Evaluation of clustering of new intramammary infections in the bovine udder, including the impact of previous infections, herd prevalence, and somatic cell count on their development.

Evidence in the literature exists to support the theory that mastitis and intramammary infection (IMI) tend to cluster within herds, within cows, and within quarters, facts which may have overarching ramifications on mastitis management in modern dairy herds. Most previous studies, however, have been carried out on prevalent IMI instead of new IMI (NIMI), although reducing incidence of NIMI is a major step toward controlling mastitis. The Canadian Bovine Mastitis Research Network (Saint-Hyacinthe, QC, Canada) has a large mastitis database derived from a 2-yr data collection on a national cohort of dairy farms, and data from this initiative were used to investigate the effect of clustering on the acquisition of NIMI. Longitudinal milk samplings of clinically normal udders taken over several 6-wk periods as well as samples from cows pre-dry-off and postcalving were used (n=73,772 quarter milk samples). Multilevel logistic models were used to evaluate the effect of location of IMI in quarters of the bovine udder previous to occurrence of an NIMI with Staphylococcus aureus, coagulase-negative staphylococci, and Corynebacterium spp. Several factors were investigated, including the number and location of quarters infected with the pathogen of interest before occurrence of an NIMI, the number of quarters infected with any other pathogen before occurrence of an NIMI (a measure of susceptibility), somatic cell count of the quarter before occurrence of an NIMI, somatic cell count of the other 3 quarters before occurrence of an NIMI, prevalence of the specific pathogen in the herd, and the average somatic cell count of the herd. The amount of variation occurring at different levels (herd, cow, and quarter) for the various pathogens was also calculated. The presence of an IMI in the ipsilateral quarter was associated with an elevated risk of an NIMI occurring for all pathogens investigated. Risk of an NIMI increased considerably as herd prevalence of the pathogen rose. Substantial clustering was found at all levels, with roughly equal amounts of variation found in all 3 levels for coagulase-negative staphylococci, most variation at the cow-level for Corynebacterium spp., and most variation found at the quarter-level for Staph. aureus. Simulation was used to calculate exact values of intraclass correlation coefficients to estimate clustering within cows and within quarters--these exact values were, for the most part, lower than estimates calculated using the latent variable approach, but also increased as pathogen prevalence and number of infections in a cow at the previous sampling increased. These results of these analyses can be used to inform approaches to preventing NIMI in modern dairy operations.

Serum resistance and the traT gene in bovine mastitis-causing Escherichia coli.

Ninety-five Escherichia coli isolates from bovine mastitis, 47 isolates from milking machine filters, 36 enterotoxigenic (ETEC) and 43 verocytotoxigenic (VTEC) isolates from cows were examined for the ability to resist the bactericidal effects of 90% gnotobiotic calf serum. There was no significant difference in the percentage of isolates in each group which demonstrated resistance. Two potential virulence traits, the traT gene and the K1 capsular antigen, previously shown to be related to serum resistance, in human E. coli pathogens, were also examined. Using colony blot hybridization there was no significant difference in the percentage of isolates in each group carrying the traT gene. A significant relationship between the presence of the traT gene and serum resistance was not found in any of the four groups of E. coli isolates tested. Only 3.2% of the bovine mastitis, 2.1% of the milk filter and 4.6% of the VTEC isolates were positive for the K1 capsular antigen. Again, no correlation between either the K1 antigen and serum resistance or between the K1 antigen and the presence of the traT gene was found in any of the four groups. None of the antimicrobial resistance patterns of the isolates were the same as those demonstrated by R plasmids known to carry the traT gene. Thus, it appears that the traT gene may not be related to serum resistance in bovine E. coli isolates.

In vitro comparison of bovine mastitis and fecal Escherichia coli isolates.

