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Objective: To evaluate seasonal patterns and risk factors for Escherichia coli O157:H7 in feces in a beef cattle herd and determine strain diversity and transition in E coli over time by use of multiple-locus variable-number tandem-repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE)., Sample Population: 456 samples of freshly passed feces collected over a 1-year period from cattle in a range-based cow-calf operation located in the foothills of the Sierra Nevada Mountains in California., Procedures: E coli O157:H7 was recovered from feces by use of immunomagnetic separation and 2 selective media. Virulence factors were detected via reverse transcriptase-PCR assay. Escherichia coli O157:H7 isolates were subtyped with MLVA and PFGE. Prevalence estimates were calculated and significant risk factors determined. A dendrogram was constructed on the basis of results of MLVA typing., Results: Overall prevalence estimate for E coli O157:H7 was 10.5%, with the prevalence lowest during the winter. Mean temperature during the 30 days before collection of samples was significantly associated with prevalence of E coli O157:H7 in feces. Nineteen MLVA and 12 PFGE types were identified., Conclusions and Clinical Relevance: A seasonal pattern was detected for prevalence of E coli O157:H7 in feces collected from beef cattle in California. Subtyping via MLVA and PFGE revealed a diversity of E coli O157:H7 strains in a cow-calf operation and noteworthy turnover of predominant types. Given the importance of accurately determining sources of contamination in investigations of disease outbreaks in humans, MLVA combined with PFGE should be powerful tools for epidemiologists.

Objective: To compare prevalence of tetracycline resistance genes in the fecal flora of conventionally raised feedlot steers and feedlot steers raised without antimicrobials., Sample Population: 61 fecal samples from conventionally raised steers and 61 fecal samples from steers raised without antimicrobials at a single feedlot., Procedures: Total DNA was extracted from each fecal sample and analyzed by means of 4 multiplex PCR assays for 14 tetracycline resistance genes., Results: At least 3 tetracycline resistance genes were identified in all 122 fecal samples. For 5 of the 14 tetracycline resistance genes, the percentage of samples in which the gene was detected was significantly higher for fecal samples from conventionally raised cattle than for fecal samples from antimicrobial-free cattle, and for 1 gene, the percent-age of samples in which the gene was detected was significantly higher for fecal samples from antimicrobial-free cattle than for fecal samples from conventionally raised cattle. The percentage of samples with > or = 11 tetracycline resistance genes was significantly higher for fecal samples from conventionally raised cattle (35/61 [57%]) than for fecal samples from antimicrobial-free cattle (16/61 [26%])., Conclusions and Relevance: Results suggested that the prevalence of tetracycline resistance genes was significantly higher in the fecal flora of conventionally raised feedlot steers than in the fecal flora of feedlot steers raised without antimicrobials and that a metagenomic approach may be useful in understanding the epidemiology of antimicrobial resistance in food animals.

Objective: To determine effects of administration of ceftiofur crystalline-free acid (CCFA) on antimicrobial susceptibility of Escherichia coli in feedlot cattle., Animals: 61 feedlot steers., Procedures: A cohort study was conducted. Steers were housed in pens (5 pens with 10 steers and 1 pen with 11 steers). Five steers in each pen were administered CCFA, and 5 served as control steers (1 pen had 6 control steers). The CCFA administration included a single-dose regimen (6.6 mg/kg, SC, on day 0), two-thirds-dose regimen (4.4 mg/kg, SC, on day 0), and 3-dose regimen (6.6 mg/kg, SC, on days 0, 6, and 13). Fecal samples were collected on days 0, 2, 6, 9, 13, 16, 20, and 28. Fecal samples were collected immediately before CCFA administration. Minimum inhibitory concentrations of 15 antimicrobials were determined for 3 E coli isolates/fecal sample. Escherichia coli were enumerated by use of direct-plating techniques., Results: Resistance to 1 or more antimicrobials was detected in 986 of 1,441 (68.4%) isolates recovered. Administration of CCFA was associated with a transient increase in the population of ceftiofur-resistant isolates. Susceptibility returned to day 0 values (ie, samples collected immediately before CCFA administration) approximately 2 weeks after completion of CCFA administration. Agreement between ceftiofur resistance and co-resistance to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline was almost perfect (kappa 0.97). We did not detect variation in susceptibility of E coli recovered from commingled control steers., Conclusions and Clinical Relevance: Administration of CCFA provided selection pressure that favored transient expansion of multiple-resistant variants.

