A framework for the animal health risk analysis of biotechnology-derived animals: a Canadian perspective.

This paper describes the framework used by the Canadian Food Inspection Agency to assess the risks to animal health associated with biotechnology-derived animals and their products. In Canada the risks to animal health associated with biotechnology-derived animals are one consideration among several other regulatory concerns (e.g. human health, the environment). The risk analysis process begins with hazard identification, includes a risk assessment for each hazard, and concludes with risk management and risk communication.

Antigenic and molecular characterization of a herpesvirus isolated from a North American elk.

OBJECTIVE: To determine whether a herpesvirus isolated from the semen of a North American elk was related to bovine herpesvirus 1 (BHV-1). SAMPLE POPULATION: Semen from 1 healthy bull elk and 2 subtypes of BHV-1 (BHV-1.1 and BHV-1.2). PROCEDURES: A virus with cytopathic and electron microscopic characteristics consistent with an alpha-herpesvirus was isolated from elk semen, using fetal bovine kidney cells. Cross-neutralization assays were performed with antisera against BHV-1 and the elk herpesvirus (EIkHV). Restriction endonuclease digests of EIkHV DNA were compared with digests of BHV-1.1 and BHV-1.2 DNA. A portion of the ElkHV DNA polymerase gene was amplified with consensus primers by use of the polymerase chain reaction and sequenced. Sequence was compared with known sequences of other herpesviruses. An immunoperoxidase monolayer assay was used to determine reactivities of 22 BHV-1-specific monoclonal antibodies (mAb) against ElkHV. In vitro neutralizing activities of the reactive mAb were determined by use of a microneutralization assay. RESULTS: Results of cross-neutralization assays indicated that ElkHV was serologically related to BHV-1. Endonuclease digestion of ElkHV DNA generated fragments that were distinct from those of BHV-1. Nucleotide sequencing confirmed that ElkHV is an alphaherpesvirus closely related to but distinct from BHV-1. Six of 22 BHV-1-specific mAb reacted against ElkHV; 2 of these 6 also neutralized in vitro infectivity of ElkHV. CONCLUSIONS AND CLINICAL RELEVANCE: ElkHV is antigenically and genetically distinguishable from BHV-1. However, the viruses are serologically related and share at least 6 antigenic determinants, one of which is a major neutralizing determinant.

Detection of antibodies to equine arteritis virus by a monoclonal antibody-based blocking ELISA.

A potent ELISA antigen was prepared from equine arteritis virus (EAV) by differential centrifugation of EAV-infected cell culture fluid, followed by solubilization of the preparation by Triton X-100 treatment. Using this antigen and a mouse monoclonal antibody against the G(L) protein of EAV, a reliable blocking ELISA (bELISA) was developed for the detection of EAV antibodies in equine sera. The bELISA was evaluated using a total of 837 test serum samples. The relative sensitivity (n = 320) of the bELISA compared to the serum neutralization (SN) test was 99.4%. The bELISA appears to be a highly specific test, the specificity of which did not appear to be adversely affected by previous exposure of horses to non-EAV-containing biologicals. Of 119 serum samples, 21 from horses without any history of exposure to EAV and 98 from racetrack Thoroughbreds, 118 were negative in the SN test and bELISA. One sample was SN-negative but suspicious with the bELISA. Based on testing 465 SN-negative field samples and 52 SN-negative samples from experimental horses, and excluding any sera giving a suspicious reaction, the relative specificity of the bELISA was 97.7%. Samples should be examined undiluted and diluted 1/10 in the bELISA because the testing of sera of high neutralizing antibody titer may be affected by a prozone-like phenomenon. The bELISA is a more rapid and cost-efficient test than the SN test for the detection of EAV antibodies in equine sera.

Efficacy of vaporized hydrogen peroxide against exotic animal viruses.

The efficacy of vapor-phase hydrogen peroxide in a pass-through box for the decontamination of equipment and inanimate materials potentially contaminated with exotic animal viruses was evaluated. Tests were conducted with a variety of viral agents, which included representatives of several virus families (Orthomyxoviridae, Reoviridae, Flaviviridae, Paramyxoviridae, Herpesviridae, Picornaviridae, Caliciviridae, and Rhabdoviridae) from both avian and mammalian species, with particular emphasis on animal viruses exotic to Canada. The effects of the gas on a variety of laboratory equipment were also studied. Virus suspensions in cell culture media, egg fluid, or blood were dried onto glass and stainless steel. Virus viability was assessed after exposure to vaporphase hydrogen peroxide for 30 min. For all viruses tested and under all conditions (except one), the decontamination process reduced the virus titer to 0 embryo-lethal doses for the avian viruses (avian influenza and Newcastle disease viruses) or less than 10 tissue culture infective doses for the mammalian viruses (African swine fever, bluetongue, hog cholera, pseudorabies, swine vesicular disease, vesicular exanthema, and vesicular stomatitis viruses). The laboratory equipment exposed to the gas appeared to suffer no adverse effects. Vaporphase hydrogen peroxide decontamination can be recommended as a safe and efficacious way of removing potentially virus-contaminated objects from biocontainment level III laboratories in which exotic animal disease virus agents are handled.

Relationship between maternal plasma progesterone concentration and interferon-tau synthesis by the conceptus in cattle.

