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Abstract

Objective

To determine efficacy of a vaccine containing modified-live bovine viral diarrhea virus (BVDV) type 1 for protecting pregnant cows and their fetuses against virulent heterologous BVDV type 1.

Design

Randomized controlled cohort study.

Animals

18 yearling beef heifers seronegative for BVDV and negative when tested for BVDV by virus isolation.

Procedure

Cattle were randomly assigned to control (unvaccinated; n = 6) or vaccinated (12) groups. Vaccinated heifers were given a combination vaccine containing modified-live BVDV type 1 comprising a cytopathic (NADL) strain. All 18 heifers were then bred and challenge-exposed between 70 and 75 days of gestation with BVDV type 1, administered intranasally. Cattle were monitored, and infection status of offspring was determined after parturition. Antibody concentrations of vaccinated and control heifers were also monitored.

Results

All 6 calves from control heifers had positive results on multiple virus isolation tests and were considered persistently infected. In comparison, only 2 calves from vaccinated cows had positive results on virus isolation tests and were considered persistently infected. One vaccinated heifer aborted, but the fetus was not persistently infected, and the abortion was not attributed to BVDV infection.

Clinical Implications

Analysis of these data indicated that a single dose of a modified-live NADL-derived BVDV type 1 vaccine will confer protection to dams and their fetuses against challenge-exposure to heterologous BVDV type 1 organisms. (Am J Vet Res 1998;59:1409–1413)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To determine whether a Pasteurella haemolytica A1 mutant that is unable to produce membrane lipoproteins has reduced susceptibility to complement-mediated killing, and to characterize the mutant strain.

Sample Population

12 sera from cattle resistant to P haemolytica challenge exposure after vaccination with P haemolytica or its antigens, or after natural exposure.

Procedures

Complement-mediated killing assays were performed, using wild-type and mutant strains and, as antibody source, various immune sera from cattle that were resistant to P haemolytica challenge exposure. Antibody response to whole-cell antigens produced by mutant and wild-type strains, production of outer membrane proteins and iron-regulated outer membrane proteins by the 2 strains, and growth of the 2 strains in various media were analyzed.

Results

Compared with wild-type P haemolytica, the lipoprotein mutant strain had increased susceptibility to bovine complement-mediated killing. Aside from the lipoproteins that are not produced by the mutant, immunoblot analysis did not reveal differences between immunoreactive antigens that are produced by the 2 strains. Some iron-regulated, outer membrane proteins, which usually are only produced by P haemolytica under iron-deficient conditions, were produced constitutively by the mutant. The mutant grew to a lower final cell density and at a lower rate under conditions likely to reflect those encountered in vivo.

Conclusions

Lack of 3 membrane lipoproteins resulted in enhanced susceptibility to bovine complement-mediated killing. Site-specific mutagenesis of genes encoding P haemolytica membrane lipoproteins alters production of iron-regulated outer membrane proteins by P haemolytica. Growth characteristics of the mutant suggested that it may have reduced capacity for survival in vivo. (Am J Vet Res 1998;59:1275-1280)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To evaluate in vitro effect of the major fraction of outer membrane proteins of Pasteurella multocida with porin-like activities on some biological functions of bovine neutrophils.

Animals

Neutrophils from 5 adult cattle.

Procedure

Variations in such biological processes as actin polymerization and chemotaxis and evaluation of hydrogen peroxide attributable to variable concentrations of P multocida were recorded and compared. Data were obtained, using the porin and lipopolysaccharide (LPS) isolated from a strain of P multocida cultivated in brain-heart infusion (BHI) broth. Various concentrations of porin and LPS were analyzed to evaluate changes in functional activation and microbicidal activity of bovine neutrophils.

Results

The 37.5-kd major polypeptide of the outer membrane of P multocida was isolated. Presence of this porin was significantly correlated with variations of some biological functions of bovine neutrophils. These immunocompetent cells had a concentration-dependent increase in actin polymerization and chemotactic activity. A concentration-dependent variation in the oxidative burst also was observed.

Conclusions

The porins of gram-negative bacteria affect several biological functions of cells involved in the immune response as well as in inflammation. Significant correlation of results of in vitro experiments also was identified between porin and LPS effect. Pretreatment of bovine neutrophils with various concentrations of porin always caused a concentration-dependent increase in examined biological activities. (Am J Vet Res 1998;59:1270–1274)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To characterize Listeria monocytogenes from tissues of channel catfish for their ability to cause hemolysis and grow intracellularly in mouse macrophages.

Samples

15 isolates from processed fillets and 15 isolates from the brain, spleen, and kidneys.

Procedure

Serotype and hemolytic activity of L monocytogenes isolates were evaluated, using plate agglutination and CAMP tests, respectively. Invasiveness of L monocytogenes was determined by inoculating each strain or isolate on J774A.1 macrophage cells. Infected cells were incubated for 0 or 3 hours and lysed; then 100 μΙ of the lysate was plated onto a brain heart infusion agar plate. Colony counts for each strain or isolate were analyzed statistically.