Objective: To estimate the association between ceftiofur use and the isolation of Escherichia coli with reduced ceftriaxone susceptibility from fecal samples of dairy cow populations., Animals: 1,266 dairy cows on 18 farms in Ohio., Procedures: Individual fecal samples from all cows in the study herds were tested for Escherichia coli with reduced ceftriaxone susceptibility. Herd antimicrobial use policy and antimicrobial treatment records were also obtained. Plasmid DNA from these isolates was tested for the presence of the AmpC beta-lactamase gene (blaCMY-2). Minimum inhibitory concentrations to a standard panel of 16 antimicrobial drugs were determined by use of a broth microdilution system., Results: Herds for which ceftiofur use was reported were more likely to have cows from which reduced susceptibility E coli was isolated than herds that did not report ceftiofur use (odds ratio, 25.0). However, at the individual cow level, no association was found between recent ceftiofur treatment and isolation of reduced-susceptibility E coli (adjusted odds ratio, 1.01). No observed linear relationship was found between the percentage of cows from which E coli with reduced ceftriaxone susceptibility was isolated and the percentage of cows in the herd recently treated with ceftiofur., Conclusions and Clinical Relevance: Our observation of a herd-level but not an individual cow-level association between ceftiofur use and isolation of E coli with reduced ceftriaxone susceptibility from fecal samples suggests that interventions to reduce the spread of antimicrobial resistance genes in agricultural animals will be most effective at the herd level.

Objective: To evaluate various sampling strategies for potential use in measuring prevalence of antimicrobial susceptibility in cattle., Sample Population: 500 isolates of non-type-specific Escherichia coli (NTSEC) isolated from the feces of 50 cows from 2 dairy farms (25 cows/farm and 10 isolates/cow)., Procedures: Diameters of inhibition zones for 12 antimicrobials were analyzed to estimate variation among isolates, cows, and farms and then used to determine sampling distributions for a stochastic simulation model to evaluate 4 sampling strategies. These theoretic sampling strategies used a total of 100 isolates in 4 allocations (1 isolate from 100 cows, 2 isolates from 50 cows, 3 isolates from 33 cows, or 4 isolates from 25 cows)., Results: Analysis of variance composition revealed that 74.2% of variation was attributable to isolates, 18.5% to cows, and 7.3% to farms. Analysis of results of simulations suggested that when most of the variance was attributable to differences among isolates within a cow, culturing 1 isolate from each of 100 cows underestimated overall prevalence, compared with results for culturing more isolates per cow from fewer cows. When variance was not primarily attributable to differences among isolates, all 4 sampling strategies yielded similar results., Conclusions and Clinical Relevance: It is not always possible to predict the hierarchical level at which clustering will have its greatest impact on observed susceptibility distributions. Results suggested that sampling strategies that use testing of 3 or 4 isolates/cow from a representative sample of all animals better characterize herd prevalence of antimicrobial resistance when impacted by clustering.

