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

Marek’s Disease Vaccine, Serotype 1, Live Virus

December 2014

Prepared by:

United States Department of Agriculture

Animal and Plant Health Inspection Service

Veterinary Services

The Center for Veterinary Biologics

_________________________________________________________________________________________________

Environmental Assessment

Field Trial of an Experimental MD Vaccine, Serotype 1, Live Virus
Environmental Assessment for Field Testing of a 

Marek’s Disease Vaccine, Serotype 1, Live Virus

I. Proposed Action

APHIS is considering granting authorization to ship an unlicensed Marek’s Disease 

Vaccine, Serotype 1, Live Virus, for field safety testing in chickens. Merial, Inc., Athens, 

Georgia, has requested authorization to conduct the field tests under conditions of 

husbandry that are typical of the commercial poultry industry in the United States. The 

vaccine is intended for use in healthy day-old chickens as an aid in the prevention of 

Marek’s disease caused by very virulent Marek’s disease virus.

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, regarding subcutaneous administration of this

single-dose vaccine in chickens, the safety risks to animals, public health, and the 

environment are low. A copy of the firm’s risk analysis with confidential business 

information redacted is available online in the Federal Register docket and by request, as 

indicated in the Federal Register Notice announcing the proposed field testing. APHIS 

considers the firm’s risk analysis, other studies and tests conducted by the firm, the 

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

vaccine’s purity, sterility, identity, and stability, and the results of literary and regulatory

review regarding this and similar previously tested or licensed products. 

Based on APHIS’ analysis of the relevant data, a preliminary determination has been 

reached 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

Marek’s disease virus (MDV) is found in chicken flocks worldwide. Virtually all flocks, 

except those kept under pathogen-free conditions, can be considered to be infected. 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.1 

For MDV, vaccination is the principal method of control for the strains common to the 

United States. The experimental vaccine to be used in the proposed field trials is a live 

virus consisting of an attenuated parental RN strain (MDV serotype 1) and an inserted 

long terminal repeat (LTR) of the reticuloendotheliosis virus (REV). Several biologics 

firms have MDV serotype 1 live virus vaccines that are licensed by the USDA.

2
Integration of an REV LTR into the genome of an MDV strain has been previously

reported to increase attenuation.3

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

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

in accordance with standard requirements published in Title 9, Code of Federal 

Regulations (9CFR), Part 103.3, concerning shipment of experimental biological 

products for use in animals.4

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 commercial 

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. However, disease can become a serious problem in individual flocks or certain 

geographic areas, most likely due to early exposure to virulent virus strains.

 

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 the 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. 

 The inserted sequence in the attenuated parental RN vaccine strain is not 

expressed and contains no known virulence genes. Like its parent, the 

recombinant strain is unable to transform T-lymphocytes in chickens.

 A reversion to virulence study was conducted in day-old chickens inoculated 

intraperitoneally. At seven days post-inoculation, blood was drawn and used to 

inoculate another group of day-old chickens. At each of four additional passages, 

naïve contact controls were placed with those receiving inoculations to monitor 

viral spread. At seven weeks post-inoculation, birds were necropsied. No lesions 

of Marek’s disease were observed and no reversion to virulence detected. 

Although the vaccine virus was recovered from four of the five backpasses, no 

virus was recovered from the contact controls. 

 Fifteen mice received a subcutaneous injection and epidermal scarification with 

the final product passage (X+5) of the MDV Serotype 1. This safety evaluation 

included removal on days 3, 7, and 14, of numerous tissues from euthanized mice

in order to attempt virus recovery. Trachea, brain, spleen, heart, liver, lungs, 
kidneys, and skin at the site of inoculation were all negative for virus growth on 

chicken embryo fibroblast monolayers.

 A safety study in non-target avian species was conducted. Groups of quail, 

turkeys, ducks, pheasant, and pigeons were inoculated with the vaccine virus and 

compared with corresponding naïve birds of each species that received a placebo. 

No seroconversion or gross lesions of Marek’s disease were detected following 

necropsy on day 46 or 47 post-inoculation in any of the birds.

 Fifty day-old chickens were administered subcutaneously a 10x dose of the 

vaccine and observed through necropsy on day 120 post-inoculation, with no 

lesions of Marek’s disease observed. Fifty each of sham-vaccinated and shamvaccinated in-contact controls were without lesions. There were no significant 

differences in body weights among the vaccinated and negative control groups, 

indicating the safety of the vaccine virus. 

B. Public Health

The risk to public health is likewise low. 

 MDV replicates only in avian-origin cells and is cell-associated.5

 MDV strains, including the RN strain (MDV serotype 1), are not considered to be 

human pathogens, and do not replicate in humans. The National Institutes of 

Health Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid 

Molecules (NIH Guidelines) indicate that MDV is not associated with disease in 

healthy adult humans; the lowest containment level (Risk Group 1) for human 

agents is recommended when MDV is used in laboratory work.6Insertion of the 

LTR gene has not increased virulence of the vaccine virus.

 Qualified personnel administering the vaccine could potentially be exposed to it

through accidental injection, with the possibility of a local reaction. Although the 

safety of this specific vaccine has not been evaluated in humans and is therefore 

unknown, accidental human injection is not expected to cause significant adverse 

effects. 

C. Environmental Safety

The risks to the environment are low. 

