UNITED STATES DEPARTMENT OF AGRICULTURE

ANIMAL AND PLANT HEALTH INSPECTION SERVICE

VETERINARY SERVICES

THE CENTER FOR VETERINARY BIOLOGICS

Environmental Assessment

For Field Testing of a

Yersinia Pestis Vaccine, Live Raccoon Poxvirus Vector

January 2013

Prepared by:

The Center for Veterinary Biologics

National Center for Animal Health

1920 Dayton Avenue

Ames, IA 50010

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Environmental Assessment 

Field Trial of an Experimental Yersinia Pestis Vaccine, Live Raccoon Poxvirus Vector
USDA-APHIS-VS-Center for Veterinary Biologics

Environmental Assessment

For Field Testing 

Yersinia Pestis Vaccine, Live Raccoon Poxvirus Vector

I. Proposed Action

The United States Department of Agriculture (USDA)-Animal and Plant Health 

Inspection Service (APHIS)-Veterinary Services (VS)-Center for Veterinary Biologics 

(CVB) is considering granting authorization to ship an unlicensed Yersinia Pestis 

Vaccine, Live Raccoon Poxvirus Vector, for field testing in wildlife. The U.S. 

Geological Survey (USGS)-National Wildlife Health Center (NWHC), has requested 

authorization to conduct a field safety trial in the United States (US) under sylvatic 

conditions that are typical of the species of interest, i.e. free-ranging prairie dogs.

Under the provisions of the Virus-Serum-Toxin Act of 1913, as amended in 1985, the 

USDA-APHIS-VS-CVB must ensure that veterinary biologics are pure, safe, potent, and 

efficacious and not worthless, contaminated, dangerous, or harmful. Accordingly, the 

CVB has conducted a risk analysis and has concluded that, regarding use of this vaccine,

the safety risks to animals, public health, and the environment are low. A copy of the 

firm’s risk analysis with confidential business information removed is available upon 

request. The CVB risk analysis includes that document, but also incorporates review of 

all the firm’s studies and tests related to the vaccine, the confirmatory testing completed 

by CVB personnel on the Master Seed Virus to confirm purity, identity, expression, and 

stability, consultation with subject matter experts, results of literary and regulatory

review, and evaluation in light of similar previously tested or licensed products.

Based on the CVB risk analysis and other relevant data, we have reached a preliminary

determination that field testing this veterinary vaccine will not have a significant impact 

on the quality of the human environment, and that an environmental impact statement 

need not be prepared. We intend to authorize shipment of this vaccine for field testing 

following the close of the comment period of the Federal Register Notice announcing the 

pending trial unless new substantial issues bearing on the effects of this action are 

brought to our attention. 

II. Background

The USGS has developed a live recombinant Yersinia pestis vaccine for oral use in wild, 

free-ranging prairie dogs, as an aid in the prevention of plague caused by Y. pestis

infection. The recombinant raccoon pox virus (RCN strain) vector expresses certain 

coding sequences of Y. pestis. The vaccine will be incorporated into a bait matrix and 

distributed by plane or vehicle to selected test sites to combat plague in prairie dogs. 

Prairie dogs are very susceptible to plague. Prairie dog colonies can experience mortality 

rates of 90 per cent or more due to plague, with large overall reductions in population and 
localized extinctions. Control of plague is necessary for prairie dog conservation as well 

as for ferret recovery programs. Prairie dogs are the primary food source of the highly 

endangered black-footed ferret, an animal also susceptible to plague. In order to manage 

the disease in ferrets, it will be necessary to control the disease in prairie dogs.

“RCN-F1-V307”, the experimental vaccine to be used in the proposed field trial, consists 

of the RCN vector derived from the attenuated Herman strain, the full coding sequence of 

the F1 capsular antigen from Y. pestis, and a truncated form of the low calcium response 

V (lcrV) gene from Y. pestis. The Herman strain has been used in several experimental 

vaccines in numerous species with no apparent harmful effects. The gene inserts were 

placed in the RCN genome at the site of the non-essential thymidine kinase (TK) gene.

