# Presentation: 889089

## Vertebrate Ecology and Biology: Non-Avian

**Vertebrate Ecology and Biology: ****Non-Avian**

**Dr. Mike Bunning **
**Centers for Disease Control and Prevention**
**Division of Vector-Borne Infectious Diseases**
**Arbovirus Diseases Branch **
**Fort Collins, Colorado **

## Objectives 

- Review vertebrate (non-avian) species
- Review of National Surveillance Data
- Research Projects
- Equine
- Canine
- Feline
- Swine
- New Information
- Serosurvey Results from 2002

## Epizootic West Nile Virus in the United States, 1999-2002

- Alpaca
- Horses
- Big Brown Bat
- Little Brown Bat
- Cat
- Dog
- Grey Squirrel
- Llama
- Alligator

- Wolf
- Fox Squirrel
- Sheep
- Eastern Chipmunk
- Rocky Mountain Goat
- Striped Skunk
- Reindeer
- Domestic Rabbit
- Harbor Seal

- *Data reported from USDA, CDC (ArboNET) and State Health and Veterinary Labs

## Equine Background 

- West Nile virus responsible for outbreaks of encephalomyelitis in humans and equids
- Concerns about equine
- Sentinel host for humans
- Dead end or amplification host
- Public Health ramifications
- Incidence in 2002:  ~12,000 cases in US
- Clinical attack rate ~ 0.1 (10%)
- Roughly 1 in 3 affected animals die or are euthanized

## Equine WNV: Initial Trials

- 12 horses: range of ages and breeds; seronegative for WNV and SLE
- Infect via bites of infected *Aedes albopictus* (horse vs crow isolates)
- Clinical observations and serum for virus assay BID for 14 days

**Published Bunning ML**, Bowen RA, Cropp CB, Sullivan KG, Davis BS, Komar N, Godsey MS, Baker D, Hettler DL, Holmes DA,
-  Biggerstaff BJ, Mitchell CJ. Experimental Infection of horses with West Nile virus . Emerg Infect Dis 2002;8:380-6.

## Slide 6

## Equine Serology/Viremia Time Line 

**IgM**

**Clinical case **

**IgG**

**Equine Serology/Viremia Time Line**** **

## Equine Clinical Case

- CNS Disease
- Ataxia
- Circling
- Hind limb weakness
- Proprioceptive deficits
- Lip droop/paralysis

## Horse 11, Tissue Virus  (Log10  PFU/gram)

- Medulla           6.8
- Cerebellum	5.0
- Frontal cortex	5.2
- Occip cortex	4.3
- Hippocampus	3.3

- Cervical cord	5.0
- Thoracic cord	4.0
- Lumbar cord	4.3
- Radial n.		neg
- Spleen		neg
- Liver		neg

## Equine Conclusions 

- Viremias
- Highest viremia
- Day-3 post infection
- 3 Log-10 Vero cell PFU/ml serum
- One clinical case
- Apparent to inapparent = 1:11
- Virus titers in brain and spinal cord, day-9
- Log 104.0 to 10 6.8  PFU/gram
- None of the virgin mosquitoes became infected

**Notes:**
Recovery of WNV from Mosquitoes Equine viremias from 1.0 to 2.7
0 of 652 mosquitoes were positive

## Equine Conclusions 

- Equines infected with WNV develop viremias of low magnitude and short duration
- Infected horses are unlikely to serve as amplifying hosts for WNV in nature
- Care should be taken on postmortem exam
- Clinical attack rate is roughly 10% in experimental and field studies
- Clinical signs usually characteristic of encephalomyelitis

## Canine Research

## Experimental Infection of Canine with WNV

- Is WNV readily transmitted to dogs by feeding of infected mosquitoes?
- What is the duration and magnitude of viremia and antibody response
- Are dogs likely to serve as amplifying hosts?

## Canine Research Results 

- There was no evidence of clinical disease
- Mild leukopenia
- Virus capable of replicating (4/4)
- Virus was not isolated from saliva
- Dogs are not likely to be amplifying hosts

