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Abstract

Objective

To determine the effect of milk and blood serum constituents on cytotoxicity of Staphylococcus aureus on mammary epithelial cells.

Design

In vitro incubation of cells with cytotoxic agents and milk and serum constituents.

Sample Population

Mammary cells, milk, and blood obtained from 3 cows.

Procedure

Staphylococcal α-toxin and culture supernatants from S aureus M60 and an α-toxin-negative mutant of M60 were incubated with bovine mammary epithelial cells in the presence of milk fractions, serum, and divalent cations. Propidium iodide fluorescence was used as a measure of cell damage.

Results

Skim milk and milk whey inhibited S aureus cytotoxic agents. Skim milk protected against α-toxin damage to a greater extent than milk whey. Serum from an adult animal was more protective than was fetal serum. Milk fat and serum albumin had no protective effect. Divalent calcium and Mg2+ were more effective inhibitors of mammary epithelial cell damage caused by α-toxin than of damage attributable to M60 culture supernatant. Divalent calcium and Mg2+ at concentrations similar to those of free Ca2+ and Mg2+ in normal bovine milk decreased cytotoxic damage attributable to α-toxin. However, concentrations similar to those of total Ca2+ and Mg2+ in normal milk were required to decrease cell damage caused by M60 culture supernatant. The α-toxin-negative mutant was less cytotoxic than the M60 parent strain.

Conclusions

Casein, as well as Ca2+ and Mg2+ in bovine milk, inhibit the cytotoxic effect of S aureus on mammary epithelial cells.(Am J Vet Res 1996;57:308-312)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To determine the scope of strain variations among Campylobacter spp associated with abortion in sheep.

Design

To examine Campylobacter spp isolated from cases of abortion for biochemical, antigenic, and genetic differences.

Sample Population

15 isolates of Campylobacter spp isolated from cases of abortion during a single lambing season.

Procedure

Isolates were examined, using biochemical tests, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins (OMP), and DNA restriction enzyme analysis (REA).

Results

Eight strain variants were detected among the 15 isolates. 14 of the isolates were C jejuni, 13 of which were biotype I and 1 biotype II, and the remaining isolate was identified as C fetus subsp fetus. Five sodium dodecyl sulfate-polyacrylamide gel electrophoresis OMP patterns had distinctive profiles for C fetus subsp fetus and C jejuni biogroup-II isolates and 3 variants within the C jejuni bio-group-I isolates. Examination of REA patterns of DNA from the 15 isolates digested with Cfo I indicated clear differences correlating with species and biogroups and 4 REA variants among biotype-l isolates.

Conclusions

Marked antigenic and genetic heterogeneity of Campylobacter isolates were associated with ovine abortion within a defined geographic area.

Clinical Relevance

Representatives of differing OMP and REA profile groups should be considered for incorporation in vaccines to optimize protection in this region and possibly other geographic areas.

Free access
in American Journal of Veterinary Research

Abstract

Objective

To test the ability of porcine respiratory coronavirus (PRCV) to induce protective immunity to antigenically related transmissible gastroenteritis virus (TGEV) in neonatal pigs.

Design

Neonatal pigs were exposed to PRCV when they were 2, 4, or 6 days old and challenge-exposed to virulent TGEV at 10 days of age.

Animals

34 hysterectomy-derived, colostrum-deprived pigs.

Procedure

After challenge exposure, clinical signs were observed, body weight, antibody response, and virus shedding were measured, and mortality was determined.

Results

After exposure to PRCV, principals had a slightly slower rate of weight gain than did controls; with 1 exception (a PRCV-exposed pig that was dyspneic for 1 day), principals and controls remained clinically normal until shortly after challenge exposure, when all pigs became listless and anorectic and developed watery diarrhea. However, by day 3, most of the pigs that had been exposed to PRCV when they were either 2 or 4 days old began to recover and most (15/18) survived. Conversely, the clinical condition of most of the other pigs worsened and most (14/16) died. Pigs exposed to PRCV when they were 2 or 4 days old also differed from all other pigs in that they had serum virus-neutralizing antibodies for PRCV and TGEV at the time of challenge exposure.

Conclusions

The PRCV can induce protective immunity to TGEV in neonatal pigs and such immunity develops at or about 6 days after exposure to PRCV. Moreover, protective immunity may be coincident with the appearance of virus-neutralizing antibody.

Clinical Relevance

Exposure to PRCV should enhance a TGE herd vaccination program.

Free access
in American Journal of Veterinary Research

Abstract

Objective

To investigate the antigenic diversity of lipo-oligosaccharides of Haemophilus parasuis.

Procedures

Immunoblot assays were done with monoclonal and polyclonal antibodies on whole-cell lysates. Individual colonies of H parasuis strains H 54, H 53, and H 128 were tested for reactivity with lipo-oligosaccharide-specific monoclonal antibodies after a single passage on chocolate agar, and colonies of strain H 54 were analyzed after 10 passages. Colony blot tests were used to screen H parasuis strains for spontaneously occurring antigenic variation in their lipo-oligosaccharides.