Results

Of 30 isolates, 19 were serotype 1 and 11 were serotype 4. Mouse J774A.1 macrophages were inoculated with catfish isolates, a wild-type (EGD) or a nonhemolytic strain of L monocytogenes. Seventy-three percent (11/15) of isolates originating from catfish organs and 100% (15/15) of isolates originating from fillets were not significantly different from the wild-type EGD strain. The nonhemolytic L monocytogenes strain used as a negative control failed to replicate. Intracellular growth of all L monocytogenes isolates decreased after an additional 3-hour incubation period with medium containing 50 μg/ml of gentamicin.

Conclusions

Similar to the wild-type EGD strain, most channel catfish L monocytogenes isolates were hemolytic, serotype 1 or 4, and were invasive for mouse J774A.1 macrophages.

Clinical Relevance

L monocytogenes growth in mouse macrophages may serve as an in vitro model for determining virulence of isolates from food products or environments. (Am J Vet Res 1998;59:1125-1128)

Free access
in American Journal of Veterinary Research

Abstract

Objectives

To characterize, on a molecular basis, variable regions of the SzP proteins of the Moore and Bryans serovars of Streptococcus zooepidemicus and specificity of opsonic responses.

Sample Population

14 Moore and Bryans serovars of S zooepidemicus.

Procedure

Using polymerase chain reaction analysis and primers from the 5’ and 3’ sequences of the prototype gene SzPW60, the SzP genes of each Moore and Bryans serovar were sequenced and translated, then the amino acid sequences were compared.

Results

Comparison of the amino acid sequences revealed 2 variations at the N terminus; a hypervariable (HV) region from residue 106 to 166, approximately; and proline-glutamic acid-proline-lysine repeats in the carboxy terminus that ranged in number from 7 to 12. Five distinct motifs, HV 1 to 5, which varied independently of the N termini were found in the internal HV region. All serovars were opsonized by antiserum to the prototype SzPW60 protein, indicating that opsonogenic epitopes are on the conserved regions of the protein.

Conclusion and Clinical Relevance

Variant motifs may be valuable in epizootiologic and pathogenesis studies of S zooepidemicus infections of the respiratory tract of young horses and in determining whether there are populations of S zooepidemicus unique to specific animal hosts. It is also clear from the opsonic responses to SzP that at least a portion of the protective responses are probably not serovar specific. (Am J Vet Res 1998;59:1129-1133)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To determine whether Staphylococcus aureus can colonize in horn flies and whether colonization is sufficiently persistent for transmission of the organism to cows by flies.

Animals

2 Jersey heifers exposed to infected horn flies.

Procedure

Staphylococcus aureus was allowed to colonize in horn flies, and duration of colonization was determined. Flies with colonized S aureus were allowed to feed on teats of uninfected heifers to determine whether intramammary infection could be transmitted from fly to heifer. Scab material from naturally infected heifers was submitted for bacteriologic culture to determine whether Saureus was present and whether scabs could serve as a possible source of S aureus for flies.

Results

Staphylococcus aureus colonized in horn flies and remained for up to 96 hours after exposure. Exposure of teats of uninfected heifers to horn flies colonized with S aureus resulted in intrammmary infection in 3 of 4 exposed teats. Culture of scab material from teats of naturally infected heifers revealed high concentration of S aureus (> 107 colony-forming units/mg), and flies without previously colonized S aureus were allowed to feed on scabs; Saureus colonized in them just as readily as it did in flies that had fed on experimentally infected blood.

Conclusions

Horn flies are capable of transmitting Saureus-induced intramammary infection to heifers, and scabs on teats are a potential source of S aureus. Fly control on dairy cows in herds with known Saureus problems is recommended as a method to help prevent these infections. (Am J Vet Res 1998;59: 1122-1124)

Free access
in American Journal of Veterinary Research

SUMMARY

Objective

To determine whether feline herpesvirus 1 (FHV-1) DNA is in the corneas of clinically normal cats and cats with eosinophilic keratitis or corneal sequestration.

Sample Population

Corneal biopsy specimens obtained from cats referred for treatment of corneal sequestration or eosinophilic keratitis.

Procedure

Corneal scraping or keratectomy specimens collected from clinically normal cats, cats with eosinophilic keratitis, and cats with corneal sequestration were evaluated for FHV-1 DNA by use of polymerase chain reaction (PCR). DNA was extracted from the tissue, and 1 μg was assayed for FHV-1 by use of a single-round (40 cycles) PCR assay with primers directed at a 322-bp region of the thymidine kinase gene. Polymerase chain reaction positivity for clinically normal and affected cats of various breeds was compared by χ2 analysis at α = 0.05.

Results

The FHV-1 DNA was detected in 5.9% (1/17) of corneas from clinically normal cats, in 55.1% (86/156) of corneal sequestra, and in 76.3% (45/59) of scraping specimens from cats with eosinophilic keratitis. Prevalence was significantly (P < 0.001) greater for cats with corneal sequestration or eosinophilic keratitis than for clinically normal cats. For cats with corneal sequestration, prevalence of FHV-1 DNA was significantly lower in Persian and Himalayan, compared with domestic shorthair and longhair breeds.

