazil

Trichophoromyia gibba Young and Arias 1994 F: P(1) Brazil

Trichophoromyia lopesi (Damasceno, Causey, and Arouck 1945) U: H Brazil

Trichophoromyia loretonensis (Llanos 1964) F: P(1) Peru

Trichophoromyia meirai (Causey and Damasceno 1945) U: H Brazil

Trichophoromyia melloi (Causey and Damasceno 1945) U: H Brazil

Trichophoromyia napoensis Young and Rodgers 1984 F: A, P(12) Ecuador

Trichophoromyia pabloi (Barreto, Burbano, and Young 2002) F: P(1) Colombia

Trichophoromyia reburra (Fairchild and Hertig 1961) F: H, A, P(2) Panama

Trichophoromyia ruii Arias and Young 1982 F: P(31) Brazil

Trichophoromyia sinuosa Young and Duncan 1994 F: H, P(1) Peru

Trichopygomyia elegans Martins, Falcao and Silva 1976 U: P(1) Peru

Trichopygomyia ferroae (Young and Morales 1987) F: H, A, P(1) Colombia

Trichopygomyia martinezi Young and Morales 1987 F: H, A, P(1) Colombia

Trichopygomyia ratcliffei Arias, Ready, and Freitas 1983 U: P(5) Brazil

Trichopygomyia triramula (Fairchild and Hertig 1952) F: H, A, P(28) Panama

Trichopygomyia wagleyi (Causey and Damasceno 1945) U: H Brazil

*Key to Repository Type:

H=holotype (1 specimen) A=allotype (1 specimen)

F=MEFSCA P=paratype (1 or more specimens)

N=neoallotype (1 specimen) T=Type (1 specimen)

U=USNMNH

†Based on types and repositories, as listed in adjacent column.
July - September 2015 43

THE ARMY MEDICAL DEPARTMENT JOURNAL

Table 1D. Types of New World sand fl ies (Phlebotominae, Psychodidae) deposited in the USNMNH and 

MEFSCA, including country of type origin (continued).

Species Repository Type*

(No. of Slides)

Country of Type

Origin†

Trichopygomyia wilkersoni Young and Rodgers 1984 F: A, P(1) Ecuador

Trichopygomyia witoto Young and Morales 1987 F: H, P(1) Colombia

Viannamyia fariasi (Damasceno, Causey, and Arouck 1945) U: H Brazil

Warileya nigrosaccula Fairchild and Hertig 1951 F: H Panama

Warileya phlebotomanica Hertig 1948 F: H Peru

Warileya rotundipennis Fairchild and Hertig 1951 F: H, A, P(6) Panama

Warileya yungasi Velasco and Trapido 1974 F: P(1); U: H, P(1) Bolivia

*Key to Repository Type:

H=holotype (1 specimen) A=allotype (1 specimen)

F=MEFSCA P=paratype (1 or more specimens)

N=neoallotype (1 specimen) T=Type (1 specimen)

U=USNMNH

†Based on types and repositories, as listed in adjacent column.

Table 2A. Types of New World sand fl ies (Phlebotominae, Psychodidae), deposited in the USNMNH and MEFSCA, with old and 

new generic and subgeneric classifi cations (continued through 2B, 2C, 2D).

New Arrangement Old Arrangement†

Bichromomyia olmeca olmeca (Vargas and Najera 1959) Lutzomyia (Nyssomyia) olmeca olmeca

Bichromomyia olmeca bicolor (Fairchild and Theodor 1971) Lutzomyia (Nyssomyia) olmeca bicolor

Bichromomyia olmeca nociva (Young and Arias 1982) Lutzomyia (Nyssomyia) olmeca nociva

Brumptomyia galindoi (Fairchild and Hertig 1947) Brumptomyia galindoi 

Brumptomyia hamata (Fairchild and Hertig 1947) Brumptomyia hamata 

Brumptomyia leopoldoi (Rodriguez 1953) Brumptomyia leopoldoi 

Dampfomyia (Coromyia) steatopyga (Fairchild and Hertig 1958) Lutzomyia (Coromyia) steatopyga 

Dampfomyia (Coromyia) vesicifera (Fairchild and Hertig 1947) Lutzomyia (Coromyia) vesicifera 

Dampfomyia (Coromyia) vespertilionis (Fairchild and Hertig 1947) Lutzomyia (Coromyia) vespertilionis 

Dampfomyia (Coromyia) viriosa (Fairchild and Hertig 1958) Lutzomyia (Coromyia) viriosa 

Dampfomyia (Coromyia) zeledoni Young and Murillo 1984 Lutzomyia (Coromyia) zeledoni 

Dampfomyia (Dampfomyia) anthophora (Addis 1945) Lutzomyia (Dampfomyia) anthophora 

Dampfomyia (Dampfomyia) rosabali (Fairchild and Hertig 1956) Dampfomyia (Dampfomyia) rosabali 

Dampfomyia (Incertae sedis) caminoi (Young and Duncan 1994) Lutzomyia caminoi 

Evandromyia (Aldamyia) sericea (Floch and Abonnenc 1944) Lutzomyia sericea 

Evandromyia (Aldamyia) williamsi (Damasceno, Causey, and Arouck 1945) Lutzomyia williamsi 

Evandromyia (Evandromyia) begonae (Ortiz and Torres 1975) Lutzomyia (Evandromyia) begonae 

Evandromyia (Evandromyia) inpai (Young and Arias 1977) Lutzomyia inpai 

Evandromyia (Evandromyia) wilsoni (Damasceno and Causey 1945) Lutzomyia wilsoni 

Hertigia hertigi Fairchild 1949 Hertigia hertigi 

Lutzomyia (Helcocyrtomyia) botella (Fairchild and Hertig 1961) Lutzomyia (Helcocyrtomyia) botella 

Lutzomyia (Helcocyrtomyia) cirrita Young and Porter 1974 Lutzomyia (Helcocyrtomyia) cirrita 

Lutzomyia (Helcocyrtomyia) hartmanni (Fairchild and Hertig 1957) Lutzomyia (Helcocyrtomyia) hartmanni 

Lutzomyia (Helcocyrtomyia) imperatrix (Alexander 1944) Lutzomyia (Helcocyrtomyia) imperatrix 

Lutzomyia (Helcocyrtomyia) noguchii (Shannon 1929) Lutzomyia (Helcocyrtomyia) noguchii 

Lutzomyia (Helcocyrtomyia) peruensis (Shannon 1929) Lutzomyia (Helcocyrtomyia) peruensis 

Lutzomyia (Helcocyrtomyia) pescei (Hertig 1943) Lutzomyia (Helcocyrtomyia) pescei 

Lutzomyia (Helcocyrtomyia) strictivilla Young 1979 Lutzomyia (Helcocyrtomyia) strictivilla 

Lutzomyia (Helcocyrtomyia) tortura Young and Rogers 1984 Lutzomyia (Helcocyrtomyia) tortura 

Lutzomyia (Incertae sedis) tanyopsis Young and Perkins 1984 Lutzomyia tanyopsis 

Lutzomyia (Lutzomyia) battistinii (Hertig 1978) Lutzomyia (Lutzomyia) battistinii 

Lutzomyia (Lutzomyia) lichyi (Floch and Abonnenc 1950) Lutzomyia (Lutzomyia) lichyi 

Lutzomyia (Tricholateralis) carvalhoi (Damasceno, Causey, and Arouck 1945) Lutzomyia (Lutzomyia) carvalhoi

*Based on WRBU22 and Galati.23

†Based on Young and Duncan4

 and various references.
44 http://www.cs.amedd.army.mil/amedd_journal.aspx

RECORDS AND DISTRIBUTION OF NEW WORLD PHLEBOTOMINE SAND FLIES (PSYCHODIDAE, DIPTERA), 

WITH SPECIAL EMPHASIS ON PRIMARY TYPES AND SPECIES DIVERSITY

Table 2B. Types of New World sand fl ies (Phlebotominae, Psychodidae), deposited in the USNMNH and MEFSCA, with old and 

new generic and subgeneric classifi cations (continued).

New Arrangement Old Arrangement†

Lutzomyia (Tricholateralis) cruciata (Coquillett 1907) Lutzomyia (Lutzomyia) cruciata 

Lutzomyia (Tricholateralis) diabolica (Hall 1936) Lutzomyia (Lutzomyia) diabolica 

Lutzomyia (Tricholateralis) falcata Young, Morales and Ferro 1994 Lutzomyia (Lutzomyia) falcata 

Lutzomyia (Tricholateralis) marinkellei Young 1979 Lutzomyia (Lutzomyia) marinkellei 

Martinsmyia gasparviannai Martins, Godoy and Silva 1962 Lutzomyia (Lutzomyia) gasparviannai 

Martinsmyia waltoni Arias, Freitas and Barrett 1984 Lutzomyia (Nyssomyia) waltoni 

Micropygomyia (Coquillettimyia ) apache (Young and Perkins 1984) Lutzomyia apache 

Micropygomyia (Coquillettimyia) stewarti (Mangabeira and Galindo 1944) Lutzomyia (Helcocyrtomyia) stewarti 

Micropygomyia (Coquillettimyia) vexator (Coquillett 1907) Lutzomyia (Helcocyrtomyia) vexator 

Micropygomyia (Micropygomyia) cayennensis cayennensis

(Floch and Abonnenc 1941)

Lutzomyia (Micropygomyia) cayennensis cayennensis 

Micropygomyia (Micropygomyia) cayennensis hispaniolae

(Fairchild and Trapido 1950)

Lutzomyia (Micropygomyia) cayennensis hispaniolae

Micropygomyia (Micropygomyia) cayennensis jamaicensis

(Fairchild and Trapido 1950)

Lutzomyia (Micropygomyia) cayennensis jamaicensis

Micropygomyia (Micropygomyia) cayennensis maciasi (Fairchild and Hertig 1948) Lutzomyia (Micropygomyia) cayennensis maciasi

Micropygomyia (Micropygomyia) cayennensis puertoriciensis

(Fairchild and Hertig 1948)

Lutzomyia (Micropygomyia) cayennensis puertoriciensis

Micropygomyia (Micropygomyia) cayennensis viequesensis

(Fairchild and Hertig 1948)

Lutzomyia (Micropygomyia) cayennensis viequesensis

Micropygomyia (Micropygomyia) cubensis (Fairchild and Trapido 1950) Lutzomyia (Micropygomyia) cubensis 

Micropygomyia (Micropygomyia) duppyorum (Fairchild and Trapido 1950) Lutzomyia (Micropygomyia) duppyorum 

Micropygomyia (Micropygomyia) hispaniolae (Fairchild and Trapido 1950) Micropygomyia (Micropygomyia) hispaniolae 

Micropygomyia (Micropygomyia) pilosa (Damasceno and Causey 1944) Lutzomyia pilosa 

Micropygomyia (Micropygomyia) xerophila (Young, Brener and Wargo 1983) Lutzomyia xerophila 

Micropygomyia (Sauromyia) atroclavata (Knab 1913) Lutzomyia (Micropygomyia) atroclavata 

Micropygomyia (Sauromyia) ferreirana (Barretto, Martins and Pellegrino 1956) Lutzomyia ferreirana 

Micropygomyia (Sauromyia) quechua (Martins, Llanos and Silva 1975) Lutzomyia quechua 

Micropygomyia (Sauromyia) quinquefer (Dyar 1929) Lutzomyia quinquefer 

Migonemyia (Blancasmyia) cerqueirai (Causey and Damasceno 1945) Lutzomyia (Evandromyia) cerqueirai 

Migonemyia (Blancasmyia) gorbitzi (Blancas 1959) Lutzomyia gorbitzi 

Nyssomyia anduzei (Rozeboom 1942) Lutzomyia (Nyssomyia) anduzei 

Nyssomyia trapidoi (Fairchild and Hertig 1952) Lutzomyia (Nyssomyia) trapidoi 

Nyssomyia ylephiletor (Fairchild and Hertig 1952) Lutzomyia (Nyssomyia) ylephiletor 

Nyssomyia yuilli Young & Porter 1972 Lutzomyia (Nyssomyia) yuilli yuilli

Oligodontomyia oligodonta (Young, Pérez and Romero 1985) Lutzomyia oligodonta 

Pintomyia (Pifanomyia) andina Osorno, Osorno-Mesa and Morales 1972 Lutzomyia andina 

Pintomyia (Pifanomyia) boliviana (Velasco and Trapido 1974) Lutzomyia boliviana 

Pintomyia (Pintomyia) christenseni Young and Duncan 1994 Lutzumyia (Pintomyia) christenseni 

Pintomyia (Pifanomyia) christophei (Fairchild and Trapido 1950) Lutzomyia christophei 

Pintomyia (Pifanomyia) gruta Ryan 1986 Lutzomyia gruta 

Pintomyia (Pifanomyia) moralesi Young 1979 Lutzomyia moralesi 

Pintomyia (Pifanomyia) odax (Fairchild and Hertig 1961) Lutzomyia odax 

Pintomyia (Pifanomyia) oresbia (Fairchild and Hertig 1961) Lutzomyia oresbia 

Pintomyia (Pifanomyia) orestes (Fairchild and Trapido 1950) Lutzomyia orestes 

Pintomyia (Pifanomyia) pia (Fairchild and Hertig 1961) Lutzomyia pia 

Pintomyia (Pifanomyia) torvida Young, Morales and Ferro 1994 Lutzomyia torvida 

Pintomyia (Pifanomyia) youngi Feliciangeli and Murillo 1985 Lutzomyia youngi 

Pressatia camposi (Rodriguez 1952) Lutzomyia (Pressatia) camposi 

Pressatia dysponeta (Fairchild and Hertig 1952) Lutzomyia (Pressatia) dysponeta 

Pressatia trispinosa (Mangabeira 1942) Lutzomyia (Pressatia) trispinosa 

*Based on WRBU22 and Galati.23

†Based on Young and Duncan4

 and various references.
July - September 2015 45

THE ARMY MEDICAL DEPARTMENT JOURNAL

Table 2C. Types of New World sand fl ies (Phlebotominae, Psychodidae), deposited in the USNMNH and MEFSCA, with old and 

new generic and subgeneric classifi cations. (continued).

New Arrangement Old Arrangement†

Psathyromyia (Incertae sedis) ignacioi (Young 1972) Lutzomyia ignacioi 

Psathyromyia (Forattiniella) barrettoi barrettoi (Mangabeira 1942) Lutzomyia barrettoi barrettoi 

Psathyromyia (Forattiniella) barrettoi majuscula (Young 1979) Lutzomyia barrettoi majuscula

Psathyromyia (Forattiniella) carpenteri (Fairchild and Hertig 1953) Lutzomyia carpenteri 

Psathyromyia (Forattiniella) runoides (Fairchild and Hertig 1953) Lutzomyia runoides 

Psathyromyia (Forattiniella) texana (Dampf 1938) Lutzomyia texana 

Psathyromyia (Psathyromyia) campbelli (Damasceno, Causey and Arouck 1945) Lutzomyia (Psathyromyia) campbelli 

Psathyromyia (Psathyromyia) cratifer (Fairchild and Hertig 1961) Lutzomyia (Psathyromyia) cratifer 

Psathyromyia (Psathyromyia) dasymera (Fairchild and Hertig 1961) Lutzomyia (Psathyromyia) dasymera 

Psathyromyia (Psathyromyia) guatemalensis Porter and Young 1986 Lutzomyia (Psathyromyia) guatemalensis 

Psathyromyia (Psathyromyia) shannoni (Dyar 1929) Lutzomyia (Psathyromyia) shannoni 

Psathyromyia (Psathyromyia) soccula (Fairchild and Hertig 1961) Lutzomyia (Psathyromyia) soccula 

Psathyromyia (Psathyromyia) souzacastroi (Damasceno and Causey 1944) Lutzomyia (Psathyromyia) souzacastroi 

Psathyromyia (Psathyromyia) undulata (Fairchild and Hertig 1950) Lutzomyia (Psathyromyia) undulata 

Psathyromyia (Psathyromyia) volcanensis (Fairchild and Hertig 1950) Lutzomyia (Psathyromyia) volcanensis 

Psathyromyia (Xiphomyia) aclydifera (Fairchild and Hertig 1952) Lutzomyia aclydifera 

Psychodopygus amazonensis (Root 1934) Lutzomyia (Psychodopygus) amazonensis 

Psychodopygus ayrozai (Barretto and Coutinho 1940) Lutzomyia (Psychodopygus) ayrozai 

Psychodopygus bispinosus (Fairchild and Hertig 1951) Lutzomyia (Psychodopygus) bispinosus 

Psychodopygus carrerai carrerai (Barretto 1946) Lutzomyia (Psychodopygus) carrerai carrerai 

Psychodopygus carrerai thula (Young 1979) Lutzomyia (Psychodopygus) carrerai thula

Psychodopygus davisi (Root 1934) Lutzomyia (Psychodopygus) davisi 

Psychodopygus fairchildi Barretto 1966 Lutzomyia (Psychodopygus) fairchildi 

Psychodopygus fairtigi (Martins 1970) Lutzomyia (Psychodopygus) fairtigi 

Psychodopygus nocticolus (Young 1973) Lutzomyia (Psychodopygus) nocticola 

Psychodopygus panamensis (Shannon 1926) Lutzomyia (Psychodopygus) panamensis 

Psychodopygus recurvus (Young 1973) Lutzomyia (Psychodopygus) recurva 

Sciopemyia nematoducta Young and Arias 1984 Lutzomyia (Sciopemyia) nematoducta 

Sciopemyia pennyi Arias and Freitas 1981 Lutzomyia (Sciopemyia) pennyi 

Sciopemyia preclara Young and Arias 1984 Lutzomyia (Sciopemyia) preclara 

Sciopemyia servulolimai (Damasceno & Causey 1945) Lutzomyia (Sciopemyia) servulolimai 

Sciopemyia sordellii (Shannon and Del Ponte 1927) Lutzomyia (Sciopemyia) sordellii 

Trichophoromyia castanheirai (Damasceno, Causey and Arouck 1945) Lutzomyia (Trichophoromyia) castanheirai 

Trichophoromyia dunhami (Causey and Damasceno 1945) Lutzomyia (Trichophoromyia) dunhami 

Trichophoromyia gibba Young and Arias 1994 Lutzomyia (Trichophoromyia) gibba 

Trichophoromyia lopesi (Damasceno, Causey and Arouck 1945) Lutzomyia (Trichophoromyia) lopesi 

Trichophoromyia loretonensis (Llanos 1964) Lutzomyia (Trichophoromyia) loretonensis 

Trichophoromyia meirai (Causey and Damasceno 1945) Lutzomyia (Trichophoromyia) meirai 

Trichophoromyia melloi (Causey and Damasceno 1945) Lutzomyia (Trichophoromyia) melloi 

Trichophoromyia napoensis Young and Rodgers 1984 Lutzomyia (Trichophoromyia) napoensis 

Trichophoromyia pabloi (Barreto, Burbano and Young 2002) Lutzomyia (Trichophoromyia) pabloi

Trichophoromyia reburra (Fairchild and Hertig 1961) Lutzomyia (Trichophoromyia) reburra 

Trichophoromyia ruii Arias and Young 1982 Lutzomyia (Trichophoromyia) ruii 

Trichophoromyia sinuosa Young and Duncan 1994 Lutzomyia (Trichophoromyia) sinuosa 

Trichopygomyia elegans Martins, Falcao and Silva 1976 Lutzomyia (Trichopygomyia) elegans 

Trichopygomyia ferroae (Young and Morales 1987) Lutzomyia (Trichopygomyia) ferroae 

Trichopygomyia martinezi Young and Morales 1987 Lutzomyia (Trichopygomyia) martinezi 

Trichopygomyia ratcliffei Arias, Ready and Freitas 1983 Lutzomyia (Trichopygomyia) ratcliffei 

Trichopygomyia triramula (Fairchild and Hertig 1952) Lutzomyia (Trichopygomyia) triramula 

Trichopygomyia wagleyi (Causey and Damasceno 1945) Lutzomyia (Trichopygomyia) wagleyi 

*Based on WRBU22 and Galati.23

†Based on Young and Duncan4

 and various references.
46 http://www.cs.amedd.army.mil/amedd_journal.aspx

RECORDS AND DISTRIBUTION OF NEW WORLD PHLEBOTOMINE SAND FLIES (PSYCHODIDAE, DIPTERA), 

WITH SPECIAL EMPHASIS ON PRIMARY TYPES AND SPECIES DIVERSITY

Table 2D. Types of New World sand fl ies (Phlebotominae, Psychodidae), deposited in the USNMNH and MEFSCA, with old and 

new generic and subgeneric classifi cations (continued).

New Arrangement Old Arrangement†

Trichopygomyia wilkersoni Young and Rodgers 1984 Lutzomyia (Trichophoromyia) wilkersoni 

Trichopygomyia witoto Young and Morales 1987 Lutzomyia (Trichopygomyia) witoto 

Viannamyia fariasi (Damasceno, Causey and Arouck 1945) Lutzomyia (Viannamyia) fariasi 

Warileya nigrosaccula Fairchild and Hertig 1951 Warileya nigrosaccula 

Warileya phlebotomanica Hertig 1948 Warileya phlebotomanica 

Warileya rotundipennis Fairchild and Hertig 1951 Warileya rotundipennis 

Warileya yungasi Velasco and Trapido 1974 Warileya yungasi 

*Based on WRBU22 and Galati.23

†Based on Young and Duncan4

 and various references.
July – September 2015 47

Since World War II, the US military has used aerial 

spray to mitigate vector-borne disease in combat situations as well as domestic postemergency scenarios. The 

benefi t of aerial application is that large areas which may 

be inaccessible from the ground can be covered rapidly, 

thus quickly breaking the cycle of infection.1

 Currently, 

USAF aerial spray operations for mosquito and other 

insect-borne disease control are conducted during daylight hours. The application platform for USAF aerial 

sprays is a C-130 H2 aircraft with a modular aerial spray 

system (MASS). Current operational parameters use a 

fl ight profi le spraying at 150 ft (45.7 m) above ground 

level (AGL) at 200 knots (370.4 km/hr) groundspeed. 

These parameters were derived from effi cacy tests coupled with the need to maintain relatively high airspeeds 

and low altitudes to mitigate the potential effect of 

ground-fi re hazards.2-4 Prior to the common use of night 

vision goggles (NVGs) to offset the unacceptable hazards of low-fl ying nighttime applications, daytime aerial 

application of pesticides was the only viable option.