In vitro methods were used to test the hypothesis that Escherichia coli from bovine mastitis are essentially no different from isolates from bovine feces. Fifty E. coli isolates from bovine mastitic milk, 50 from feces of mastitic cows and 50 from feces of healthy cows were compared with respect to biochemical properties and certain potential virulence factors. There were no significant differences among the groups in tests for biotype; production of colicins, colicin V, or Vero cell cytotoxicity; and growth in 90% gnotobiotic calf serum or 90% normal milk whey. Resistance to killing in 90% gnotobiotic calf serum varied from 66 to 84%. Most isolates grew in normal whey: the percentage in a group varied from 86 to 96. Mastitic milk isolates were significantly different from the fecal isolates in adonitol fermentation (P < or = 0.006), production of aerobactin (P < or = 0.026), and ability to grow in 90% mastitic whey (P < or = 0.00004). However, only 40% of mastitis E. coli fermented adonitol and only 20% produced aerobactin. Ninety-six percent of mastitic milk E. coli grew in mastitic whey, whereas 64% and 60%, respectively, of mastitic fecal and normal fecal isolates grew in this medium. It is concluded that none of the properties that were investigated constitute potential virulence factors or markers for ability to induce mastitis; the data are consistent with the hypothesis that mastitic E. coli are simply opportunistic pathogens.

Analysis of the immunodominant antigens of Corynebacterium pseudotuberculosis.

Antibodies to seven antigens in a whole cell lysate of Corynebacterium pseudotuberculosis ranging in molecular mass from 22 to 120 kilodaltons (kDa) were present in sera of 40 sheep and goats infected with C. pseudotuberculosis. Three antigens of about 120, 68, and 31.5 kDa in size were consistently detected with sera from all animals and twenty-two sera had antibodies to 64, 43, 40, and 22 kDa antigens. None of these antigens were detected by sera from 160 sheep in a C. pseudotuberculosis-free research flock. An NaCl extract of C. pseudotuberculosis cells contained one major protein of about 31.5 kDa and four minor proteins of 68, 64, 43, and 22 kDa in molecular mass as shown by Coomassie Blue staining. Immunoblot analysis demonstrated that the three immunodominant antigens identified in the whole cell extract were contained in the NaCl extract. The 31.5-kDa protein was purified from the NaCl extract by fast-protein liquid chromatography gel filtration to near homogeneity. The purified 31.5-kDa protein showed phospholipase D activity as indicated by synergistic hemolysis with Rhodococcus equi factors and sphingomyelinase activity. The 31.5-kDa protein reacted with antibodies in serum from a sheep naturally infected with C. pseudotuberculosis. This serum also had phospholipase D neutralizing activity. On the basis of its molecular mass, biological activity, N-terminal amino acid sequence analysis, and immunoreactivity, the 31.5-kDa protein was identified as the phospholipase D exotoxin of C. pseudotuberculosis.

The use of a microagglutination assay for the detection of antibodies to Corynebacterium pseudotuberculosis in naturally infected sheep and goat flocks.

Two goat flocks comprising 326 animals and four sheep flocks comprising 343 animals, all with a previously recognized problem of abscesses due to Corynebacterium pseudotuberculosis, were examined for the presence of abscesses and antibody titers to C. pseudotuberculosis as detected by direct microagglutination assay. In sheep there was a strong positive relationship between age and titer (p less than 0.0001). However, the relationship in goats between age and titer could not be determined due to a strong interaction between flock and age. When the relationship between abscesses and titer was examined, it was found that goats with abscesses had higher titers than those that did not (p less than 0.05), whereas there was no difference in titer between sheep with abscesses and those without (p = 0.5753). The sensitivity of the microagglutination test was poor to good for both species (52.3% for goats and 89.7% for sheep). The specificity of the test was fair to poor (64.9% for goats and 21.7% for sheep). Given a disease prevalence of 13.5% for goats and 8.5% for sheep the predictive value of the positive test was very poor (18.9% for goats and 9.6% for sheep) but the predictive value of the negative test was good to excellent (89.7% for goats and 95.8% for sheep). The poor specificity of the test and therefore the positive predictive value may be due in part to the criterion of classification of presence of disease, i.e. presence of an abscess at the time of sampling.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of a guarded bronchoscopic method for microbial sampling of the lower airways in foals.

