In this study the tonB2 gene was cloned from Actinobacillus pleuropneumoniae JL01 (serovar 1) and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli BL21(DE3). The GST fusion protein was recognized by antibodies in serum positive for A. pleuropneumoniae by Western blot analysis. Purified soluble GST-TonB2 was assessed for its ability to protect BALB/c mice against A. pleuropneumoniae infection. Mice were vaccinated with GST-TonB2 subcutaneously and challenged intraperitoneally with either approximately 4.0 × 10(5) colony-forming units (CFU) or approximately 1.0 × 10(6) CFU of A. pleuropneumoniae 4074. They were examined daily for 7 d after challenge. The survival rate of the TonB2-vaccinated mice was significant higher than that of the mice given recombinant GST or adjuvant alone. These results demonstrate that A. pleuropneumoniae TonB2 is immunogenic in mice and should be further assessed as a potential candidate for a vaccine against A. pleuropneumoniae infection. In addition, an indirect enzyme-linked immunosorbent assay (ELISA) based on the GST-TonB2 recombinant protein was developed. Compared with the ApxIVA ELISA, the TonB2 ELISA provided earlier detection of antibodies in pigs at various times after vaccination with A. pleuropneumoniae live attenuated vaccine. When compared with an indirect hemagglutination test, the sensitivity and specificity of the TonB2 ELISA were 95% and 88%, respectively. The TonB2 ELISA provides an alternative method for rapid serologic diagnosis of A. pleuropneumoniae infection through antibody screening, which would be especially useful when the infection status or serovar is unknown.

Objective—To identify and characterize cytochrome P450 enzymes (CYPs) responsible for the metabolism of racemic ketamine in 3 mammalian species in vitro by use of chemical inhibitors and antibodies. Sample—Human, canine, and equine liver microsomes and human single CYP3A4 and CYP2C9 and their canine orthologs. Procedures—Chemical inhibitors selective for human CYP enzymes and anti-CYP antibodies were incubated with racemic ketamine and liver microsomes or specific CYPs. Ketamine N-demethylation to norketamine was determined via enantioselective capillary electrophoresis. Results—The general CYP inhibitor 1-aminobenzotriazole almost completely blocked ketamine metabolism in human and canine liver microsomes but not in equine microsomes. Chemical inhibition of norketamine formation was dependent on inhibitor concentration in most circumstances. For all 3 species, inhibitors of CYP3A4, CYP2A6, CYP2C19, CYP2B6, and CYP2C9 diminished N-demethylation of ketamine. Anti-CYP3A4, anti-CYP2C9, and anti-CYP2B6 antibodies also inhibited ketamine N-demethylation. Chemical inhibition was strongest with inhibitors of CYP2A6 and CYP2C19 in canine and equine microsomes and with the CYP3A4 inhibitor in human microsomes. No significant contribution of CYP2D6 to ketamine biotransformation was observed. Although the human CYP2C9 inhibitor blocked ketamine N-demethylation completely in the canine ortholog CYP2C21, a strong inhibition was also obtained by the chemical inhibitors of CYP2C19 and CYP2B6. Ketamine N-demethylation was stereoselective in single human CYP3A4 and canine CYP2C21 enzymes. Conclusions and Clinical Relevance—Human-specific inhibitors of CYP2A6, CYP2C19, CYP3A4, CYP2B6, and CYP2C9 diminished ketamine N-demethylation in dogs and horses. To address drug-drug interactions in these animal species, investigations with single CYPs are needed.

Objective—To determine whether canine tumor cell lines express functional tissue factor and shed tissue factor-containing microparticles. Sample—Cell lines derived from tumors of the canine mammary gland (CMT12 and CMT25), pancreas (P404), lung (BACA), prostate gland (Ace-1), bone (HMPOS, D-17, and OS2.4), and soft tissue (A72); from normal canine renal epithelium (MDCK); and from a malignant human mammary tumor (MDA-MB-231). Procedures—Tissue factor mRNA and antigen expression were evaluated in cells by use of canine-specific primers in a reverse transcriptase PCR assay and a rabbit polyclonal anti-human tissue factor antibody in flow cytometric and immunofluorescent microscopic assays, respectively. Tissue factor procoagulant activity on cell surfaces, in whole cell lysates, and in microparticle pellets was measured by use of an activated factor X-dependent chromogenic assay. Results—Canine tissue factor mRNA was identified in all canine tumor cells. All canine tumor cells expressed intracellular tissue factor; however, the HMPOS and D-17 osteosarcoma cells lacked surface tissue factor expression and activity. The highest tissue factor expression and activity were observed in canine mammary tumor cells and pulmonary carcinoma cells (BACA). These 3 tumors also shed tissue factor-bearing microparticles into tissue culture supernatants. Conclusions and Clinical Relevance—Tissue factor was constitutively highly expressed in canine tumor cell lines, particularly those derived from epithelial tumors. Because tumor-associated tissue factor can promote tumor growth and metastasis in human patients, high tissue factor expression could affect the in vivo biological behavior of these tumors in dogs.

