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SUMMARY

Six monoclonal antibodies (mab) to virulent Mycobacterium bovis ATCC 19210 were produced, using a suspension of heat-inactivated whole cells. Immunoglobulin isotype for mab VMB6, VMB73, and VMB93 was IgG1, and for VMB31, VMB99, and VMB119, it was IgG2a. Monoclonal antibodies were examined for cross-reactivity to M tuberculosis, M kansasii, M fortuitum, M paratuberculosis, M avium serovars 1, 2, 4, 8, and 10, M chelonei, M phlei, M scrofulaceum, M smegmatis, Nocardia asteroides, and Rhodococcus equi. Monoclonal antibodies could be grouped on the basis of binding activity by elisa and immunoblot analysis, in which mab VMB6, VMB31, and VMB119 had binding activity to M bovis; mab VMB93 and VMB99 detected M bovis and M tuberculosis antigens, and mab VMB73 reacted with other mycobacterial species, as well as with N asteroides and R equi. Apparent molecular mass of antigens was 30 to 25 kilodaltons (kD) for VMB6, VMB31, and VMB119 and 63 kD for VMB93 and VMB99, and ranged from > 200 to 31 kD for VMB73, as estimated by immunoblot analysis. Monoclonal antibody binding activity to 18 field isolates of M bovis was evaluated, using elisa. Each of 18 field isolates was detected, using mab VMB6, VMB31, or VMB119; 10 isolates were detected, using mab VMB93/VMB99, and 14 were detected by use of mab VMB73. Use of mab in elisa failed to detect antigens from M bovis strain AN-5.

Free access
in American Journal of Veterinary Research

SUMMARY

Latency and reactivation of pseudorabies virus in swine was studied. Thirty-one pigs were assigned to 5 groups and were given 1 of 4 vaccines; 10 remained unvaccinated controls. All pigs were then challenge exposed with a sublethal dose of virulent pseudorabies virus. One hundred one days after challenge exposure, all pigs were treated with dexamethasone to reactivate the virus. Virus-positive tonsil and nasal mucus isolates were recovered from 29 of the 31 pigs over a 12-day period. Frequency and duration of virus-positivity were significantly (P < 0.05) and consistently lower among vaccinated pigs than among the unvaccinated controls. It was concluded that vaccination before challenge exposure had little or no effect on the rate of establishment of virus latency, but that vaccination reduced shedding after subsequent reactivation of the virus.

Free access
in American Journal of Veterinary Research

Summary

Because of the importance of environmental survival of pseudorabies virus to proposals to eradicate the virus from swine in the United States, survival of the virus was studied in various diluents and on combinations of diluents and solid fomites at 25 C. Suspensions of the virus in phosphate-buffered saline and saline G solutions remained infectious for at least 10 days. Infectivity of other virus/diluent suspensions decreased to <10 plaque-forming units/ml in 14 days (swine urine), 7 days (well water), 4 days (swine saliva), 2 days (lagoon water and swine nasal washings), and 1 day (swine pit effluent, chlorinated water, and bile). Suspensions of pseudorabies virus in saline G solution and on the solid fomites, whole corn, and steel remained infectious for at least 7 days. Infectivity of other virus/diluent/fomite combinations decreased to <10 plaque-forming units/ml in 7 days. The role of the fomites as vehicles for transmission of infection is discussed.

Free access
in Journal of the American Veterinary Medical Association

Summary

A technique for detection of bovine viral diarrhea virus (bvdv) from circulating blood leukocytes, using the polymerase chain reaction, is described. The published nucleotide sequences of 2 strains of bvdv and that of hog cholera virus were aligned and the information was used to design oligonucleotides coding for 2 regions of amino acid homology. The oligonucleotides were a mixed population including all possible codons for the conserved amino acids. These degenerate oligonucleotides were used in the polymerase chain reaction to detect viral rna in cells infected in vitro, or in circulating blood leukocytes from infected animals. Virus was detected in over 60 samples from diverse isolates. The detection of bvdv by the polymerase chain reaction is a rapid, sensitive, and specific technique, which represents an improvement over existing technology.

Free access
in American Journal of Veterinary Research

Summary

The effects of alkalinizing agents, administered prior to feeding colostrum, on blood-gas and acid-base values and on absorption of IgGl were determined in 40 newborn Holstein calves. Two treatments, sodium bicarbonate (3 mEq/kg of body weight, IV) and doxapram HCl (2 mg/kg, IV), were evaluated, using a randomized complete-block experimental design. These treatments resulted in significant (P< 0.01) alteration of blood-gas and acid-base values, generally in the direction of normal values for adult cattle. Significant least squares mean effects were detected for sodium bicarbonate treatment on blood pH ( + 0.04 units, P < 0.01), Pco2( + 4.1 mm of Hg, P <0.01), and HCO3 concentration ( + 4.4 mEq/L, P < 0.01). Significant least squares mean effects were detected for doxapram HC1 treatment on blood pH ( + 0.06 pH units, P <0.01) and Pco2(–5.2 mm of Hg, P <0.01). Absorption of colostral IgGl was not affected by the treatments given or by the altered blood-gas and/or acid-base status.