A novel method to reduce contamination of the bronchoscope during microbial sampling of the lower airways of foals was evaluated. Methylene blue (MB) was used as a nasopharyngeal dye marker to assess the relative contamination from the upper airways of bronchoalveolar lavage (BAL) specimens obtained by standard bronchoscopy (SB) and a "guarded" bronchoscopic method (GB). For GB, a clear sterile cellulose sheath was fitted over the bronchoscope in an effort to protect the endoscope tip and channel from contamination. Methylene blue was detected visually in seven of eight BAL samples from foals following SB, but in none of the samples recovered by GB (p less than 0.001). Significantly less MB was detected in BAL by spectrophotometry in the GB group as well (p less than 0.02). The GB was next employed to study the microbial flora in the lower airways of healthy weaned foals (n = 30). Bacteria were isolated from 29 of 30 (97%) BAL samples, and in moderate or large numbers from 26 of 30 (87%) of the foals. Potential pathogens, including Bordetella bronchiseptica, Streptococcus zooepidemicus, Staphylococcus aureus, Mycoplasma felis and Streptococcus pneumoniae, were cultured from the lower airways of foals. In conclusion, the bronchoscope and bronchoalveolar lavage specimens were readily contaminated by a dye marker placed in the nasopharynx of foals, and the degree of contamination was significantly reduced by sheathing the endoscope. This contamination during bronchoscopy may obscure the interpretation of isolates from BAL specimens from foals, which may possess a bacterial flora in the lower airways without cytological evidence of inflammation.

A field trial to evaluate a whole cell vaccine for the prevention of caseous lymphadenitis in sheep and goat flocks.

A field trial to evaluate a whole cell vaccine for the prevention of caseous lymphadenitis (CLA) in sheep and goats was performed in one goat herd and one sheep flock over a period of three years. In goats, there was a nonstatistically significant trend for fewer cases of CLA in the vaccinated animals compared to the controls. In sheep, from six months to 36 months postinitial vaccination, the proportion of vaccinated sheep that developed CLA was significantly less (p less than 0.05) than in the control sheep. The antibody titers to Corynebacterium pseudotuberculosis as detected by microagglutination assay were significantly different (p less than 0.0001) at all times except at the initial vaccination. Swellings occurred at the vaccination site at an incidence level of 29.6% in goats and 34.1% in sheep. The vaccine appeared to be efficacious in reducing the proportion of sheep that developed CLA when challenged naturally in a field situation.

Relation of lipid content and exotoxin production to virulence of Corynebacterium pseudotuberculosis in mice.

Twenty-five isolates of Corynebacterium pseudotuberculosis from lesions of caseous lymphadenitis in goats were examined for cell wall lipid content, exotoxin production, and virulence in mice. The mean percentage of lipid content was 6.52% and significant (P less than 0.005) differences were demonstrated between some isolates. Exotoxin in broth culture filtrates of the 25 isolates was measured by the staphylococcal beta-hemolysin inhibition (BHI) test and a radioassay for phospholipase D. All isolates were positive for exotoxin in both tests and 1 isolate had higher activity than the others. Because the correlation between the BHI test and radioassay was poor, it may be that the radioassay detects only enzymatically active exotoxin, whereas the BHI test detects active and inactive exotoxin. All isolates were virulent in mice at 2 dosage levels used to produce subacute-chronic and acute disease. Differences in virulence among isolates were detected only at the lower dosage level. There was a direct relationship between percentage of lipid and induction of chronic abscessation in mice, but not between lipid content and mortality. Although exotoxin production in vitro could not be related quantitatively to virulence, the results of inoculating mice with bacterial cells and broth culture supernatant were consistent with a role for exotoxin in disease.

Sensitivity and specificity of bronchoalveolar lavage and protected catheter brush methods for isolating bacteria from foals with experimentally induced pneumonia caused by Klebsiella pneumoniae.