Benefi ts of night-spray capability are multiple. Application of pesticides concurrent to night feeding mosquito 

(or other) vector activity will potentially maximize vector mortality and reduce human pathogen transmission. 

It is generally considered that the optimal timing for 

application of a fast-acting pesticide is when the target 

insect is active and in search of a blood meal or other 

specialized fl ight periods.5

 Based on this concept, the 

need for a nighttime aerial spray application capability 

becomes self-evident. The need for this capability was 

reinforced when West Nile virus (WNV) quickly spread 

through the Americas beginning in 1999. Though most 

of the endemic encephalitides were well documented to 

be vectored by mosquitos of the genus Culex, the introduction of WNV galvanized the public health fi eld with 

respect to the importance of controlling populations of 

these vectors.6

 The Culex and some Aedes species responsible for transmitting viruses like WNV to humans 

are reportedly far more active in the crepuscular and 

night hours rather than the daytime hours.7

 Because of 

this, a majority of aerial adulticiding conducted by civilian mosquito control agencies in the United States is 

conducted in the evening and nighttime hours.

Benefi ts of night spraying also include minimizing exposure of diurnal benefi cial insects to pesticides. The 

potential for pollinator and other benefi cial insect mortality is a signifi cant concern with aerial spray pesticide 

application. In the case of honeybees, standard operating 

procedure for daytime aerial spray application of pesticides includes notifying local beekeepers so that they 

can cover and isolate their hives prior to any application. 

Nighttime application does not remove the responsibility from the applicators to inform the beekeepers, but it 

reduces potentially negative effects of pesticide application (bee kill) as honeybees are normally naturally ensconced in a protected hive at time of application. The 

same level of protection may not be as well known for 

other diurnal benefi cial insects, and nocturnally active 

benefi cial insects might not be so protected from night 

pesticide applications.

Development of Air Force Aerial Spray

 Night Operations: High Altitude Swath

 Characterization

 Lt Col Karl A. Haagsma, BSC, USAFR

 Lt Col Mark S. Breidenbaugh, BSC, USAFR

 Kenneth J. Linthicum, PhD

 Robert L. Aldridge

 Seth C. Britch, PhD

ABSTRACT

Multiple trials were conducted from 2006 to 2014 in an attempt to validate aerial spray effi cacy at altitudes 

conducive to night spray operations using night vision goggles (NVGs). Higher altitude application of pesticide 

(more than 400 ft (121.9 m) above ground level (AGL)) suggested that effective vector control might be possible 

under ideal meteorological conditions. A series of lower altitude daytime applications (300 ft (91.4 m) AGL) 

demonstrated effective and repeatable mortality of target sentinel insects more than 5,000 ft (1,524 m) downwind, and control of natural vector populations. From these results we believe further pursuit of aerial night 

applications of pesticide using NVGs at 300 ft (91.4 m) AGL by this group is warranted.
48 http://www.cs.amedd.army.mil/amedd_journal.aspx

Night spraying also leverages favorable meteorological 

conditions that may reduce effects of unwanted pesticide drift. Convective atmospheric activity during daytime aerial pesticide applications has always plagued 

uniform pesticide application. Differential heating may 

cause convective surface currents to blow away sections 

of pesticide application plumes at altitude, thereby potentially leaving signifi cant application gaps. Lowered 

convective activity during nighttime applications may 

reduce this effect. At night, surface wind currents generally become more laminar in nature and pesticide drift 

may become more predictable and effective (conversation with H. Thistle, January 2009).

Despite these benefi ts of nighttime aerial spray application, the obvious signifi cant danger to night application 

is the lack of ability to easily see and identify objects 

that present collision hazards at low altitude. In addition 

to natural obstructions, man-made obstructions such as 

buildings, towers, or antennae present formidable obstacles to low-level aircraft navigation. In the contiguous 

United States, 68% of all surface obstructions are less 

than 300 ft (91.4 m) AGL, 75% of all surface obstructions are less than 350 ft (106.7 m) AGL, and 5% of all 

surface obstructions are greater than 500 ft (152.4 m) 

AGL (conversation with Lt Col John Kochansky, September 2014). Thus, fl ying at higher altitudes (300+ ft 

AGL) presents signifi cantly less risk than fl ying a 150 ft 

(45.7 m) ground profi le at night. Although NVGs have 

mitigated some hazards of night fl ying, they have limitations regarding which objects are visible at night.

In an attempt to increase the military spray capability 

to include nighttime application, the USAF aerial spray 

fl ight investigated the feasibility of pesticide application 

fl ying on NVGs. This investigation was especially challenging as the fl ight profi le was outside the parameters 

of the standard low-level NVG routines which are most 

exclusively employed by special operations C-130 missions using a modifi ed-contour scenario fl ying at 300 ft 

(91.4 m) AGL on NVGs. Aerial spray mission requirements for low-level NVG operations required a new, 

unique set of operating conditions that had not been previously considered.

Proposals including a point paper to convince USAF leadership of the benefi ts of aerial spray nighttime operation 

were initiated in 2004. Following a series of inquiries 

and safety reviews, and with endorsement from agencies 

including the US Centers for Disease Control and Prevention, the Armed Forces Pest Management Board, the 

Offi ce of the Surgeon General of the Air Force, and the 

support of several US mosquito and vector control agencies, permission was granted to the USAF aerial spray 

fl ight to begin low-level NVG training activities specifi c 

to night-time aerial spray operations in 2014.

The potential operational capability by the USAF aerial 

spray fl ight to conduct night spray missions must be supported by evidence of functional capability. This would 

include demonstrations that night pesticide applications 

were effective against target medically important insect 

vectors of signifi cant disease while operating within an 

acceptable USAF operational scenario. Herein, we report the results of multiple daytime aerial pesticide application trials conducted to determine pesticide droplet 

size and effective swath width, including fi eld mosquito 

sentinel mortality, at a variety of altitudes and with a variety of spray nozzle sizes to support USAF NVG operations for night-spray capability at 300 ft (91.4 m) AGL.

METHODS AND MATERIALS

Each of the trials detailed below, from earliest to most 

recent, was designed as a standalone evaluation, generally without replication. The aerial spray platform for all 

trials was a USAF C-130 H2 aircraft equipped with a 

MASS, with a variety of nozzle sizes and confi gurations 

on 2 fuselage booms as detailed below. Meteorological 

conditions were measured using a Swath Kit weather 

monitoring station (Droplet Technologies, College Station, PA) (trial sets 1-3) or a Kestrel NV4500 logging 

portable weather station (Nielsen-Kellerman, Boothwyn, PA) (trial sets 4-6). Trial sets 7 and 8 used 4 Kestrel NV4500 logging portable weather stations placed at 

various intervals along each sampling line, and meteorological data were averaged between data derived from 

each station over the course of each test.

Trial Set 1

The trials were conducted January 13-18, 2006, at Avon 

Park Training Range, Avon Park, Florida (Figure 1).

On January 14, 10 aerosol impingers (spinners) (John 

Hock Company, Gainesville, FL) were placed in an eastwest orientation on Kissimmee Road. Impingers were 

positioned in a linear array with each sampling station separated by 2500 ft (762 m). Each impinger was 

equipped with 2 Tefl on-coated 25mm by 75mm microscope slides to collect droplets from the aerosol cloud 

released from the aircraft as the cloud drifted downwind 

past each sampling station.

The fuselage booms were fi tted with a total of 6 fl atfan TeeJet 8008 Nozzles (Spraying Systems Company, 

Wheaton, IL). BVA-13 oil was used to simulate pesticide, 

applied at 4.5 gallons of material per minute (17.03 L/

min) at an operating system pressure of 83 psi. Three 

north-south overlapped (crosswind) passes of the aircraft 

DEVELOPMENT OF AIR FORCE AERIAL SPRAY NIGHT OPERATIONS:

HIGH ALTITUDE SWATH CHARACTERIZATION
July – September 2015 49

THE ARMY MEDICAL DEPARTMENT JOURNAL

were made to increase the droplet concentrations at the 

sampling stations. The spray-on time for each pass was 

40 seconds, with spraying beginning 30 seconds prior to 

the aircraft’s point of intersection with Kissimmee Road. 

The aircraft fl ew directly over the upwind sampling station at 500 ft (152.4 m) AGL.

On January 15, 10 impingers were aligned on Frostproof 

Road (Figure 1). Sampling stations were placed 1,700 ft 

(518 m) apart, with the fi rst sampling station located on 

the northwest point in the road. Three adjacent southwest-northeast passes at 500 ft (152.4 m) AGL were 

made over the fi rst sampling station, with each pass 

commencing and ending spray 30 seconds before and 

after the aircraft’s intersection with Frostproof Road. 

The MASS sprayed BVA-13 oil at 4.75 gal/min (17.98 L/

min) at an operating pressure of 56 psi.

On January 16, 10 impingers were placed in a northsouth orientation along Van Eegan Road (Figure 1). 

Sampling stations were positioned 2,500 ft (762 m) apart 

with the fi rst sampling station located at the southern 

point of the road. The BVA-13 oil spray fl ow rate was 4.7 

gal/min. Four overlapped west-east passes were fl own at 

500 ft (152.4 m) AGL, with each pass commencing and 

ending spray 30 seconds prior to and after intersection 

with the road at the fi rst sampling station.

Following each set of sprays on the 3 trial days, Tefl on 

slides were collected 45 minutes after the last pass and 

viewed under a compound microscope. Up to 50 droplets (if available) were counted per slide at each one of 

the sampling stations. Droplets were measured for volume median diameter (VMD), calculated using the Yeomans method.8

Figure 1. Map of Avon Park Air Force Range (Florida) with testing locations highlighted.

Van Eegan Rd

Kissimmee

Frostproof Rd
50 http://www.cs.amedd.army.mil/amedd_journal.aspx

Trial Set 2

These trials were conducted at the Avon Park Training Range 13-17 February 2006. In general, the methods were similar to those in Trial set 1. All trials were 

conducted with the aircraft spraying at 500 ft (152.4 m) 

AGL. The spray booms were fi tted with 6 fl at-fan TeeJet 8008 nozzles, three on each boom and in each trial, 

the MASS delivered approximately 4.5 gal/min (17.03 

L/min) of BVA-13 oil with the system operating at approximately 50 psi. In these tests, an optical whitener 

(Uvitex OB, Ciba Specialty Chemicals, Tarrytown, NY) 

was added to the BVA-13 oil in an attempt to differentiate between potential contamination of the Tefl on slides 

with ambient atmospheric aerosols versus the spray 

coming from the aircraft. A fl uorescent compound microscope was used to examine droplet collection slides. 

The Uvitex in the BVA-13 oil caused droplets collected 

from the spray mission to fl uoresce vigorously, making identifi cation unambiguous. Four overlapping spray 

passes for each trial in this series were conducted on a 

course perpendicular to the impingers, directly overfl ying the fi rst sampling station in the series. Each pass 

totaled 30 seconds of spray-on time, 15 seconds before 

and 15 seconds following intersection with the road(s) 

and the fi rst sampling station. Slides were collected and 

droplet data were calculated as in Trial Set 1, with the 

exception that droplet density (droplets/cm2

) was also 

recorded for each of the sampling stations.

On February 14, a trial was conducted with impingers 

set up on a northwest-southeast orientation along Frostproof Road (Figure 1) with sampling stations separated 

by 1,700 ft (518.1 m). The fi rst sampling station was located at the southeast end of the road.

On February 15, a test was conducted on an east-west 

orientation on Kissimmee Road (Figure 1) with sampling stations separated by 2,500 ft (762 m). The fi rst 

sampling station was at the east end of the road.

On February 16, a north-south linear array of sampling 

stations was installed on Van Eegan Road (Figure 1). 

Sampling stations were separated by 1,742 ft (531 m) 

with the fi rst station located at the junction of Kissimmee and Van Eegan Roads.

Trial Set 3

Trials were conducted December 4-8, 2006, at the Avon 

Park Training Range. In these trials, a sentinel mosquito 

bioassay was used in conjunction with the droplet collection system described above. The fuselage booms were 

fi tted with a total of 20 fl at-fan TeeJet 8005 nozzles, (10 

on each side), which produced fl ow rates approaching 7.2 

gal/min (27.25 L/min) at an operating system pressure 

of 50 psi. The pesticide used for these trials was Dibrom 

(Naled; AMVAC Chemical Corp., Los Angeles, CA) 

organophosphate adulticide, applied at 500 ft (152.4 m) 

AGL. In each test, 10 impinger sampling stations were 

collocated with 10 bioassay cages consisting of cardboard rings (approximately 3.175 cm by 15.25 cm), with 

open ends covered with fi ne plastic mesh. In each cage 

was placed approximately 25 adult female Culex quinquefasciatus from the laboratory colony at the US Department of Agriculture Agricultural Research Service 

Center for Medical, Agricultural, and Veterinary Entomology (USDA-ARS CMAVE), Gainesville, FL. Sampling arrays each consisting of an impinger and a sentinel cage were deployed at 0.5 mile (804.6 m) intervals 

along Van Eegan Road (Figure 1) on December 6 and 

7. The fi rst sampling array locations for both trials were 

at the intersection of Kissimmee and Van Eegan Roads. 

Four control sentinel mosquito cages were located approximately 0.5 miles (804.6 m) north (upwind) of the 

fi rst sampler/cage and exposed for the duration of each 

trial to ambient conditions. The aircraft conducted 2 

overlapping passes for each trial on a course perpendicular to the sampling stations, fl ying directly over the fi rst 

sampling station. Spray-on time was 30 seconds before 

and 30 seconds after overfl ying this location. The aerosol cloud was allowed to drift and settle for 45 minutes, 

after which time the Tefl on slides and the bioassay cages 

were collected. Droplets were measured as described for 

the previous Trial Sets, and VMD, numerical median 

diameter (NMD), and droplet density were calculated 

for each sampling station. Mosquitoes were aspirated 

from the bioassay cages and transferred to clean cages 

equipped with cotton balls soaked with a 10% sugar water solution. Mosquito mortality was determined at 2, 12, 

and 24 hours postspray, and corrected with Abbott’s formula9

 relative to mortality in the controls.

Trial Set 4

Trials were conducted December 6-10, 2009, at the Avon 

Park Training Range. Ten impinger sampling stations 

equipped as before were deployed along Van Eegan 

Road (Figure 1) on December 8 and 9 at 0.25 mile (402.3 

m) intervals for the fi rst mile (1,609.3 m) of the sampling 

array, followed by 0.5 mile (804.6 m) intervals for the remaining 4.5 mile (7,242 m) sampling line. The spray platform was outfi tted with 7 fl at fan TeeJet 8005 nozzles on 

each spray boom, producing a fl ow rate of approximately 

7.2 gal/min (27.25 L/min) at an operating pressure of 50 

psi. BVA-13 oil was used to simulate pesticide, and application altitude was 300 ft (91.4 m) AGL. The aircraft 

conducted 2 overlapping spray passes per trial perpendicular to the sampling stations, with each pass totaling 

60 seconds of spray-on time (30 seconds prior to and 30 

seconds after intersection with fi rst sampling location). 

DEVELOPMENT OF AIR FORCE AERIAL SPRAY NIGHT OPERATIONS:

HIGH ALTITUDE SWATH CHARACTERIZATION
July – September 2015 51

THE ARMY MEDICAL DEPARTMENT JOURNAL

The fi rst sampling station was located at the intersection 

of Kissimmee and Van Eegan Roads. Tefl on slides were 

collected as described in the above Trial Sets. One hundred drops (if available) were counted from each slide, 

and VMD, NMD, and droplet density were recorded.

Trial set 5

This trial series was conducted at the Avon Park Training Range the week of January 23, 2012.

On January 24, 10 impinger sampling stations separated by 0.5 miles (804.6 m) were placed in an east-west 

orientation on Kissimmee Road (Figure 1). Fuselage 

spray booms on the aircraft were fi tted with 8 TeeJet 

8005 nozzles, four on each side, for a fl ow rate of 4.5 

gal/min (17.03 L/min) at an operating pressure of 83 

psi. Three north-south overlapped perpendicular passes 

were made by the aircraft beginning directly over the 

fi rst sampling station in the array. Spray-on times were 

20 seconds prior to and 20 seconds after intersection of 

aircraft fl ight path and Kissimmee Road. Altitude of the 

application was 400 ft (121.9 m) AGL and BVA-13 oil 

was used to simulate pesticide.

On January 25, the January 24 trial was repeated, however, spray booms were outfi tted with 10 TeeJet 8005 

nozzles, 5 on each side, for a fl ow rate of 4.75 gal/min 

(17.98 L/min) at an operating pressure of 56 psi. The fi rst 

4 sampling stations were 0.25 miles (402.3 m) apart, with 

the remainder positioned at 0.5 mile (804.6 m) intervals.

Trial parameters from January 25 were repeated on January 26. However, the sampler array was aligned in a 

north-south orientation along Van Eegan Road (Figure 

1) and aircraft spray passes were from west-east. Flow 

rate during this test was 4.6 gal/min (17.4 L/min) with 

a pressure of 45 psi. Droplet size and density data were 

collected as described for the January 24 trial set.

Trial Set 6

On October 17, 2012, 2 trials were performed in conjunction with an operational aerial spray at Parris Island Marine Corps Recruit Depot (MCRD), South Carolina. In 

each of these trials, 9 bioassay cages similar to those described in Trial Set 3, each with approximately 20 adult 

female Cx. quinquefasciatus, were placed in a linear array along Wake Blvd. The bioassay cages were clamped 

to wooden dowels at approximately 2 ft (0.61 m) AGL 

and fi tted with cotton balls saturated with 10% sugar solution. The fi rst 5 cages were separated from each other 

by 250 ft (76.2 m), with each of the remaining 4 cages 

separated by 500 ft (152.4 m). The spray booms were 

outfi tted with 18 TeeJet 8003 fl at-fan nozzles, 9 on each 

boom. In each trial in the set, the aircraft fl ew a swath 

perpendicular the bioassay sampling line, dispensing Dibrom (Naled) at 300 ft (91.4 m) AGL. Flow rate and pressure recorded from the MASS were 3.3 gal/min (12.5 L/

min) at approximately 60 psi. Spray-on times were 20 

seconds before and 20 seconds following intersection of 

the aircraft at the fi rst bioassay station. An additional 3 

bioassay cages were included as controls for each trial 

and exposed to ambient conditions during the trials, approximately 0.5 miles (804.6 m) upwind of the application spray line. Bioassay cages were collected and initial knockdown was recorded approximately 35 minutes 

after each trial. Mosquitoes were then transferred into 

clean holding cups fi tted with cotton balls saturated with 

10% sugar water solution. Mortality was recorded at 1, 

12, and 24 hours postspray, and all mortality was corrected with Abbott’s formula as described in Trial Set 3.

Trial Set 7

Two independent bioassay trials were conducted on April 

9, 2013, at Parris Island MCRD in conjunction with an 

operational aerial application of the island, and were the 

result of a cooperative effort between the USAF aerial 

spray unit and personnel from USDA-ARS-CMAVE. 

Two tests were conducted using linear arrays of bioassay 

cages placed either along Wake Blvd (Figure 2A) or the 

Causeway (Figure 2B). One dispensing swath was used 

per test. For each test, fuselage booms were equipped 

with 14 TeeJet 8003 fl at fan nozzles, 7 on each boom.

For the fi rst application, 20 bioassay cages identical to 

those described in Trial Set 6 were placed along Wake 

Blvd at approximately 500 foot (152.4 m) intervals. The 

fi rst bioassay cage was positioned at the southwest terminus of Wake Blvd. The aircraft dispensed Dibrom 

(Naled) adulticide at 300 ft (91.4 m) AGL perpendicular 

to the sampling array, with an approximately 300 foot 

(91.4 m) upwind offset. Spray-on and off times were 20 

seconds before and after the aircraft intersection with 

the fi rst sampling station. The MASS parameters were 

2.7 gal/min (10.2 L/min) at 52 psi. Five sentinel (control) 

mosquito control cages were placed at the golf course 

on the southernmost point on the island outside of the 

treatment area and exposed to ambient conditions for 

the duration of each spray test.

For the second test, 25 bioassay cages identical to those 

described in Trial Set 6 were deployed along the Causeway at approximately 300 foot (91.4 m) intervals, with 

the fi rst bioassay cage at the northwestern terminus of 

the road and an additional 5 sentinel (control) cages 

placed in the open at the golf course upwind from the 

spray application. The aircraft dispensed adulticide at 

300 ft (91.4 m) AGL on a swath perpendicular to the 

causeway, directly overfl ying the last sampling station.
52 http://www.cs.amedd.army.mil/amedd_journal.aspx

Initial knockdown from the bioassay cages was recorded after a 40 minute hold following each application, 

and the sentinel cages were placed in ice chests. Sentinel mosquito mortality was subsequently recorded at 4 

and 12 hours postspray, and all mortality was corrected 

using Abbott’s formula as described above.

Trial Set 8

Two bioassay tests were conducted on October 9, 2014, 

at the Parris Island MCRD, SC, with parameters similar 

to those in Trial Set 7. In both tests, 30 sentinel mosquito 

cages were deployed along Wake Blvd, each separated 

by approximately 250 ft (76.2 m), with an additional 

set of 10 sentinel cages for each trial located upwind 

of the spray area on the southern end of the island. The 

fi rst sampling location in both trials was positioned at 

the southwest terminus of Wake Blvd. Fuselage booms 

were equipped with 15 fl at-fan TeeJet 8003 nozzles, 7 

on the left boom and 8 on the right boom. The MASS 

parameters during the tests were a fl ow rate of 2.8 gal/

min (10.6 L/min) and an operating system pressure of 40 

psi in the fi rst trial and 52 psi in the second trial. Postapplication protocols for tallying mortality data collection 

were the same as those described in Trial Set 7.

In the fi rst trial, 2 roughly overlapping passes (swaths) 

were made as the aircraft dispensed adulticide perpendicular to the sampling line over sampling locations 10 

and 13 (Figure 3A). Spray-on times were 20 seconds 

prior to and after intersection of the aircraft with the 

sampling points on the ground.

In the second trial, 2 swaths were applied with similar spray-on times as in the fi rst test, with the aircraft 

DEVELOPMENT OF AIR FORCE AERIAL SPRAY NIGHT OPERATIONS:

HIGH ALTITUDE SWATH CHARACTERIZATION

Figure 2A. Caged mosquito mortality at 12 hours after pesticide application and meteorological data for pesticide release

April 9, 2013, on sampling array located on Wake Blvd, Parris Island Marine Corps Recruit Depot, SC. Flight path of aircraft is

depicted by green arrow.
July – September 2015 53

THE ARMY MEDICAL DEPARTMENT JOURNAL

dispensing adulticide while fl ying over sampling locations 1 and 7, essentially skipping a 1,000 foot (304.8 m) 

swath (Figure 3B).