This study investigated bovine coronavirus (BCV) in both beef calves direct from the ranch and commingled, mixed-source calves obtained from an auction market. The level of BCV-neutralizing antibodies found in the calves varied among ranches in 2 different studies in a retained-ownership program (ROP), from the ranch to the feedlot. Calves with low levels of BCV-neutralizing antibodies (16 or less) were more likely to be treated for bovine respiratory disease (BRD) than those with higher titers. In 3 studies of commingled, mixed-source calves, BCV was recovered from calves at entry to the feedlot and the infections were cleared by day 8. The BCV was identified in lung samples [bronchoalveolar lavage (BAL) collection] as well as in nasal swabs. Calves with low levels of BCV-neutralizing antibodies at entry were most likely to be shedding BCV. Bovine coronavirus was isolated from both healthy and sick calves, but not from sick calves after 4 d arrival at the feedlot. Bovine coronavirus (BCV) should be considered along with other bovine respiratory viruses in the diagnosis of etiologies in bovine respiratory disease, especially for animals that become sick shortly after arrival. If approved vaccines are developed, it would be best to carry out vaccination programs before calves are weaned, giving them sufficient time to gain active immunity before commingling with other cattle.

Objective—To evaluate a commercially available modified-live Streptococcus equi subsp equi vaccine for safety and persistence in vaccinated ponies and to detect recombination or reversion events in the vaccine strain. Animals—5 ponies that were 1.5 to 8 years old (group 1) and 4 ponies that were 6 months old (group 2). Procedures—Ponies were vaccinated, with a subsequent booster vaccination 2 to 3 weeks later, and monitored for 50 days. At booster vaccination, an equal amount of a tracycline-resistant wild-type strain of S equi was administered. Recovery of all strains was performed by use of bacteriologic culture and PCR assays. Results—Ponies in group 1 had background antibody titers against S equi antigen before vaccination despite the lack of known exposure to S equi. Ponies in group 2 were immunologically naïve. Increases in anti-S equi antibody titers were detected in both groups. Ponies in group 1 did not have clinical signs of disease caused by S equi. In group 2, all ponies developed abscesses in retropharyngeal lymph nodes; 1 pony developed severe clinical disease and was euthanized. The vaccine strain was recovered from ponies in group 2 for up to 24 days after vaccination. Conclusions and Clinical Significance—Although the vaccine was successful in inducing IgG antibodies against S equi in all ponies, findings suggested that the vaccine may have caused substantial morbidity and some deaths in the young ponies. In young ponies, the vaccine strain persisted in tissues for weeks; however, no evidence of recombination was detected.

Objective—To evaluate the safety and efficacy of an experimental adjuvanted DNA-plasmid vaccine against West Nile virus (WNV) in red-tailed hawks (Buteo jamaicensis). Animals—19 permanently disabled but otherwise healthy red-tailed hawks of mixed ages and both sexes without detectable serum antibodies against WNV. Procedures—Hawks were injected IM with an experimental WNV DNA-plasmid vaccine in an aluminum-phosphate adjuvant (n = 14) or with the adjuvant only (control group; 5). All birds received 2 injections at a 3-week interval. Blood samples for serologic evaluation were collected before the first injection and 4 weeks after the second injection (day 0). At day 0, hawks were injected SC with live WNV. Pre- and postchallenge blood samples were collected at intervals for 14 days for assessment of viremia and antibody determination; oropharyngeal and cloacal swabs were collected for assessment of viral shedding. Results—Vaccination was not associated with morbidity or deaths. Three of the vaccinated birds seroconverted after the second vaccine injection; all other birds seroconverted following the live virus injection. Vaccinated birds had significantly less severe viremia and shorter and less-intense shedding periods, compared with the control birds. Conclusions and Clinical Relevance—Use of the WNV DNA-plasmid vaccine in red-tailed hawks was safe, and vaccination attenuated but did not eliminate both the viremia and the intensity of postchallenge shedding following live virus exposure. Further research is warranted to conclusively determine the efficacy of this vaccine preparation for protection of red-tailed hawks and other avian species against WNV-induced disease.

Escherichia coli O157:H7 remains a threat to humans via cattle-derived fecal contamination of food and water. Preharvest intervention strategies represent a means of reducing the pathogen burden before harvest. In this study, the efficacy of a commercially produced type III secreted protein (TTSP) vaccine was evaluated with the use of a commingled experimental calf infection model (30 placebo-treated animals and 30 vaccinates). The calves were vaccinated on days 0, 21, and 42 and infected with 109 colony-forming units (CFU) of E. coli O157 by oral–gastric intubation on day 56. Fecal shedding was monitored daily for 14 d. Serologic assessment revealed a robust immune response to vaccination; the serum titers of antibodies against EspA, Tir, and total TTSPs were significantly higher in the vaccinates than in the placebo-treated animals on days 21, 42, 56, and 70. Significantly less (P = 0.011) of the challenge organism was shed by the vaccinates than by the placebo-treated animals on days 3 to 10. Peak shedding occurred in both groups on days 3 to 6; during this period the vaccinates showed a mean log reduction of 1.4 (P = 0.002) and a mitigated fraction of 51%. The number of animals shedding was significantly lower among the vaccinates compared with the placebo group on days 3 to 6 (P ≤ 0.05), with a mean prevented fraction of 21%. No differences in the duration of shedding were observed. Owing to the low challenge shedding in both groups on days 11 to 14 (mean CFU/g < 10; median = 0), no significant differences were observed. These data indicate that TTSP vaccination had protective effects through significant reductions in the number of animals shedding and the number of challenge organisms shed per animal and provides evidence that TTSP vaccination is an effective preharvest intervention strategy against E. coli O157.

