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Protective responses generated by vaccination with an immuno-stimulating complex (ISCOM)-based vaccine for equine influenza (EQUIP F), containing a new 'American lineage' H3N8 virus, were studied. Seven ponies in the vaccine group received two intramuscular injections of EQUIP F given 6 weeks apart. Aerosol challenge with an A/eq/Newmarket/1/93 reference strain 4 weeks after booster vaccination resulted in clinical signs of infection and viral shedding in 7 influenza-naive control animals whereas the vaccinated ponies were significantly protected from both clinical signs and virus excretion. Influenza virus-specific IgG responses in serum following immunisation with the ISCOM vaccine were predominantly of the IgGa and IgGb sub-isotypes, a pattern similar to that generated by equine influenza virus infection. However, in contrast to the response following infection, virus-specific antibody responses in nasal washes following immunisation were characterised by the presence of IgG but not IgA.These results demonstrated that an ISCOM-based vaccine containing A/eq/Kentucky/98 provides strong protective immunity against challenge with an 'American lineage' H3N8 reference virus.

The efficacy of a live attenuated anti-coccidial vaccine, Paracox-5, administered to 1-day-old chicks was investigated by assessing protection against changes in weight gain following virulent challenge. Vaccinated birds were challenged independently 28 days later with each of the component species (Eimeria acervulina, Eimeria maxima, Eimeria mitis or Eimeria tenella), and protection was demonstrated against associated reduction in weight gain and lesion formation. In addition, an improvement in bird performance, in terms of feed conversion ratio, was also observed following vaccination. Furthermore, under conditions designed to more closely mimic those in the field and using hatchery spray administration, protection against a mixed virulent challenge introduced by 'seeder birds' was demonstrated evenly across a flock of broiler birds within 21 days after vaccination. These data demonstrate that Paracox-5 vaccine will protect broiler chickens against the adverse effects on performance induced by Eimeria spp.

Twenty-five Ayrshire/Friesian cows were vaccinated once with a new combined vaccine against rotavirus, coronavirus and Escherichia coli F5 (K99) or given a saline placebo 31 days before the first expected calving date. Blood samples were taken from the cows at intervals from vaccination until seven days after calving and from their calves up to 28 days after birth, and colostrum and milk samples were collected from the cows at intervals for 28 days after calving. There was a significant increase in the mean specific antibody titre against all three antigens in the serum of the vaccinated animals (even in the presence of pre-existing antibody) which was accompanied by increased levels of protective antibodies to rotavirus, coronavirus and E coli F5 (K99) in their colostrum and milk for at least 28 days.

In order to investigate the ability of an oil adjuvanted vaccine containing bovine coronavirus antigen to enhance lactogenic immunity in the calf, pregnant cows and heifers were vaccinated and specific virus neutralising antibody levels determined in serum, colostrum and milk. Pre-existing antibody titres (as a result of natural infection) in the serum of these animals were found to be significantly increased as a result of a single shot vaccination carried out between 2 and 12 weeks before calving. This was reflected in a similar increase in the titre and duration of specific antibody in milk and colostrum that was passed on to the calves. The overall response observed was highly dependent on an adequate antigen payload being incorporated within the single dose vaccine. No abnormal local or systemic reactions were observed as a result of vaccination. It is hoped that this approach will lead to the production of a superior commercial vaccine for the protection of neonatal calves against enteric coronavirus infection.

AIMS--To evaluate the clinical performance of enzyme immunoassays for IgG and IgM antibodies to Toxoplasma gondii based on enhanced chemiluminescence. METHODS--Classification of routine clinical samples from the originating laboratories was compared with that obtained using the chemiluminescence based assays. Resolution of discordant results was achieved by testing in alternative enzyme immunoassays (IgM) or by an independent laboratory using the dye test (IgG). RESULTS--Compared with resolved data, the IgM assay was found to be highly specific (100%) with a cut off selected to give optimal performance with respect to both the early detection of specific IgM and the detection of persistent levels of specific IgM (sensitivity 98%). Compared with resolved data, the IgG assay was shown to have a sensitivity and a specificity of 99.4%. CONCLUSIONS--The Amerlite Toxo IgM assay possesses high levels of sensitivity and specificity. Assay interference due to rheumatoid factor like substances is not a problem. The Amerlite Toxo IgG assay possesses good sensitivity and specificity, but is less sensitive for the detection of seroconversion than methods detecting both IgG and IgM.