One indication for referral of horses to veterinary hospitals is for diagnosis of the microbiologic cause of pneumonia, particularly when the initial treatment fails. Although endoscopic methods have long been available for microbiologic sample collection, accuracy of these methods under these conditions have not been studied in detail. We compared the bacteria isolated from samples obtained by bronchoalveolar lavage (BAL) with those obtained by protected catheter brush (PCB) from foals with unilateral pneumonia induced by inoculation with Klebsiella pneumoniae. As part of previously described clinical trials, foals were administered antimicrobial therapy IM (n = 15) or vehicle IM (n = 7), and collection of distal airway secretion samples was conducted during the treatment period. Sensitivity and specificity of the sample collection methods were assessed by comparison of the isolates from BAL or PCB samples with isolates from tissue of the inoculated lung lobe, which was the most severely affected lung region. Sensitivity and specificity of BAL for recovery of K pneumoniae (challenge strain) and Streptococcus zooepidemicus (common secondary pathogen) was 90 and 69%, respectively, compared with 76 and 85%, respectively, for the PCB method. Sensitivity was significantly (P = 0.03) higher for BAL (100%) than for PCB (69%) for recovery of K pneumoniae (P = 0.03) from lungs. However, difference in the sensitivity of these methods for recovery of S zooepidemicus was not significant. In conclusion, BAL was a more reliable method for recovery of bacteria from the lungs in chronically infected foals that received antimicrobial treatment.

Evaluation of sulbactam plus ampicillin for treatment of experimentally induced Klebsiella pneumoniae lung infection in foals.

Efficacy of sulbactam, a beta-lactamase inhibitor, in combination with ampicillin, was evaluated for treatment of experimentally induced pneumonia caused by beta-lactam-resistant Klebsiella pneumoniae. Infection was experimentally induced in 18 healthy weanling foals that were randomly allocated to 3 treatment groups: sulbactam plus ampicillin (S/A, 3.3 and 6.6 mg/kg of body weight, respectively), ampicillin (6.6 mg/kg), or vehicle only. Foals were treated daily for 7 days; the observer was unaware of treatment status. Compared with ampicillin and vehicle, treatment with S/A resulted in a statistically significant (P less than 0.05) decrease in severity of pneumonia, with regard to bronchoalveolar lavage cytologic findings (decreased total cell and neutrophil numbers, and increased lymphocyte numbers) and extent of macroscopic lesions in lung tissue of the noninoculated regions. Marked trends toward improvement of S/A-treated foals were observed for quantitative results of bacteriologic culture of bronchoalveolar lavage fluid samples (P less than 0.07), macroscopic pathologic features of the whole lung (P less than 0.1), and histopathologic variables (P less than 0.07), compared with ampicillin- and vehicle-treated foals. Treatment effects were not observed for radiographic, hematologic, and blood gas abnormalities that resulted from infection. In conclusion, the combination of sulbactam plus ampicillin was found to have synergistic effects in vivo, to reduce the extent and severity of experimentally induced gram-negative lung infection in foals.

Bacteriological and histological profile of turkeys condemned for cyanosis.

Canadian Food Inspection Agency (CFIA) has adopted the term cyanosis to describe a category of condemnation for poultry that is dark but has no other condemnable lesions. Two case-control studies (n = 30 pairs; n = 65 pairs) of 18-wk-old tom turkeys were conducted. A case was defined as a carcass condemned by the veterinary inspector for cyanosis, and a control carcass was one that passed inspection. Microbiological tests were conducted on samples of Pectoralis major and Gastrocnemius lacteralis. A modified Rappaport Vassiliadis medium was used for Salmonella, and a Petrifilm method was used to assess aerobic counts, coliform counts, and Escherichia coli. The Salmonella (qualitative) test was negative for all cases and controls, and there were no significant differences between the aerobic counts, coliform counts, and E. coli counts of case and control carcasses. Two pathologists conducted a blind histopathological study: there were no lesions compatible with those of septicemia-toxemia, as defined by CFIA and the USDA, nor any significant histopathological differences between the skin, P. major, G. lateralis, kidney, liver, spleen, small intestine, pancreas, lung, and heart of cases and controls. The inter-rater agreement between pathologists ranged from good to excellent (Kappa = 0.7 to 1.0). In the absence of important lesions and microbial contamination, carcasses with this color change alone should be suitable for human consumption.