Objective: To evaluate the effects of using retention-pond water for dust abatement on performance of feedlot steers and carriage of Escherichia coli O157 and Salmonella spp., Design: Matched cohort studies., Animals: 2 groups of feedlot steers comprising 3,510 (pathogen carriage) and 3,737 (performance) animals housed in a large commercial feedlot in the Texas Panhandle., Procedure: Steers were systematically allocated to treatment pens approximately 60 days after arrival (pathogen carriage) or at arrival (performance). For evaluation of pathogen carriage, feces and hide swab specimens were collected from 25 animals in each pen within 10 days of slaughter. Samples were submitted for bacterial culture for E. coli O157 and were tested with a polymerase chain reaction-based assay for Salmonella spp. For evaluation of performance, pen weights of animals were obtained at arrival and slaughter and feed delivered to each pen was recorded. The exposure of interest for both studies was application of retention-pond water through fixed high-pressure sprinklers., Results: Carriage of E. coli O157 and Salmonella spp and animal performance were not adversely affected by exposure to retention-pond water. Prevalences of E. coli O157 in feces, on hides, and either in feces or on hides for those exposed to retention-pond water were 8.3%, 8.9%, and 15.4%, respectively; prevalences for those unexposed to retention-pond water were 11.4%, 15.4%, and 22.6%, respectively., Conclusions and Clinical Relevance: Results suggest that use of retention-pond water for dust abatement in feedlot pens does not adversely affect pathogen carriage or animal performance.

Objectives: To compare stability, antigenicity, and aggregation characteristics of Moraxella bovis cytolysins among isolates from geographically diverse areas., Study Population: 8 isolates of M. bovis., Procedure: Filter-sterilized broth culture supernatants of M. bovis were concentrated, diafiltered, and chromatographed. The endotoxin and cytolysin activities in samples were measured. Chromatographed cytolysins of M. bovis were examined by immunoblotting. Hemolytic and leukotoxic activities were measured from samples collected at each step of purification and before and after storage. Hemolysis was measured directly by use of washed bovine erythrocyte targets. Leukotoxicity was measured by use of a 51Cr release assay., Results: Cytolysin was retained by a filter with 100-kd nominal molecular weight limit. Hemolytic activity, leukotoxic activity, and endotoxin were eluted together in void volume of a gel-filtration column (molecular mass exclusion limit = 4 X 10(7) d). Gel-column chromatographed diafiltered retentate had the greatest specific cytolytic activity and the highest endotoxin-to-protein ratio. Frozen diafiltered retentate(-80 degrees C, 4 months) was cytolytic after thawing. Immunoblots of gel-column chromatographed cytolysin contained 4 proteins with molecular masses between 90 and 68 kd. Fractions with high lytic activities also had additional protein bands with molecular masses of 98 and 63 kd. Immunoblots of gel-column chromatographed diafiltered retentate revealed proteins with molecular masses between 90 and 68 kd., Conclusions and Clinical Relevance: Diafiltered M. bovis cytolysin is aggregated with endotoxin. Antigenicity and cytolytic activities in diafiltered retentate are conserved among M. bovis isolates. Diafiltration could be useful for bulk semipurification of M. bovis cytolysin. Cytolysin-enriched vaccines of M. bovis could be contaminated by endotoxin.

Objective: To evaluate the effectiveness of various sampling techniques for determining antimicrobial resistance patterns in Escherichia coli isolated from feces of feedlot cattle., Sample Population: Fecal samples obtained from 328 beef steers and 6 feedlot pens in which the cattle resided., Procedure: Single fecal samples were collected from the rectum of each steer and from floors of pens in which the cattle resided. Fecal material from each single sample was combined into pools containing 5 and 10 samples. Five isolates of Escherichia coli from each single sample and each pooled sample were tested for susceptibility to 17 antimicrobials., Results: Patterns of antimicrobial resistance for fecal samples obtained from the rectum of cattle did not differ from fecal samples obtained from pen floors. Resistance patterns from pooled samples differed from patterns observed for single fecal samples. Little pen-to-pen variation in resistance prevalence was observed. Clustering of resistance phenotypes within samples was detected., Conclusions and Clinical Relevance: Studies of antimicrobial resistance in feedlot cattle can rely on fecal samples obtained from pen floors, thus avoiding the cost and effort of obtaining fecal samples from the rectum of cattle. Pooled fecal samples yielded resistance patterns that were consistent with those of single fecal samples when the prevalence of resistance to an antimicrobial was > 2%. Pooling may be a practical altemative when investigating patterns of resistance that are not rare. Apparent clustering of resistance phenotypes within samples argues for examining fewer isolates per fecal sample and more fecal samples per pen.