The objective of this study was to determine the relationship between maternal progesterone concentration and conceptus synthesis of interferon-tau as an index of conceptus viability at the time of maternal recognition of pregnancy. Heifers of mixed beef breeds were randomly assigned to receive 1 of 2 treatments: 1) intramuscular injection of 1500 IU hCG on Day 5 after artificial insemination (AI; n = 12) or 2) intramuscular injection of saline on Day 5 after AI (n = 17). Ovaries were scanned daily by transrectal real-time ultrasonography. Progesterone concentrations were determined from daily blood samples collected from the jugular vein. Heifers were slaughtered on Day 18 after AI and conceptus tissues were collected. These were incubated individually at 37 degrees C in RPMI medium, and supernatant collected after 24 h. Conceptus secretory products in the supernatant were analyzed for interferon concentration by antiviral assay using vesicular stomatitis virus. Transrectal ultrasonography showed all heifers that received hCG had at least 1 extra corpus luteum (CL) in addition to the spontaneous CL formed from the previous ovulation (10 with 2 CL, 2 with 3 CL). A significant increase in plasma progesterone concentration was detected in pregnant heifers treated with hCG (n = 9) vs pregnant control heifers (n = 11; P < 0.001). There was a tendency for an increase (P = 0.059) in synthesis of interferon-tau by conceptuses from hCG-treated heifers compared to control heifers. Maternal plasma progesterone concentrations were correlated with interferon-tau production by the conceptuses (r = 0.593, P < 0.006), suggesting that higher maternal progesterone may provide a more suitable environment for the developing conceptus.

Antiviral action of interferon-alpha against porcine transmissible gastroenteritis virus.

Swine testis (ST) cell cultures were treated with various doses of recombinant human interferon-alpha 2a (IFN), and assayed for 2',5' oligoadenylate synthetase (2-5 A synthetase) activity. Treatment with 100 or 1000 units/ml of IFN resulted in increased 2-5 A synthetase activity, but there was no significant response to 1 unit/ml of IFN. Titres of porcine transmissible gastroenteritis virus (TGEV) were reduced between 6 and 15 hours post-infection in ST cells treated with 1000 or 2500 units/ml of IFN. Polyacrylamide gel electrophoresis of lysates of TGEV-infected ST cells, and of lysates immunoprecipitated with anti-TGEV antibodies, revealed that the synthesis of the N and S proteins of TGEV was reduced in cells treated with 100 or 1000 units/ml of IFN. Viral RNA production, as determined with a probe which hybridized to the S gene of TGEV, was found to be reduced in ST cells treated with 1000 units/ml of IFN, but not in cells treated with 100 units/ml. It was concluded that, in IFN-treated ST cells, TGEV protein production may be decreased in the absence of reduced viral RNA production, and that 2-5 A synthetase may not be a significant factor in the antiviral activity of IFN against TGEV.

Interferon induction in swine lymphocyte antigen-defined miniature pigs.

Interferon was induced in two groups of swine lymphocyte antigen (SLA)-defined miniature pigs with polyinosinic: polycytidylic acid complexed with poly-L-lysine and carboxymethylcellulose. The group 1 pigs were low antibody-response phenotypes (SLAa/a, SLAa/c, SLAc/c), and the group 2 pigs were high antibody-response phenotypes (SLAd/d, SLAd/g, SLAg/g). Six hours after induction the antiviral tires were not influenced by the SLA group, but higher titres were observed in females. Higher antiviral titres were found in group 2 pigs before treatment and 24 hours after treatment, and higher titres were found in female pigs. The antiviral titres before and after treatment were also influenced by the sire. Group 2 pigs had a lower total leucocyte counts before treatment, and there was a significant reduction in leucocyte numbers in both groups six hours after induction, due mainly to a large reduction in lymphocyte counts.

Antiviral activity of interferon against transmissible gastroenteritis virus in cell culture and ligated intestinal segments in neonatal pigs.

Segments of jejunum in 5 to 6 days old piglets were surgically ligated, inoculated with transmissible gastroenteritis virus (TGEV) and 18 hours later the segments were fixed for histology or suspensions were prepared for plaque assay in swine testis (ST) cell cultures to determine the yield of virus. When the virulent Purdue strain of TGEV was used, villous atrophy was seen and TGEV antigen was demonstrated immunohistochemically in the villous enterocytes. The Miller M6 strain of virus produced less extensive lesions in the segments, but since it was titratable by plaque assay it was used in the subsequent yield reduction assays to determine the antiviral activity of interferon. When intestinal segments were inoculated simultaneously with either 3200 units of natural porcine interferon-alpha or up to 1000,000 units of recombinant human interferon-alpha 2 a, and TGEV, there no reductions in virus yield, although the same cytokines exerted an antiviral effect in ST cells treated in a similar way. However, virus yields were significantly reduced in intestinal segments in piglets treated parenterally with the synthetic interferon inducer polyinosinic: polycytidylic acid 6 hours before challenge of the segments with TGEV. There was also a trend for the antiviral effects of interferon induction before challenge to be augmented by the inclusion of interferon with the virus inoculum. It was concluded that interferon would be ineffective as a therapeutic for TGEV, although it might be useful prophylactically.