Environmental Assessment

For Field Testing 

Avian Influenza-Marek’s Disease Vaccine, 

H5 Subtype, Serotype 3, Live Marek’s Disease Vector

I. Proposed Action

APHIS is considering granting authorization to ship an unlicensed Avian InfluenzaMarek’s Disease Vaccine, H5 Subtype, Serotype 3, Live Marek’s Disease Vector, for 

field safety testing. Biomune Company, Lenexa, Kansas, has requested authorization to 

conduct field tests under conditions of husbandry that are typical of the commercial 

poultry industry in the United States.

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

USDA must ensure that veterinary biologics are pure, safe, potent, and efficacious and 

not worthless, contaminated, dangerous, or harmful. Accordingly, APHIS has conducted 

a risk analysis and has concluded that 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 APHIS risk analysis includes this 

document, but also incorporates review of all the firm’s studies and tests, the 

confirmatory testing completed by APHIS personnel on the Master Seed Virus to confirm 

purity, identity, and expression of immunogenic protein, consultation with subject matter 

experts as needed, results of literary and regulatory review, and comparison with similar 

previously tested or licensed products.

II. Background

Marek’s disease virus (MDV) can be found in chicken flocks worldwide. It is said that 

every flock, except those kept under stringent pathogen-free conditions, can be presumed 

to be infected with this herpesvirus (1). This infection may manifest itself as clinical 

disease consisting of depression, ataxia, enlarged nerves, and death, or as a subclinical 

decrease in egg production and growth rate. Early exposure to very virulent virus strains 

may be an important cause of significant losses due to Marek’s disease (MD). 

Avian influenza virus (AIV) infects domestic poultry and wild birds, and may cause mild 

respiratory disease or drops in egg production (2); these are known as low pathogenicity 

avian influenza (LPAI) viruses. Occasionally, AIV can cause severe systemic disease 

that results in high mortality; these are described as high pathogenicity viruses, or HPAI. 

The HPAI viruses can arise through mutations in LPAI viruses of H5 or H7 subtype and 

quickly cause severe and widespread disease. Quarantine and depopulation are used to 

control their spread. 

The experimental vaccine to be used in the proposed field trials is a live recombinant 

virus consisting of a turkey herpesvirus (HVT) backbone that expresses a gene of AIV. 
The product is intended for in ovo vaccination of 18 day old chick embryos or for 

subcutaneous vaccination of healthy day-of-age chicks, as an aid in the prevention of MD

and avian influenza (AI).

HVT is considered to be nonpathogenic for turkeys, chickens, and other animals. The 

HVT parent strain has been used commercially for at least two decades in chickens as a 

vaccine against the closely-related MDV without adverse effects on human health. The 

current HVT vector is the same as for two USDA-licensed vaccines: Bursal DiseaseMarek’s Disease Vaccine, Serotype 3, Live Marek’s Disease Vector and Marek’s 

Disease-Newcastle Disease Vaccine, Serotype 3, Live Marek’s Disease Vector.

The proposed field safety test will be conducted in at least two different geographical 

locations and the product will be used according to instructions on the product circular. 

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

release sites is low. The personnel to conduct the study are experienced in poultry health 

management.

III. Need for the Proposed Action

MDV is a ubiquitous and economically important cause of neoplasm in poultry. The 

advent of vaccination has dramatically reduced losses due to MDV in broiler and layer 

flocks. HVT-vectored vaccines have been shown to be effective against virulent MDV. 

For AIV, H5 and H7 subtype vaccination is restricted in the U.S. and requires specific 

approval granted by state authorities and the USDA. Commercial poultry are under 

surveillance for AIV and exposure is reportable to the USDA; most exposed flocks are 

depopulated. In several countries around the world, vaccination is used to control H5 

AIV.

This experimental vaccine represents an attempt by Biomune Company to produce an 

efficacious and safe vaccine against MDV and AIV that protects chicks early in life with 

one vaccination.

IV. Areas of Concerns

The three areas of concern to APHIS are: 1) animal safety, 2) public health, and 3) 

environmental safety. APHIS 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. 18 day-old embryos or day-of-age chicks were inoculated in ovo or subcutaneously, 

respectively, with a 10X dose and observed for 120 days. No clinical signs of MD or 
AI were seen and average body weights were not statistically different from the 

negative control group.

2. Three efficacy studies using vaccinated chicks of varying age that were subsequently 

challenged by either HPAI or the MDV GA strain indicated no adverse effects during 

the observation periods.

3. Safety of the rHVT/A1-H5 virus in chickens was tested in a backpassage study. Five 

passes in chickens followed by testing for reversion to virulence and genetic stability 

revealed no adverse reactions or clinical signs of MD or AI, out to 45 days after the 

fifth backpassage.

4. Purity testing of the master seed virus (MSV) indicated the absence of extraneous 

agents, including chicken anemia virus and reticuloendotheliosis virus.

5. The MSV was passed five times in vitro to generate MSV+5, the highest level to be 

used in production. Molecular testing by Southern blot, PCR/sequencing, Western 

blot, and black plaque assay indicated the genetic and phenotypic stability of the 

recombinant during cell culture. These same tests were used to indicate the in vivo

stability of the recombinant in the backpassage study, following five passes chickens.