Objective: To determine effects of vaccination prior to transit and prophylactic administration of florfenicol at time of arrival at a feedyard on health of cattle and colonization of the nasopharynx by Mannheimia haemolytica (MH)., Animals: 121 steers from Tennessee and 84 steers from New Mexico., Procedure: Half of the steers were vaccinated before transport to a feedyard. Steers from Tennessee were vaccinated with MH bacterin-toxoid, and steers from New Mexico were vaccinated intranasally with modified-live leukotoxin-deficient MH. Half of the vaccinates and nonvaccinates were randomly selected to receive florfenicol on arrival at the feedyard. Steers were observed daily for respiratory tract disease (RTD)., Results: Administration of florfenicol at time of arrival reduced the incidence of RTD, delayed the interval before onset of RTD, and reduced the incidence of MH colonization of the nasopharynx for at least 4 days, but vaccination did not have any effect. Vaccination elicited an increase in serum antibody titers to MH. Administration of florfenicol at time of arrival reduced the development of serum antibody titers in intranasally vaccinated steers and both groups of nonvaccinated steers, but intranasal vaccination did not affect colonization by wild-type MH., Conclusions and Clinical Relevance: Administration of florfenicol at time of arrival decreased the incidence of MH organisms in the nasopharynx and delayed the onset of RTD. Prophylactic use of suitable antibiotics is likely to reduce the incidence of acute RTD in calves for several days after arrival at feedyards, which is the period when they are most susceptible to infectious organisms.

Objective: To serotype an enterotoxin gene from Escherichia coli isolated from cows, pigs, and chickens in Korea., Sample Population: Isolates from 37 cows with mastitis, 51 diarrheic pigs, and 5 diarrheic chickens., Procedure: Serogroups and serotypes were identified by slide agglutination testing, using pathogenic E coli sera. Detection of E coli enterotoxins by use of reversed passive latex agglutination and ELISA was compared by proving existence of the gene by polymerase chain reaction (PCR) analysis., Results and Conclusions: Detection of E. coli enterotoxin by either method was positive for 1 strain (O20:H10; heat-labile enterotoxin [LT+], heat-stable enterotoxin [STa+]; isolation rate, 2%) and 3 other strains (O111:H10, O119:H9, and O125:H6, STa+; isolation rate, 5.9%) isolated from fecal specimens obtained from diarrheic pigs. The E coli enterotoxin genes were identified by use of PCR analysis in 1 strain containing the 417- and 163-base pair (bp) genes (LT+, Sta+; O20:H10) and in 3 strains containing only the 163-bp gene (STa+; O111:H10, O119:H9, and O125:H6)., Clinical Relevance: Serotyping of E coli enterotoxin may be used to analyze patterns of transmission among species of domestic animals.

Objective: To compare muscle fluid with serum samples for detection of antibodies to Salmonella lipopolysaccharide., Sample Population: Muscle fluid and serum samples from 2 cattle populations: 1 from the island of Bornholm with no history of salmonellosis (n = 39), and the other from the S dublin-enzootic areas of Jutland (n = 144)., Procedure: Salmonella dublin (O:1,9,12), S typhimurium (O:1,4,5,12), and Salmonella O:9-blocking ELISA were used for testing the samples., Results: In the S dublin ELISA, all serum and muscle fluid samples from cattle on the island of Bornholm had OD450 values well below the cutoff value (0.5). For samples obtained from cattle in the enzootic areas of Jutland, high correlation was found between serum and muscle fluid samples (rs = 0.89, P < 0.001). In addition, 19% (28/144) of the cattle had ELISA-positive muscle fluid and serum samples; 2% (3/144) had positive results for muscle fluid only, whereas 1 animal had positive results for serum only (kappa = 0.91, P < 0.0001; sensitivity and specificity of 97%). The same samples had similar significant correlation in the S typhimurium ELISA (rs = 0.88, P < 0.001, kappa = 0.7, P < 0.001; sensitivity of 73% and specificity of 98%) and the O:9-blocking ELISA (rs = 0.49, P < 0.001)., Conclusion and Clinical Relevance: Muscle fluid samples taken at slaughter can be used as a practical alternative to serum samples for surveillance of Salmonella infections in cattle.