## Feline Research

##  Cats as West Nile virus hosts

**Notes:**
Each of the four cats exposed to WNV by feeding of infected mosquitoes developed viremia 12 to 36 hours after exposure (Table 1).  The peak titers of virus in serum ranged from 103.0 to 104.0 pfu/ml, and virus was not isolated from any samples collected more than 4.5 days after mosquito feeding.  Three of the four cats (numbers 1, 2, and 4) displayed both obtundation and a fluctuant febrile response (Figure 1), but neurologic signs were not observed in any of the cats. Statistically significant elevations of rectal temperature occurred from days 1 through 6.  A number of isolated significant deviations from baseline in hematologic parameters were identified, but meaningful trends were not observed.  Cat 4 was anemic on arrival (hematocrit 21), became more severely anemic during the first week after challenge (hematocrit as low as 13), then rebounded by days 9 to 14  (hematocrit 18 to 22).  WNV was not isolated from any of the oral swab samples. At necropsy on day 50, gross abnormalities were seen in cat 2 (pale, reticulated liver) and cat 4 (hydrocephalus and a nodular, fibrous spleen).  These were considered incidental findings, unrelated to WNV infection.  
Clinical signs, including pyrexia and neurologic abnormalities, were absent throughout the study period in cats inoculated with WNV via ingestion of three (n=2) or one (n=2) infected mice.  Cats ingesting three mice (cats 14 and 16) developed viremia within 24 hours of ingesting the first mouse (these cats were not bled at 12 hours), and viremia resolved by day 6 (Table 1). The peak viremia in these two animals was 103.7 and 103.9 pfu/ml serum, respectively.  Cohort mice of those fed at the first of the three feedings yielded 109.5 and 109.7 pfu per gram of brain.  Cats 17 and 18 each ingested a single mouse.  Those mice were euthanized approximately two days earlier in the course of their infection than those fed to cats 14 and 16, and virus titration of their cohorts revealed 105.3 and 106.5 pfu per gram of brain.  Cats fed a single mouse had viremia of similar duration but delayed onset (day 4 and 2.5 for cats 17 and 18 respectively), and lower peak titer (102.5 and 102.2 pfu/ml serum) than occurred in the cats ingesting mice with higher virus content (Table 1).   Three of the four cats fed infected mice developed neutralizing antibody titers to WNV of at least 1:10 by day 14 post-exposure (range 1:10 to 1:40, 90% neutralization).  Cat 18, which had a neutralizing titer <1:10 on day 14 had a titer of 1:40 on day 21.

## Clinical Disease in Feline 

- Mild clinical disease, lasting two to three days

## Oral Transmission

- 2 cats fed a West Nile virus infected mouse, one daily for 3 days
- 2 cats fed a single West Nile virus infected mouse
- Clinical exams, BID serum for virus isolation

## Feline Oral Transmission Studies

## Feline Oral Infections

- Cats were readily infected by consuming infected mice (4/4)
- No clinical disease
- Occasional mosquitoes may become infected by feeding on infected cats

## Feline Research Results 

- Virus capable of replicating (12/12)
- Virus not isolated from saliva
- Mild, nonspecific disease
- Inconsistent hematologic disturbances, fever
- Cats develop a level of viremia that may be capable of infecting mosquitoes

## Swine Research 

## Alligators 

- Epizootic characterized by neurologic disease which occurred at a 9,000-head alligator farm in Florida.
- Approximately 300 alligators (*Alligator mississippiensis*) died during this outbreak

- Picture courtesy
- of Elliott R Jacobson

- *Information provided by University of Florida, preliminary data

## Alligators 

- Of the tissues sampled, liver had the highest viral loads (maximum 108.9log10 pfu/0.5cm3)
- Brain and spinal cord had the lowest viral loads (maximum 106.6log10 pfu/0.5cm3) each
- Viral loads in plasma ranged from 103.6 to 106.5 log10 pfu/mL

## Slidell, Louisiana:  West Nile Virus Mammal Serosurvey -2002

## Slide 26

## Serosurvey

- First hot spot of summer 2002, ending with:
- ~ 37 human cases in St. Tammany Parish, La
- ~ 18 human cases in Slidell, La
- August - October serosurvey teams were assembled to work in Slidell, LA for collection of specimens from a wide array of species

## WNV Mammal Serosurvey Team Members

- Nick Panella

- Stan Langevin

- Sarah Lasater

- Gabrielle Dietrich

- James Kile

- John Montenieri

- Kaci Klenk

## Acknowledgements

**Acknowledgements**

**Colorado State University**
**Laura Austgen**
**Paul Gordy**
**Max Teehee**
**Dale Baker**
**Dick Bowen **
**United States Department of Agriculture**
**Louisiana State Department of Health**
**Raoult Ratard**
**Anne Buff**
**Erin Brewer **
**Slidell Memorial Hosp**
**Department of Fish and Wildlife **
**Department of Agriculture**

- CDC
**Carl Mitchell****	****Nick Komar**
**Brent Davis****	****Aaron Kipp**
**Derek Thomas****	****Jenn Leheman**
**Jeff Chang****	****	****Steve Stephens**
**Sherif Zaki****	****	****John Montenieri**
**Nick Panella****	****Stan Langevin **
**Kaci Klenk****	****	****Sarah Lasater **
**James Kile****	****	****Manuel Amador**
**Leah Colton****	****Gary Clark**
**Duane Gubler****	****John Roehrig**
**Lyle Petersen****	****Grant Campbell**
**Andrea Vicari****	****Dan O’leary**
**Sue Montgomery****	****Cathy Chow**
**Administrative Staff**