RESULTS AND DISCUSSION

Trial Set 1

No data were collected from the fi rst test conducted on 

January 14, because no droplets were visible on the microscope slides. Wind speed and direction at the time of 

the test was between 9 and 15 knots from 290 degrees 

measured at ground level. We speculate that the wind 

speed was too high, and at the height the simulant was 

applied combined with the 20 degree crosswind component, the material may have missed the sampling array 

altogether. Wind speed and direction on the January 15 

test was 2-4 knots at 310 degrees. Droplet VMD ranged 

from approximately 32 μm 1,700 ft (518.2 m) from the 

aircraft release point, increasing to approximately 50 

μm at 3,400 ft (1036 m) and 5,100 ft (1,554.5 m), followed by a roughly linear decrease in VMD at 13,600 

ft (4,145.3 m) from release point. Droplets were not detected at sampling stations greater than this distance, or 

at the sampling location directly beneath the aircraft 

fl ight path. Wind speed and direction on the January 

16 test was 6-7 knots from 188 degrees. Droplets collected at the sampling location 2,500 ft (762 m) from 

the release point were approximately 68 μm, with the 

remainder of droplets collected by the samplers ranging 

from 30-50 μm up to 22,500 ft (6858 m) from the release 

point. Droplet density increased from approximately 

0.75 drops/cm2

 at the 2,500 foot (762 m) sampling location, to over 2.25 drops/cm2

 at 10,000 ft (3,048 m), and 

then decreased to between 0.5 and 1.0 drops/cm2

. For 

both ofthese tests, no droplets were collected directly 

Figure 2B. Caged mosquito mortality at 12 hours after pesticide application and meteorological data for pesticide release

April 9, 2013, on sampling array located on Causeway, Parris Island Marine Corps Recruit Depot, SC. Flight path of aircraft is 

depicted by green arrow.
54 http://www.cs.amedd.army.mil/amedd_journal.aspx

beneath the release point. As evidenced by the data from 

the January 15 trial, a lighter wind condition may have 

resulted in a tighter and more normal distribution of 

droplet sizes. No droplets were collected at the far limits 

of the sampling array. This is in contrast with the January 16 trial, where increased wind speed apparently carried the simulant cloud past the limits of our detection 

devices. Thus, in light wind conditions it appears that 

the material drift can be reasonably defi ned, whereas 

spraying from 500 ft (152.4 m) AGL may not be optimal 

in terms of controlling or defi ning drift when sprayed at 

higher wind speeds.

Trial Set 2

The fi rst test on February 14 yielded no data, possibly 

due to light and variable winds. Ground wind speed was 

less than 2 knots and the wind direction shifted more 

than 180 degrees several times during the test. Conditions were signifi cantly better on February 15. Surface 

winds ranged from 1.5 to 3 knots and remained consistent in direction from approximately 070 degrees. The 

highest droplet densities (10-14 drops/cm2

) were seen 

at stations 3 and 4 at 4,224 (1,287.5 m) and 6,336 ft 

(1,931.2 m) downwind from the release point, respectively. Densities dropped dramatically by sampling station 4, and remained low from then on. The VMD of 

droplets was greatest at sampling station 1, exceeding 

50 μm, and ranged from 28-42 μm at the remainder of 

the stations. Because higher droplet densities are generally correlated with greater insect mortality, it appears 

the portion of the swath with the greatest potential for 

effective mosquito control may in this case be an effective swath of approximately 2,000 ft (609.6 m), beginning approximately 3,000 ft (914.4 m) downwind of the 

DEVELOPMENT OF AIR FORCE AERIAL SPRAY NIGHT OPERATIONS:

HIGH ALTITUDE SWATH CHARACTERIZATION

Figure 3A. Caged mosquito mortality at 12 hours after pesticide application and meteorological data for multiple pass pesticide

release October 8, 2014, on sampling array located on Wake Blvd, Parris Island Marine Corps Recruit Depot, SC.
July – September 2015 55

THE ARMY MEDICAL DEPARTMENT JOURNAL

release point. While this test was encouraging, the lack 

of a concurrent bioassay data renders this observation 

unsubstantiated. Ground conditions on the February 16 

test remained favorable, with surface winds averaging 

1.5 to 4 knots, though wind direction was slightly more 

variable in nature, ranging from 360 to 030 degrees. 

Winds at altitude were 11 knots at 060 degrees. Droplets 

were only recovered downwind as far as station 6 (8,712 

ft downrange (2655.4 m)). Droplet sizes were greatest 

at station 2 (3,485 ft (1,062.2 m)), though droplet density in the aerosol cloud was relatively low (less than 

5 drops/cm2

). Droplet densities were greatest at station 

1 (1,742 ft (530.9 m) downrange). Discrepancies in the 

data from the 2 tests might have been attributed to the 

signifi cant crosswind component at the 500 ft AGL release point, where winds were almost 090 degrees from 

the theoretical direction that could have led to greater 

drift and dispersion. In this case it appears only a small 

leading edge portion of the aerosol cloud was effectively 

sampled. While both trials effectively indicated that an 

aerosol cloud generated at 500 ft (152.4 m) AGL can descend to ground level, they also suggest there might be a 

fairly low tolerance for crosswind components, making 

spraying from this altitude much more unpredictable.

Trial Set 3

Ground wind conditions for the December 6 test were 

3-5 knots at 045-060 degrees for 30 minutes prior to the 

test, but wind direction subsequently shifted to 080-092 

degrees at the time of application. For this test, postapplication mosquito mortality was 100% at the second 

sampling station 2,500 ft (762 m) downrange of release 

point, and then dropped dramatically to approximately 

20% at 5,000 ft (1524 m) downrange. Droplet size and 

Figure 3B. Caged mosquito mortality at 12 hours after pesticide application and meteorological data for skipped swath test 

October 8, 2014, on sampling array located on Wake Blvd, Parris Island Marine Corps Recruit Depot, SC.
56 http://www.cs.amedd.army.mil/amedd_journal.aspx

density remained consistent at both of these sampling 

locations, with approximate VMDs of 45 and droplet 

densities of 85-100 drops/cm2

. Data from the December 7 test yielded signifi cantly different results. Surface 

winds during the test ranged from 1.2 to 3.5 knots at 

340-360 degrees, while winds at altitude during the application averaged 2 knots at 324 degrees. As occurred 

the day before, insect mortality was 100% at the second sampling station 2,500 ft (762 m) downrange, and 

dropped to approximately 75% and 20% at sampling stations 3 and 4 (1,524 m, and 2,286 m), respectively. Droplet diameters and droplet densities were very different 

from the previous test: VMDs at sampling stations 2 and 

3 were approximately 11 μm, and droplet densities were 

20 drops/cm2

 and 10 drops/cm2 at the sampling stations 

2,500 (762 m) and 5,000 ft (1,524 m) downrange. No 

droplets were collected past the third sampling station. 

It appears that in the fi rst test the wind shift encountered during the application may have resulted in only 

a small portion of the aerosol cloud coming in contact 

with the sampling stations and caged mosquitoes. We 

speculate that perhaps only the leading edge of the spray 

with the associated larger and faster depositing droplets 

encountered the sampling array. Conversely, it would 

appear that wind shift prior to application in the second trial may have resulted in the heavier fraction of the 

spray moving perpendicular to the sampling array, with 

only the fi ner fractions which dispersed by diffusion or 

entrainment in the wingtip vortices contacting the remainder of sampling stations. Thus, while it appears that 

application at 500 ft (152.4 m) AGL can result in insect 

mortality at ground level, it is apparent that the vagaries 

of environmental conditions might be greatly amplifi ed 

while spraying at higher altitudes.

Trial Set 4

Surface wind conditions were ideal during the December 8 trial, with wind speed averaging 3.9 knots from a 

steady 360 degree direction. Droplet size (VMD) ranged 

from approximately 43 μm at the sampling station 2500 

ft (762 m) downwind, generally decreasing to 27 μm 

10,000 ft (3048 m) downrange. Droplet density peaked 

at the fi rst sampling point (1,250 ft (381 m) past release 

point) at 25 drops/cm2

. Droplet density decreased at all 

further sampling stations with the exception of sampling 

station 3, which was located 3,750 ft (1,143 m) from 

simulant release point. Trial 2 on December 9 produced 

generally similar results. Surface winds were again 

relatively light. Wind speed was 3-5 knots with directions ranging from 340-016 degrees. The highest droplet 

density was seen at sampling station 2 (2,500 ft (762 m) 

downrange) with corrected densities of approximately 

27 drops/cm2

. Droplet densities fell to roughly 15 drops/

cm2

 from station 3 (3,750 ft (1,143 m) downrange), to 

station 6 (7,500 ft (2,286 m) downrange), after which 

densities fell dramatically. Droplet size was fairly consistent, in the 40-50 μm range for the fi rst 5,000 ft (1,524 

m) downwind, after which it tapered off to approximately 28 μm at the end of the sampling array. Based on relative similarities and consistencies of these 2 tests, the 

data would suggest under similar circumstances that a 

nominal swath width of perhaps up to 5,000 ft (1,524 m) 

may be warranted when spraying at 300 ft (91.4 m) AGL, 

although that had not been empirically tested with bioassays at this point. Unfortunately, in these trials, there 

was no sampling station at the release point, and as such, 

we cannot speculate on any outcomes from the spray 

release point out to 13,500 ft (4,114.8 m) downwind. Perhaps the most important point from these trials is that it 

appears detection levels of pesticide (or simulant) when 

dispensed at a lower altitude (300 ft (91.4 m) AGL), display a much more concentrated effect in terms of droplet 

densities and VMD than those previously demonstrated 

in higher altitude tests. Previous trials dispensing at 500 

ft (152.4 m) AGL indicated some detection of pesticide/

simulant past 18,000 ft (5,486.4 m) downwind. These 

trials suggest that while material may well indeed drift 

over 2 miles (3.21 km), the potential for effective mosquito control may be within 5,000 ft (1,524 m) downwind or less of the release point.

Trial Set 5

The surface conditions on January 24 had ground winds 

of 6-7 knots from 290 degrees. Droplet size ranged from 

42 μm to approximately 31 μm in diameter, with most 

of the VMD size classes around 40 μm out to 4,000 ft 

(1,219.2 m) downwind of application. Droplet density 

was greatest at 1,250 ft (381 m) downwind at approximately 5.8 drops/cm2

, and then decreased in a semilinear 

fashion to where droplet densities were almost zero at 

5,000 ft (1,524 m) downwind. Conditions on January 25 

were surface winds averaging 4 knots from 280 degrees. 

As expected, VMD was greatest at the fi rst 2 sampling 

stations (750 ft (228.6 m) and 1,250 ft (381 m)), and then 

decreased at 5,000 ft (1,524 m) downrange. Droplet densities were greatest at the fi rst sampling station at 12.7 

droplets/cm2

, and decreased to approximately 2.0 drops/

cm2

 by sampling station 4 at 2,750 ft (838.2 m) downrange. The January 26 surface conditions were 6-7 knot 

winds at a direction of 350 degrees. Droplet size (VMD) 

was greatest at 750 ft (228.6 m) downrange (approximately 60 μm) and then dropped until 2,750 ft (838.2 m) 

downrange where it peaked again at 61 μm. Droplet density was maximized at 1,250 ft (381 m) downrange at approximately 14.0 drops/cm2

. Droplet density approached 

zero at 2,750 ft (838.2 m) postrelease point. From these 3 

trials at an application altitude of 400 ft (121.9 m) AGL, 

it appears that the droplets dispensed from the aircraft 

DEVELOPMENT OF AIR FORCE AERIAL SPRAY NIGHT OPERATIONS:

HIGH ALTITUDE SWATH CHARACTERIZATION
July – September 2015 57

THE ARMY MEDICAL DEPARTMENT JOURNAL

did show a fairly predictable swath in terms of width 

and droplet size. Though the range of detectability appeared acceptable, we consider that the droplet density 

was not—all measured statistics were a result of 3 passes 

in these test iterations versus a single pass as would be 

used in a normal mosquito control operation calibration 

trial. Again, we speculate that while a certain amount a 

material drifts to the ground or is pushed to the ground 

by the dynamic effects of the aircraft, perhaps much of 

the material, that is, the smaller fractions of the aerosol cloud, may be simply drifting away. However, this 

hypothesis has not been substantiated by fi eld sentinel 

bioassay data under these conditions.

Trial Set 6

In this bioassay trial, both tests were conducted on the 

same day, October 12. Surface conditions on the ground 

during these fi eld tests was an average wind speed of 

1-5 knots from 050 degrees, and remained consistent for 

both trials. No swath characteristics were recorded. In 

the fi rst trial, 100% mosquito mortality at 1 hr, 12 hr, 

and 24 hour was witnessed at the sampling station 500 ft 

(152.4 m) downwind of release point. This was true also 

at the station located 1,000 ft (304.8 m) downwind, after which mosquito mortality dropped to approximately 

20% at 1,500 ft (457.2 m) downwind, and to almost zero 

thereafter. The second trial indicated similar, but slightly skewed results. Mosquito mortality of 100% was observed 750 ft (228.6 m) downwind of application, and 

was continued to 1,500 ft (457.2 m) downrange, at which 

time mortality dropped to approximately 30% at 2,000 

ft (609.6 m) downrange. Interestingly, mortality increased signifi cantly at stations 2,500 ft (762 m) or more. 

This result was unexpected, and we have no explanations for the apparent anomaly. In these conditions, both 

of these tests would suggest an effective swath from 300 

ft (91.4 m) AGL release height to be at minimum 1,000 ft 

(304.8 m), with the effective edge at approximately 500 

ft (152.4 m) for these particular conditions. Of particular 

note, however, is that, under these conditions, mosquito 

mortality was quite low at least after 2,000 ft (609.6 m) 

downwind of application. The benefi t of these bioassay 

data from a single-pass application is that we might determine a minimum baseline for functionality of aerial 

application under these conditions when pesticides are 

applied at 300 ft (91.4 m) AGL.

Trial Set 7

Field sentinel cage insect mortality for a bioassay conducted on April 9, 2013, located on Wake Blvd, Parris 

Island MCRD is shown in Figure 2A. Wind direction 

was approximately 160 degrees and wind speed ranged 

from 2-6 knots. There was zero mortality (Abbott corrected) at all stations when examined at 15 minutes 

postspray. At 4 hours postspray, the fi rst 5 stations in 

the sampling array exhibited 100% mortality. The remainder of the stations showed zero to less than 10% 

corrected mortality. The situation at 12 hours postspray 

was not signifi cantly different than that the 4 hour observation. It appears that in this test our effective swath 

was approximately 2,000 ft (609.6 m), which is somewhat expected in that due to a last minute wind shift, 

the prevailing wind was almost parallel to the fl ight 

path of the aircraft, as opposed to the theoretically optimal crosswind application condition to maximize drift. 

What appears to have happened is that the crosswind 

component (approximately 10-40 degrees) essentially 

allowed the applied spray to cut the angle and brought 

the aerosol cloud in contact with the fi rst few bioassay 

cages and very little else. The increased altitude may 

have also caused some signifi cant diffusion of the cloud 

before it actually reached ground level. Figure 2B shows 

fi eld sentinel cage mortality data and meteorological 

data for the sampling array located on Causeway, Parris 

Island MCRD on April 9, 2013. Winds at spray release 

were from approximately 190 degrees and ranged from 

2-8 knots. When observed at 15 minutes postapplication, 

no mortality was noted in any of the test cages. When 

reexamined at 4 hours postapplication, all test cages 

from station 4 to 24 along the causeway indicated 95% 

to 100% mortality of caged mosquitos. In this test, no 

mortality was noted greater than approximately 6,000 ft 

(1,828.8 m) downwind of aircraft fl ight path. No mortality was noted at station 25, which was offset from the 

aircraft fl ight path by approximately 1,000 ft (304.8 m). 

No signifi cant differences between 4 hour and 12 hour 

mortality were observed. Wind direction was much 

more favorable for this trial than the previous trial conducted on Wake Blvd. Direction was much closer to perpendicular to the fl ight path of the aircraft, and for this 

test under these conditions suggests that an effective 

swath in terms of highest mosquito mortality at or near 

ground level may be up to 4,000 ft (1,219.2 m). Also, at 

altitude dispensed with relatively moderate wind speeds, 

an offset of approximately 1,000 ft (304.8 m) may be 

anticipated when applying in almost direct crosswind 

conditions. We were encouraged by these results as they 

provided an indication that good control over fairly wide 

areas may be achieved when applying pesticide at 300 

ft (91.4 m) AGL.

Trial Set 8

Figures 3A and3B show fi eld sentinel cage mortality data from the trials conducted on October 8, 2014. 

These trials were slightly different than those in Trial 

Set 7 in that it was a combined application of 2 adjacent swaths as opposed to a single swath test. Winds 

at release were from 200-208 degrees (averaged from 
58 http://www.cs.amedd.army.mil/amedd_journal.aspx

4 weather stations), and wind speed varied from 2-8 

knots at the time of the test. As before, no mortality was 

noted in caged mosquitoes 15 minutes after application, 

however, mortality at 4 hour postspray was substantial 

among all downwind cages. Figure 3A shows mortality 

measured 12 hours postapplication, which was not signifi cantly different than mortality noted at 4 hours postapplication. Mortality of 90% to 100% was observed in 

all cages from station 22 to station 43, an approximate 

distance of 6,000 ft (1,828.8 m) downwind of the fi rst 

spray swath fl own. Unfortunately, our sampling stations 

did not extend beyond this point, so it is unknown if the 

effective swath may be greater than 6,000 ft (1,828.8 m). 

Interestingly, near 100% mortality was noted at cages 

which were located directly below the aircraft, which 

was not expected as the previous single-swath trial under similar conditions suggested a 1,000 foot (304.8 m) 

offset might be expected. Though we have no defi nitive 

explanation for this phenomenon, it is possible that the 

relative open area associated with the rifl e range may 

have resulted in local unpredictable air movement at the 

interface of open and wooded areas, or areas dominated 

by other structures.

Field sentinel cage mortality data for the second trial 

conducted on October 8, 2014, is shown in Figure 3B. 

This trial was set up similarly to the previous trial, with 

a 2-pass application, but the second swath was offset 

2,000 ft (609.6 m) from the fi rst. Wind direction averaged from 195-200 degrees, and wind speed ranged 

from 2-7 knots. Mortality of 90% to 100% was observed 

at all stations from sampling site 20 to sampling site 

43, which encompassed an area approximately 7,000 

ft (2,133.6 m) downwind of release of pesticide. Again, 

it is unknown if the effectiveness of the pesticide may 

have gone farther downrange, as this was the limit of 

the sampling array. In this second trial it is interesting to 

note the apparent lack of effi cacy of the second applied 

swath, as there was very little mortality noted in sampling locations 13 through 18. We speculate that either 

the interface of the water and land produced some cryptic wind conditions, perhaps an extremely localized offshore breeze, or conditions were such that the pesticide 

drifted farther downrange before ultimately settling. 

Regardless, it again appears that pesticide application at 

this altitude can have signifi cant effi cacy against target 

insects at or near ground level, which is the optimal outcome of aerial sprays of this type.

GENERAL CONCLUSIONS

This article summarizes outcomes from a series of trials using a variety of aerial spray confi gurations in an 

array of meteorological conditions that provide select 

empirical data to investigate effi cacy of higher altitude 

pesticide applications that may be required for effective 

nighttime aerial pesticide missions. In general, based on 

our swath characterizations and bioassays, we believe 

that while pesticide applications at altitudes of 500 ft 

(152.4 m) and 400 ft (121.9 m) AGL may be effective 

under absolutely ideal meteorological conditions, lower 

altitude application will achieve more consistent results 

with regard to target insect mortality and allow more 

reliable prediction of where pesticide may drift. We believe the results of these trials, in particular the 300 ft 

(91.4 m) AGL mosquito sentinel tests, provide supporting evidence that nighttime applications of pesticides 

may be very effi cacious at lower altitudes. Given that we 

are developing these innovative, emerging techniques, 

we acknowledge that these data only provide preliminary quantifi cation of effi cacy and drift, which may be 

different when pesticides are applied in nighttime hours 

as opposed to daylight. Additional future research and 

testing will be required to fully validate aerial nighttime pesticide application. However, the research trials 

described here provide the key baseline data to guide 

future research.

ACKNOWLEDGEMENTS

The authors thank Mr Jerry Kerce and colleagues at 

Camp Blanding Joint Training Center, Starke, FL, Mr 

Jim Clark and colleagues at US Marine Corps Recruit 

Depot Parris Island, Beaufort, SC, for study site availability and expert ground support, and Mr Matthew Hazen Brown at USDA-ARS-CMAVE for providing mosquitoes for use in fi eld bioassay cages.

REFERENCES

1. Eldridge BF. Strategies for surveillance, prevention, and control of arboviral diseases in 

Western North America. Am J Trop Med Hyg. 

1987;37(suppl.):77S-86S.

2. Mount GA, Lofgren CS, Pierce NW, Baldwin KF, 

Ford HR, Adams CT. Droplet size, density, distribution and effectiveness in ultra-low volume aerial 

sprays dispersed with TeeJet nozzles. Mosq News. 

1970;30:589-599.

3. Burkett DA, Biery TL, Haile DG. An operational 

perspective on measuring aerosol cloud dynamics. 

J Am Mosq Control Assoc. 1996;12:380-383.

4. Cecil PF, Young AL. Operation FLYSWATTER: A 

war within a war. Env Sci Pollut Res. 2007;15:3-7.

5. World Health Organization. Space Spray Application of Insecticides for Vector and Public Health 

Pest Control: A Practitioner’s Guide. Geneva, 

Switzerland: World Health Organization; 2003.

6. Komar N. West Nile virus: epidemiology and 

ecology in North America. Adv Virus Res. 

2003;61:185-234

DEVELOPMENT OF AIR FORCE AERIAL SPRAY NIGHT OPERATIONS:

HIGH ALTITUDE SWATH CHARACTERIZATION
July – September 2015 59

THE ARMY MEDICAL DEPARTMENT JOURNAL

7. Guimarães AE. Gentile C, Lopes CM, de Mello RP. Ecology of mosquitoes (Diptera: Culicidae) in areas of Serra do 

Mar State Park, State of São Paulo, Brazil. III. Daily biting rhythms and lunar cycle infl uence. Mem Inst Oswaldo 

Cruz. 2000;95(6):753-760.