The RCN strain is a member of the Poxviridae family containing a linear and nearly 200 

kb double-stranded DNA genome with a hairpin loop at each end. Like other poxviruses, 

RCN replicates in the cytoplasm and uses its own transcription systems. Poxviruses have 

been used many times as live vectors to express foreign genes for vaccine development. 

Examples of USDA-licensed poxvirus vaccine vectors include vaccinia virus, fowlpox 

virus, and canarypox virus. RCN has been used previously as a live vector to express 

foreign genes in a recombinant vaccine, although those constructs have not received

USDA licensure. For this current vaccine construct, no genetic manipulation was made 

to the wild type RCN parent strain prior to insertion of the Y. pestis genes. RCN is 

considered to be nonpathogenic for raccoons. It was first isolated from the upper 

respiratory tract of a raccoon with no obvious clinical symptoms by Y.F. Herman in 

Aberdeen, Maryland, in 1961-1962. The F1 gene and truncated lcrV gene (V307) were 

inserted into the RCN TK locus, further attenuating the wild type parent strain. The CVB 

conducted confirmatory testing on the Master Seed for this vaccine with satisfactory

results for identity, purity, extraneous agents, expression, stability, and genetic analysis. 

The proposed field studies to assess the safety of the sylvatic plague vaccine in prairie 

dogs and non-target animals will potentially be conducted at numerous sites in the US,

under State and Federal approval and oversight, in accordance with standard 

requirements published in Title 9, Code of Federal Regulations (CFR), Part 103.3, 

concerning shipment of experimental biological products for use in animals.

1

The 

potential for escape and dispersal of the experimental vaccine from the proposed release 

sites is low. The USGS-NWHC personnel to conduct the study are experienced in 

wildlife health management.

III. Need for the Proposed Action

The prairie dog’s range has been reduced by habitat loss and poisoning to 1 to 2 percent 

of its former range, leaving colonies smaller and less centralized, thus more susceptible to 

elimination by plague. The prairie dog and its burrows are highly significant in the 

grassland ecosystem, being a major source of food and shelter for other species of 

wildlife.
Inactivated vaccines do not generate an adequate immune response when given orally. 

Live baited vaccines may have pathogenicity in some species or fail to provide sufficient 

protection to others. Ferrets were protected by inoculation of the RCN-vectored Y. pestis 

vaccine both in the laboratory and in the field. But, inoculation of free-ranging animals is 

not practical for field use. This experimental vaccine represents an attempt by the USGS

to produce an efficacious and safe baited vaccine against plague that protects prairie dogs

using oral vaccination, and in so doing help preserve the endangered black-footed ferret, 

as well. 

IV. Areas of Concerns

The three areas of concern to the CVB regarding use of this live recombinant vaccine are: 

1) animal safety, 2) public health, and 3) environmental safety. The CVB has conducted 

its own risk analysis to assess whether risks are associated with the proposal to field test 

this experimental vaccine. The safety characteristics of this vaccine have been 

thoroughly evaluated. The conclusions derived from the risk analysis for each of the 

areas of concern are summarized below.

A. Animal Safety

The risk to animals is low. 

1. In three experimental studies using oral RCN-vectored plague vaccines in prairie 

dogs, the target species, no adverse effects were observed. The data were published 

in peer-reviewed journals. 

2. In two field safety studies of the oral baited RCN-F1-V307 at two remote, enclosed 

sites, a high percentage of sampled prairie dogs ingested baits but no morbidity or 

mortality was detected. Biomarker analysis of hair and whisker samples indicated 

that certain rodent species consumed the baits but without any associated adverse 

effects.

3. The RCN virus has been reported to be avirulent for various domestic and wild 

animals. Safety studies have been conducted in raccoons, dogs, bobcats, cotton rats, 

striped skunks, gray foxes, rabbits, domestic cats, American mongooses, sheep, 

swine, patas (African) and cynomolgus (Asian) monkeys.

2, 3

 However, rabbits 

inoculated intradermally with RCN and a recombinant RCN developed skin lesions 

but no edema or hemorrhage. 

