**Environmental Assessment**

**for Field Testing**

**Marek's Disease-Newcastle Disease Vaccine, Serotypes 2&3, Live Virus**

**Live Marek's Disease Vector**

**I**. **Proposed Action**

APHIS is considering granting authorization to ship an unlicensed
Marek's Disease-Newcastle Disease Vaccine, Serotypes 2&3, Live Virus,
Live Marek's Disease Vector, for field safety testing. Intervet Inc.,
Millsboro, Delaware, 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 risk analysis with confidential
business information removed is available upon request.

**II. Background**

Marek's disease virus (MDV) is isolated from chicken flocks worldwide.
Every flock, except those kept under stringent pathogen-free conditions,
can be presumed 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.
Newcastle disease virus (NDV) can be either neurotropic or
viscerotropic. The neurotropic viruses cause respiratory and nervous
signs. The viscerotropic viruses, which are most common, result in
respiratory signs, diarrhea, and swelling of the head and neck. For both
of these diseases, 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.
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. In the case of this vaccine, it also serves as a live
viral vector for a gene from a lentogenic strain of NDV. 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 pathogenic), mesogenic
(moderately pathogenic), and velogenic (highly pathogenic). This vaccine
also contains a strain of MDV serotype 2, thus providing broader
protection against MDV.

The resulting live recombinant vaccine is for use in chicks as an aid in
the prevention of disease caused by NDV and MDV. The proposed field
safety test will be conducted in at least three different geographical
locations 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. However, disease can become a
serious problem in individual flocks or certain geographic areas, most
likely due to early exposure to very virulent virus strains. Protection
against NDV generally requires repeated vaccination throughout life, the
frequency depending on the level of exposure. This experimental
recombinant vaccine represents an attempt by Intervet 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 a 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. The recombinant viral fraction was injected
into 18-day-old embryonated SPF eggs at more than ten times the vaccine
dose, without untoward effects in hatchability, survival, or lesions in
the chickens. None of the unvaccinated hatchmates serving as contact
controls developed lesions. There was no significant difference between
average weight of vaccinated and sham-inoculated control chickens.

Virus isolations were performed on tissues harvested from chickens
vaccinated with a high dose of the recombinant or the HVT parent strain.
There was no difference in tissue tropism or dissemination in the target
species found between the parent and recombinant.

The recombinant was shown to be genetically and phenotypically stable
through five successive backpassages in chickens and through the highest
passage level to be used in production. This was demonstrated by the
lack of reversion to virulence, by the lack of genomic rearrangement of
the insert, and by the continued expression of the inserted NDV gene.

The vaccine was safely administered *in ovo* to 18-day-old embryonated
SPF eggs and provided protection to the hatched vaccinated chickens when
challenged with virulent MDV and NDV.

Non-target animal safety was shown by inoculation with over ten times
the recommended dose of the recombinant viral fraction in turkeys,
ducks, and pigeons. The recombinant replicated in turkeys but not ducks
and pigeons, and there were no adverse effects. Also, two rabbits were
inoculated ocularly with one dose of the recombinant without the
development of clinical signs, conjunctivitis, or viremia.

**B. Public Health**

The risk to public health is low. There are no indications that special
safety measures should be taken to conduct this study. Human exposure
will be limited to the qualified personnel administering the vaccine and
the people handling the poultry.

Both HVT and MDV are classified as Biosafety Level (BL) 1 agents by CDC
and NIH guidelines, as they do not pose a health risk to humans. NDV is
a BL2 agent and can produce a transitory conjunctivitis in humans.
However, only one gene from NDV will be present in this recombinant
vaccine, so the human health risk is negligible.

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. 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. The potential for escape and
dispersal of this experimental vaccine is low. The shed/spread
capabilities of the vaccine used in chickens are limited even under
direct contact exposure. The vaccine was determined to be safe in
chickens, the target species, and in turkeys, ducks, pigeons, and
rabbits, which are non-target animals. There are no expected adverse
ecological effects associated with the proposal to conduct field testing
with this experimental vaccine, which is derived from a commercial
vaccine strain.

Recombination in tissue culture between MDV serotype 1 and MDV serotype
2 was reported once in the literature. A cell line containing latent MDV
serotype 1 was infected with a serotype 2 and a recombinant virus was
produced. However, this event could not be reproduced, suggesting its
rarity. Recombination between the recombinant HVT and another MDV
serotype has low potential to occur because the HVT backbone of the
recombinant has not been modified by deletion. It should be no more
likely to recombine with wild-type HVT or MDV than a conventional live
HVT vaccine. The cloning site for the inserted NDV gene has been used
safely in the past to encode immunogens and immunomodulators.

There are no apparent substantial issues with adverse environmental
impacts concerning this agent.

**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 Environmental
Assessment, 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).