8. Yeomans AH. Directions for Determining Particle Size of Aerosols and Fine Sprays. Washington, DC: US Department of Agriculture Bureau of Entomology and Plant Quarantine; 1945. Available at: https://archive.org/stream/

directionsfordet00unit#page/2/mode/2up. Accessed June 4, 2015.

9. Healey MJR. A table of Abbott’s Correction for natural mortality. Ann Appl Biol. 1952;39:211-212.

AUTHORS

Lt Col Haagsma is Research Entomologist, Air Force Aerial Spray Unit, 757th Airlift Squadron, 910th Airlift Wing, 

Youngstown Air Reserve Station, Vienna, OH.

Lt Col Breidenbaugh is Chief Entomologist, Air Force Aerial Spray Unit, 757th Airlift Squadron, 910th Airlift Wing, 

Youngstown Air Reserve Station, Vienna, OH.

Dr Linthicum is Director, USDA-ARS Center for Medical, Agricultural, and Veterinary Entomology, Gainesville, FL.

Mr Aldridge is Biological Sciences Technician, USDA-ARS Center for Medical, Agricultural, and Veterinary Entomology, Gainesville, FL.

Dr Britch is Research Entomologist, USDA-ARS Center for Medical, Agricultural, and Veterinary Entomology, Gainesville, FL.

Announcing

The 2015 Spurgeon Neel

Annual Award Competition

The Army Medical Department Museum Foundation is pleased to announce the 2015 Spurgeon 

Neel Annual Award competition for a paper of 5,000 words or less that best exemplifi es the history, legacy, and traditions of the Army Medical Department.

Named in honor of Major General (Retired) Spurgeon H. Neel, fi rst Commanding General of 

Health Services Command (now US Army Medical Command), the award competition is open to 

all federal employees, military and civilian, as well as nongovernmental civilian authors. More 

information about MG (Ret) Neel can be found at htt p://en.wikipedia.org/wiki/Spurgeon_Neel.

The AMEDD Museum Foundation will present a special medallion award and a $500 monetary 

prize to the winner at a Foundation-sponsored event early in 2016. The winning submission 

will be published in the AMEDD Journal during 2016.

All manuscripts must be submitt ed to the AMEDD Museum Foundation by September 30, 2015. 

At the time of submission, a manuscript must be original work and not pending publication in 

any other periodical. It must conform to the Writing and Submission Guidance of the AMEDD 

Journal, and must relate to the history, legacy, and/or traditions of the Army Medical Department. Manuscripts will be reviewed and evaluated by a six-member board with representatives 

from the AMEDD Museum Foundation, the AMEDD Center of History and Heritage, and the 

AMEDD Journal. The winning manuscript will be selected and announced in December 2015.

Submit manuscripts to amedd.foundation@att.net. Additional details concerning the Spurgeon 

Neel Annual Award may be obtained by contacting Mrs Sue McMasters at the AMEDD Museum 

Foundation, 210-226-0265.
60 http://www.cs.amedd.army.mil/amedd_journal.aspx

In 2007, the National Academy of Sciences (NAS) published a comprehensive report, “Toxicity Testing in the 

21st Century: A Vision and a Strategy”1

 in response to 

a US Environmental Protection Agency request to the 

National Research Council to develop a plan using innovative methods to advance toxicity testing. Toxicity 

determination in the previous half-century required animal testing of all new chemicals (medicine, food additives, industrial, consumer, and agricultural chemicals) 

for potential to cause cancer, birth defects, and other adverse health effects. Animal testing is slow, expensive, 

uses many animals, and often requires assumptions and 

controversial extrapolations.1

The NAS vision discussed harnessing technologies developed in emerging fi elds such as systems biology (eg, 

use of computational models fused with in vitro laboratory data) and bioinformatics (computational models 

to analyze massive data sets), with high throughput 

screening assays of effects of chemicals on human cells, 

cellular components, and tissues, at low levels, which 

are typically more relevant.1

 The report recommended 

identifi cation not only of changes at the molecular level, but of signal transduction and other pathways that, 

when perturbed, lead to adverse effects. Study of these 

infl uences may lead to improved predictability of health 

effects in human populations. Mapping such “toxicity 

pathways” and then discerning actual or predicted perturbations by means of computational models and high 

throughput screening of chemicals:

could reduce the backlog of the large number of industrial chemicals that have not yet been evaluated under the 

current testing system.1(p30)

The new approach would most likely reduce animal use 

as well. However, many of these methods are new; their 

value remains to be validated in predicting effects or determining safe levels of exposure for humans.

In 1985, the Offi ce of The Surgeon General (OTSG) 

designated the Army Institute of Public Health (AIPH), 

now part of the Army Public Health Command, as lead 

agent of the Health Hazard Assessment Program.2

 In 

fulfi llment of that mission, the AIPH Toxicology Portfolio is charged with evaluating the toxicity of specifi c 

military-unique chemicals in materials entering the 

Army supply system.3

 The primary goals have been 

(1) to identify health hazards associated with exposure 

to new substances used in military applications, (2) to 

provide a technical foundation for approvals (or disapprovals) to eliminate or control hazards associated with 

manufacturing-related exposures and use and disposal 

of weapons, equipment, clothing, training devices and 

other materials.3

 The Toxicity Clearance (TC) is the instrument used for this evaluation. It provides a technical basis to help the acquisition program manager make 

important life cycle decisions. Solvents, fi re extinguishing agents, repellents, fabric fi nishes, refrigerants, explosives, energetics, propellants, pyrotechnics, hydraulic fl uids, metals/alloys, and pest control agents are 

examples of substances used in systems that have been 

evaluated. Toxicity Clearances are provided for specifi c 

applications and are generally not applicable to systems 

with different use conditions.2

In accordance with the NAS recommendations and Department of Defense mandates to improve effi ciency in 

research, development, and acquisition, the Toxicology 

Portfolio has implemented a phased approach to toxicity testing and has expanded its toolbox of in silico, in 

vitro, as well as in vivo methods to better evaluate potential health and environmental threats, ideally, before 

a new substance is approved for entry into the Army 

supply chain. Used in a relative manner, these emerging 

methods can be used in side-by-side comparison to determine which substance is likely to cause health effects 

from exposure and use and which would present a lower 

hazard risk.

The phased approach to toxicity testing is intended to 

identify and characterize occupational and environmental human toxicity concerns as early as possible in the 

science and technology (S&T) phase of research, prior 

to transition to an advanced developer. Even before a 

new material has been synthesized, its properties and 

performance can be estimated and evaluated using 

computer modeling techniques that allow toxicity and 

physical properties to be assessed.4

 Modifi cation, reformulation, or even substitution at the S&T stage of development, generally in budget activity levels (BA) 1 to 3 

Toxicity Testing in the 21st Century:

 Refining the Army’s Toolbox

 LTC Erica Eggers Carroll, VC, USA

 Mark S. Johnson, PhD, DABT
July – September 2015 61

would most likely be signifi cantly less time-consuming 

and less costly than comparable changes at BA stages 

4 to 8. An example of the phased approach to toxicity 

testing is underway with the upgrade for the 2.75-inch 

Hydra rocket:

The Hydra is one of the most extensively used munitions 

in the Army, but environmental concerns are associated 

with it. The training warhead for the rocket contains 

perchlorate. The propellant contains lead...The phased 

approach to environmental safety and occupational 

health (ESOH) was used to replace components of the 

Hydra with safer formulations. These replacement compounds are now entering the fi nal stages of approval and 

implementation.4

Another success story involved the reformulation of the 

propellant for the M-115/116/177 (whistle, bang, fl ash) 

simulators. The original formulation used ammonium 

perchlorate as the propellant that resulted in signifi cant 

contamination of training ranges where used. The new 

formulation contained a more traditional black powder 

mix that was just as effective on the ranges, without 

resulting in toxic environmental residues, loss of performance, or signifi cant addition to cost. Both formulations were evaluated side-by-side and recommendations 

made using this approach.

Rather than waiting for an upgrade, phased toxicity testing should be a prerequisite to reach a given Technology Readiness Level (TRL) for new products or systems 

in initial development. The type of toxicity assessment 

would be selected to be compatible with the stage of 

development. For example, at the risk of repetitiveness, 

even before a new material has been synthesized, properties and performance of the substance can be initially 

evaluated using computer modeling techniques to identify potential toxicity. More sophisticated (and more extensive) tools would be used later in development, but 

still prior to transition to a weapon system or platform. 

For example, in silico assays that provided reasonable 

estimates of confi dence regarding toxicity would be 

followed by in vitro assays, including measures of mutagenesis, genotoxicity, and cytotoxicity assays. These 

measures provide data that help address targets of toxicity and potential mechanisms for extrapolation to soldiers, civilians, and the environment. Focused animal 

testing would be reserved for those candidates selected 

as most likely to be effi cacious and safe, in preparation 

for transition to a program executive offi cer or advanced 

developer like the US Army Medical Materiel Development Activity.

Currently, a Programmatic Environmental, Safety, and 

Occupational Health Evaluation (PESHE) is not required 

until Milestone B, at which the product must be at TRL 

6. New S&T products are often considered for transition 

about the BA-3 level at roughly TRL 3-4. It is generally 

accepted that a primary cause of failure to transition includes “lack of technical maturity.” The National Environmental Policy Act of 1969 (Pub L No. 91-90, 83 Stat 

852 (1969)) mandates full disclosure of possible impacts, 

alternatives and environmental mitigation measures. 

Moreover, if not evaluated alongside S&T, the program 

manager runs the risk of development of a system that 

may cause injury to the Warfi ghter or worker, or not lend 

itself for sustainable use at testing and training ranges. 

Therefore, it behooves the S&T community to examine 

the potential for toxicity as part of technology maturity 

determination, prior to or at least as part of the transition 

process into an acquisition program of record. Codifying the need for appropriate toxicity assessment in the 

technology transfer agreement is therefore important. 

Additionally, since the PESHE is currently the fi rst requirement addressing toxicity and is not required prior 

to the product advancement to TRL 6 and Milestone B, 

some products may require reformulation or replacement due to toxicity. This would be fairly late in the acquisition pipeline, by which time the Army might have 

already committed hundreds of thousands of dollars to a 

product. This practice is inconsistent with Executive Order 13514 (“Federal Leadership in Environmental, Energy and Economic Performance, 2009) which required:

...minimizing the generation of waste and pollutants...

[and] reducing and minimizing the quantity of toxic and 

hazardous chemicals and materials acquired...5(p3)

There is no guidance on what data are needed to help 

make environmental safety and occupational health 

(ESOH) decisions for the PESHE or elsewhere regarding toxicity testing, therefore, program managers accept 

risks based solely on available ESOH data.6

 The proposed phased approach:

...seeks to make an ESOH evaluation compatible with 

each stage of the development process by applying appropriate assessment tools...[and] adds a data requirement to each stage for which managers can plan and 

program,...6(p53)

A proposed requirement for appropriate toxicity data at 

each BA level is illustrated in the Figure.

In addition to a phased approach to early toxicity testing, the Toxicology Portfolio has implemented a number 

of targeted assays to help research, development, test 

and evaluation (RDT&E) scientists and managers make 

funding decisions based upon ESOH risks. Moreover, individuals in the Toxicology Portfolio have been working 

with the Technical Cooperative Program, Key Technical 
62 http://www.cs.amedd.army.mil/amedd_journal.aspx

Recommended process using the phased approach in the development of toxicity data to support environmental, safety 

and occupational health (ESOH) decision making.

 ?  ? X

 ? X

 ? X

 ? X

 ? X

Is ESOH

Risk

Acceptable?

BA4: Advanced

Component

Development &

Prototypes

(TRL 6, 7) BA3: Advanced

Technology

Development

(TRL 4, 5, 6)

BA2: Applied

Research

(TRL 2,3)

BA1: Basic

Research

(TRL 1)

BA5: System

Development &

Demonstration

(TRL 8)

BA7: Operational

System

Development

(TRL 9)

A B C

Materiel Solution

Analysis

Technology

Development

Engineering and

Manufacturing

Development

Production

and

Deployment

Operations

and

Support

Material

Development

Decision

IDEAL PARADIGM

can currently be used for notional new substances in 

RDT&E where only small amounts are available. High 

throughput methods can determine if substances have 

characteristics with a high probability of causing illness in soldiers and workers, or of adversely affecting range sustainment. Computer projections can be 

made by comparing the molecular structure of a new 

compound with databases of compounds in which 

structure has been correlated with toxicity. Such projections typically provide relative measures of confi -

dence in estimates, and can also help identify pathways of toxicity. The latter inform future study design. 

Affi rmation of functionality of a new substance also 

helps identify potential toxic endpoints. This, in turn, 

helps industrial hygienists and occupational health 

physicians conduct meaningful surveillance in the 

workforce.

The AIPH Toxicology Portfolio attempts to assess 

high priority military-related substances using the 

above philosophy wherever possible. In Fiscal Year 14 

alone, it completed 39 Toxicity Clearances, 21 technical reports (of which 10 are Toxicity Assessments), 

and 17 peer-reviewed manuscripts. Doctoral level 

subject matter experts (SMEs) in endocrine disruption, ecotoxicity, developmental toxicity, genotoxicity, 

Area 4-42 to develop internationally harmonized methods for use in the development of new weapon systems 

or platforms to ascertain ESOH hazards.7

Although currently a “hot topic” in discussions of toxicity testing and foreseen in the NAS vision as a valuable in silico method, most high throughput methods 

are still in the research stage. Much work remains to 

validate their ability to determine maximum safe exposure levels. Use of high throughput methods will require 

refi ned dose metrics for calculation of a safe level of exposure using in vitro results. However, these methods 

TOXICITY TESTING IN THE 21ST CENTURY: REFINING THE ARMY’S TOOLBOX
July – September 2015 63

THE ARMY MEDICAL DEPARTMENT JOURNAL

immunotoxicity, and quantitative structure activity relationships make up the 2 branches of the Portfolio (Toxicity Evaluation Program; Health Effects Research Program).6 Technical experts in inhalational toxicity testing, 

dermal sensitization, mutagenicity and novel methods 

team with the SMEs to develop and execute good laboratory practices-compliant protocols using rodents and 

select sentinel species.

The Toxicology Portfolio is funded by a combination of 

core Defense Health Program funds and investments by 

other Department of Defense organizations in collaborative arrangements. As important as toxicity testing 

is, the contribution of the AIPH Toxicology Portfolio is 

not widely known. Years ago, new products and materials were fi elded based on effi cacy and the ability to 

meet military operational requirements. Only relatively 

recently have regulatory guidelines mandated that new 

military products be not only effective (can it function as 

designed?), but also safe for Soldiers. Some reports state 

that human male fertility in certain developed nations 

declined in the 20th century,8

 although others dispute 

the claim. Regardless, regulatory guidelines for safety 

assessment of pharmaceuticals and chemicals currently 

include screens for effects on reproduction and fertility, 

including stage-aware histopathological examination 

of the testis, as a sensitive method for detecting disturbances in spermatogenesis.9-11 These are services which 

AIPH Toxicology Portfolio personnel routinely perform 

as part of their mission.

As the nation grows increasingly motivated to spend taxpayer dollars effi ciently and to protect the environment 

to preserve our future, the need for phased and targeted 

toxicity assessment will most likely become a prerequisite for all R&D investments, and it is reasonable to 

expect that the AIPH Toxicology Portfolio will remain a 

leader in this effort.

REFERENCES

1. National Research Council of the National Academies. Toxicity Testing in the 21st Century: A Vision 

and a Strategy. Washington, DC: National Academies Press; 2007. Available at: http://www.nap.edu/

openbook.php?record_id=11970. Accessed May 14, 

2015.

2. Mughal MR, Houpt J, Kluchinsky TA. Health hazard assessment and the toxicity clearance process. 

US Army Med Dep J. July-September 2014:59-60. 

3. McCain WC, Salice CJ, Johnson MS, et al. USACHPPM toxicology: maintaining readiness and 

protecting the environment. US Army Med Dep J.

July-September 2004:10-14.

4. Johnson MS. Phased approach to ESOH assessment streamlines search for replacement substances. One Health. Spring, 2012:19-20.

5. Executive Order 13514: Federal Leadership in 

Environmental, Energy, and Economic Performance. 74 Federal Register 194 (2009). Available at: https://www.whitehouse.gov/assets/docu

ments/2009fedleader_eo_rel.pdf. Accessed May 

14, 2015.

6. Eck WS, Watts K, Lieb NJ, Johnson MS. Avoiding environmental risk. Army AL&T. April-June 

2013:50-56. Available at: http://asc.army.mil/web/

wp-content/uploads/2013/04/April-June2013_

army_al.pdf. Accessed May 14, 2015.

7. Brochu S, Hawari J, Monteil-Rivera F, et al. Assessing the Potential Environmental and Human 

Health Consequences of Energetic Materials: A 

Phased Approach. The Technical Cooperation Program; April 2014. TTCP Technical Report CP 4-42 

[TR-WPN-TP04-15-2014]. 

8. Creasy DM. Evaluation of testicular toxicity in safety evaluation studies: the appropriate 

use of spermatogenic staging. Toxicol Pathol. 

1997;25(2):119-131.

9. International Conference on Harmonisation of 

Technical Requirements for Registration of Pharmaceuticals for human Use. Guideline for Industry: 

Detection of Toxicity to Reproduction for Medicinal Products. Geneva, SUI: ICH Secretariat; 1994. 

ICH-S5A. Available at: http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryIn

formation/Guidances/ucm074950.pdf. Accessed 

May 14, 2015.

10. Organisation for Economic Cooperation and 

Development. OECD Guideline for Testing of 

Chemicals: Reproduction/Developmental Toxicity 

Screening Test. Paris, France: OECD; 1995. OECD 

Test Guideline 421.

11. Organisation for Economic Cooperation and Development. OECD Guideline for Testing of Chemicals: Combined Repeated Dose Toxicity Study 

with the Reproduction/Developmental Toxicity 

Screen Test. Paris, France: OECD; 1996. OECD 

Test Guideline 422.

AUTHORS

LTC Carroll is Chief, Division of Toxicologic Pathology, 

Toxicology Portfolio, Army Institute of Public Health, 

US Army Public Health Command, Aberdeen Proving 

Ground, Maryland.

Dr Johnson is Director, Toxicology Portfolio, Army Institute of Public Health, US Army Public Health Command, Aberdeen Proving Ground, Maryland.
64 http://www.cs.amedd.army.mil/amedd_journal.aspx

It is known across the Department of Defense (DoD) 

that the Secretary of the Army is the Executive Agent 

for DoD Veterinary Public and Animal Health Services, 

the functional responsibilities for which are assigned to 

US Army Veterinary Services.1,2 A critical component 

of the Veterinary Services mission is the assurance of 

safe food and water for DoD personnel and their dependents assigned throughout the world, even extending to 

some US embassies and consulates in the most austere 

environments.

However, much less is known about the behind-thescenes, complex mechanisms required to provide those 

safe food and water sources, achieved only by the consistent and elaborate coordination among military and 

civilian public health authorities, medical and veterinary personnel, logistics personnel, and foreign customs 

authorities. It is probably safe to say that the average 

consumer at an overseas Defense Commissary Agency 

commissary or an Army and Air Force Exchange Service facility is only vaguely aware of the intensive efforts made on their behalf to ensure constant availability 

of US food items.

The US European Command (USEUCOM) area of responsibility is a prime example of the enormous complexities involved with exporting and shipping foodstuffs to DoD’s multiple locations around the world. As 

DoD locations span Europe, US government-owned 

subsistence transits the breadth of the continent, crossing many borders before reaching outlying installations. To support that mission, the processes involved 

have historically required sound logistics, communication, and teamwork across multiple services. In recent 

years, however, it has also required strategic diplomacy 

to integrate those processes into compliance with the 

European Union (EU) Trade Control and Expert System 

(TRACES), designed to track animal and animal origin 

product EU imports.

This article highlights the progression of US military’s 

incorporation into and compliance with TRACES. It 

outlines the broader strategic implications of United 

States engagement in that program to ensure food safety 

and quality assurance of public health for DoD personnel assigned throughout USEUCOM while, at the same 

time, ensuring, and even strengthening, diplomatic ties 

and trust with European partners.

As the EU was coming together as a fully functioning 

regulatory and authoritative body representing its member states, around the year 2000 the European Commission Directorate General for Health and Consumers 

began to pay attention to animal origin products from 

the US that were entering the EU bound for US military installations. Movement of such products had been 

ongoing decades, but the backdrop of a number of high 

impact, economically costly animal disease outbreaks 

in the EU, combined with emerging awareness of and 

concerns about the use of growth hormones, antibiotics, 

and genetically modifi ed organisms by the US agricultural industry, served to elevate attention even further.

The fi rst of these animal disease outbreaks in the EU began in 1996 with cases of bovine spongiform encephalopathy (BSE), otherwise known as “mad cow disease,” 

occurring in Britain. The scientifi c community associated human cases of the variant form of Creutzfeld-Jakob 

disease with the consumption of BSE-contaminated beef, 

and the resultant economic loss to the United Kingdom 

(UK) reached an estimated $6.4 billion by early 2001.3

The BSE outbreak was quickly followed by an epidemic 

of classical swine fever which began with the fi rst case 

reported from Germany in January, 1997 and presumably spreading from there to the Netherlands, then on 

to Italy, Spain and eventually to Belgium.4

 In 2001, the 

UK’s livestock industry was again hit, this time by a 

foot-and-mouth disease outbreak which crippled exports 

and cost the UK an estimated £8 to £8.6 billion ($12 to 

The New US Military Role in the European

 Union’s Import Program: Strategic

 Implications Ensuring Safe Food for

 the European Theater

 MAJ Michael McCown, VC, USA

 Jacob L. Hall, II

 Megan McCormick, DVM, MPH

 Lt Col Henry H. Triplett, III, USAF
July – September 2015 65

$13 billion at current exchange rates) to resolve.5,6 Collectively, these diseases resulted in devastating effects 

on health, economic, and consumer confi dence. It had 

become obvious that there was enormous potential economic risk posed by the introduction of foreign animal 

diseases into the EU. This awareness, undoubtedly, contributed to shaping the comprehensive import regulations currently being developed and implemented.

There was, however, another signifi cant change as a result of the 1996 association of human disease with the 

consumption of potentially BSE-contaminated beef. 

