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 – Newcastle Disease Vaccine, 

Serotype 3, Live Marek’s Disease Vector

October 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 – ND Vaccine, Serotype 3, Live MD Vector
Environmental Assessment for Field Testing of a Marek’s Disease –

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

I. Proposed Action

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

Newcastle Disease Vaccine, Serotype 3, Live Marek’s Disease Vector, 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 18-day-old or 

older embryonated eggs or day-old chickens as an aid in the prevention of Marek’s 

disease and Newcastle disease.

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 in ovo or subcutaneous administration of 

this single-dose vaccine in embryonated eggs or 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 expression of 

immunogenic protein, 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 

Newcastle disease virus (NDV) can be neurotropic and/or viscerotropic, affecting the 

respiratory, nervous, and digestive systems. NDV occurs in many species of birds, is 

transmitted by inhalation and ingestion, and displays wide variation in pathogenicity
among strains. NDV strains are classified as asymptomatic, lentogenic (mildly virulent), 

mesogenic (moderately virulent), and velogenic (highly virulent). Respiratory signs are 

most common in low virulence infections. Nervous signs usually follow but may occur 

simultaneously with the respiratory signs in neurotropic velogenic disease. Respiratory

signs with depression, diarrhea, and swelling of the head and neck are common to the 

most virulent form of NDV (viscerotropic velogenic Newcastle disease or VVND), but 

nervous signs may be seen as well.2

For both MDV and NDV, vaccination is the principal method of control for the strains 

common to the United States. 

The herpesvirus of turkeys (HVT), an avirulent strain related to MDV, has been widely 

used commercially as a heterotypic vaccine against MDV.1 HVT, classified as MDV 

serotype 3, is not pathogenic in chickens and has minimal risk of environmental 

dispersal, less so than the MDV strains of serotypes 1 and 2. The experimental vaccine to 

be used in the proposed field trials is a live virus consisting of the HVT backbone 

expressing an immunogenic protein from NDV. Several recombinant HVT vaccines have 

been used in recent years featuring the insertion of genes from other poultry viruses 

including NDV, infectious bursal disease virus, and infectious laryngotracheitis virus.

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.3 

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. Protection 

against NDV generally requires repeated vaccination throughout life, the frequency

depending on the level of exposure. This recombinant vaccine represents an attempt by 

Merial, Inc., to produce an efficacious and safe vaccine against MDV and NDV 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 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 HVT backbone of the vaccine is well-characterized and has been safely used 

previously in numerous licensed vaccines. The NDV gene insert is the same as 

that expressed without issue in a licensed recombinant NDV-fowl pox vectored 

vaccine, licensed in 1995.4The NDV-HVT vectored vaccine has similar 

attenuated characteristics as those of the parental HVT, as demonstrated in 

shed/spread and tissue tropism studies conducted by the firm.

 In a reversion to virulence study in chickens conducted according to Veterinary

Services Memorandum 800.201, following five passes in one-day-old chickens, 

no lesions were observed in any of the birds at seven weeks post-inoculation. 

Virus was recovered from all passages, but no virus was recovered from contact 

controls. There was no loss of expression of the NDV gene after the five passes.

 Non-target animals: A safety study in mice indicated no virus recovery after 

subcutaneous injection and epidermal scarification of 15 mice. The safety study in 

non-target avian species consisted of groups of 40 quail, turkeys, ducks, 

pheasants, and pigeons that received 10x doses of the vaccine either in ovo or 

subcutaneously. The groups were observed for 120 days until necropsy. No MDV 

clinical signs or lesions were seen.

 Target animals: A safety study was conducted in chickens per 9CFR, Part

113.330, using a 10x dose for the subcutaneous route in day-old chicks and the in 

ovo route for 18-day-old embryos. The chickens were observed for 120 days, then 

weighed, euthanized, and necropsied. No MDV lesions and no weight loss, when 

compared to sham-inoculated controls, were detected.

 Chicks vaccinated by the subcutaneous route and chicks hatched from embryos 

vaccinated in ovo were challenged at 28 days with NDV or MDV and were 

protected against disease.

B. Public Health

The risk to public health is likewise low. 

 MDV strains, including the HVT strain (MDV serotype 3), 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 and the lowest containment level (RG1) for human agents is 

recommended when used in laboratory work.5Insertion of the NDV 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. 

 Millions of doses of various live HVT-vectored vaccines have previously been 

administered to chickens. No significant adverse events have been reported for 

those vaccines.

C. Environmental Safety

The risks to the environment are low. 

 HVT replicates only in avian-origin cells and is cell-associated.6 A study

performed by the firm indicated that the NDV-HVT vaccine virus did not spread 

to HVT infected or naïve turkey poults, or to HVT infected or naïve chicks when 

administered subcutaneously to day-old SPF chicks.

 In a comparison study conducted by the firm, the parent and vaccine strains were 

inoculated into birds and on days 7 and 28 post-inoculation, various organs were 

isolated and evaluated for the presence of virus. Results of the study indicated the 

tissue tropism was the same for both parental HVT and vaccine strain NDV-HVT.

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

HVT and the NDV-HVT on sterile shavings at room temperature (~25oC). 

Neither virus was recovered after the initial day of inoculation.

 The HVT backbone virus, the NDV gene and its site of insertion, the promoter, 

and the polyadenylation signal sequence have been used in previously licensed 

products. There have been no significant adverse effects to the environment as a 

result of their usage.

 No bacterial DNA sequences, including no antibiotic resistance genes, have been 

inserted in the vaccine virus during its construction.

 Confirmatory testing was conducted at CVB to determine purity, sterility, 

identity, expression of immunogenic NDV protein, and stability of the vaccine 

virus, with satisfactory results.

 The NDV gene did not react in the National Veterinary Services Laboratories 

surveillance testing for detection and differentiation of velogenic NDV and will 

not falsely test positive as exotic NDV.

 Recombination or horizontal gene transfer between HVT (serotype 3) and other 

MDV strains (serotypes 1 and 2) has not been observed, perhaps due to the lower 

homology among the serotypes. The firm conducted a study to determine if coinoculation of the NDV- HVT vaccine with virulent MDV or with serotypes 1 and 

2 would perhaps result in recombination and a change in virulence. No increase in 

virulence was observed and no indication of recombination was detected.

 

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

NDV-HVT 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. Miller, P.J. 2010. Newcastle 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 January 2014: 

http://www.merckmanuals.com/vet/poultry/newcastle_disease_and_other_paramyxovirus

_infections/newcastle_disease_in_poultry.html

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

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

4. 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

5. 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

6. 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.