Free access
in American Journal of Veterinary Research

Summary

Chlamydia psittaci proteins capable of binding eukaryotic cell membranes were identified and antigenically characterized. Cell membrane proteins (cmp) of noninfected cells were labeled with biotin (b-cmp), then were extracted with 1% Triton X-100. Nitrocellulose membrane strips containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of chlamydial elementary bodies (eb) were reacted with the b-cmp extract, followed by addition of streptavidin-conjugated horse radish peroxidase. Among the various strains of chlamydiae examined, a protein of approximately 16 to 18 kDa consistently bound b-cmp. A second larger protein, ranging in molecular mass from 24 to 32 kDa, also bound b-cmp. Immunoblotting techniques were used to analyze the reactions of antisera from immunized and experimentally infected animals to these proteins. A rabbit polyclonal antiserum produced against the 18-kDa adhesin of a serovar-1 strain of C psittaci (B577) reacted strongly with 18-kDa proteins of all C psittaci strains, but weakly with that of C trachomatis. Mouse antisera raised against the serovar-2 (FC-Stra) 28-kDa protein reacted only with proteins of the homologous serovar. Sera from experimentally infected animals did not react with the C trachomatis 18-kDa adhesion protein, but did react in 2 patterns with related and nonrelated C psittaci isolates. Two rabbits inoculated with infective serovar-1 eb and 1 rabbit inoculated with a serovar-2 strain reacted specifically with the 18-kDa proteins of their homologous serovars. In contrast, 2 other rabbits inoculated with the same serovar-2 strain produced antisera that reacted with all C psittaci 18-kDa proteins, as did serum from a similarly inoculated bull. The reason for the 2 types of responses to infection remains to be determined.

Free access
in American Journal of Veterinary Research

SUMMARY

Eight strains of Chlamydia psittaci isolated from swine with pneumonia, pleuritis, pericarditis, and enteritis were characterized through analysis of the major outer membrane protein gene ompA by a two-step polymerase chain reaction, by their interactions with cells in culture, and the morphologic features and ultrastructure of intra-cellular inclusions. Amplified chlamydial ompA dna fragments were differentiated by restriction endonuclease digestion. Chlamydial isolates were separated into 2 types on the basis of ompA restriction fragment length polymorphism. Strains of type L71 had finely granular inclusions, whereas those of type 1710S contained pleomorphic reticulate bodies (rb) in the inclusions, which are characteristic of aberrant chlamydial developmental forms. Chlamydial types L71 and 1710S required centrifuge-assisted inoculation for efficient infection of cell cultures. Cultivation in cell culture medium containing cycloheximide increased the numbers of chlamydial inclusions about 1.5-fold. These strains formed few elementary bodies in yolk sac cells of chicken embryos. Ultrastructurally, unique doublet rb were observed, particularly in strains of the ompA type L71. These doublets consisted of 2 rb, bounded by a cytoplasmic membrane, contained within a common cell wall and an extended periplasmic space. Ultrastructural examination of strains of the ompA type 1710S confirmed the aberrant chlamydial developmental forms, but evidence of viral infection of the rb as a cause of these aberrant forms was not found. The strain S45 isolated from intestinal sites of swine was a trachoma restriction fragment length polymorphism type. With the mouse biotype, it represented the second isolate from animals of Chlamydia trachomatis.

Free access
in American Journal of Veterinary Research
in Journal of the American Veterinary Medical Association

Abstract

Objective—To monitor by use of 5-color flow cytometry the antigen-specific responses of subsets of peripheral T cells in cattle inoculated with a killed Mycobacterium avium subsp paratuberculosis (MAP) vaccine and to compare results with those for 2 established cell-mediated immunity assays.

Animals—45 female Holstein cattle with negative results for MAP in skin tests conducted at time of inoculation with MAP.

Procedures—Cattle were allocated to 4 groups. Cattle of group 1 (n = 12) were 0 to 3 months old and inoculated with a killed MAP vaccine. The 10 cattle of group 2 were the same age as those in group 1 but were not inoculated with MAP vaccine. The 11 cattle of group 3 were 9 to 12 months old and inoculated with killed MAP vaccine. The 12 cattle of group 4 were the same age as those in group 3 but were not inoculated with MAP vaccine.

Results—Flow cytometry identified T-cell subsets that responded specifically to the recall antigen. Results of assays for CD25 expression and wholeblood interferon-γ had the strongest correlation with results for skin tests as well as results with each other. Intracellular expression of interferon-γ was not correlated as well with results for the other tests.

Conclusions and Clinical Relevance—Flow cytometry can be useful for characterizing the immune response after administration of MAP vaccine and should be evaluated with regard to its sensitivity and specificity when used in detecting cattle naturally infected with MAP.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To assess the status of antimicrobial resistance (AMR), identify extraintestinal virulence factors (VFs) and phylogenetic origins, and analyze relationships among these traits in extraintestinal pathogenic Escherichia coli (ExPEC) isolates from companion animals.

Sample—104 E coli isolates obtained from urine or genital swab samples collected between 2003 and 2010 from 85 dogs and 19 cats with urogenital infections in Japan.

Procedures—Antimicrobial susceptibility of isolates was determined by use of the agar dilution method; a multiplex PCR assay was used for VF gene detection and phylogenetic group assessment. Genetic diversity was evaluated via randomly amplified polymorphic DNA analysis.

Results—Of the 104 isolates, 45 (43.3%) were resistant to > 2 antimicrobials. Phylogenetically, 64 (61.5%), 22 (21.2%), 13 (12.5%), and 5 (4.8%) isolates belonged to groups B2, D, B1, and A, respectively. Compared with other groups, group B2 isolates were less resistant to all tested antimicrobials and carried the pap, hly, and cnf genes with higher frequency and the aer gene with lower frequency. The aer gene was directly associated and the pap, sfa, hly, and cnf genes were inversely associated with AMR. Randomly amplified polymorphic DNA analysis revealed 3 major clusters, comprised mainly of group B1, B2, and D isolates; 2 subclusters of group B2 isolates had different VF and AMR status.

Conclusions and Clinical Relevance—Prevalences of multidrug resistance and human-like phylogenetic origins among ExPEC isolates from companion animals in Japan were high. It is suggested that VFs, phylogenetic origins, and genetic diversity are significantly associated with AMR in ExPEC.

Full access
in American Journal of Veterinary Research