Historically, the US military had procured beef for the 

European theater’s needs predominantly from US approved European sources (primarily from the UK as it 

was economically the most competitively priced). But 

once the protection of human health was at risk according to the best scientifi c information available, the US 

military categorically shifted to exclusively procuring 

beef from US sources via import, as the US beef industry 

largely escaped infection with BSE and was considered 

safe. This monumental change of sourcing was to have 

far-reaching effects approximately a decade later when 

EU attention focused on US military meat importation.

By 2008, the EU community’s internal implementation 

and compliance with import regulation had reached maturity and the spotlight shifted to US military animal origin imports which were not in compliance with existing 

regulations. Historically, US military importation had 

been permitted with the recognition that its purpose was 

to supply military forces outside the European economy, 

so installations were treated as “foreign soil” versus host 

nation. But that “exemption” was coming under increasing scrutiny, and the United States was approached to 

initiate an effort to comply with new EU requirements. 

Initial meetings, panels, working groups, and senior 

leader engagements failed to make progress toward this 

end, however, and US representatives gradually concluded that the problem would be more diffi cult to solve 

than originally anticipated, despite the best intentions.

Preventing the construction of a solution framework 

was a signifi cant legal hurdle. From the inception of the 

issue, legal experts in multiple organizations insisted 

that no governmental organization outside of the Offi ce 

of the Secretary of Defense (OSD) or the Department of 

State had the authority to initiate any such agreement. 

Over many years, multiple legal opinions were issued 

that effectively prevented any work toward an achievable solution. The chief legal concern was that while the 

United States and each of the individual member states 

in the EU are themselves sovereign nations, the EU itself, 

while a legislative body, is not recognized as a sovereign 

body and thus has no standing from which to negotiate.

By 2011, with multiple initials attempts failed and no 

traction toward compliance in sight, the EU’s patience 

was wearing thin and US animal origin product imports 

were being threatened with refusal at ports of entry. It 

was clear that the US military had not responded rapidly 

enough and that EUCOM had to step in to reach a resolution. Finally, in the fall of 2012, senior leaders from 

EUCOM and other agencies including the Principal 

Deputy Assistant Secretary of Defense for Logistics and 

Materiel Readiness and the Joint Staff’s J4 and J5 met 

and concluded that this problem required urgent solution as product procurement was in imminent jeopardy. 

Ultimately, the OSD determined that because this issue 

primarily affected forces serving in the EUCOM area 

of responsibility, the EUCOM Directorate of Logistics 

could serve as the lead agency to develop and implement 

policy governing a successful solution.

POINT OF ENTRY: EU BORDER INSPECTION POSTS

First, and most critically, a successful solution was required to address the arrival and acceptance of shipments at EU Border Inspection Posts (BIPs), the importation entry points located at ports, certain borders, 

and airports. Then, the EU required that US military 

shipments be tracked from entry to receipt at destination 

using the EU’s electronic tracking database, TRACES. 

This system is used, along with the information provided in accompanying animal health certifi cates and other 

import documents, to produce the EU transit health certifi cate called a Common Veterinary Entry Document 

(CVED) which accompanies a shipment. Upon receipt 

at destination, a shipment is either processed by an exit 

BIP as having left the EU in the case of transiting goods 

or, in the case of goods bound for retail operations with 

consumer sales, its status updated to refl ect arrival at 

its fi nal tracking destination. Thirty days are allowed 

from the time a shipment is logged at the entry point 

until it must be closed out. Failure to meet requirements 

potentially jeopardizes future shipments for a specifi c 

importer as compliance history is monitored and a poor 

track record can result in subsequent refusals of entry.

PROGRESS TOWARDS SOLUTION

Once authority was granted for the involved players to 

engage with the EU toward constructing a workable 

framework for US compliance, progress was slow, but 

at least there was movement. While the US would no 

longer be permitted to import goods with an entirely 

free rein, it was recognized that shipments intended for 

US installations were, in fact, not freely entering the EU 
66 http://www.cs.amedd.army.mil/amedd_journal.aspx

food economy as were most other imported goods. So, 

despite the fact that US military imported animal origin 

products that often do not meet EU standards (eg, are 

not from EU approved sources, are not antibiotic free or 

may contain unapproved growth hormone), the EU level 

veterinary authority made the determination that US 

import shipments would be considered to be transiting 

the EU until they arrived at designated US installations 

which were to be considered “non-EU” destinations. 

This decision took advantage of the existing allowances in EU regulations that permitted the legal transit of 

goods bound for actual non-EU countries, such as from 

EU ports through the EU, then out via an EU exit BIP 

where the shipment is closed out of the TRACES tracking system. This decision, however, did require the offi cial designation of US installations as exit BIPs and 

the identifi cation and training of personnel at these locations to be able to perform this function.

As the oversight and authority for importation of animal 

origin products falls under the purview of EU veterinarians and the DoD veterinary mission belongs to the US 

Army, the US Army Public Health Command RegionEurope (PHCR-E) was delegated authority by USEUCOM to provide direct support and program implementation. The PHCR-E controlled most veterinary assets 

in Europe and its deputy (the senior veterinary offi cer) 

was appointed the Competent Veterinary Authority and 

granted limited authority to engage the EU directly regarding the importation program, thus ensuring appropriate professional engagement with the European veterinary authorities.

A decision was also made regarding prime vendor warehouse facilities located in the EU which could not technically be considered US military installations. They were 

assessed and categorized as nonconforming warehouses 

(permitted under EU legislation). They could function 

to process transiting goods provided they maintained 

US government owned goods strictly isolated from 

goods intended for the EU, and they shipped only to fi -

nal destinations that included ships’ supply, US military 

installations, or other locations actually outside the EU, 

such as those under the US Central Command or Africa 

Command. European Union veterinarians continued to 

be involved with these facilities which function as both 

entry and exit BIPs as subsequent shipments are moved 

on EU generated transit paperwork (“daughter CVEDs” 

based on the original CVED), then closed out when they 

reach their ultimate destinations at US installations.

US INTERAGENCY COOPERATION

Before an export shipment of animal origin product 

leaves the United States, the Department of Agriculture’s 

(USDA) Food Safety and Inspection Service (FSIS)* certifi es the goods and issues health certifi cates confi rming 

their quality. The FSIS is the public health agency within 

the USDA responsible for ensuring the nation’s commercial supply of meat, poultry, and egg products is safe, 

wholesome, and correctly labeled and packaged—in other words, safe for human consumption per US standards. 

Once approved by USDA, a shipment can be loaded at a 

supplier’s location, then transported to the east coast for 

transoceanic movement to European BIPs.

The USDA Foreign Agricultural Service (FAS)† links 

US agriculture to the world, enhancing export opportunities and global food security, as well as expanding 

and maintaining access to foreign markets for US agricultural products by removing trade barriers and ensuring US rights under existing trade agreements. The FAS 

also works with foreign governments, international organizations, and the Offi ce of the US Trade Representative to establish international standards and rules to improve accountability and predictability for agricultural 

trade. For the DoD, the US Foreign Agricultural Service 

Mission to the EU, headquartered in Brussels, Belgium, 

has played an absolutely key role in assisting with issues 

related to importation of animal origin products to the 

EU. They were critical in overcoming challenges with 

the implementation of TRACES and continue to assist 

with ongoing and emerging situations.

APPROACHING A STABLE END STATE

In 2013, the multiyear-long process of US military integration into the EU import program culminated with 

the release of USEUCOM European Command Instruction 4506.01: USEUCOM Guidance Regarding the EU 

TRACES, which codifi ed the rules of engagement for 

the working levels of all service components and affected agencies to participate in the European import 

program. This guidance serves as the cornerstone for 

ensuring animal origin products including fresh meat 

and meat products from the US are available at commissaries, dining halls, food courts, schools, day care 

centers, and other installation food sources.

Combatant commands such as EUCOM are organized 

and structured for strategic military oversight and direction. As such, it is unusual that EUCOM was designated 

as the lead to pull together multiple service components 

and both DoD and non-DoD agencies to safeguard the 

US European theater’s access to US animal origin products. Although progress remains ongoing, it is far enough 

along to be assessed and considered a success story.

*http://www.fsis.usda.gov/About_FSIS/index.asp

†http://www.fas.usda.gov/aboutfas.asp

THE NEW US MILITARY ROLE IN THE EUROPEAN UNION’S IMPORT PROGRAM:

STRATEGIC IMPLICATIONS ENSURING SAFE FOOD FOR THE EUROPEAN THEATER
July – September 2015 67

THE ARMY MEDICAL DEPARTMENT JOURNAL

ACKNOWLEDGEMENT

The research involved in the preparation of this article 

was supported in part by an appointment to the Knowledge Preservation Program at the US Army Public 

Health Command administered by the Oak Ridge Institute for Science and Education through an interagency 

agreement between the US Department of Energy and 

the Army Public Health Command.

REFERENCES

1. Department of Defense Directive 6400.04E: DoD 

Veterinary Public and Animal Health Services. 

Washington, DC: US Dept of Defense; 2013.

2. Army Regulation 40-905: Veterinary Health Services. Washington, DC: US Dept of the Army; 2006.

3. Regmi A, ed. Changing Structure of Global Food 

Consumption and Trade. Washington, DC: US 

Dept of Argiculture Economic Research Service; 

2001:60. Agriculture and Trade Report WRS01-1. Available at: http://www.ers.usda.gov/media/293645/wrs011_1_.pdf. Accessed April 13, 

2015.

4. Greiser-Wilke I, Fritzemeier J, Koenen F, et al. Molecular epidemiology of a large classical swine fever epidemic in the European Union in 1997–1998. 

Vet Microbiol. 2000;77(1-2):17-27.

5. Donaldson A, Lee R, Ward N, Wilkinson K. Five 

Years On: The Legacy of the 2001 Foot and Mouth 

Disease Crisis for Farming and the British Countryside. Newcastle, England: Centre for Rural 

Economy; February 2006:4. Centre for Rural Economy Discussion Paper Series No. 6. Available at: 

http://www.ncl.ac.uk/cre/publish/discussionpapers/

pdfs/dp6.pdf. Accessed April 22, 2015.

6. National Cattlemen’s Beef Association. Fact Sheet: 

Industry Economics. FootAndMouthDiseaseInfo.

org web site. Available at: http://www.footandmouthdiseaseinfo.org/factsheetindustryeconomics.

aspx. Accessed April 22, 2015.

AUTHORS

MAJ McCown is Chief, Veterinary Services Division, 

US Army Public Health Command Region-Europe, 

Landstuhl, Germany.

Mr Hall is a Traffi c Management Specialist, Air Force 

Security Assistance and Cooperation Directorate, 

Wright-Patterson Air Force Base, Ohio.

Dr McCormick is the EU TRACES Specialist, US Army 

Public Health Command Region-Europe, Landstuhl, 

Germany.

Lt Col Triplett is assigned to Headquarters, US European 

Command, Stuttgart, Germany.
68 http://www.cs.amedd.army.mil/amedd_journal.aspx

Surpassed only by heart disease, cancer is the second 

highest cause of all deaths, accounting for 1 in every 4 

deaths in the United States. According to the American 

Cancer Society, there will be more than 1.66 million new 

cancer diagnoses and an estimated 590,000 Americans 

will die of cancer in 2015.1

 These fi gures are similar to 

those reported by the Surveillance, Epidemiology, and 

End Results Program for 2014.2

 In its most recent Cancer Trends Progress Report – 2011/2012 Update, the National Cancer Institute reports that death rates for the 4 

leading types of cancer as well as all cancers combined 

have been declining, yet incidence rates of some cancers 

are on the rise.3

 Worldwide, cancer is a leading cause 

of both morbidity and mortality, with approximately 14 

million newly diagnosed cases and more than 8 million 

deaths attributed to cancer in 2012.4

The evidence indicating a connection between occupational and environmental exposures and cancer has been 

growing in recent years.5

 This is of particular concern 

because such cancers are theoretically avoidable, as 

measures can be taken to avoid these nongenetic risk 

factors. The World Health Organization estimates that 

19% of all cancers are attributed to environmental factors, accounting for 1.3 million deaths per year.6

 The military population presents a unique opportunity to study links between environmental exposures 

and cancer. Advantageous aspects of studying cancer 

among military personnel include well characterized 

person-time, occupation, and, though not always the 

case, environmental hazards. Access to routine healthcare including recommended cancer screenings at no 

cost to the service member and robust electronic medical record systems also facilitate assessments of cancer 

outcomes in the military population. Furthermore, exposures associated with military deployments may infl uence cancer risk among military personnel.7

 Possible 

deployment-related exposures have been documented by 

the Department of Defense,8,9 to include potential carcinogens (eg, industrial solvents, jet fuel, air pollution, 

radiation). Behavioral changes during deployment, such 

as increased tobacco use, have also been documented.10

It is thus plausible that military deployment and associated exposures may be risk factors for subsequent cancer among warfi ghters.

CANCER IN THE MILITARY

Vietnam War

Historically, there has been concern regarding military 

service-related hazards and potential long-term health 

implications following military deployment. Postdeployment cancer risk is often at the forefront of the issue, 

as was the case after the Vietnam War.11-12 As Richards 

describes in an article reviewing responses to militaryassociated environmental and occupational exposures:

During the latter half of the 20th Century, medical 

knowledge of and concern about carcinogens grew, and 

human experimentation guidelines became more stringent. During the Vietnam era, concern for troop exposure to environmental contaminants evolved beyond 

acute exposures and experimentation to encompass 

long-term occupational and environmental hazards encountered on the battlefi eld.13

By far, the most prominent exposure in terms of health 

concern generated during this confl ict is the herbicide 

commonly referred to as Agent Orange. Many veterans 

of the Vietnam confl ict between 1965 and 1972 attribute 

poor postdeployment health outcomes, including rare 

cancers, to 2,3,7,8-Tetrachlorodibenzodioxin, an extremely toxic dioxin compound that contaminated one 

of the compounds used to make the herbicide Agent Orange.14 The scientifi c evidence linking postdeployment 

cancer to Agent Orange exposure during the Vietnam 

War varies. Some studies have not found higher rates of 

mortality for outcomes such as soft tissue sarcomas,15

Hodgkin’s disease,16 non-Hodgkin lymphoma, or testicular cancer in Vietnam veterans.17,18 Another study of 

participants of the Agent Orange Registry had similar 

results, showing no difference in prevalence for any 

type of cancer when comparing Vietnam veterans to 

non-Vietnam veterans.17 However, the CDC Selected 

Cancer Study reported a higher risk of non-Hodgkin’s 

lymphoma among Vietnam veterans when compared to 

other men.19 Frumkin summarized the existing literature on Agent Orange and cancer, reporting consistent 

Evaluation of Postdeployment Cancers

 Among Active Duty Military Personnel

 Jessica M. Sharkey, MPH

 Joseph H. Abraham, ScD
July – September 2015 69

to fairly consistent negative results for increases of soft 

tissue sarcomas, Hodgkin’s disease, and gastrointestinal 

and brain cancers, but inconsistent results of increases 

in respiratory and prostate cancers among Vietnam veterans.20 Still yet, in the current Institute of Medicine Report of the health effects of herbicides used in Vietnam, 

Veterans and Agent Orange: Update 2012,

21 the committee found suffi cient evidence of an association between 

soft tissue sarcomas, non-Hodgkin lymphoma, chronic 

lymphocytic leukemia, and Hodgkin lymphoma, and 

limited/suggestive evidence of an association with laryngeal, lung, bronchus, trachea, and prostate cancers 

as well as multiple myeloma.

1991 Gulf War

Similar to those of the Vietnam confl ict, many veterans 

of the 1991 Gulf War are also concerned about the specter 

of cancer and possible links to hazards associated with 

their deployment. Notable hazards of concern to service 

members during the Gulf War include depleted uranium, 

petroleum products, pesticides, and chemical and biological warfare agents.22 However, scientifi c literature 

shows mixed fi ndings regarding potential associations 

between Gulf War exposure and postdeployment cancer 

risk. A particular exposure event of interest during the 

Gulf War was the destruction of chemical munitions at 

Khamisiyah, Iraq. While Bullman et al indicated an increased risk of brain cancer mortality among US Army 

Gulf War veterans who were potentially exposed to 

low-level chemical warfare agents at Khamisiyah when 

compared to Gulf War veterans who were not exposed,23

a later study by Young et al found no excess in brain 

cancer.24 In his report on a study on testicular cancer following Gulf War deployment, Levine stated:

...testicular cancer was found to be the only signifi cantly 

increased malignancy among deployed Persian Gulf War 

veterans. The increase became apparent 2 to 3 years after 

the Persian Gulf War and peaked 4 to 5 years afterward.11

Yet, Knoke et al found that although there was an initial 

increase in testicular cancer immediately following deployment among Gulf War veterans compared to nondeployed Gulf War era veterans, the difference was no 

longer observed by 4 years postdeployment.25 Kang et al 

described “very small rate differences (less than 1.0%) 

between Gulf veterans and non-Gulf veterans” for both 

skin cancer and other cancers, with higher rates among 

the Gulf War veterans.26 Kang and Bullman reported

...no signifi cant excess of overall cancer deaths or deaths 

from cancer at any specifi c site among Gulf veterans 

compared with non-Gulf veteran controls.27

In a 2005 report, Gulf War and Health, an Institute of 

Medicine committee found suffi cient evidence of an association between combustion products and lung cancer 

and limited/suggestive evidence of an association between combustion products and nasal, oral, laryngeal, 

and bladder cancers and between hydrazines and lung 

cancer. There was inadequate/insuffi cient evidence to 

support conclusions regarding potential associations 

between fuels, combustion products, hydrazines, and 

nitric acid for numerous types of cancers.28

Operations Enduring and Iraqi Freedom

Deployment-related exposures are now causing the 

same concerns regarding cancer among service members following support of Operations Enduring Freedom 

(OEF) and Iraqi Freedom (OIF). Since 2001, in excess of 

2 million US military personnel have deployed to Southwest Asia,29,30 with environmental hazards including but 

not limited to pollutants from local industry; militaryproduced exhaust from vehicles, machinery, and generators; open air burn pit emissions and fumes from fi res; 

high levels of indigenous ambient particulate matter; 

munitions and weapons; depleted uranium; and radiation.7,31-39 Potential relationships between exposures in 

theater and cancer diagnoses subsequent to deployment 

are again a priority for researchers and public health 

professionals in the military community.

BASELINE CANCER RATES

In the population of OIF and OEF veterans, one expects 

a certain amount of cancer to occur, irrespective of deployment history and associated deployment-related environmental exposures. Understanding baseline rates of 

cancer in the military population is useful when evaluating whether cancer among service members with a history of deployment in support of OIF and/or OEF occurs at excessive rates. Cancer investigations in military 

populations typically focus on specifi c types of cancer 

or are specifi c to a single service branch. This was the 

case when Yamane reported on cancer incidence from 

1989-2002 among Airmen. In comparison to the general 

US population, he found standardized incidence ratios 

for all cancers to be lower than expected among male 

Air Force service members and as expected among female Air Force service members.40 Zhu et al later compared incidence rates of a select group of cancers (lung, 

colorectal, prostate, breast, testicular, and cervical cancers) across the military to US civilians. The authors 

reported lower incidence rates of colorectal, lung, and 

cervical cancers, and higher rates of prostate and breast 

cancers.7

 Although these comparisons provide valuable information, knowledge of rates across all service 

branches for all types of cancers is important.

In June 2012, the Armed Forces Health Surveillance 

Center published a report describing incident diagnoses of cancers and cancer-related deaths in active duty 
70 http://www.cs.amedd.army.mil/amedd_journal.aspx

military personnel from 2000-2011. Results for the 12-

year surveillance period showed a crude incident rate of 

55.2 per 100,000 person-years, with the lowest annual 

incidence rate of 50.3 per 100,000 person-years occurring in 2003 and the highest annual incidence rate of 60.1 

per 100,000 person-years occurring in 2009. The data 

indicated no apparent increasing or decreasing trends 

in overall or site-specifi c incident cancer diagnoses. Of 

note, rates of cancer diagnoses among active duty military members have remained stable since 2000.41

IDENTIFYING CARCINOGENS

More than 900 agents have been evaluated by the International Agency for Research on Cancer for determination of potential to cause cancer. A group of four 

different categories is utilized to classify every agent: 

carcinogenic to humans (Group 1), probably or possibly carcinogenic to humans (Group 2A and Group 2B, 

respectively), unclassifi able as to carcinogenicity in humans (Group 3), and probably not carcinogenic to humans (Group 4). In excess of 125 agents have been classifi ed into Group 1.42 It is suspected or known that some 

of these environmental carcinogens can be found in the 

deployment environment.

IDENTIFYING CANCERS

The concern for postdeployment cancer due to potential 

exposure environmental carcinogens in theater has been 

raised by service members and veterans alike, as demonstrated by advocacy groups such as Burnpits360 and 

Operation Purple Heart, which allow for self-reported 

cancer diagnoses on website registries.43,44 While these 

concerns are reasonable and recognized by public health 

professionals in the military community, they have yet to 

be supported by epidemiologic studies using appropriate 

populations and suitable comparison groups. However, 

there are many factors that should be considered when 

approaching a study intended to establish whether a history of deployment in support of OIF or OEF is associated with subsequent incidence of postdeployment cancer.

Age

Age is an important factor to consider when designing 

any epidemiologic investigation pertaining to postdeployment cancers among service members and veterans. 

Incidence rates of many types of cancers are known to 

increase with age. As pointed out by the Armed Forces Health Surveillance Center, generally speaking, US 

military personnel are younger than the general population.41 When focused on a chronic disease such as cancer 

that is known to increase with age, in a younger population, priority should be given to cancers that typically 

occur with highest incidence falling during the young 

adult years.

Latency Periods

The empirical latent period for cancers consists of 2 parts: 

an induction period ranging from the time between the 

action of a given component cause (ie, an exposure of 

interest) and the action of the last causal component (ie, 

biological onset of the cancer) and a subsequent period 

ranging from the biological onset of the cancer to the 

clinical detection of the cancer. Minimum empirical latency periods must be taken into account when deciding which cancers to evaluate in service members and 

veterans postdeployment, as they must be consistent 

with study hypotheses. Latency periods vary by different type of cancer of interest, with some cancers having a typical latency period of 15 to 20 years or longer, 

while some cancers typically have latency periods that 

are considerably shorter. In the former, these types of 

cancers would be better suited for postdeployment cancer evaluations among veteran populations of wars that 

occurred at least that far in the past, such as Vietnam or 

the fi rst Gulf War, yet they would not be appropriate for 

OIF/OEF veterans as that much time has not yet passed 

since exposure. On the other hand, it would be prudent 

to study the latter types of cancers in a population of 

OIF/OEF deployed service members because time since 

deployment and typical latency periods align.

