**Environmental Assessment**

**for Field Testing**

**Mannheimia Haemolytica - Pasteurella Multocida Vaccine, Avirulent Live
Culture**

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

APHIS is considering granting authorization to ship an unlicensed
Mannheimia Haemolytica -- Pasteurella Multocida Vaccine, Avirulent Live
Culture, for field safety testing. Schering-Plough Animal Health
Corporation, Elkhorn, Nebraska, has requested authorization to conduct
field tests under conditions of husbandry that are typical of the
commercial cattle 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**

Bovine respiratory disease (BRD) in the form of 'shipping fever' (SF)
pneumonia is a significant cause of morbidity (approaching 35%) and
mortality (5-10%) in North American cattle feedlots, resulting in major
economic loss to the industry.^1^ Although a number of factors and
multiple pathogens are involved in the disease complex, *M. haemolytica*
is the primary microorganism isolated from SF calves with pneumonia. *P.
multocida* is less frequently isolated but is an important and common
contributor to disease.

To prevent SF, it is important to reduce stressors that contribute to
its development, such as prolonged transport, poor nutrition, excessive
heat, and crowding. Vaccination against the agents involved is
recommended at 2-3 weeks before transport and can be repeated on arrival
at the feedlot. Vaccines for the SF bacterial pathogens generally
include bacterins and subunits (toxoids). A live vaccine can potentially
improve protection against disease by stimulating both cell-mediated and
humoral immune response in vaccinated animals.

The vaccine under consideration consists of two genetically modified
live bacterial strains. The *M. haemolytica* strain is characterized as
biotype A, serotype 1 (A1). It was derived from a field isolate that was
attenuated by gene deletion. The *P. multocida* strain is biotype A,
serotype 3 (A3) and was also derived from a field isolate and attenuated
by deletion.

The resulting avirulent live vaccine is for use in healthy cattle as an
aid in the prevention and/or reduction of pneumonic lesions associated
with bovine pneumonic pasteurellosis, commonly known as shipping fever.
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 who will conduct the study are experienced in cattle health
management.

**III. Need for the Proposed Action**

This experimental vaccine represents an attempt by Schering-Plough
Animal Health Corp. to produce an efficacious and safe vaccine against
SF that protects cattle with one dose administered subcutaneously.

**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 two strains were safety tested
separately and then together in a final product formulation.

Regarding the *M. haemolytica* deletant strain:

1\. It was found to be safe in calves following subcutaneous or
intranasal administration of a single dose. For the recommended
subcutaneous route, there was no shedding, no dissemination from the
injection site, and no spreading to nonvaccinated calves. Calves
vaccinated intranasally, however, can shed the organism and spread it to
other calves by nose-to-nose contact. The intranasal route will not be
used in the field trials.

2\. It was found to be stable genetically and phenotypically at Master
Seed (MS)+20 *in vitro* passages and MS+5 *in vivo* passes in calves
following intranasal administration.

3\. Virulence of the deletant strain in non-target animals (sheep, mice)
was much reduced compared to that of the parent strain. Safety following
intranasal administration to sheep, the most susceptible non-target
species, was tested. The organism was not detected in the sheep 14 days
after inoculation and no evidence of clinical disease due to the
deletant strain was observed.

Regarding the *P. multocida* deletant strain:

1\. It was found to be safe for use in calves following subcutaneous
administration of a single typical field dose.

2\. When given at higher doses (not to be used in the proposed field
trials), it caused some undesirable adverse reactions such as fever,
lethargy, rapid breathing, and large injection site reactions. The
calves recovered rapidly from the clinical signs without treatment but
still had visible injection site reactions 21 days after vaccination.
There was no detectable shedding or spread to other susceptible calves.
At 7 and 21 days, the strain was isolated from the injection site and
pre-scapular lymph nodes (not the blood, nasal passages, or lung).

3\. In the host animal reversion-to-virulence study, the deletant strain
was given intranasally rather than subcutaneously, to optimize potential
replication. Minimal lung lesions were observed in the first and last
passages only. Such lesions did not occur with the recommended
subcutaneous route (see 2. above). The deletant strain given
intranasally was not isolated from lung tissue in any of the calves.

4\. The deletant strain was found to be genetically and phenotypically
stable at MS+5 *in vivo* and MS+20 *in vitro* passage levels, as
determined by PCR analysis of the deletion and by phenotypic testing.

5\. The deletant strain is susceptible to commonly used antibiotics for
cattle.

6\. Safety of the deletant strain following intranasal administration to
sheep, rats, rabbits, and chickens was demonstrated by the absence of
clinical signs and no isolation of the *P. multocida* deletant strain.
These species are known to be susceptible to *P. multocida*.

7\. Intraperitoneal administration of the strain to mice to determine
the LD~50~ indicated that the deletant strain was 400 times less
virulent in mice than the parent strain.

**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 cattle.

Regarding the *M. haemolytica* A1 deletant strain, its host range is
essentially restricted to ruminants. Accidental human exposure, e.g. by
injection, is not expected to cause adverse effects.

Regarding the *P. multocida* A3 deletant strain, cattle isolates of *P.
multocida* are not generally implicated in human infections. Moreover,
this attenuated strain is not pathogenic to otherwise susceptible
non-target animals and does not contain any genetic material not already
present in the environment. Accidental human injection is not expected
to cause adverse effects.

Although the safety of this experimental vaccine containing the two
attenuated strains has not been specifically evaluated in humans, and is
therefore unknown, no safety hazards to the public health are expected.

C.  **Environmental Safety**

The risks to the environment are low. Both parental bacteria are present
in the environment.

Regarding the *M. haemolytica* deletant strain:

1\. *M. haemolytica* wildtype strains have wide distribution and are
readily found in ruminants. The deletant strain has reduced virulence,
no plasmids or mobile chromosomal elements, and is expected to be much
less of an environmental threat than the naturally occurring field
strains.

2\. The stability of the deletant strain at various temperatures and
conditions was determined to be similar to that of the parent strain.

3\. The deletant strain was found to be cleared from the site of
injection within 2 weeks following vaccination and is more sensitive to
a wider array of antibiotics than current circulating field isolates.

Regarding the *P. multocida* deletant strain:

1\. *P. multocida* is a commensal organism of the respiratory and
digestive tract of many domestic and wild animals, and is common to the
nasopharynx of healthy cattle.

2\. The survival of the deletant strain was found to be similar to the
parental organism, which is not normally a long period of time in the
natural environment. At cooler temperatures and following drying of the
reconstituted vaccine, there is some survival.

Regarding the dual-fraction vaccine containing both attenuated strains:

1\. There are no apparent substantial issues with adverse environmental
impacts concerning this vaccine. The potential for escape and dispersal
of the experimental vaccine is very low because the rate of shedding of
the vaccine in both target and susceptible non-target species is very
low. Also, its ability to survive in adverse conditions of temperature
and drying is limited.

2\. The firm reported that for calves vaccinated subcutaneously with the
dual-fraction vaccine using single dose, 10-fold overdose, or 2-dose at
21-day interval regimens, transient elevations in rectal temperatures
and local reactions at the injection site were observed. They believed
this to be consistent with administration of a Gram-negative bacterial
vaccine. The extent of these reactions can be better determined in the
proposed field trials.

**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).

**References**

1\. Baker, J.C. 1998. Respiratory Diseases of Cattle. pp. 1068-1080. In:
The Merck Veterinary Manual, Eighth Edition. S.E. Aiello, (Ed), Merck &
Co., Inc., Philadelphia.