The enteritis complex in domestic rabbits: A field study.

A study of the causative agents of enteritis in domestic rabbits from 44 different accessions is described. In descending order of frequency, the organisms most commonly demonstrated were intestinal and hepatic coccidia (Eimeria species), Escherichia coli, Clostridium spp., Salmonella, Bacillus piliformis, and rotavirus. The species of Eimeria identified included those moderately pathogenic and coccidia of low pathogenicity. Using seven antisera against known enterpathogenic strains of E. coli, only one strain, O15, was identified in three cases. Clostridium perfringens or C. spiroforme was demonstrated in the intestinal contents in 11 cases, and lesions compatible with clostridial enteropathy were identified on gross and histopathology. In a serological survey, over 50% of 200 fryer rabbits submitted to Ontario abattoirs and of animals from commercial rabbitries had detectable antibody to rotavirus, indicating the widespread distribution of rotaviral infections in this species. In the cases of enteritis studied, two or more potentially pathogenic organisms were frequently identified, emphasizing that several different organisms may be acting in concert to produce clinical disease.

Development of a novel protein microarray method for serotyping Salmonella enterica strains.

An antibody microarray assay was developed for Salmonella serotyping based on the Kauffmann-White scheme. A model (8 by 15) array was constructed using 35 antibodies for identification of 20 common Salmonella serovars and evaluated using 117 target and 73 nontarget Salmonella strains. The assay allowed complete serovar identification of 86 target strains and partial identification of 30 target strains and allowed exclusion of the 73 nontarget strains from the target serovars.

Antimicrobial susceptibility testing of veterinary clinical isolates with the Sceptor System.

The Sceptor System (Becton Dickinson) was compared with an agar dilution method for antimicrobial susceptibility testing of veterinary clinical isolates. The results indicate that the Sceptor System may be used to test gram-positive and fastidious gram-negative bacteria.

Evaluation of the Sceptor system for identification of bacteria of veterinary origin.

The Sceptor system (Becton Dickinson Diagnostic Instrument Systems, Towson Md.) was assessed for its ability to identify veterinary clinical isolates. A total of 605 bacteria, including 315 isolates of the family Enterobacteriaceae, 191 gram-negative nonenteric bacteria, and 99 gram-positive bacteria, were tested. Overall, 534 (88.3%) were correctly identified, 28 (4.6%) were not identified, 12 (2.0%) were incorrectly identified at the genus levels, and 32 (5.3%) were incorrectly identified at the species level. The Sceptor system correctly identified 292 (92.7%) isolates of Enterobacteriaceae, 165 (86.4%) gram-negative nonenteric bacteria, and 77 (77.8%) gram-positive bacteria. One hundred thirty organisms not contained in the data base were tested with the Sceptor system to assess the possibility of expanding the data base. The Sceptor system was an acceptable method for the identification of isolates of Enterobacteriaceae but not gram-negative nonenteric and gram-positive bacteria of animal origin. Development of a veterinary isolate-specific data base would improve the utility of the Sceptor system in veterinary diagnostic bacteriology.

Characterization of strains of corynebacterium pseudotuberculosis.

Twenty-five strains of Corynebacterium pseudotuberculosis isolated from lesions of caseous lymphadenitis in goats were examined for their biochemical characteristics, antimicrobial susceptibility, and phospholipase D activity. The strains were uniform in biochemical reactions, cultural characteristics, and susceptibility to antimicrobial agents. Presence of urease and phospholipase D and absence of pyrazinamidase were valuable criteria in the identification of C. pseudotuberculosis.