Biologic Plausibility

When selecting cancer outcomes of interest, the focus 

should be on cancers that are biologically plausible. For 

example, the following cancers were selected for an 

upcoming collaborative study between the US Army 

Public Health Command, the Navy and Marine Corps 

Public Health Center, and the Department of Veterans 

Affairs: melanoma, leukemia, lymphoma, and brain, 

thyroid, testicular, and breast cancers. Those cancers 

have peak incidence during young adult years, which 

matches the demographics of our service members with 

potential exposure(s) of interest.45 These selections were 

also made based on suspected or known occupational 

or environmental risk factors.46-49 The latent periods of 

these cancers are also in accordance with investigating 

the association between in-theater environmental exposures and postdeployment cancer among service members formerly deployed to OIF or OEF.50,51

KARSHI-KHANABAD: AN EXAMPLE

Recent efforts to understand possible associations between environmental exposures in theater and postdeployment cancer diagnoses include an investigation conducted at the US Army Public Health Command, which 

explored multiple cancer outcomes among service members formerly deployed to Karshi-Khanabad, an air base 

located in southeastern Uzbekistan used to support missions in neighboring Afghanistan during OEF.39 Active 

EVALUATION OF POSTDEPLOYMENT CANCERS AMONG ACTIVE DUTY MILITARY PERSONNEL
July – September 2015 71

THE ARMY MEDICAL DEPARTMENT JOURNAL

duty members of the US armed forces were located at 

the Karshi-Khanabad Air Base, also known as K2 or 

Camp Stronghold Freedom, between 2001 and 2005. 

General conditions were known to be harsh. Historically, the site was used by the Soviet military to support 

operations in Afghanistan in the late 1970s. During this 

time, the Soviet Air Force maintained a fl eet of various 

bomber aircraft at K2, which required an underground 

fuel distribution system. Furthermore, construction of 

military equipment (including missiles) in the Soviet era 

used materials such as asbestos and radioactive material. 

An occupational and environmental survey performed 

at K2 in November 2001 by the Center for Health Promotion and Preventive Medicine-Europe.found underground jet-fuel plumes and surface dirt contaminated 

with asbestos and radioactive uranium.38 Periodic high 

levels of dust and other particulate matter (PM) in the air 

due to seasonal dust storms was also noted.

Although efforts for remediation of the environmental 

health risks present at K2 were made (eg, covering the 

contaminated areas with clean soil and declaring these 

areas “off-limits”), exposure to the toxicants mentioned 

above during deployment to K2 was plausible. In other 

settings, exposure to jet fuel plumes, asbestos-contaminated soil, radioactive materials, and/or dust and PM 

have resulted in documented adverse health outcomes, 

including both acute and chronic disease. As such, this 

investigation focused on identifying the frequency of 

postdeployment medical encounters for health outcomes 

consistent with exposure to the identifi ed toxicants, with 

an emphasis on cancer due to the various types among 

personnel previously deployed to K2.52-61

At the request of a US Central Command Force Health 

Protection Offi cer, an evaluation of health outcomes 

among active duty military personnel with a history of 

deployment to K2 was conducted to address concerns 

for exposure(s) of health consequence among Army, Air 

Force, and Marine Corps personnel deployed to the air 

base. The Army Public Health Command subsequently 

conducted a comparative health assessment using one 

year of postdeployment medical follow-up. In the context of the above discussion regarding latency periods 

for cancer outcomes, the US Army Special Operations 

Command Surgeon later requested that the original 

analysis be repeated to incorporate up to 10 years of 

follow-up, using all available postdeployment medical 

encounter data. In response to this request, a retrospective cohort study was conducted in order to assess postdeployment health status among service members formerly deployed to K2. This was accomplished by linking K2 deployment rosters from 2001-2005 with postdeployment inpatient and outpatient medical records from 

2001-2011. Additionally, a reference group of personnel 

stationed in South Korea during the same period was 

selected for comparison. The results are presented in the 

Table.

The results of this analysis are somewhat mixed, with 

relative risks lower than one for about half of the specifi c cancer type outcomes and relative risks higher than 

one for the other half. The only statistically signifi cant 

fi ndings were for malignant melanoma and neoplasms 

of lymphatic and hematopoietic tissues (excluding 

Non-Hodgkin Lymphoma and Leukemia; highlighted 

in bluein the Table), indicating a risk approximately 

3.7 times greater and 5.6 times greater among those 

deployed to K2 compared to those stationed in Korea. 

Concern for postdeployment cancer at K2 is warranted, 

given the relative risks above one, irrespective of statistical signifi cance and the limitations of this particular 

analysis. Although the environmental hazard risk profi le 

may differ somewhat between K2 and other OIF/OEF 

locations, these results bolster the rationale for conducting broader studies to evaluate incidence of cancers following military deployment.

CHALLENGES AND LIMITATIONS

Long latency periods, low incidence rates of most types 

of cancer, and appropriate selection of nondeployed 

controls present challenges for investigators wishing to 

evaluate postdeployment cancer risk. Only very recently 

has a suffi cient amount of time elapsed in order to assess cancer incidence following OIF and OEF deployments. Given the low incidence rates of most types of 

cancers, researchers must take care to ensure that study 

sample sizes are large enough to provide adequate statistical power to detect associations, should they exist. 

Epidemiologic studies comparing cases to controls with 

respect to OIF/OEF deployment status presents a challenge due to a high prevalence of deployment for any 

military personnel serving between 2001 and 2014. As 

such, a large well-powered study is imperative.

Additional challenges include a lack of data on individual environmental exposures over time as well as a lack 

of exact locations of each service member during military deployments. As a result, deployment in general 

is typically used as a proxy for deployment-associated 

exposures. Also limiting to epidemiologic studies such 

as these is the lack of information on behavioral habits 

such as smoking, which can have signifi cant effects on 

certain types of cancer.

Cancer case defi nitions are often based on ICD-9-CM 

coded medical encounter data from military medical record databases. Using administrative records to 
72 http://www.cs.amedd.army.mil/amedd_journal.aspx

ascertain cancer cases may result in false positives. For 

example, not only are some cancers not well defi ned, 

but some require several encounters, sometimes with 

multiple specialists or requiring different medical procedures, in order to make a defi nitive diagnosis. In such 

circumstances, an ICD-9-CM code may refl ect a true 

case of cancer or the medical encounter may signify 

that a patient is in the process of fulfi lling diagnostic 

evaluations necessary to rule out cancer. Using medical 

encounter data for case ascertainment presents another 

limitation of this study: whereas medical encounter data 

capture is complete for service members who remain in 

service, the same cannot be said for personnel who leave 

military service. This becomes particularly problematic 

when studying chronic health outcomes such as cancer, 

with the latency periods often years after exposure, beyond the average time of military service. Investigators 

are currently attempting to establish methodology for 

linking medical encounter records from military service with medical encounter records from the Veterans 

Administration (VA) in order to minimize loss of follow 

up due to attrition from military service. However, this 

methodology will still fail at perfect case capture, as a 

certain portion of veterans are not VA benefi ciaries or 

simply choose to obtain healthcare services outside the 

VA health system. It has been suggested that state cancer registries be used as additional sources of data in 

postdeployment cancer studies, however, the feasibility 

of this approach has yet to be explored.

Although many challenges are presented to researchers 

seeking to determine whether or not cancer incidence is 

elevated among military service members and veterans 

formerly deployed in support of OIF and OEF relative 

to personnel without a history of deployment, it is an 

important topic that is worthy of public health efforts 

and resources.

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Age-Adjusted Relative Risks and Corresponding 95% Confi dence Intervals for Cancer Outcomes, Comparing US Military 

Personnel Deployed to K2 to US Military Personnel Stationed in Korea

Outcome

K2 Korea

Age-Adjusted*

Age Age

Young Old Young Old

n%n%n%n % RR 95% CI

All cancer 11 0.39 50 1.21 41 0.28 133 1.00 1.23 0.92-1.65

Brain cancer 1 0.04 4 0.10 0 0.00 8 0.06 2.04 0.68-6.09

Cervical cancer 0 0.00 0 0.00 1 0.01 0 0.00 0 --

Leukemia 0 0.00 1 0.02 5 0.03 4 0.03 0.43 0.05-3.63

Malignant melanoma 1 0.04 7 0.17 3 0.02 5 0.04 3.68 1.35-10.04

Neoplasm of bone/connective tissue/skin/breast 1 0.04 3 0.07 5 0.03 9 0.07 1.06 0.35-3.22

Neoplasm of colon/rectum 2 0.07 3 0.07 2 0.01 9 0.07 1.6 0.57-4.51

Neoplasm of digestive organs/peritoneum 0 0.00 1 0.02 1 0.01 6 0.05 0.48 0.06-3.95

Neoplasm of female breast 1 0.04 3 0.07 1 0.01 9 0.07 1.35 0.43-4.24

Neoplasm of genitourinary organs 1 0.04 4 0.10 2 0.01 8 0.06 1.74 0.60-5.08

Neoplasm of lip/oral cavity/pharynx 1 0.04 3 0.07 0 0.00 6 0.05 2.18 0.64-7.49

Neoplasm of lung/bronchus 0 0.00 4 0.10 0 0.00 0 0.00 -- --

Neoplasm of lymphatic and hematopoietic tissue 2 0.07 5 0.12 6 0.04 0 0.00 5.64 1.70-18.70

Neoplasm of respiratory/intrathoracic organs 0 0.00 0 0.00 0 0.00 2 0.02 0 --

Neoplasm of testis 1 0.04 2 0.05 8 0.05 12 0.09 0.57 0.17-1.91

Non-Hodgkin lymphoma 0 0.00 3 0.07 4 0.03 8 0.06 0.89 0.25-3.26

Prostate cancer 0 0.00 4 0.10 0 0.00 18 0.14 0.71 0.24-2.10

Neoplasm of other and unspecified sites 0 0.00 3 0.07 3 0.02 27 0.20 0.33 0.10-1.09

Neoplasm of uncertain behavior (plasma cells) 0 0.00 0 0.00 0 0.00 0 0.00 -- --

*RR indicates relative risk. CI indicates confidence intervals.

EVALUATION OF POSTDEPLOYMENT CANCERS AMONG ACTIVE DUTY MILITARY PERSONNEL
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AUTHORS

Ms Sharkey and Dr Abraham are Epidemiologists, Environmental Medicine, US Army Public Health Command, 

Aberdeen Proving Ground, Maryland.
76 http://www.cs.amedd.army.mil/amedd_journal.aspx

BACKGROUND

During the fall of 2014, a US medical liaison offi cer 

from the Offi ce of The Surgeon General of the Army, 

stationed in the United Kingdom, made an inquiry to the 

Army Hearing Program (AHP), US Army Public Health 

Command (USAPHC) regarding noise induced hearing 

loss (NIHL) in military musicians (W. Startz, e-mail, 

September 16, 2014). The USAPHC AHP conducted a 

literature search for studies on noise exposure and hearing loss in military band members; however, the search 

yielded limited specifi c information on hearing loss in 

military musicians. As a result, a multidisciplinary team 

formed at the USAPHC to carry out a preliminary analysis on hearing loss in military band members.

Noise exposure is a known occupational health hazard 

to those serving in the military.1,2 The effect of hazardous noise, however, can vary signifi cantly depending on 

the type of noise (impulse versus steady state), intensity and duration of exposure, and the degree of effort 

to mitigate the effects. As a result of the variability in 

noise exposure, some military occupations may be more 

at risk for NIHL than others. For example, it has been 

estimated that Soldiers serving in combat arms units 

have a 30% chance of experiencing a hearing loss.3,4

Previous epidemiology studies have shown that infantry, gun crews, and seamanship specialists are 1.4 to 2 

times more likely to suffer a signifi cant threshold shift 

(change in hearing) than other military occupations.5,6

Although several military occupations have been identifi ed in previous military epidemiology studies, musicians have not been specifi cally mentioned. Previous 

noise measurements collected in rehearsal halls and 

during performance venues suggest that noise exposure for musicians can range from 83 to 120 A-weighted 

decibels (dBA).7-14 We presume that military musicians 

will be at risk for similar noise exposures as their civilian counterparts and may be more at risk for NIHL than 

other military occupations.

Noise exposure in the performance of duties as a military musician varies depending on the type of instrument, composition of the band or orchestra, venue, 

A Preliminary Analysis of Noise Exposure

 and Medical Outcomes for Department

 of Defense Military Musicians

 Cindy Smith Thomas Helfer, PhD

 Sharon Beamer, AuD Timothy A. Kluchinsky, Jr, DrPH

 Shane Hall, MS

ABSTRACT

Noise exposure is a known occupational health hazard to those serving in the military. Previous military 

epidemiology studies have identifi ed military occupations at risk of noise induced hearing loss (NIHL); however, musicians have not been specifi cally mentioned. The focus of military NIHL studies is usually on those 

service members of the combat arms occupations. This project was a preliminary examination of Department 

of Defense (DoD) active duty military musicians in regard to their noise exposure, annual hearing test rates, 

and hearing injury rates using available data sources. The analysis concluded that DoD military musicians are 

an underserved population in terms of hearing conservation efforts. Noise surveillance data extracted from 

the Defense Occupational and Environmental Health Readiness System-Industrial Hygiene showed that every 

musician similar exposure group (SEG) with noise survey data from 2009 to 2013 exceeded the occupation 

exposure level adopted by DoD Instruction 6055.12. However, only a small percentage of all DoD active duty 

military musicians (5.5% in the peak year of 2012) were assigned to a SEG that was actually surveyed. Hearing 

test data based on Current Procedural Terminology coding extracted from the Military Health System revealed 

that the percentage of musicians with annual hearing tests increased over the 5 years studied in all services 

except the Air Force. During 2013, the data showed that the Navy had the highest percentage of musicians 

with annual hearing tests at 70.9%, and the Air Force had the lowest at 11.4%. The Air Force had the highest 

percentage of hearing injuries of those musicians with annual hearing tests for all 5 years analyzed. Although 

noise surveillance and annual hearing tests are being conducted, they occur at a much lower rate than required 

for a population that is known to be overexposed to noise.
July – September 2015 77

duration of exposure and proximity to other musicians. 

Also, as the number of years of military service increases, the likelihood of developing a noise-induced hearing 

loss increases. There is evidence of NIHL of 15 dB or 

greater at 4000 or 6000 Hertz in at least one ear in 45% 

of student (nonmilitary) musicians aged 18 to 25 years. 

Student musicians who practiced more than 2 hours a 

day were more likely to exhibit a decrease in hearing at 

some frequency than those who reported practicing for 

less hours.15 Another recent study examining incidence 

of hearing loss among professional musicians in Germany, suggests musicians have 3.51 times higher incidence 

rate of noise induced hearing loss and 1.45 times higher 

incidence rate of tinnitus than the general German population.16 The incidence of hearing loss for musicians increases with length of time of exposure.17,18 Professional 

symphony orchestra musicians in Denmark were found 

to have better hearing than the general population, but 

were considered at risk for occupational noise-induced 

hearing loss after prolonged exposure.19 Brazilian military musicians were found to be 14.54 times more likely 

to experience hearing loss when compared to their nonexposed counterparts, with a further decline in hearing 

noted as years of music exposure increases.20 However, 

among British Army musicians with 8 to 12 years of 

military service, the risk of developing hearing loss did 

not appear to be any greater than their nonmusician 

counterparts.21

 Despite confl icting results of studies with regard to the 

effects of music on hearing,11,22 musicians are generally 

considered to be at risk for NIHL and efforts to prevent 

hearing loss among this group of military personnel is 

essential. The military musician serves not only in their 

chosen occupation but also performs other military duties, such as weapons fi ring, placing them at even greater risk of hearing loss from noise exposure compared to 

nonmilitary musicians.

PURPOSE

A multidisciplinary team consisting of audiologists, industrial hygienists, and a statistician formed to analyze 

NIHL in military musicians based on a review of available data sources. The purpose of this project was to 

determine the noise exposure of Department of Defense 

(DoD) military musicians, the percentage of DoD military musicians receiving annual hearing tests, and the 

percentage of DoD military musicians that received an 

annual hearing test and was diagnosed with a hearing 

injury.

This collection of information and its analysis was initiated and completed as a component of operational public health investigations and was not, therefore, subject 

to review by a human protections board such as an Institutional Review Board.

METHODS

Population

The population used for the analysis consisted of active 

duty musicians serving in the Air Force, Army, Marine 

Corps, and Navy as identifi ed in Defense Enrollment 

Eligibility Reporting System (DEERS) by DoD occupational codes 145000 (enlisted) and 271400 (offi cer 

and warrant offi cers) during calendar years 2009 to 

2013. The DEERS data were extracted using the Military Health System (MHS) Management and Analysis 

Reporting Tool (M2).

Ascertainment of Noise Exposure

The Defense Occupational and Environmental Health 

Readiness System-Industrial Hygiene (DOEHRS-IH) 

was queried for the personal noise dosimetry conducted 

during calendar years 2009 to 2013 by the installation 

industrial hygiene program. The noise survey results 

were converted to an 8-hour time weighted average 

(TWA) using a 3 dB exchange rate as required by the 

DoD Instruction 6055.1223 to identify personnel who 

were at risk of occupational exposure to hazardous noise. 

The DOEHRS-IH personal noise survey results were 

considered to be representative samples of the similar 

exposure groups (SEG) in which the sampled musicians 

were assigned. The use of SEGs is a well-established 

strategy employed by industrial hygienists to conduct 

occupational exposure assessments. A SEG is a group 

of workers who have the same general exposure profi le 

for an agent, such as noise, because of the similarity 

and frequency of the tasks they perform.24 The assigned 

SEG population identifi ed in the DOEHRS-IH was confi rmed for active duty status, catchment area, and DoD 

occupational code through DEERS.

Ascertainment of Audiology Procedures

 Data were extracted from the MHS using the M2 for 

the population using the Current Procedural Terminology (CPT) codes captured during direct care encounters. 

The CPT codes and their defi nitions are listed in Table 1. 

Table 1. Current Procedural Terminology Codes for Hearing 

Test Surveillance

CPT

Code

Defi nition

92552 Pure tone audiometry (threshold)

92555 Speech audiometry threshold 

92556 Speech audiometry threshold with speech recognition

92557 Comprehensive audiometry threshold evaluation and 

speech recognition

92559 Audiometric test of groups 
78 http://www.cs.amedd.army.mil/amedd_journal.aspx

If one or more of these CPT codes were indicated in the 

fi rst 5 procedures for an encounter, the patient was considered to have had an audiogram. Persons with multiple 

encounters with audiograms within the calendar year 

were counted once for having an annual hearing test.

Ascertainment of Hearing Injury

Data for the population were extracted from the MHS 

direct care via the M2 using the International Classifi -

cation of Diseases, 9th Revision, Clinical Modifi cation 

(ICD-9 CM) codes identifi ed as a diagnosis of a hearing injury. The ICD-9 CM codes identifi ed for hearing 

injuries and their corresponding defi nitions and hearing 

injury categories are listed in Table 2. If one of the hearing injury-identifi ed ICD-9 CM codes was indicated in 

the fi rst 5 diagnoses for the direct care encounter, the 

diagnosis was identifi ed for the person for the calendar 

year. A person diagnosed with one or more hearing injury ICD-9 CM codes within a calendar year is considered to have a hearing injury and was counted once for 

the calendar year. A person with one or more hearing 

injury ICD-9 CM codes within a hearing injury category (NIHL, tinnitus, sensorineural hearing loss (SNHL), 

and signifi cant threshold shift (STS)) is counted once 

for each category for the calendar year. For this project, 

hearing injuries were analyzed for those who had an annual hearing test as identifi ed through CPT codes.

ANALYSIS

Noise Surveillance

From 2009 to 2013, a total 174 personal noise dosimetry samples were taken across the services. These 174 

samples were representative of 38 different SEGs for 

musicians ranging from general characterization such 

as “band” to more specifi c characterization of a specifi c 

musical genera of “rock,” “marching,” and “concert” 

bands. The population of a SEG ranged from one to 165 

personnel. Consistent with SEG assessment strategy, if 

one of the personal noise dosimetry samples of a given 

SEG is determined to be over the occupational exposure 

limit (OEL) of 85 dBA 8-hour TWA adopted by DoD 

Instruction 6055.12,

23 then all personnel assigned to the 

SEG are considered to be over the OEL and at risk of exposure to hazardous noise. All DoD personnel exposed 

to noise levels greater than the OEL are identifi ed on the 

command’s roster for inclusion in the Hearing Conservation Program (HCP); therefore, requiring personnel to 

be placed in a hearing testing surveillance program and 

have an audiogram conducted at least annually.23

Hearing Test Surveillance

H earing test data were compared across services within 

a given year. A chi-square test, followed by the Marascuilo procedure, was used to determine if the Navy, Marines Corps, or Air Force had a signifi cantly different 

proportion of service members with an annual hearing 

test compared to the Army. Statistical signifi cance was 

defi ned as P<.05. The Army was chosen as the reference group because Army Pamphlet 40-50125 requires 

every Soldier to undergo an annual hearing test that 

is recorded in both DOEHRS-Hearing Conservation 

(HC) and the MHS during a direct care encounter. Data 

from the DOEHRS-HC system is not linked into military treatment data; therefore, this project used the CPT 

coding as a surrogate for the DOEHRS-HC hearing test 

data.

Hearing Injury

Hearing injury data analysis was restricted to those 

in the population that received an annual hearing test. 

Hearing injuries were compared across the services and 

evaluated at the DoD level for total injuries and for each 

diagnosis.