Results

Eight H parasuis strains were separated into 4 lipo-oligosaccharide serovars on the basis of immunoblot reactions with 3 polyclonal rabbit antisera. Nine monoclonal antibodies against lipo-oligosaccharides of a lipo-oligosaccharide-serovar I strain reacted with all tested serovar I strains but failed to react with other H parasuis strains.

Conclusions

Variations in the antigenic reactivity after 1 or 10 passages on chocolate agar were not observed. The serovar I lipo-oligosaccharide strains included virulent as well as avirulent H parasuis strains, indicating that these epitopes do not correlate directly with virulence properties of H parasuis. (Am J Vet Res 1996;57:63-67)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To investigate in vitro antigenic relations, in vivo cross-protection, and isotype antibody responses to a winter dysentery (WD) and calf diarrhea strain of bovine coronavirus (BCV).

Design and Animals

Gnotobiotic and colostrum-deprived calves were inoculated oronasally with a WD (DBA) or a calf diarrhea (DB2) BCV, and were challenge exposed with the heterologous BCV.

Procedure

Nasal swab and feces specimens and blood samples were collected. Fecal and nasal specimens were assayed for BCV shedding by antigen-capture ELISA or immune electron microscopy. Bovine coronavirus antigens were detected in nasal epithelial cells by immunofluorescence. Antibody titers to BCV in serum were assayed by virus neutralization (VN), and BCV antibody isotype titers in feces and sera were quantitated by ELISA.

Results

All calves developed diarrhea and shed BCV nasally and in feces, then recovered and were protected from BCV-associated diarrhea after challenge exposure with the heterologous BCV. After challenge exposure with either strain, fecal shedding of DBA was detected in 1 of 4 calves and nasal shedding of DB2 was detected in 2 of 4 calves. Immunoglobulin M was the principal coproantibody to BCV early, followed predominantly by IgA. Immunoglobulin G1 coproantibody titers to BCV were low, but increased after challenge exposure. Immunoglobulin G1 antibodies were predominant in serum. After challenge exposure, all serum antibody isotype titers increased except IgG2. The VN antibody responses paralleled serum IgG1, antibody responses.

Conclusions and Clinical Relevance

Immunoglobulin A coproantibodies at challenge exposure were associated with protection against diarrhea. Nasal shedding of BCV after challenge exposure confirmed field data documenting reinfection of the respiratory tract of cattle, suggesting that, in closed herds, respiratory tract infections constitute a source of BCV transmission to cows (WD) or young calves. (Am J Vet Res 1996;57:48-53)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To evaluate antimicrobial agents for treatment of models of acute and persistent leptospirosis caused by Leptospira interrogans serovar pomona.

Design

Randomized trials comparing dosages and regimens of various antimicrobial agents for treatment of acute and persistent leptospirosis.

Animals

245 Golden hamsters to model acute leptospirosis and 121 mixed-breed swine to model persistent leptospirosis.

Procedure

Hamsters and swine were inoculated with L interrogans serovar pomona. Antimicrobial agents were given to hamsters for 3 or 5 days after inoculation, with necropsy at 14 days after inoculation. Swine were treated for 1, 3, or 5 days beginning at 3 weeks after inoculation, and were necropsied 7 to 10 days after completion of antimicrobial agent treatment. Hamster tissue and swine tissue and urine specimens were examined by culture, fluorescent antibody testing, and histologic examination for presence of leptospires.

Results

All untreated control hamsters became infected and manifested clinical signs and lesions of acute leptospirosis. Leptospires were not detected in hamsters treated with dihydrostreptomycin/penicillin G (25 mg/kg of body weight). Administration of ampicillin at all dosages reduced the number of hamsters infected, as confirmed at necropsy; the other agents tested required dosages greater than label recommendations to reduce the number infected. All untreated control swine became infected and shed leptospires in urine through the time of necropsy. Leptospires were not detected in kidneys or urine of swine treated with dihydrostreptomycin/penicillin G (25 mg/kg) for 1, 3, or 5 days, or in swine treated with oxytetracycline (40 mg/kg for 3 or 5 days), tylosin (44 mg/kg for 5 days), or erythromycin (25 mg/kg for 5 days). Treatment with ceftiofur and ampicillin was not effective in elimination of L interrogans serovar pomona in swine.

Conclusions

Dihydrostreptomycin/penicillin G is effective for treatment of acute and persistent leptospirosis. Differences between the effectiveness of antimicrobial agents in the acute and persistent model of leptospirosis emphasize the importance of using the appropriate model for treatment evaluation. Antimicrobial agents evaluated for treatment of persistent leptospirosis in swine required the use of dosages above those recommended by the manufacturer.

Clinical Relevance

Use of antimicrobial agents at extra-label dosages for treatment of persistent leptospirosis may cause residue problems in food animals; however, these regimens may be useful for treatment of breeding stock or animals destined for import/export. (Am J Vet Res 1996;57:59-62)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To determine prevalence and relevance of coagulase-positive Staphylococcus hyicus and S intermedius intramammary infections (IMI) in dairy cows and determine the ability of the 4-hour tube coagulase (TC) test to differentiate the coagulase-positive staphylococci (CPS).