 A study was performed by the firm to compare the survivability of the 

experimental vaccine and a licensed Rispens vaccine on sterile shavings at room 

temperature (~25oC). Neither virus was recovered after the initial day of 

inoculation.

 The firm demonstrated genotypic stability using Southern blot and sequencing of 

the master seed virus (MSV), the fifth cell culture passage from the MSV (X+5), 

and the fifth back passage in chickens with identical results for the three samples.

 Field isolates of MDV serotype 1 infect chickens by the respiratory tract, whereas 

the vaccine will be inoculated subcutaneously. This limits genetic transfer of the 

vaccine insert into field strains because recombination requires co-infection of the 

same cell and subsequent replication by the two viruses in this cell. Also, the 
administered vaccine induces an immune response that should limit co-infection 

of chickens with a field strain.

 The firm conducted a study to evaluate potential recombination in groups of 50 

day-old chicks each inoculated subcutaneously with the experimental serotype 1 

vaccine alone, in combination with GA 22 (another serotype 1 strain), or in 

combination with HVT (a serotype 3 strain), then compared to the groups 

vaccinated with GA22 or HVT alone. On day 35 post-inoculation, the groups 

were necropsied, weighed, and observed for gross MD lesions. The combinationGA22 group had no increase in virulence over the GA22 group. There were no 

lesions in the two HVT-containing groups. There was no significant difference in 

body weight in the group comparisons.

 Although there is widespread use of combination vaccines containing multiple 

MDV serotypes, recombination among the MDV viruses has not been reported.

This is likely due to the lack of sufficient homology among the three serotypes of 

MDV. Even if recombination should occur between serotype 1 strains, the 

resulting virus should still be more attenuated in comparison to the parental 

wildtype strain that is co-infecting the birds, because the vaccine strain is 

attenuated.

 The replication kinetics of a commercial MDV serotype 1 Rispens vaccine has

been reported in the literature.7 In a dissemination study conducted by the firm, 

chickens vaccinated subcutaneously with the experimental MDV vaccine showed 

less replication and viral recovery in tissues and in feather follicles. At days 14, 

21, and 28, tracheal and cloacal swabs were taken from all vaccinates, and 

primary feather follicles were collected from two vaccinates in each group. All 

tracheal and cloacal swabs were negative for virus isolation, with virus recovery

from feather follicles of vaccinates only on day 21. On study days 21 and 49, 

birds were necropsied and spleens tested for virus recovery and by PCR with 

negative results. No shed/spread to in-contact nonvaccinated birds was detected, 

as indicated by the lack of clinical disease and no virus isolation from spleens of 

in-contact nonvaccinated birds. If virus was potentially spread to the environment 

from feather follicles, it was not sufficient to infect other chickens.

 MDV typically proliferates in lymphoid cells in various tissues and organs, 

primarily blood and spleen. One study performed by the firm examined the 

spleen, liver, lung, kidney, and gonads from five birds on each of days 7 and 28 

post-inoculation. The vaccine virus was isolated from only one spleen of the five 

vaccinated birds on day 28 post-inoculation. In another study, vaccinated birds 

were tested for virus isolation from multiple tissues, spleen, liver, lungs, kidneys, 

feather tips, and blood on days 7, 14, and 49. More tissues showed virus recovery

at day 14, but none was detected on day 49.

 A PCR test was developed that can differentiate the recombinant vaccine from 

other MDV serotype 1 strains including the parental vaccine virus. Thus, should 

an issue arise in the field, surveillance testing can potentially distinguish the 

vaccine virus from prevalent strains.

The information above provides support to the claims made regarding the safety of the 

MDV serotype 1 vaccine to animals, public health, and the environment. There are no 
apparent substantial issues with adverse environmental impacts concerning use of 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. Dunn, J. 2010. Marek’s Disease in Poultry. In: The Merck Veterinary Manual, Tenth 

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

last full review/revision July 2013: 

http://www.merckmanuals.com/vet/poultry/neoplasms/mareks_disease_in_poultry.html#

v3342416

2. USDA Veterinary Biological Products, Licensees and Permittees. 2014, p 41. 

Available online: 

http://www.aphis.usda.gov/animal_health/vet_biologics/publications/CurrentProdCodeB

ook.pdf

3. Sun, A., Xu, X., Petherbridge, L., Zhao, Y., Nair, V., Cui, Z. 2010. Functional 

evaluation of the role of reticuloendotheliosis virus long terminal repeat (LTR) integrated 

into the genome of a field strain of Marek’s disease virus. Virol 397:270-276.

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

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

5. Purchase, H.G., Burmester, B.R., and Cunningham, C.H. 1971. Responses of cell 

cultures from various avian species to Marek’s disease virus and turkey Herpesvirus. Am 

J Vet Res 32:1811-1823.
6. The National Institutes of Health Guidelines for Research Involving Recombinant or 

Synthetic Nucleic Acid Molecules. March 2013, p 45. Available online:

http://osp.od.nih.gov/sites/default/files/NIH_Guidelines_0.pdf

7. Baigent, S.J., Smith, L.P., Currie, R.J.W., and Nair, V.K. 2005. Replication kinetics of 

Marek’s disease vaccine virus in feathers and lymphoid tissues using PCR and virus 

isolation. J Gen Vir 86:2989-2998.