RESULTS

Noise Surveillance

All of the 38 different SEGS had at least one personal noise dosimetry sample over the OEL, resulting in all those assigned to the 38 SEGs being classifi ed as being “overexposed.” The percentage of DoD military musicians assigned to 

the 38 SEGs during the 5-year period represents 

both those musicians under noise surveillance 

and those classifi ed as being overexposed. Figure 1 shows the percentage of the DoD military 

musicians by each service that was assigned to 

SEGs during the noise dosimetry testing. There 

was limited information available for the personnel assigned to SEGs in 2009 because DOEHRSIH was still being incorporated into DoD-wide 

Table 2. ICD-9 CM Codes for Hearing Injury

Hearing Injury

Category

ICD-9 Defi nition

Noise induced

hearing loss

388.10 Noise effect-ear not otherwise specified (NOS)

388.11 Acoustic trauma 

388.12 Hearing loss D/T noise

Tinnitus 388.30 Tinnitus NOS

388.31 Subjective tinnitus

388.32 Objective tinnitus

Sensorineural

hearing loss

389.10 Sensorineural hearing loss NOS

389.11 Sensory hearing loss, bilateral

389.15 Sensorineural hearing loss, unilateral

389.16 Sensorineural hearing loss, asymmetrical

389.17 Sensory hearing loss, unilateral

389.18 Sensorineural hearing loss, bilateral

 Significant

threshold shift

794.15 Abnormal auditory function study

A PRELIMINARY ANALYSIS OF NOISE EXPOSURE AND MEDICAL OUTCOMES FOR

DEPARTMENT OF DEFENSE MILITARY MUSICIANS
July – September 2015 79

THE ARMY MEDICAL DEPARTMENT JOURNAL

use. From a surveillance perspective, the percentage of 

musicians being surveyed is far below what is expected 

given that all SEGs were over the OEL. The 2 services 

with the highest percentage of personnel in SEGs with 

noise surveillance were the Air Force and the Army, 

with peaks of 12% for the Air Force in 2013 and 8% for 

the Army in 2012. The percentage of military musicians 

under noise surveillance DoD wide (Figure 2) showed a 

steady increase from 2009 to 2013; however, the highest percentage in the 5 years was 5.5%. Given that all 

SEGS within all years were considered overexposed, 

it appears that regardless of how many SEGs or which 

SEG the industrial hygienists surveys, they will be 

classifi ed as overexposed.

Hearing Test Surveillance

As shown in Table 3, the Army consistently had the 

highest (or nearly the highest) proportion (54% to 69%) 

of musicians with annual hearing tests from 2009 to 

2013. In these 5 years, Army had a signifi cantly greater 

proportion of musicians with an annual hearing test 

compared to the Air Force and Marine Corps (P<.05). 

Both the Navy (31% to 71%) and Marine Corps (21% 

to 51%) had an increased proportion of musicians with 

annual hearing tests from 2009 to 2013, noting that 

from 2011 to 2013, the Navy had nearly identical proportions as the Army. The Air Force started with the 

lowest percentage tested in 2009 (18%) with no improvement observed from 2010 to 2013.

The 5-year trend is presented in Figure 3. Although 

some services, notably the Army and Navy, had higher 

testing rates, all services were not in compliance with 

the 100% testing requirement. No service had above 

71% of its musicians tested within a given year.

Hearing Injury

As shown in Table 4, hearing injury rates are highest in 

the Air Force for all 5 years; however, the Air force has 

the least percentage of musicians with annual hearing 

tests. The Army showed the second highest injury rates 

followed by the Marine Corps and Navy, respectively. 

The most common hearing injury diagnosis among 

DoD military musicians is SNHL, with tinnitus as the 

second most common diagnosis. Signifi cant threshold 

shift comes in third and NIHL fourth. The rates of 

these diagnoses fl uctuated 

between 2009 and 2013, 

but only marginally.

The comparison of each 

diagnosis across the services (Figures 4, 5, 6, 7), 

revealed that the Air Force 

had much higher injury 

rates for NIHL, SNHL, 

and tinnitus than the other 

3 services for all 5 years. 

Figure 1. Percentage of active duty musicians by military service 

assigned to SEGs during noise surveillance.

0%

2%

4%

6%

8%

12%

14%

10%

Air Force Army Navy Marine Corps

2012

2013

2011

2010

2009

Table 3. Number and Percentage by Service of DoD Military Musicians With an Annual Hearing 

Test from 2009 to 2013

Military

Service

2009 2010 2011 2012 2013

Counta % Counta % Counta % Counta % Counta %

Air Force 134 17.7%b 136 18.4%b 95 13.3%b 89 13.1%b 78 11.4%b

Army c 1076 54.6% 1268 61.6% 1129 55.1% 1170 58.5% 1363 69.0%

Marine Corps 206 21.1%b 352 36.1%b 398 43.1%b 368 41.3%b 455 51.2%b

Navy 223 31.1%b 326 44.6%b 405 55.7%b 412 58.3% 498 70.9%

Data from the Military Health System Management and Analysis Reporting Tool.

aCount indicates the number of persons with one or more of the designated CPT codes during the calendar year.

bSignificantly different proportion of hearing injuries compared to the reference group (Army).

cReference Group

Figure 2. Percentage of active duty DoD musicians assigned to 

SEGS during noise surveillance.

2%

1%

0%

4%

3%

6%

5%

2009 2010 2011 2012 2013
80 http://www.cs.amedd.army.mil/amedd_journal.aspx

The Air Force’s injury rate for SNHL 

was at least 3 times those in the Army. 

With exception of 2012, the Air Force’s 

injury rate for tinnitus was 4 times the 

Army injury rate. The injury rates for all 

the services for NIHL, SNHL, and tinnitus had little fl uctuation during the 5 

years.

The noise surveillance data from DOEHRS-IH demonstrates that a portion of the 

DoD musicians are exposed to hazardous occupational noise related to their 

jobs as musicians in the military. A large 

percentage of DoD military musicians 

were not assigned to a SEG and not under noise surveillance; however, the 

noise surveillance documented shows 

overexposure. The annual hearing test 

results do not refl ect that which would 

be expected for a population with known 

hazardous occupational noise exposure. 

DoD Instruction 6055.1223 requires personnel in hazardous noise environments 

to have an annual hearing test. Among the services, the 

Army and the Navy showed the highest percentage of 

musicians receiving their annual hearing test; however, 

even these 2 services are well below 100%. The Air 

Force’s injury rates for hearing injuries in general and for 

NIHL, SNHL, and tinnitus specifi cally were higher than 

the other services. Additionally, the Air Force had the 

lowest percentage of musicians with annual hearing tests.

There are multiple strengths of this analysis. The interdisciplinary team brought different perspectives that 

enhanced the public health performance evaluation 

methods promulgated by the Institute of Medicine.26

The data sources used (DEERS, MHS, and DOEHRSIH) provided a broad-spectrum approach using the best 

available data, which provided an analysis that incorporated multisource data integration techniques including 

demographic details data, noise exposure data, medical 

procedures data (as a surrogate for hearing test surveillance), and medical outcome diagnoses data associated 

with procedure data.

There are limitations of the analysis as well. National 

Guard and Reserve service member data were not included and the injury data do not include purchase care 

visits. The 5 years of data was not determined to be suffi cient to determine trends in the 

longitudinal data. Since the data set 

in this analysis is limited, the sample may not truly be representative 

of the entire population of military 

musicians; therefore, generalization 

to the entire population may not be 

feasible.

CONCLUSION AND

RECOMMENDATIONS

This article demonstrates that DoD 

military musicians are an underserved population with regard to 

hearing conservation efforts. While 

noise surveillance and annual hearing tests are conducted, they occur 

Figure 3. Percentage of military musicians within each service with an annual 

hearing test, 2009 to 2013.

40%

50%

0%

30%

60%

20%

70%

10%

80%

2009 2010 2011 2012 2013

Air Force

Marine Corps

Navy

Army

Table 4. Number and Percentage of Injuries by Service and Diagnosis Among DoD Musicians Who Received an Annual Hearing Test from 2009 to 2013

Military

Service

2009a 2010a 2011a 2012a 2013a

Count % Count % Count % Count % Count %

Air Force 25 18.7% 31 22.8% 25 26.3% 17 19.1% 17 21.8%

Army 73 6.8% 91 7.2% 92 8.1% 101 8.6% 93 6.8%

Marine Corps 13 6.3% 16 4.5% 25 6.3% 33 9.0% 33 7.3%

Navy 11 4.9% 14 4.3% 19 4.7% 30 7.3% 24 4.8%

Diagnosis

NIHL 12 0.7% 8 0.4% 10 0.5% 13 0.6% 7 0.3%

SNHL 88 5.4% 92 4.4% 90 4.4% 110 5.4% 117 4.9%

STS 38 2.3% 66 3.2% 72 3.6% 54 2.6% 35 1.5%

Tinnitus 74 4.5% 85 4.1% 79 3.9% 101 5.0% 106 4.4%

Data from the Military Health System Management and Analysis Reporting Tool.

aCount and percentages are representative only of those injuries that occurred among military 

personnel who had an annual hearing test. Denominator for counts and percentages is the total 

number of military personnel tested.

A PRELIMINARY ANALYSIS OF NOISE EXPOSURE AND MEDICAL OUTCOMES FOR

DEPARTMENT OF DEFENSE MILITARY MUSICIANS
July – September 2015 81

THE ARMY MEDICAL DEPARTMENT JOURNAL

at a much lower rate than required for a population that is 

overexposed to noise. Although the focus of this analysis 

was on a single population that is relatively small in comparison with other military occupations, a systematic approach is recommended to improve the hearing conservation efforts that would affect DoD military musicians.

The ultimate responsibility for compliance with annual hearing tests and the requirement for the HCP lie 

with the commanders. The hearing conservation efforts should be a high-priority item evaluated during all 

command safety assessments and inspector general inspections. Making hearing conservation efforts a leadership priority will require commanders to engage the 

professional fi elds responsible for the different aspects 

of hearing conservation (industrial hygiene, preventive 

medicine, occupational health, hearing conservation, 

audiology, and safety). Command situational awareness and command emphasis that identifi es and characterizes hearing health challenges sets a foundation 

for hearing injury prevention planning and execution 

at all levels.

ACKNOWLEDGMENTS

We thank Kevin Wisniewski and Monica Ahuna-Williams of the Army Institute of Public Health (AIPH) 

Industrial Hygiene and Medical Safety Management 

Program, Ralph Rogers and Brian Grace of the AIPH 

Industrial Hygiene Field Services Program, and Charles 

Jokel of the Army Hearing Program for their support in 

this analysis.

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9. Emmerich E, Rudel L, Richter F. Is the audiologic 

status of professional musicians a refl ection of the 

noise exposure in classical orchestral music? Eur 

Arch Otorhinolaryngol. 2008;265(7):753-758.

1.5%

4.0%

3.5%

3.0%

2.5%

2.0%

1.0%

0.5%

0.0%

2012

2013

2011

2010

2009

Air Force Army Navy Marine Corps

Figure 4. Percentage of DoD military musicians by service with annual hearing test and NIHL diagnosis.

Figure 5. Percentage of DoD military musicians by service with annual hearing test and SNHL diagnosis.

0%

2%

4%

6%

8%

12%

14%

16%

18%

10%

Air Force Army Navy Marine Corps

2012

2013

2011

2010

2009
82 http://www.cs.amedd.army.mil/amedd_journal.aspx

10. Royster,JD, Royster LH, Killion MC. Sound exposures and hearing thresholds of symphony musicians. J Acoust Soc Am. 1991;89:2793-2803.

11. Sataloff RT. Hearing loss in musicians. Am J Otol. 

1991;12(2):122-127.

12. Royster JD, Royster LH, Killion MC. Sound exposures and hearing thresholds of symphony orchestra 

musicians. J Acoust Soc Am. 1991;89(6):2793-2803.

13. Early KL, Hurstman SW. Noise exposure to musicians during practice. Appl Occup Environ Hyg. 

1996;11(9):1149-1153.

14. Chasin M. Musicians and the Prevention of Hearing Loss. San Diego, CA: Singular Publishing 

Group. 1996

15. Phillips SL, Henrich VC, Mace ST. Prevalence of 

noise-induced hearing loss in student musicians. 

Int J Audiol. 2010;49: 309-316.

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Incidence and relative risk of developing hearing 

disorders in professional musicians. Occup Environ Med. 2014;71(7):472-476.

17. Schmuziger N, Patscheke J, Probst R. Hearing in 

non-professional pop/rock musicians. Ear Hear. 

2006;27:321-330.

18. Morais D, Benito JI, Almaraz A. Acoustic trauma 

in classical music players. Acta Otorhinolaryngol. 

2007;58:401-407.

19. Schmidt JH, Pederson ER, Paarup HM, Christensen-Dalsqaard J, Andersen T, Poulsen T, Baelum J. Hearing loss in relation to sound exposure 

of professional symphony orchestra musicians. Ear 

Hear. 2014;35(4):448-460.

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D. Auditory thresholds among military musicians: conventional and high frequency. Codas. 

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Int J Audiol. 2010;49:54-64.

23. Department of Defense Instruction 6055.12: Hearing Conservation Program. Washington, DC: US 

Dept of Defense; 2010.

24. Bullock WH, Ignacio JS. A Strategy for Assessing 

and Managing Occupational Exposures. Farfax, 

VA: American Industrial Hygiene Association; 

2006.

25. Department of the Army Pamphlet 40-501: Army 

Hearing Program. Washington DC: US Dept of the 

Army; 2015.

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Health Performance in the Public Sector. Washington, DC: National Academy Press; 2015.

AUTHORS

Ms Smith is an Industrial Hygienist with the US Army 

Public Health Command, Aberdeen Proving Ground, 

Maryland.

Dr Beamer is assigned to the Navy Bureau of Medicine 

and Surgery, Falls Church, Virginia. Previously she was a 

Hearing Conservation Consultant with US Army Public 

Health Command, Aberdeen Proving Ground, Maryland.

Mr Hall is a Statistician with the US Army Public Health 

Command, Aberdeen Proving Ground, Maryland.

Dr Helfer is a Hearing Conservation Consultant with the 

US Army Public Health Command, Aberdeen Proving 

Ground, Maryland.

Dr Kluchinsky is Manager, Health Hazard Assessment 

Program, US Army Public Health Command, Aberdeen 

Proving Ground, Maryland.

Figure 6. Percentage of DoD military musicians by service with annual hearing test and STS diagnosis.

0%

2%

1%

4%

3%

6%

5%

Air Force Army Navy Marine Corps

2012

2013

2011

2010

2009

0%

5%

15%

20%

25%

10%

Air Force Army Navy Marine Corps

2012

2013

2011

2010

2009

Figure 7. Percentage of DoD military musicians by service with annual hearing test and Tinnitus diagnosis.

A PRELIMINARY ANALYSIS OF NOISE EXPOSURE AND MEDICAL OUTCOMES FOR

DEPARTMENT OF DEFENSE MILITARY MUSICIANS
July – September 2015 83

Historically, health physics support to the Combined 

Joint Operations Area-Afghanistan (CJOA-A) was administered by a nuclear medical science offi cer (NMSO) 

assigned to Task Force-Medical built around a medical 

brigade. This offi cer was responsible for managing the 

theater health physics program and had no other health 

physics experts to serve as assistants. In late spring 2013, 

the NMSO (military occupational specialty [MOS] 72A) 

position was eliminated to meet force size reduction requirements. The US Forces Afghanistan (USFOR-A) 

Safety Offi ce initiated a hiring action for a civilian radiation safety professional at this time, but the hiring 

action was never approved. An NMSO working as the 

senior medical planner at the International Security Assistance Force Joint Command Headquarters for most 

of 2013 was available to provide consultation assistance. 

However, the loss of the dedicated NMSO position left 

the CJOA-A without a full-time health physics expert 

when a surge in the health physics mission workload and 

a corresponding heightening of ionizing radiation exposure risks occurred in early 2014.

The reasons for the surge in health physics mission requirements in 2014 were varied. A primary cause was 

the acceleration of retrograde operations which led to 

progressively larger quantities of radioactive commodities arriving for processing and shipping at the 

Bagram Airfi eld (BAF) and Kandahar Airfi eld (KAF) 

Redistribution Property Accountability Team (RPAT) 

yards. The arrival rates of these commodities far exceeded removal rates, thus overtaxing the RPAT yard’s 

management capabilities and creating problems in storage of such items. Another leading cause was a dramatic 

increase in base closures and increase in demolitions of 

structures. These activities led to the discovery of orphan sources (unwanted and uncontrolled radioactive 

materials) on the installations and also necessitated 

radiological surveys of foreign military equipment on 

some of the closing bases to clear for demilitarization 

and removal. Additionally, safety concerns about exposures to x-ray and gamma radiation sources in mobile 

vehicle and cargo inspection systems (MVACISs) found 

at installation entry control points spurred a requirement to begin monitoring and inspecting the operation 

of these systems.

Filling the health physics capability gap left by the departure of the NMSO forced medical planners in the 

CJOA-A to devise a solution within the mandated force 

management level constraints at the time. Under these 

constraints, reestablishing the lost NMSO billet was not 

deemed a viable option due to the General Offi cer level of 

approval necessary for such action. The developed solution was to substitute 2 health physics specialists (HPSs) 

for preventive medicine specialists projected to arrive 

in June of 2014 with the incoming 172nd Preventive 

The Benefits of Deploying Health Physics

 Specialists to Joint Operation Areas

 LTC Scott Mower, MS, USA

 MAJ Joshua D. Bast, MS, USA

 MAJ Margaret Myers, MS, USA

ABSTRACT

Preventive Medicine Specialists (military occupational specialty [MOS] 68S) with the health physics specialist 

(N4) qualifi cation identifi er possess a unique force health protection skill set. In garrison, they ensure radiation exposures to patients, occupational workers and the public from hospital activities such as radioisotope 

therapy and x-ray machines do not to exceed Federal law limits and kept as low as reasonably achievable. 

Maintaining suffi cient numbers of health physics specialists (HPSs) to fi ll authorizations has been a consistent 

struggle for the Army Medical Department due to the rigorous academic requirements of the additional skill 

identifi er-producing program. This shortage has limited MOS 68SN4 deployment opportunities in the past and 

prevented medical planners from recognizing the capabilities these Soldiers can bring to the fi ght. In 2014, for 

the fi rst time, HPSs were sourced to deploy as an augmentation capability to the 172nd Preventive Medicine 

Detachment (PM Det), the sole PM Det supporting the Combined Joint Operations Area-Afghanistan. Considerable successes in bettering radiation safety practices and improvements in incident and accident response 

were achieved as a result of their deployment. The purposes of this article are to describe the mission services 

performed by HPSs in Afghanistan, discuss the benefi ts of deploying HPSs with PM Dets, and demonstrate to 

senior medical leadership the importance of maintaining a health physics capability in a theater environment.
84 http://www.cs.amedd.army.mil/amedd_journal.aspx

Medicine Detachment (PM Det). As a stopgap measure, 

an HPS who had deployed as a Battle Noncommissioned Offi cer with the 31st Combat Support Hospital 

(Task Force 31) in February 2014, was employed to perform crucial health physics mission requirements on a 

part-time base until arrival of the 172nd PM Det.

The substitution of HPSs for PMS was made possible by 

the fact that HPSs are former preventive medicine specialists. The only difference between the 2 types of specialists from a qualifi cations standpoint is the HPSs have 

completed a 20-week course to earn N4 skill identifi er. 

The HPS may be considered MOS qualifi ed to perform 

PMS tasks and as such executed these tasks on a routine 

basis in Afghanistan. However, HPSs are consistently in 

short supply due to the rigors of their academic training 

and are usually assigned to health care facilities to support health physics programs. Their small number and 

high garrison demand have made long-duration deployments rare and deprived them the opportunity to prove 

their value in the combat theater environment. Therefore, their deployment to the CJOA-A beginning in 2014 

was a unique event providing an opportunity for lessons 

to be learned about how to best employ them.

MISSION OVERVIEW

The HPSs assigned to the 172nd PM Det served on PM 

teams at BAF and KAF. The HPS at BAF supported 

Regional Command (RC)-North, RC-East, RC-West, 

and RC-Capital, while the other supported RC-South 

and RC-Southwest. When not performing health physics missions, they were engaged in standard preventive 

medicine technician duties such as sanitation inspections and water quality monitoring. The most common 

health physics and radiation safety missions performed 

are summarized in the Table. It is important to note these 

missions could have been executed by a NMSO. The 

medical imaging oversight mission was performed by a 

2-person team comprised of a NMSO and a HPS from 

US Army Public Health Command (USAPHC) brought 

in for a 3-week period. The remaining missions shown 

in the Table were performed by the HPSs assigned to 

the PM Det.

MVACIS Inspections

Many entry control points used MVACIS to image local national vehicles for weapons and explosives prior 

to permitting their entry onto installations. The Nuclear 

Regulatory Commission (NRC) regulates the MVACIS 

radioactive sources in the United States. The Communications-Electronics Command (CECOM) holds the 

NRC license for these sources before they are shipped 

overseas. Although the NRC does not have jurisdiction, 

the MVACIS sources are managed by an Army Radiation Authorization given to USFOR-A by CECOM. 

Appointments as Radiation Safety Offi cers for NRC licenses and Army Radiation Authorizations are routinely 

held by NMSOs, civilian health physicists, or very specialized trained individuals.

United States and International Security Assistance 

Force (ISAF) uniformed personnel assigned security responsibilities for installations served as the operators of 

these systems. Many operators had no experience with 

MVACIS operations prior to their deployments and had 

never been enrolled in a radiation dosimetry program. 

Contractors were used by ISAF and USFOR-A to maintain and service these systems. The contractors would 

position contract service representatives (CSRs) at major bases and designate coverage areas for the representatives to support. The CSRs would travel to the bases 

to administer safety and operator training, and issue and 

collect thermoluminescent dosimeters. They also visited 

entry control points at a specifi ed frequency to examine 

the daily exposure by reading records kept by operators, 

collect and reissue dosimeters, and troubleshoot any 

problems. Upon the completion of the visit, they would 

offer recommendations and advice to the site operators 

on improving work practices. The effectiveness of their 

visits was limited since they lacked the authority to hold 

operators accountable for failing to follow proper safety 

procedures.

THE BENEFITS OF DEPLOYING HEALTH PHYSICS SPECIALISTS TO JOINT OPERATION AREAS

Health Physics and Radiation Safety Missions

Type Frequency Description

Mobile Vehicle and Cargo Inspection 

System (MVACIS) inspections

Quarterly Quarterly inspections of MVACIS at entry control points to ensure safe system 

operation.

Radioactive commodity retrograde 

support

Continuous Consultation on the establishment and operation of a consolidated storage locations, offering radiation safety training to the workforce, leak testing turned-in 

items, and surveying packaged items prior to shipment.

Radiation surveys As needed Survey with specialized measurement equipment areas where a suspected radiation 

release and/or exposure has occurred, followed by an assessment of health risks.

Orphan source management As needed Provide assistance and consultation on how to manage items unexpectedly found 

on installations. These discoveries could prompt a radiation survey mission.