Objective-To develop and evaluate a sandwich ELISA incorporating rabbit antiserum specific for canine surfactant protein A (SP-A) for use in measuring concentrations of SP-A in serum of dogs Sample-Serum samples obtained from 6 healthy dogs and 3 dogs with pulmonary disease. Procedures-Rabbit antiserum was prepared against purified canine SP-A. The IgG fraction was isolated via protein G affinity chromatography and was then biotinylated. The sandwich ELISA was performed by use of anti-SP-A antibody (IgG) preabsorbed with sera from healthy dogs. Validity of the ELISA was confirmed by determination of the detection limit, precision, reproducibility, and accuracy. Serum SP-A concentrations were measured in 6 healthy dogs and 3 dogs with pulmonary disease. Results-Detection limit of the ELISA was 2.0 ng/mL. Within- and between-assay coefficients of variation ranged from 3.8% to 14.1% and from 15.5% to 35.6%, respectively. The observed-to-expected recovery ratio ranged from 77.1% to 89.9%. Serum SP-A concentrations measured by use of the ELISA were ≤ 2.3 ng/mL in the 6 healthy dogs, 25.6 ng/mL in a dog with severe cardiac pulmonary edema, 8.3 ng/mL in a dog with pneumonia, and 10.1 ng/mL in a dog with lung lobe torsion. Conclusions and Clinical Relevance-The sandwich ELISA was found to be useful for measuring purified canine SP-A concentrations and canine SP-A concentrations in serum samples. The ELISA was precise, reproducible, and accurate. The ELISA may be beneficial in assessing serum concentrations of canine SP-A as a potential biomarker of pulmonary diseases in dogs.

Objective—To assess and compare the expression of perinuclear antineutrophilic cytoplasmic antibodies (pANCA) in sera obtained from dogs with inflammatory bowel disease (IBD) and dogs with intestinal lymphoma. Animals—104 dogs with IBD and 23 dogs with intestinal lymphoma. Procedures—Each ill dog had persistent gastrointestinal signs (> 3 weeks in duration) and absence of response to diet changes or antimicrobial treatments. Gastrointestinal endoscopy was performed in ill dogs to obtain intestinal biopsy specimens for histologic confirmation of IBD or lymphoma. A serum sample was obtained from each ill dog. Neutrophils were isolated from a blood sample from the healthy dog; neutrophil-bearing slides were incubated with serum from each ill dog and examined for expression of pANCA by use of an indirect immunofluorescence technique. Detection of cells that had a perinuclear fluorescence pattern was considered a positive result. Results—The 2 groups of dogs did not differ with regard to breed and sex but did differ with regard to age. Expression of pANCA was detected in 38 of the 104 (36.5%) dogs with IBD and 4 of the 23 (17.4%) dogs with intestinal lymphoma. Although the frequency of pANCA expression was higher in dogs with IBD, compared with findings in dogs with intestinal lymphoma, the difference was not significant. Conclusions and Clinical Relevance—Results indicated that circulating pANCA are present in some dogs with IBD or intestinal lymphoma. However, pANCA detection does not seem to be useful for distinguishing dogs with IBD from dogs with intestinal lymphoma.

Objective-To investigate shedding of chlamydiae from conjunctiva and genital tracts of cats without clinical signs of conjunctivitis or other infectious disease in relation to their titers of serum antibodies against chlamydiae and to serum amyloid A (SAA) and serum alpha1-acid glycoprotein (AGP) concentrations. Animals-62 healthy cats. Procedures-Serum from each cat was analyzed for antibodies against chlamydiae and for SAA and AGP concentrations. Swab samples from the conjunctival sac and genital tract were analyzed with a real-time PCR assay for Chlamydiaceae. Results-4 of 8 of cats with high antibody titers (ie, 1,600) shed chlamydiae, but only from the conjunctiva. Chlamydiae could not be detected in samples from cats with lower antibody titers nor from any genital tract samples. In cats with antibody titers of 1,600, mean +/- SD SAA concentration was significantly higher when chlamydiae were detected in conjunctival swab samples (3.9 +/- 1.0 mg/L) than when no chlamydiae were detected (1.4 +/- 1.0 mg/L). However, SAA concentration was greater than the limit for an acute-phase response in only one of those cats. There was no significant difference in serum AGP concentrations between cats with high titers that were or were not shedding chlamydiae. Nine of 30 (30%) cats (5 with and 4 without detectable serum antibodies against chlamydiae) that had been mated developed reproductive disorders. Conclusions and Clinical Relevance-Clinically normal cats with high chlamydiae-specific antibody titers can shed and thus transmit chlamydiae. Venereal spread from cats without clinical signs of infection is likely not common.