6. Chickens inoculated with either a 10X dose of the vaccine or the HVT parent strain 

were evaluated for possible changes in tissue tropism. There were no apparent 

clinical signs, reactions, or lesions. The vaccine and HVT strain demonstrated the 

same tropism in the vaccinated chickens; both were isolated from purified white 

blood cells from the blood, spleen, thymus, and bursa.

7. Evaluation of vaccine safety in non-target avian species consisted of inoculation of 

turkeys, quail, pheasants, and pigeons with either the vaccine or the HVT parent, and 

a comparison made of clinical signs, gross lesions, adverse vaccine reactions, and 

virus isolation. These non-target avian species had no signs, lesions, or reactions. 

The recombinant and parent displayed a similar nonpathogenicity in these other avian 

species and a similar host range.

8. To evaluate the safety of the vaccine in non-avian species, three mammalian cell lines 

(mouse, porcine, and canine) were inoculated with either the recombinant or the HVT 

parent but, after five passes, the viruses had not induced cytopathic effect. Previous 

attempts by others to replicate the HVT parent in mammalian cell culture (3, 4) and 

mammalian species such as newborn hamsters, rhesus monkeys, and marmoset 

monkeys (5, 6) were reported to be unsuccessful, as well.

B. Public Health

The risk to public health is low. 
1. There are no indications that special safety measures should be taken to conduct this 

study. Human exposure will be limited to qualified personnel administering the 

vaccine and handling the poultry. 

2. Accidental human injection is not expected to cause adverse effects. The HVT parent 

strain from which the recombinant is derived has been used safely for decades as a 

vaccine in chickens.

3. The National Institutes of Health (NIH) Guidelines indicate that MDV is not 

associated with disease in healthy adult humans and classify MDV in Risk Group 1 

(7). HVT is not specifically mentioned in the Guidelines but is also not associated 

with human disease. 

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

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

expected.

C. Environmental Safety

The risks to the environment are low. 

1. The host range of the HVT parent is limited to certain avian species. The potential 

for escape and dispersal of this experimental vaccine from poultry houses is low. The 

transmission capabilities of the vaccine used in ovo or subcutaneously in chickens 

under normal husbandry conditions are limited.

2. In a shed/spread study conducted by the firm, chickens were vaccinated in ovo with a 

10X dose of the vaccine or an equivalent amount of the HVT parent. Upon hatching, 

for each virus group, chicks were placed with a group of non-vaccinated chicks to 

evaluate shed/spread. Virus isolation was attempted at 10, 14, and 21 days of age, 

from the blood of all treatment groups. Virus was reisolated from the vaccinated 

groups but not from any control animal, suggesting that the vaccine and the parent 

were not transmitted to the in-contact controls.

3. Regarding the potential for horizontal gene transmission, recombination between 

HVT and AIV has not been reported. Recombination between rHVT/AI-H5 and field 

MDV is considered unlikely. Vaccines commonly mix serotypes without apparent 

recombination among these or field MDV strains.

4. The firm conducted environmental stability studies on rHVT/AI-H5 and the HVT 

parent in sterile wood shavings and cotton swabs at 37±3o

C or 25±3oC. No 

significant difference between the two was noted. 

There are no apparent substantial issues with adverse environmental impacts concerning 

this vaccine.
V. Alternatives

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

preferred action alternative is not to approve the proposed field tests, 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 tests 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. Marek’s Disease. In: The Merck Veterinary Manual, Tenth Edition, 2010. C.M. 

Kahn, (Ed), Merck & Co., Inc., Whitehouse Station, N.J. Available online: 

http://www.merckvetmanual.com/mvm/index.jsp

2. Avian Influenza: Introduction. In: The Merck Veterinary Manual, Tenth Edition, 

2010. C.M. Kahn, (Ed), Merck & Co., Inc., Whitehouse Station, N.J. Available 

online: http://www.merckvetmanual.com/mvm/index.jsp

3. Muelemans, G. et al. Susceptibility of mammalian and avian cell cultures to infection 

with cell-free turkey herpes virus. J. Comp. Pathol. 83:605-608. 1973.

4. Witter, R. L. and Sharma, J. M. Transient infectivity and heterokaryon formation in 

hamster cell cultures inoculated with cell-associated stocks of Marek’s disease virus 

and herpesvirus of turkeys. J. Natl. Canc. Inst. 53:1731-1742. 1974.

5. Witter, R. L. and Schat K. A. Marek’s disease. In: Diseases of Poultry, Eleventh 

Edition, 2003. Y.M. Saif et al (Eds), Iowa State University Press, Ames, IA. pp. 407-

465.

6. Sharma, J. M. et al. Lack of pathogenicity of Marek’s disease virus and herpesvirus 

of turkeys in marmoset monkeys. J. Natl. Canc. Inst. 49:1191-1197. 1972.

7. NIH Guidelines for Research Involving Recombinant DNA Molecules. Appendix BV. NIH Recombinant DNA Advisory Committee, September 2009, p 43. Available 

online: http://oba.od.nih.gov/oba/rac/guidelines_02/NIH_Gdlines_2002prn.pdf