Medical imaging system oversight Annually Inspection of HCF imaging equipment that emits ionizing radiation.
July – September 2015 85

THE ARMY MEDICAL DEPARTMENT JOURNAL

In order to address radiation exposure concerns and improve 

radiation safety operational practices, in mid-2014 USFOR-A issued a directive implementing

MVACIS inspection program 

(Figure 1). The inspection checklist was developed with input from 

the HPSs, USAPHC health physics experts, and the civilian radiation safety offi cer employed by 

the largest MVACIS contractor in 

Afghanistan. Prior to starting the 

quarterly inspections, an initial 

site assistance visit was completed 

at each ECP by the HPSs or uniformed PM personnel at bases not 

supported by the 172nd PM Det. 

The non-HPS inspectors received 

training by the CSRs on how to 

perform these inspections before 

initiating the initial site assistance 

visits. The scrutiny and attention 

garnered by the inspections improved operator adherence to safety policies and procedures. The inspections also improved the completeness 

of occupational and environmental health site assessments since preventive medicine elements had previously not been populating the Defense Occupational and 

Environmental Health Readiness System’s (DOEHRS) 

DoD Deployment Surveillance Portal with information 

concerning the occupational hazards associated with 

MVACIS radiation emissions.

Radioactive Commodity Retrograde Support

Over the course of 13 years of continuous military operations, signifi cant quantities of US equipment containing radioisotopes had been brought into Afghanistan. 

Examples of some of the more common items included 

weapon system optics, compasses, and chemical agent 

detection alarms. The processes and procedures for retrograding items varied based on the manager of the commodity and the type of radioisotope it contained. Many 

items had to be tested through the collection and submission of wipe tests to the US Army Test, Measurement, 

and Diagnostic Equipment laboratory in Pirmasens, 

Germany, to prove they had been surveyed with a radiation detection device and were free of leaks before fi nal 

packaging and shipment. Prior to arrival of the HPSs, 

there was only one contractor in all of Afghanistan stationed at KAF qualifi ed to perform wipe sampling.

When the push to retrograde commodities began, management and shipping processes were not suffi ciently 

mature to handle the infl ux of 

turned-in items. The problem was 

further magnifi ed by a lack of 

trained personnel to support these 

processes. Unlike the drawdown 

from Operation Iraqi Freedom, 

there was no Army Contaminated 

Equipment Team, a special team 

deployed by the Army Material 

Command, to lead the radioactive 

commodities retrograde effort in 

Afghanistan. Efforts to overcome 

personnel shortfalls were further 

hampered by delays in civilian 

hiring actions; stringent force 

management level constraints 

governing the number of contractors, service members, and Department of Defense employees 

permitted in country; and diffi culties in modifying infl exible scopes 

of work to permit contractors already involved with incountry retrograde operations to participate 

in radioactive commodity retrograde support activities.

By necessity, the HPSs were used to support the retrograde effort even though retrograding activities are doctrinally the responsibility of logistics rather than medical authorities. Their assistance was broad in scope and 

evolved throughout the deployment as new challenges 

were identifi ed and previous problems were solved. The 

effect of their assistance was greatly amplifi ed by their 

exceptional knowledge base in health physics, strong 

oral and written communication skills, and the credibility boost offered by their ranks as noncommissioned 

offi cers. Those areas of assistance where the HPSs had 

the most meaningful effect on retrograde support were 

in the administration of safety awareness training to 

retrosort yard personnel, reviewing and coauthoring 

pertinent standard operating procedures and policy 

documents, relieving the wipe sample collection backlog through additional sampling, surveying prepped 

shipments, participation in installation radiation safety 

working groups, and providing consultative services to 

aid the establishment of a consolidated radioactive commodities storage area on BAF.

Radiation Surveys

During the course of their deployment, the HPSs performed surveys of areas and equipment possibly contaminated with radioactive material. Some of these 

surveys were of an urgent nature and required prompt 

execution, while others were less time-sensitive. The 

Figure 1. A nuclear medical science offi cer examining a mobile vehicle and cargo inspection 

systems at Bagram Airfi eld on March 28, 2015. 

(Photo courtesy of the authors.)
86 http://www.cs.amedd.army.mil/amedd_journal.aspx

urgent surveys were typically high profi le and garnered 

intense interest from senior commanders. One such incident resulted after a report was received about possible 

acute radiation exposures from a malfunctioning X-ray 

emitting MVACIS received by North Atlantic Treaty 

Organization (NATO) soldiers manning an entry control point at the ISAF Headquarters in downtown Kabul. 

The guards were evacuated to a nearby NATO-run hospital and kept under medical observation for symptoms 

of acute radiation sickness after it was discovered that 

one of the 3 x-ray tubes of the MVACIS was not functioning properly, and high radiation readings were allegedly read from a radiation detection meter.

Within one hour of notifi cation of the incident, a HPS 

was fl own by air ambulance from BAF to the scene to 

investigate. Much to the relief of all parties involved, 

this investigation conclusively proved that no medically 

signifi cant radiation releases had occurred. The malfunctioning x-ray tube was determined to be burned-out, 

meaning it could not emit any x-ray radiation. The alleged high radiation measurements were due to a 3 order 

of magnitude instrument reading error. As an additional 

precautionary measure, the HPS performed a radiation 

survey of the entry control point and found no radiation 

readings above normal background levels.

Another high-profi le radiation survey was performed at 

the site of an MI-17 helicopter fi re within a hangar at the 

New Kabul Afghanistan International Airport (Figures 

2 and 3). The Russian-made helicopter was the property 

of the Afghanistan Air Force (AAA) and was equipped 

with instrumentation that contains radioisotopes. The 

hangar where the helicopter was parked was an important rotary wing aircraft maintenance location and also 

served as a training area where US military experts 

administered hands-on training to AAA maintenance 

recruits. Senior AAA and ISAF leadership wanted to 

remove the burned out helicopter hulk and resume hangar operations as soon as possible; however, a radiation 

survey was fi rst required to assess the dangers posed 

from any radiation contamination. The survey results 

from the HPS found the hangar free of contamination, 

thus clearing the way for recovery operations.

Most radiation surveys performed by the HPS were not 

as spectacular as the two previous examples. The majority of surveys supported retrograde efforts and included 

surveys checking for contaminated areas at radioactive 

commodity storage locations and the aforementioned 

surveys of items prepped for shipment out of the CJOAA. The HPSs also surveyed items, often of foreign make, 

earmarked for demilitarization to certify those items 

were radioisotope free.

Orphan Source Management

There were several instances where the HPSs were 

called upon to provide assistance and consultative support in dealing with orphan sources discovered on ISAF 

installations. Examples of such orphan sources included 

a Soviet-era ice detector containing strontium-90, a beta 

emitter, found at BAF (Figure 4); thorium nitrate, an alpha emitter, encapsulated in concrete within a 5 gallon 

bucket in an abandoned building that once served as an 

East German Pharmaceutical Plant on Camp Phoenix 

(Figure 5); and 2 ion chamber survey meters improperly 

discarded, presumably by a contractor, in a dumpster at 

BAF. Though all the orphan source discovery incidents 

were judged by the HPS to pose low health risks, they 

were, nevertheless, documented and archived within 

DOEHRS.

THE BENEFITS OF DEPLOYING HEALTH PHYSICS SPECIALISTS TO JOINT OPERATION AREAS

Figure 2. Smoke pours from a burning Afghan MI-17 helicopter inside of a maintenance hangar at the Kabul International 

Airport. The helicopter in foreground is another MI-17 not involved in the fi re. (Photo courtesy of the authors.)

Figure 3. Damage inside of the maintenance hangar at the Kabul International Airport following removal of burned Afghan 

MI-17 helicopter. (Photo courtesy of the authors.)
July – September 2015 87

THE ARMY MEDICAL DEPARTMENT JOURNAL

Medical Imaging System Oversight

One of the basic responsibilities for HPSs in a nondeployed environment is to ensure medical imaging systems (x-rays, fl uoroscopy machines, and computed tomography scanners) at medical treatment facilities, dental clinics, and veterinary clinics are functioning properly. The highly specialized equipment necessary to 

perform these checks was not available in Afghanistan. 

As such, the oversight role of medical imaging systems 

for HPSs within Afghanistan was limited to checking 

on TLD wearing and monitoring system operator work 

practices to determine their adherence to as low as reasonably achievable radiation exposure work practices. In 

order to satisfy the regulatory requirement for an annual 

check of imaging systems, a request for assistance was 

submitted by USFOR-A in collaboration with US Army 

Central Command to the US Army Medical Command 

for a team to perform these checks. This team spent 3 

weeks in country checking 17 systems at 6 installations.

RECOMMENDATIONS

The health physics support provided by the HPSs proved 

to be of great benefi t across multiple staff support areas 

(safety, logistics, and force health protection). As a fi rst 

of its kind deployment, there was initially some confusion as to how best utilize them. The confusion abated 

as duties and responsibilities became better defi ned and 

benefi cial partnerships with other organizations and 

commands were established. The following paragraphs 

offer several recommendations on how to best prepare, 

equip, and employ HPs should they be used for future 

long-term deployments.

Preparation

Since HPSs are not assigned to PM Dets in garrison, 

they should be afforded an opportunity to participate in 

the PM Det’s predeployment certifi cation training exercise. Doing so allows them to meet their deployment 

teammates, relearn basic PMS tasks, and advance their 

understanding of health physic mission requirements in 

the deployed environment before arriving at the deployment destination. The HPSs selected to deploy should 

be knowledgeable in performing medical imaging system surveys and conducting contamination surveys, and 

skilled in decontamination practices. Given the strong 

possibility of being asked to provide retrograde operation support and MVACIS inspections, completion 

of Class 7 shipment training and familiarization with 

MVACIS radiation safety fundamentals prior to deployment is advisable. In addition, they should have the necessary rank, experience, and initiative to successfully 

establish and maintain an effective health physics program in the absence of a NMSO or other offi cers well 

versed in health physics.

Equipment

With the exception of the medical imaging radiation surveys, all other surveys performed by the HPSs required 

use of an AN/PDR 77 radiac set plus its accessories to 

measure and detect radiation levels. This radiac set is 

not an item found in the modifi ed table of organization 

and equipment of a PM Detachment. One of the 2 sets 

used in Afghanistan by the HPSs was a loaner from a 

USAPHC, while the other was acquired through logistic 

channels after submitting an operational needs statement. Had these radiac sets not been available, the scope 

and the overall effectiveness of the services provided 

by the HPSs would have been severely diminished. A 

commercial off-the-shelf portable gamma spectroscopy 

Figure 4. A Soviet aviation ice warning device containing strontium-90, a beta particle emitter, found at Bagram Airfi eld. 

(Photo courtesy of the authors.)

Figure 5. A metal bucket containing thorium nitrate which had A metal bucket containing thorium nitrate which had 

been encapsulated in concrete at the now closed Camp Phoe- een encapsulated in concrete at the now closed Camp Phoenix in the Kabul base cluster. (Photo courtesy of the authors.) ix in the Kabul base cluster. (Photo courtesy of the authors.)
88 http://www.cs.amedd.army.mil/amedd_journal.aspx

instrument will also be useful in a theater of operations 

since it can identify common radioisotopes, is more sensitive than the AN/PDR-77 in detecting gamma-emitters, 

and can detect some neutron emissions.

Employment

Since the bulk of the HPS mission services occurred at 

the retrograde hubs at BAF and KAF, this was logically 

the best location to station them. While stationed here, 

they had ample time to gain an understanding of the retrograde processes and learn where their services were 

needed most to support retrograde efforts. Also, BAF 

and KAF were installations where the 172nd PM Det 

had the lead for providing most PM support. This meant 

the HPSs were able to perform PMS mission work when 

there were lulls in the health physics missions.

Currently, there is only one HPS for the 224th PM Det 

which replaced the 172nd PM Det. This soldier is located at BAF where the majority of the health physics 

operations are concentrated. The USFOR-A is preparing to designate an Air Force Industrial Hygienist as 

their Radiation Safety Offi cer (RSO), in absence of an 

NMSO, and the HPS as the alternate RSO. A NMSO 

recently arrived in Kuwait and provides long-distance 

support to Afghanistan and other countries in the area 

of operations. The NMSO serves as a bridging solution 

until the safety community develops a permanent solution for the Radiation Safety Program.

CONCLUSION

Maintaining a capability to execute health physics 

missions is critical and likely to grow in urgency and 

magnitude during the drawdown phase of an overseas 

military deployment operation. In the absence of an 

available NMSO, medical planners should give strong 

consideration to deploying HPSs to assist with fi lling 

health physics and radiation safety capability gaps. An 

ideal place to assign HPSs is within a deploying PM Det. 

As members of the detachment, they are well-positioned 

to execute their health physics missions while also being available to perform routine PMS duties. The HPSs 

deployed to the CJOA-A in 2014 executed a multitude of 

crucial missions. Some of the missions were extremely 

high profi le with signifi cant diplomatic implications. 

Based on the notable success of their Afghanistan deployment, the HPSs have proven their worth many times 

over and should be considered for future deployments.

AUTHORS

LTC Mower was the International Security Assistance 

Force Joint Command and US Forces-Afghanistan (USFOR-A) Force Health Protection Consultant from January through October 2014 at North Kabul Afghanistan 

International Airport. He is an Environmental Science 

and Engineering Offi cer and Registered Environmental Health Specialist assigned to XVIII Airborne Corps, 

Fort Bragg, North Carolina.

MAJ Bast is an Army Medical Entomologist and the 

Commander of the 172nd Preventive Medicine Detachment, which deployed to Afghanistan from June 2014 

through February 2015. He also served as the USFOR-A 

Force Health Protection Consultant from October 2014 

through January 2015.

MAJ Myers is currently serving in Kuwait as the Theater 

Radiation Safety Offi cer at US Army Central Command 

Headquarters (Forward). Her duties require frequent 

travel to Afghanistan.

THE BENEFITS OF DEPLOYING HEALTH PHYSICS SPECIALISTS TO JOINT OPERATION AREAS

Articles published in the Army Medical Department Journal are indexed 

in MEDLINE, the National Library of Medicine’s (NLM’s) bibliographic 

database of life sciences and biomedical information. Inclusion in the 

MEDLINE database ensures that citations to AMEDD Journal content will 

be identifi ed to researchers during searches for relevant information using any 

of several bibliographic search tools, including the NLM’s PubMed service.
July – September 2015 89

The US Army has made education of its Soldiers regarding behavioral health one of its foremost considerations. 

This emphasis has resulted in the Borden Institute’s publication of the fourth Textbook of Military Medicine in 

the past 20 years to address the behavioral health of the 

Soldier: Forensic and Ethical Issues in Military Behavioral Health.

The writing of the fi rst two behavioral health books,

Military Psychiatry: Preparing in Peace for War (1994) 

and War Psychiatry (1995), was led by COL (Ret) 

Franklin D. Jones. These volumes were published soon 

after the end of the Gulf War in 1991. COL (Ret) Elspeth 

Ritchie was the Senior Editor for Combat and Behavioral Health, published in 2011, as well as this newly published volume. COL Ritchie’s books were published as 

Operations Iraqi Freedom and Enduring Freedom were 

winding down. All four of these books present insights 

into the emotional aspects of war and the role of behavioral health care providers within the context of the US 

Army. Each volume reinforces the understanding that 

the stresses of military life can be signifi cant. Forensic

and Ethical Issues differs from the other books in that 

it focuses on the practice of forensic psychology and 

psychiatry and their application to medical issues arising within the military legal system. Covering a broad 

range of topics, this book provides an easily accessible 

reference for readers wishing to understand the implications of a Soldier’s behavior and how care providers 

specializing in psychological and psychiatric care can 

help Soldiers in trouble.

The men and women who serve in the military responded without hesitation to the challenge of terrorism provoked by the events of September 11, 2001. Yet, now that 

military operations conducted in response are winding 

down, many Soldiers and their families have been left 

with the psychological wounds of war. Our military specialists in behavioral health focus on helping Soldiers 

cope with these mental injuries and associated behaviors.

In this book, psychiatrists, psychologists, scientists with 

expertise in behavioral health, and lawyers who specialize in military law, from all the military services, have 

coauthored chapters detailing how the sciences of psychology and forensic psychiatry apply to behavioral issues in the context of the legal system. The intent is to 

show the insight and understanding that forensic specialists add to the military justice system as Soldiers 

face great challenges in their lives. Throughout the 

book are descriptions of the very broad roles that behavioral health professionals play in attempting to help 

jurists better understand why Soldiers commit crimes. 

The work of these professionals adds an element of fairness to the evaluation of Soldiers in crisis. The content 

of each chapter refl ects topical issues in today’s military 

world.

Suicide, sexual assault, and posttraumatic stress disorder (PTSD) are the primary behavioral health issues facing the military. To begin addressing the fi rst of these, 

the authors discuss ways to stop a determined individual 

from committing suicide. When a suicide occurs, the 

authors detail the military’s investigative plan, which 

provides a “psychological autopsy” of the events leading 

to the act. When suicides are perceived to occur in clusters within a military unit or location, an epidemiologic 

review of the cases and identifi cation of common forces 

(if any) that drive these events are described.

Sexual assaults are discussed in a similarly straightforward manner. The need for the sexual education of men, 

particularly where it applies to the issue of competent 

consent, and the underlying psychological environment 

A New Volume in the Borden Institute

Textbooks of Military Medicine Series

Forensic and Ethical Issues in

Military Behavioral Health

COL (Ret) Elspeth Cameron Ritchie, MC, USA

Senior Editor

LTC Daniel E. Banks, USA

 COL (Ret) Edward Lindeke, USA
90 http://www.cs.amedd.army.mil/amedd_journal.aspx

leading to a culture in which sexual assaults occur is 

recognized and addressed.

The authors also provide an understanding of the psychiatric diagnosis of PTSD. Flashbacks, sleep disturbances, 

and a labile mood in the context of previous combat experience form the core of the diagnosis. Yet the chapter 

proceeds to address unresolved questions about PTSD, 

with the recognition that it is a very diffi cult injury to 

treat; available therapies are not as effective as we would 

wish. Not infrequently Soldiers with PTSD come to the 

attention of care providers because of legal diffi culties, 

during struggles in navigating the Veterans Administration Disability System, or following accusations of 

malingering. Although there is a recognition that PTSD 

may invoked as a possible defense for crimes, no clear 

conclusion is presented to show whether a diagnosis is 

likely to affect trial outcomes. However, this defense 

strategy may be a more powerful mitigating factor in a 

military, as opposed to a civilian, courtroom. Furthermore, care providers continue to struggle with optimal 

PTSD therapy, and their numerous, widely disparate approaches help us realize how diffi cult overcoming this 

illness can be. Resilience training, cognitive behavioral 

therapies, and medications are all a part of care. Not infrequently, alternative and complementary methods are 

also tried. Although PTSD is labelled an anxiety disorder, overlapping therapies of antipsychotics and antidepressants remain the primary approach to medical 

therapy.

The training of forensic psychologists and psychiatrists, 

both within and outside of the military, is outlined, providing insight into the formal education of these care 

providers. These experts must be prepared to help Soldiers interact with the sanity board process, help recognize mitigating factors in the defense of a Soldier facing 

criminal charges, and explain behavioral health issues 

in a Soldier’s disability hearing by describing how he or 

she has faced mental health and disciplinary issues. In 

addition, the role of these care providers in two specialized and specifi c criminal arenas is discussed. The fi rst 

is the role of the forensic specialist in cases where Soldiers are charged with substance abuse; the second is the 

rare but intense situation when capital murder is alleged 

and the death penalty is sought.

Another chapter draws a clear line defi ning the expected 

(and required) behavior of psychiatrists in their role as 

care providers of detainees. A key point is that the psychiatrist or psychologist is not an interrogator, but he or 

she may act as a behavioral health care provider to the detainee or a as consultant to the interrogators (never serving in both roles). In the latter role, the focus is to help 

interrogators better recognize features of mental illness 

and the psychological effects of interrogation techniques.

The book also discusses how to design a safe and secure 

psychiatric facility. A clear description of how to house 

those with long-term forensic psychiatric illnesses is detailed, as illustrated by the new Saint Elizabeths Hospital in Washington, DC, maximizing both security and 

therapeutic concerns. The principles presented can be 

used by all in the mental health fi eld.

The book closes with an intriguing chapter addressing the relationship of mefl oquine, a drug prescribed 

to Soldiers for malaria prophylaxis, with development 

of mood changes, psychosis, and in the context of underlying PTSD. The author recommends that all service 

members should be screened for the use of mefl oquine. 

If the service member has been exposed to the medication and is facing legal charges, the situation should be 

articulated by the defense.

Forensic and Ethical Issues in Military Behavioral 

Health makes a signifi cant contribution to the body 

of work identifying the lessons learned by Army care 

providers following the wars in Iraq and Afghanistan. 

Throughout the book, Dr Ritchie and the contributing 

authors identify and address many of the issues now in 

the forefront of care provided by the Army’s behavioral 

medicine specialists. The authors show a strong understanding of the relationship between war and a Soldier’s 

mental health and help us recognize approaches that 

must be in place as we go forward to treat service members of the next generation, lessons that will be relevant 

for many years to come.

AUTHOR

LTC Banks is the Director of the Borden Institute, Joint 

Base Fort Sam Houston, Texas.

COL (Ret) Lindeke is the Assistant Director of the Borden Institute, Joint Base Fort Sam Houston, Texas.

FORENSIC AND ETHICAL ISSUES IN MILITARY BEHAVIORAL HEALTH

A NEW VOLUME IN THE BORDEN INSTITUTE TEXTBOOKS OF MILITARY MEDICINE SERIES

This book is available in PDF and will soon be available in iPad and Kindle formats for download on your electronic reader. The book can be ordered at no cost by those on active duty, in 

the National Guard, and in the Reserve by following the instructions on the Borden Institute 

website, http://www.cs.amedd.army.mil/borden/. It will soon be available for purchase from the 

US Government Printing Offi ce.
July – September 2015 91

THE ARMY MEDICAL DEPARTMENT JOURNAL
92 http://www.cs.amedd.army.mil/amedd_journal.aspx

The US Army Health Readiness Center of Excellence

The Army Medical Department Center and School

ENVISION, DESIGN, TRAIN, EDUCATE, INSPIRE

Joint Base San Antonio Fort Sam Houston, Texas
SUBMISSION OF MANUSCRIPTS TO THE ARMY MEDICAL DEPARTMENT JOURNAL

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All manuscripts will be reviewed by the AMEDD Journal’s Editorial Review Board and, if required, forwarded to the appropriate 

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