4. One report indicated poor antibody response to a recombinant RCN expressing a

rabies virus glycoprotein (rRCNV-G), when given orally to sheep. This implied that 

if ingested by sheep the vaccine would not be harmful. In addition, transmission of 

the virus between vaccinated and in-contact controls was not observed or detected by 

serology.

4

Intradermal inoculation of sheep did result in mild erythematous skin 

lesions with edema, but not systemic disease.

5. In studies comparing the pathogenicity of RCN, a rRCNV-G construct, the 

Copenhagen strain of vaccinia virus (vv), and a vv-rabies virus glycoprotein (vv-G) 

construct, the rRCNV-G construct was 100- fold less pathogenic than RCN, 100- to 

1,000-fold less pathogenic than vv-G, and 1,000- to 10,000-fold less pathogenic than 
vv. This supports the claim of viral attenuation by foreign gene insertion and the 

relative avirulence of RCN when compared to the Copenhagen strain of vaccinia. 

The results were determined using rabbit skin histopathology, suckling mouse 

lethality, and oral infection of monkeys and mice.5

6. One case was reported of natural infection with RCN in a domestic Canadian cat.6

The cat presented with an ulcerative lesion on the forepaw that was determined to be 

caused by a raccoon poxvirus infection using electron microscopy, PCR, and 

sequencing. The cat was not systemically affected and recovered fully from the 

infection.

7. USGS studies indicate that the vaccine is avirulent in various species of prairie dogs 

and in all non-target animals tested thus far. Infection via the oral route in prairie 

dogs and deer mice results in localized infection that does not become systemic.

8. The anticipated field dose is 5 x 107pfu. Vaccine doses as high as 108pfu have been 

injected or fed to animals then comparing results with wild type and TK-deleted RCN 

virus, with no adverse effects in mice or prairie dogs.7, 8

9. Backpassage studies in prairie dogs indicated no reversion to virulence of the vaccine 

with passage.

B. Public Health

The risk to public health is low. 

1. Special safety measures will be taken to conduct the field trials including isolated or 

closed test sites and project signage providing safety information placed along trails 

and roads near the sites. 

2. Human exposure will be limited to the qualified personnel handling the vaccine 

during distribution, and they will be using personal protective equipment (latex 

gloves). 

3. There is one reported case of human exposure, caused by a needle prick to the finger 

of a scientist administering a recombinant raccoon poxvirus expressing Y. pestis F1 

capsular antigen during an experiment. A very small blister developed within 9 days 

and was fully healed within four weeks.9 

4. Although the safety of this experimental vaccine in humans has not been fully

evaluated and is therefore unknown, no significant safety hazards to the public health 

are expected following oral baited vaccination of the intended target species. It is 

reported that the baits disappear rapidly when deposited in a prairie dog colony, 

within 3-4 days. There are few baits available for people to encounter and the 

vaccine has low virulence in humans; thus, the risk to public health is expected to be 

low. Accidental human exposure is not expected to cause adverse effects.

C. Environmental Safety

The risk to the environment is low. 

1. Bait ingredients are food grade, FDA-approved when used within the regulatory

limits, and flavored with peanut butter. USGS has found that the baits are not 
attractive to wild pronghorn, mule deer, elk, fox, coyote, badger, skunk, and not 

highly palatable for captive hoofed stock (cattle, horses), and domestic dogs. 

2. The potential for dispersal of this recombinant vaccine is low. Shed/spread studies of 

the oral vaccine in prairie dogs indicated most (9/10) shed virus orally, but fecal 

shedding was not detected. No horizontal transmission of the virus from co-housed 

vaccinated prairie dogs to in-contact unvaccinated prairie dogs was detected.

3. The survivability of the recombinant in the field has not yet been tested. However,

USGS reports that the experimental baited vaccine was tested for virus stability at 

various temperatures in the laboratory, with no loss of RCN titer for at least 28 days 

at 4oC (39oF), 14 days at 20oC (68oF), and 7 days at 28oC (82oF). For proposed field 

trial distribution during the summer and fall months, what remains of the vaccine 

after day three will be largely degraded within seven days. 