SUMMARY Using an ELISA for the detection of virus-specific immune complexes, ten cows were found to be shedding bovine enteric coronavirus. The shedding patterns from five of these animals were followed for a period of 12 weeks, and all were found to be chronically shedding virus. Despite the presence of both faecal and serum antibody the infection was not cleared; therefore, the role of cell-mediated immunity (CMI) was investigated by immunosuppressing the chronically shedding cows with dexametha- sone. No major role for CMI in maintaining the chronic infection could be determined, although immunosuppression did result in a temporary reduction in the shedding of virus-specific immune complexes. INTRODUCTION Coronaviruses are associated with a wide variety of diseases in both humans and animals (Tyrrell et al., 1978) and many members of the coronavirus family appear to be capable of establishing chronic infections in their hosts. These infections include chronic central nervous system disease in mice (Bailey et al., 1949) and pigs (Mengeling & Cutlip, 1976), chronic nephritis in chickens (Alexander et al., 1978) and chronic disease of the eye in rats (Lai et al., 1976). The detection of coronaviruses associated with enteritis for several months after infection has also been reported in both humans (Moore et al., 1977) and pigs (Underdahl et al., 1975). Bovine enteric coronavirus (BEC) has been shown to be a primary pathogen in neonatal calf diarrhoea (Mebus et al., 1973). The incidence of BEC-associated enteritis in naturally occurring outbreaks of diarrhoea in young calves has been reported to vary from 15 to 70% (Crouch et al., 1984; Langpap et al., 1979; Marsolais et al., 1978; Morin et al., 1976) and serological evidence indicates that the virus may be widespread (Rodak et al., 1982), at least in Western Canada. Little information is available concerning the infection of cows by BEC, and the possible role they may play in the epidemiology of infection. We have previously shown that the virus can be detected in the faeces from over 70% of clinically normal cows from a single herd (Crouch & Acres, 1984). This report describes the shedding pattern and immune responses of clinically normal cows chronically shedding BEC in their faeces over a period of several months. METHODS Virus. The P.Q isolate of BEC (originally obtained from S. Dea, Department of Pathology and Microbiology, Faculty of Veterinary Medicine, University of Montreal, St Hyacinthe, Quebec, Canada) grown in Madin-Darby bovine kidney (MDBK) cells as described previously (Crouch & Raybould, 1983) was used throughout this study. Preparation oJJaecal samples. Faecal samples were collected and stored at 4 °C until diluted 1 in 3 (w/v) in 0.01 M- phosphate-buffered saline pH 7-2 (PBS) containing 0.05 % Tween 20. Following low-speed centrifugation at 3000 r.p.m. (MSE Chilspin) for 20 min, the partially clarified supernatants were removed and stored at -20 °C until tested. 0000-6410 © 1985 SGM

The majority of mice inoculated with a mixture of a lethal dose of virulent Semliki Forest virus (SFV) strain ts+ and defective-interfering (DI) SFV remained completely health, with virus infectivity levels in brain tissue reduced by 99.9%. The results of previous studies had suggested that these effects were primarily the result of the intrinsic interfering capacity of DI virus rather than of host defense responses. Because SFV strain ts+ and an avirulent strain of SFV have clearly distinguishable histopathologic effects in brain tissue, the capability of DI virus to change the virulent into the avirulent form of the disease was examined. Modulation of strain ts+ virus infection by DI virus was accompanied by a complete absence of histopathologic changes despite significant levels of infectious virus and thus differed qualitatively from infection with avirulent SFV. These results provide further evidence that the interference is not mediated through stimulation of an immune cell infiltration.

Monoclonal antibodies reactive with three different viral polypeptides were evaluated singly and in combination as the capture antibody(s) in an enzyme-linked immunosorbent assay system for the detection of bovine enteric coronavirus. Similar levels of sensitivity were found for all combinations tested. A sensitive, highly specific, and reproducible assay for the detection of bovine enteric coronavirus was developed, using a mixture of two of these monoclonal antibodies reactive with antigenic components either external or internal to the virion. These monoclonal antibodies were bound indirectly to 96-well plates via rabbit anti-mouse immunoglobulin. After sample application and incubation, virus was detected by using rabbit anti-coronavirus peroxidase conjugate followed by enzyme substrate and chromagen. Fecal samples from a single herd of cows were screened for the presence of coronavirus by this assay. Five percent of clinically normal cows were found to be shedding coronavirus.

Summary This study demonstrates that populations of defective interfering Semliki Forest virus (DI SFV) are heterogeneous particularly in respect of their interference properties. Interference was quantified by two assays, one measuring inhibition of the yield of infectious progeny virus, and the other measuring reduction in virus-directed RNA synthesis; for 11 different DI SFV preparations a ratio of the two interference titres was calculated. These ratios varied up to 46-fold indicating that each DI virus preparation contained an interference activity that varied independently of the other. However, sister stocks made from the same parental inoculum had similar properties. The effects of different DI virus preparations on other parameters (virus polypeptide synthesis, yield of DI virus and yield of infectious virus) were investigated using inocula with interference titres standardized by either assay. Co-inoculation of L929 cells with 50 p.f.u. SFV showed that these parameters varied independently of each other and of the DI virus inoculum. There was no correlation with the number of undiluted passages each DI stock had received. Direct evidence of physical heterogeneity was demonstrated by metrizamide density gradient centrifugation. Although infecting virus sedimented as a narrow band, DI SFV was distributed over a broad region of the gradient. Its position on the gradient indicated that DI SFV has a higher nucleic acid: protein ratio than standard virus. DI virus progeny obtained by using fractions of the gradient as inoculum were as heterogeneous as the unfractionated parent, confirming that DI viruses retain heterogeneity on passage.

