**UNITED STATES DEPARTMENT OF AGRICULTURE ANIMAL AND PLANT HEALTH INSPECTION SERVICE** 

**VETERINARY SERVICES THE**  **CEN TER FOR VETERINARY B**  **IOLOGICS**



# Environmental Assessment **For Field Testing of a Marek’s Disease**  **Vaccine ,**  **Serotype 1**  **, Live**  **Viru s** 

Dec em b er 201 4 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, Seroty pe 1 , Live V i r

us**Environmental Assessment f**  **or Fi**  **eld Testing of a**  **Marek’s Disease Vaccine, Serotype**  **1 , Live V**  **irus I**  . **Proposed Action** APHI S is considering granting authorization to ship an unlicensed Marek’s Disease Vaccine, Serotype 1 , Live Virus , for field safety testing in chickens . Merial , In c., Athens, Georgia, has requested authorization to conduct the field tests under conditions of husbandry

that are ty

pical of the commercial poultry industry in the United States. The vaccine is intended for use in health y day- old chickens as an aid in th e prevention of Marek’s disease caused by very virulent Marek’s disease virus . Under the provisions of the Virus - S erum - 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 h armful. Accordingl y, APHIS has conducted a risk anal ys is and has concluded th at, 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 analy sis with confidential business information re dact ed is available onli ne 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 anal ys is, o ther studies and tests conducted by the firm, the confirmatory

testing completed by

APHI S 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 APHI S ’ anal ys is 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 N otice announcing the pending trial unless new substantial issues bearing on the effects of this action are brought to our attent ion. **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 consider ed to be infected. This infection may

manifest itself as clinical disease consist ing 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 u sed 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 serotyp e 1 live virus vaccines that are licensed b y the USDA . 2Integration 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 locati ons . T he 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 ( 9 CFR), 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 po ultry health management. **III. Need for the Proposed Action**  MD V is a ubiquitous and economicall y important cause of neoplasm in poultry. The advent of vaccination has dramaticall y 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 earl y exposure to virulent virus strains. **IV. Areas of Concerns**  The three areas of concern to APHI S are: 1) animal safety, 2) public health , and 3) environmental safety . APHI S has conducted its own risk anal ys is to assess whether risks are associated with the proposal to field t est this experimental vaccine. The safety characteristics of th e vaccine h ave been thoroughly evaluated. The conclusions derived from the risk anal ys is 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. Li ke its parent, the recombinant strain is unable to transform T - ly mphocy tes in chickens.  A reversion to v irulence study was conducted in day- old chickens inoculated intraperitoneall y. At seven day s 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 necro psied. No lesions of Marek’s disease w ere 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 w ith the final product passage (X+5) of the MDV Serotype 1 . This safet y 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,kidney s, 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, turkey s, ducks, pheasant, and pigeons were inoculated with the vaccine vi rus 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 necrops y on day 46 or 47 post - inoculation in any of the birds .  Fifty day- old chickens wer e administered subcutaneously a 10x dose of the vaccine and observed through necrops y on day 120 post - inoculation , with no lesions of Marek’s disease observed. Fifty each of sham - vaccinated and sham vaccinated in - contact controls were without lesions. Ther e were no significant differences in b ody

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 seroty pe 1 ) , are not considered to be human pathogen s , and do not replicate in humans. The National Institutes of Health Guidelines for Research Involving Recombinant or Sy nthetic Nucleic A cid 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. 6 Insertion of the LTR gene has not inc reased virulence of the vaccine virus.  Q ualified 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 (~25 o C). Neither virus was recovered after the initial day of inoculation.  The firm demonstrat ed genoty pic 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 seroty pe 1 infect chickens by

the respiratory

tract, whereas the vaccine will be inoculated subcutaneousl y. This limits genetic transfer of the vaccine insert into field strains because recombination requires co - infection of the same cell and subsequent replicatio n by the two viruses in this cell . Also, theadministered 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 subcutaneousl y 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 pos t - inoculation, the groups were necropsied, weighed, and observed for gross MD lesions. The combination GA22 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 serot ypes, recombination among the MDV viruses has not been reported. This is likely

due to the lack of sufficient homology among the three seroty

pes of MDV . Even if recombination should occur between seroty pe 1 strains , the resulting virus should still be more attenuated in comparison to the parental wildty pe strain that is co - infecting the birds , because the vaccine strain is attenuate d .  The r eplication kinetics of a commercial MDV seroty pe 1 Rispens vaccine ha s been reported in the literature. 7 Ina 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 day s 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 ne gative for virus isolation, with virus recovery from feather follicles of vaccinates only on day 21 . On study

day s 21 and 49 , birds were necropsied and spleens tested for virus recovery and b y PCR with negative results . No shed/spread to in - contact nonvacc inated birds was detected , as indicated b y the lack of clinical disease and no virus isolation from spleens of in - contact nonvaccinated birds . If virus wa s potentially spread to the environment from feather follicles, it was not sufficient to infect other chickens.  MDV ty

pically proliferates in ly

mphoid cells in various tissues and organs, primarily blood and spleen. One study perform ed by the firm examined the spleen, liver, lung, kidney , and gonads from five birds on each of day s 7 and 28 post - inoculation. T he v accine virus was isolated from only one spleen of the five vaccinated birds on day

28 post - inoculation. Inanother study , vaccinated birds were tested for virus isolation from multiple tissues, spleen, liver, lungs, kidney s, feather tips, and blood on day s 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 serotyp e 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 serot ype 1 vaccine to ani mals, publi c health, and the environment. There are noapparent substantial issues with adverse environmental impacts concerning use of this

vaccine . **V. Alternatives**  Two alternatives were considered. T he onl y alternative considered other than the preferr ed 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 p otential environmental impact. Based upon the res ults of our risk analy sis and the potential applications for this vaccine in disease control, APHI S adopts the alternative that the proposed field tests be approved. **VI. Conclusion**  Based upon the risk analysis documented in this EA, APHI S 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 . 201 0 . Marek’s Disease in Poultry . In: The Merck Vet erinary 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. Fun ctional 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

Sy nthetic 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., Curri e, R.J.W., and Nair, V.K. 2005. Replication kinetics of Marek’s disease vaccine virus in feathers and ly mphoid tissues using PCR and virus isolatio n. J Gen Vir 86:2989 - 2998.