4. There are no expected adverse ecological effects associated with the proposal to 

conduct field testing with this experimental vaccine, which is derived from a strain 

that already exists in nature, in raccoon populations that are not clinically affected by 

its presence. One earlier report from Maryland, in the area where RCN was isolated, 

indicated that 23.8% (22/92) of raccoons had antibodies to RCN, suggesting the virus 

was present in nature in that region.10 The vaccine candidate is more attenuated than 

the parental strain, suggesting its risk to the environment is minimal.

5. The potential for horizontal gene transfer is low and the risk of recombination 

between the RCN-F1-V307 construct and wild type (wt) poxviruses is low.

There are no apparent substantial issues with adverse environmental impacts concerning 

this agent. The two completed penned field trials have reinforced the estimation of its 

safety. As there are no apparent significant adverse effects on animal, human, or 

environmental safety associated with the use of the RCN-F1-V307 vaccine, this supports 

a finding of no significant impact for conducting the proposed field trials.

V. Alternatives

Two alternatives were considered. The only alternative considered other than the 

preferred action alternative is not to approve the proposed field test, the “no action” 

alternative. We have considered the applicants’ goals in light of the agency’s public 

interest and responsibilities and any potential environmental impact. Based upon the 

results of our risk analysis and the potential applications for this vaccine in disease 

control, APHIS adopts the alternative that the proposed field test be approved.

VI. Conclusion

Based upon the risk analysis documented in this EA, APHIS has determined that 

implementation of the proposal would not significantly affect the quality of the human 

environment and that the preparation of an Environmental Impact Statement is not 

required (Finding of No Significant Impact).
References

1. Code of Federal Regulations, Washington, DC. Available online: 

http://www.gpoaccess.gov/cfr/index.html

2. Esposito JJ, Chandler FW, and Baer GM. Oral immunization of animals with raccoon 

poxvirus expressing rabies virus glycoprotein. In: Vaccines 89. (ed. R Lerner, H 

Ginsberg, RM Chanock, F Brown) Cold Spring Harbor Laboratory Press, NY, p 403-

408, 1989.

3. Esposito JJ, Sumner JW, Brown DR, Ebert JW, Shaddock JH, He BX, Dobbins JG, 

and Fekadu M. Raccoon poxvirus rabies-glycoprotein recombinant oral vaccine for 

wildlife: further efficacy and safety studies and serosurvey for raccoon poxvirus. In: 

Vaccines 92. (ed. F Brown, RM Chanock, HS Ginsberg, RA Lerner) Cold Spring 

Harbour Laboratory Press, NY, p 321-329, 1992.

4. DeMartini JC, Bickle HM, He BX, and Esposito JJ. Raccoon poxvirus rabies virus 

glycoprotein recombinant vaccine in sheep. Arch Virol 133:211-222, 1993.

5. Esposito JJ. Live poxvirus-vectored vaccines in wildlife immunization programmes: 

the rabies paradigm. Res Virol 140:480-482. 1989.

6. Yager JA, Hutchison L, Barrett JW. Raccoonpox in a Canadian cat. Vet Dermatol 

17:443-8, 2006. 

7. Rocke TE, Iams KP, Dawe S, Smith SR, Williamson JL, Heisey DM, and Osorio JE. 

Further development of raccoon poxvirus-vectored vaccines against plague (Yersinia 

pestis): Vaccine 28:338-344, 2010.

8. Rocke TE, Pussini N, Smith SR, Williamson J, Powell B, and Osorio JE. 

Consumption of baits containing raccoon pox-based plague vaccines protects blacktailed prairie dogs (Cynomys ludovicianus). Vector-borne & Zoonotic Dis 10:53-58, 

2010.

9. Rocke TE, Dein FJ, Fuchsberger M, Fox BC, Stinchcomb DT, and Osorio JE. Limited 

infection upon human exposure to a recombinant raccoon pox vaccine vector. Vaccine 

22:2757-2760, 2004. 

10.Alexander AD, Flyger V, Herman YF, McConnell SJ, Rothstein N, and Yager RH. 

Survey of wild mammals in a Chesapeake Bay area for selected zoonoses. Jour 

Wildlife Dis 8:119-126, 1972.