Design

Prevalence of CPS was determined for primiparous cows (point prevalence and prevalence at first parturition) and multiparous cows (point prevalence) of 2 herd groups: < 6% CPS IMI prevalence = low prevalence (LP); > 10% CPS IMI prevalence = high prevalence (HP).

Sample population

For prevalence, cows of 22 dairy herds. For TC, 1,038 CPS strains isolated from cow milk.

Procedure

Speciation of CPS from aseptically collected composite milk samples was performed. Coagulase-positive isolates from 4 cow groups were tested for their ability to coagulate rabbit plasma by 4 hours: LP and HP primiparous cows at parturition, and LP and HP cows any time after first parturition.

Results

Of 487 CPS in the prevalence study, 82.1% were S aureus, 17.7% were coagulase-positive S hyicus, and 0.2% were S intermedius. Of all CPS IMI in LP herds, 34% were coagulase-positive S hyicus; of all CPS IMI in HP herds, 9% were coagulase-positive S hyicus. Coagulase-positive S hyicus appeared to persist to the end of lactation in 4 cows (mean linear somatic cell count = 3.7). The TC test was ≥ 97% sensitive, ≤ 33% specific, and had a predictive value positive range of 60 to 97% for S aureus isolates.

Conclusion

Coagulase-positive S hyicus appears capable of inducing chronic, low-grade IMI. Staphylococcus intermedius does not appear to be an important mastitis pathogen. The TC test is not valid to use as the sole method to differentiate CPS species. (Am J Vet Res 1996;57:54-58)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To evaluate the ability of bovine complement to kill a variety of field isolates and laboratory strains of Brucella abortus.

Design

The experimental approach was to determine the sensitivity of B abortus isolates to killing by bovine serum, and to document the role of complement in brucellacidal activity.

Sample population

Six laboratory isolates and 12 field isolates of B abortus were tested.

Procedure

The ability of B abortus to survive exposure to undiluted bovine serum for 2 hours at 37 C was assessed. The role of complement in killing was determined by examining the ability of heat (56 C for 60 minutes) and cobra venom factor to obliterate the activity in serum, and by detecting binding of the ninth component of bovine complement to serum-sensitive target cells.

Results

Isolates of B abortus that were resistant to the bactericidal activity of normal bovine serum were revealed. These included field isolates and laboratory strains. Furthermore, the study confirmed earlier reports that bovine serum-mediated killing of B abortus is caused by the complement cascade.

Conclusions

Some isolates of B abortus, like other gram-negative bacteria, were resistant to complement-mediated killing. Resistance was associated with smooth colony morphology. Isolates lacking detectable O antigen were serum sensitive.

Free access
in American Journal of Veterinary Research

SUMMARY

Fetal infectivity of Ehrlichia risticii was investigated in 19 ponies that were E risticii negative on the basis of results of an indirect fluorescent antibody (ifa) test. Thirteen pregnant ponies were infected by iv administration of E risticii between 90 and 180 days of gestation. Six pregnant ponies served as noninfected controls. Each infected pony had clinical signs of equine monocytic ehrlichiosis, was confirmed to be ehrlichemic, and developed an ifa titer to E risticii. Two infected ponies became recumbent, were unresponsive to supportive care, and were euthanatized. After recovery from clinical illness, the remaining ponies were observed throughout gestation for reproductive abnormalities. On abortion, each fetus was necropsied and tissue specimens from the liver, bone marrow, spleen, colon, and mesenteric lymph nodes were inoculated into canine monocyte cell cultures. Six infected ponies aborted at a mean 217 days of gestation, which was between postinoculation days 65 and 111. Five fetuses were recovered for evaluation, and E risticii was isolated from 4 of them. All 5 fetuses recovered had similar histologic findings, including enterocolitis, periportal hepatitis, and lymphoid hyperplasia with necrosis of the mesenteric lymph nodes and spleen. All 5 fetuses tested negative for IgG to E risticii, although 3 had low IgM titer to E risticii. The remaining 5 infected ponies had normal parturition. Presuckle ifa titer to E risticii was measured in 4 of the term foals, and results for 3 were positive. Two foals from infected ponies were monitored for 6 months and daily gain in body weight was comparable to that of a control foal. None of the control ponies became ill or seroconverted during the clinical illness phase, and none aborted throughout gestation. Two control ponies seroconverted to E risticii 6 weeks before parturition. Results of this study indicate that E risticii is a primary abortifacient under experimental conditions.

Free access
in American Journal of Veterinary Research

SUMMARY

Mycobacterial culture was performed on colostrum, milk, and feces from 126 clinically normal cows of a single herd with high prevalence of Mycobacterium paratuberculosis infection. Thirty-six (28.6%) cows were determined to be shedding the organism in the feces. Of the 36 fecal culture-positive cows, M paratuberculosis was isolated from the colostrum of 8 (22.2%) and from the milk of 3 (8.3%). Cows that were heavy fecal shedders were more likely to shed the organism in the colostrum than were light fecal shedders.

Free access
in American Journal of Veterinary Research