A field trial was designed to determine the efficacy of a combination rotavirus-coronavirus/Escherichia coli vaccine on dairy farms in southwestern Ontario. In Part A of the trial, 321 cows on 15 farms were randomly assigned to either vaccination or placebo groups. On eight farms, 50% of the dams were vaccinated, while on the other seven farms, 80% of the dams were vaccinated. In Part B of the trial, 26 farms were randomly assigned to either a total vaccination program or to no vaccination program. Mortality, disease occurrence and weight gains were recorded on all calves for the first two weeks of life. In Part A, 23.5% of all calves were treated in the first two weeks of life, 20.9% were treated specifically for scours and 3.6% of live-born calves died. Enteropathogenic E. coli was identified on 13 of the 15 farms, rotavirus on 11 and coronavirus on ten. At least one of the three potential pathogens was found on every farm. There were no significant differences between calves from placebo-treated and vaccine-treated dams with regard to the proportion treated for all diseases, or for scours, or the proportion which died. Neither were there differences in days to first treatment for all diseases (seven days on average), days to first scour (6.7 days), duration of treatments (3.9 days for all diseases, 3.7 days for scours), or estimated weight gains (0.5 kg/day to 14 days). These results were not altered when the presence or absence of enteropathogenic E. coli, rotavirus or coronavirus on the premises was accounted for.(ABSTRACT TRUNCATED AT 250 WORDS)

SUMMARY We describe a simple, rapid and reproducible assay for defective-interfering Semliki Forest virus (DI SFV) which is based on the inhibition of synthesis of virus-specified RNAs in SFV-infected cells. Using the assay, we have been able to show that DI virus is generated by a single passage in baby hamster kidney (BHK) cells in an inoculum which contained no detectable DI virus and we have calculated the u.v. target size of the interfering activity.

Spleen cells from a calf hyperimmunized with bovine enteric coronavirus were fused with nonproducer mouse plasmacytoma cells. Stable hybridoma lines secreting bovine immunoglobulins were obtained. One line secreted monoclonal bovine immunoglobulin G2, which reacted specifically with bovine enteric coronavirus in an enzyme-linked immunosorbent assay, inhibited virus hemagglutination, and precipitated a structural polypeptide with a molecular weight of 26,000 daltons.

SUMMARY A serial study of rotavirus infection in gnotobiotic piglets is described. They were infected when 7 days old and the course of infection was followed for 21 days, by immunofluorescence and histological examinations of the intestinal epithelium and by titration of the virus content of the gut lumen. There appeared to be two stages of recovery of the intestinal wall, the earlier stage involving non-immune mechanisms and the later involving antibody.

Production of diarrhea in neonatal calves by enterotoxigenic Escherichia coli depends on its ability to attach to the epithelial cells of the intestine via surface adhesins called pili or fimbriae and to secrete enterotoxins. The most important of these fimbriae are designated K99 and F41. We produced and characterized a murine monoclonal antibody specific to F41. This monoclonal antibody and a K99-specific monoclonal antibody were used to develop sensitive and specific passive hemagglutination and capture enzyme-linked immunosorbent assays (ELISAs) for detection and quantitation of F41 and K99 antigens in E. coli cultures and culture supernatants. The capture ELISA systems exhibited excellent sensitivity and specificity, whereas the passive hemagglutination systems appeared to be oversensitive. The ability of the capture ELISAs to detect K99 and F41 fimbrial antigens in fecal specimens from calves was evaluated. Fimbrial antigens were detected in six of six specimens from scouring calves but not in four of four specimens from nonscouring calves.

Purified coronavirus, detergent extracts of purified coronavirus, and virus-infected Madin-Darby bovine kidney cells were evaluated as antigen substrates in enzyme-linked immunosorbent assay (ELISA) and passive hemagglutination systems. Only detergent-extracted and -unextracted, purified viruses were reactive as antigen substrates in ELISA, whereas all three antigen preparations could be used for sensitization of erythrocytes in the passive hemagglutination assay. The passive hemagglutination system with infected cell extracts exhibited a similar level of sensitivity and specificity to the ELISA system employing purified coronavirus but enabled 300 times more tests to be performed per volume of virus-infected cell culture.

The prevalence of rotavirus and coronavirus shedding by adult cows was investigated using capture enzyme-linked immunosorbent assays. Fecal samples from 121 cows in a single herd were tested for the presence of rotavirus and coronavirus, either free or complexed with immunoglobulin. Free rotavirus was not detected in any samples while rotavirus-immunoglobulin complexes were detected in 53 of 121 (44%) samples tested. In contrast, free coronavirus was detected in six (5%) samples and coronavirus-immunoglobulin complexes were detected in 85 (70%) of the samples tested. Thus it appears that subclinical infection of cows by either of these viruses is common, possibly providing a source for infection of the neonate. These assays may therefore provide important information regarding the epidemiology of enteric virus infections and suggest means of improving management to prevent epidemics of neonatal diarrhea.