Conclusion

Data strongly imply involvement of FHV-1 in the pathogenesis of eosinophilic keratitis and corneal sequestration. In Persian and Himalayan breeds, however, other nonviral factors also appear to be involved.

Clinical Relevance

Feline herpesvirus 1 must be considered when treating cats with corneal sequestration or eosinophilic keratitis. (Am J Vet Res 1998;59:856–858)

Free access
in American Journal of Veterinary Research

SUMMARY

Objective

To screen supernatants of Pasteurella haemolytica cultures grown in 4 serum-free culture media for maximal leukotoxin (LKT) production and minimal protein concentration as an optimal source of LKT for purification.

Sample Population

One strain of P haemolytica biotype A serotype 1 originally isolated from the pneumonic lung of a calf.

Procedure

Pasteurella haemolytica was grown in brain-heart infusion (BHI) broth, yeast-tryptone broth, RPMI-1640 medium, and McCoy's modified 5A medium. Culture biomass and protein concentration, LKT activity, and LKT concentration in culture supernatants were measured. Effects of media pH and supplementation with metal cations and glucose on growth rate of P haemolytica and culture supernatant parameters were evaluated.

Results

Pasteurella haemolytica cultivated in BHI broth or RPMI-1640 medium containing 0.1M phosphate (pH 6.8) produced the highest concentrations of LKT. Supplementation of RPMI-1640 medium with 0.36 mM FeCl3 or 1.0 mM MgSO4 further increased specific activity of LKT in culture supernatant, but addition of 1 % glucose did not enhance LKT production. Leukotoxin production in MgSO4-supplemented RPMI-1640 medium was comparable to that in serum protein-supplemented medium.

Conclusions

Although BHI broth was superior to RPMI-1640 medium for P haemolytica growth and LKT production, the higher protein concentration and lower LKT specific activity made BHI broth a less desirable medium, compared with RPMI-1640 medium. Growth rate and LKT production with minimal protein content was optimal in pH 6.8 phosphate-buffered MgSO4-supplemented RPMI-1640 medium. This medium can serve as a source of culture supernatant for purification of LKT. (Am J Vet Res 1998;59:851–855)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To determine systemic and mucosal antibody responses in calves to Pasteurella haemolytica 1:A and to 2 major outer membrane proteins (OMP) and 1 major iron-regulated OMP of P haemolytica 1:A.

Animals

23 crossbred calves.

Procedure

2 experiments were performed. In the first experiment, 6 calves were vaccinated and challenge exposed intranasally with an aerosol of P haemolytica 1:A and 6 calves were only challenge exposed. In the second experiment, 8 calves were vaccinated in the area of the tracheal bifurcation with an aerosol of P haemolytica 1:A and 3 calves were used as controls. Serum, nasal secretions, and bronchoalveolar lavage (BAL) samples were collected, and IgG1, IgG2, IgA, and IgM titers were determined. Nasal secretions and BAL samples were also submitted for bacterial culture.

Results

Serum antibody responses in the 2 groups were similar. Antibody titers in nasal secretions and BAL samples increased in calves vaccinated intranasally. In calves vaccinated in the area of the tracheal bifurcation, antibody titers increased in BAL samples but not in nasal secretions. Antibody responses did not correlate with results of bacterial culture.

Conclusions

Results indicated that intranasal administration of P haemolytica 1:A may be a better method for stimulating protective immune responses in the upper portion of the respiratory tract than lung administration. The single dilution ELISA provided a reliable and economical method for determining antibody titers. (Am J Vet Res 1998;59:727-732)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To evaluate polymerase chain reaction (PCR) for detection of Lawsonia intracellularis DNA in feces and an indirect fluorescent antibody test (IFAT) for detecting serum IgG antibodies in pigs exposed to L intracellularis.

Animals

15 seven-week-old pigs and 42 three-week-old pigs.

Procedure

During 3 experiments, 23 pigs were inoculated with a pure culture of L intracellularis, 31 pigs served as noninoculated controls, and 3 pigs were used as sentinels. Fecal shedding of L intracellularis was monitored by use of PCR analysis at 7-day intervals. At euthanasia, the ileum was obtained for PCR and histologic analyses. Serum was obtained at 7-day intervals for use in the IFAT.

Results

Polymerase chain reaction analysis detected L intracellularis DNA in the feces of 39% of the inoculated pigs; by postinoculation days 21 to 28, 90% of inoculated pigs developed IgG antibodies detected by IFAT. Neither L intracellularis DNA nor IgG antibodies were detected in any of the noninoculated control pigs at euthanasia. Sera from pigs inoculated with enteric pathogens other than L intracellularis did not contain detectable antibodies that reacted with L intracellularis by use of the IFAT.

Conclusion

The IFAT for L intracellularis IgG antibody detection appeared to be a more sensitive antemortem test for detecting pigs experimentally infected with L intracellularis than was a PCR method for direct detection of the organism in the feces.

Clinical Relevance

Not all animals that are infected with L intracellularis shed the organism in feces at detectable amounts. (Am J Vet Res 1998;59:722-726)

Free access
in American Journal of Veterinary Research