We defined methods for use of luminol-dependent chemiluminescence (LDCL) and superoxide anion (O2-) production as parameters of the oxidative metabolism of neutrophils isolated from 1.5- to 5-week-old neonatal calves. We determined how variations in blood sample handling, agonist preparation, individual variability, and age of calves influenced the LDCL and O2- responses to certain agonists, and defined concentrations of soluble and particulate agonists that maximally stimulated the oxidative metabolism of bovine neutrophils. Oxidative responses, particularly LDCL, were characterized by marked day-to-day variability, differed greatly within and between calves, were partially age-dependent, and were partially dependent on the individual agonist. Superoxide anion production had substantially less variability. We compared the in vitro oxidative (LDCL and O2-) responses of neutrophils isolated from neonatal calves stimulated by defined concentrations of the agonists--latex, phorbol myristate acetate, calcium ionophore, and opsonized zymosan--with responses to formylated oligopeptides and zymosan-activated serum, and to live, dead, live opsonized, and dead opsonized Pasteurella haemolytica organisms. Opsonization of particulates, pathogenic or nonpathogenic, enhanced the LDCL and O2- responses of stimulated neutrophils although P haemolytica was a less potent stimulant of oxidative functions than were nonbiological agonists. We conclude that the generation of reactive oxygen species by bovine neutrophils in response to P haemolytica is highly dependent on the presence of opsonins and is greatly enhanced in live vs killed bacteria. Furthermore, the in vitro generation of reactive oxygen species, including O2- by stimulated neutrophils, may be of biologic importance if similar events occur in vivo, and could have a major role in the pathogenesis of the acute lung injury associated with pneumonic pasteurellosis.

Restriction endonuclease patterns of genomic fragments separated by use of pulsed-field gel electrophoresis were used to differentiate Brucella abortus strain RB51, a rifampin-resistant mutant of the standard virulent strain 2308, from other brucellae. Results were compared with results obtained by use of standard methods for characterizing brucellae. Electrophoretic patterns of the ATCC type strains allowed identification of the strains to the level of species. Genomic profiles of B abortus biovars 1, 2, and 4 were similar, as were those of biovars 5, 6, and of biovar 3 was similar to that of biovars 5, 6, and 9, except for a missing band at 93 kb and additional bands at 65 and 67 kb. A different fingerprint was detected in B abortus strain RB51, using the pulsed-field gel electrophoresis patterns of genomic DNA digested with restrictive endonuclease Xba I. The profile of B abortus strain RB51 contained a band at 104 kb, as opposed to a 109-kb fragment within profiles of B abortus isolates from naturally infected cattle, bison, and elk. Despite known biochemical and biological differences between RB51 and its parent strain (2308), restriction endonuclease analysis results were similar.

Two hundred ninety-six Escherichia coli isolates from feces or intestines of calves with diarrhea were hybridized with 7 gene probes. One probe (the eae probe) was derived from the eae gene coding for a protein involved in the effacement of the enterocyte microvilli by the group of bacteria called attaching and effacing E coli (AEEC), and 2 probes were derived from genes coding for the Shiga-like toxins (SLT) 1 and 2 produced by the verocytotoxic E coli (VTEC). The other 4 probes were derived from DNA sequences associated with the adhesive properties of enteroadherent E coli (EAEC) to cultured cells (the EAF probe for the localized adherence pattern, probes F1845 and AIDA-1 for the diffuse adherence pattern, and the Agg probe for the aggregative adherence pattern). Hybridization results for the eae probe were in agreement, for all but 1 of the 8 isolates, with previously published phenotypic results of microvilli effacement. The latter was previously reported as effacing the microvilli of calf enterocytes, but was eae probe-negative. Two classes of isolates hybridized with the eae probe. Members of a first class (60 isolates) additionally produced a positive signal with 1 or both of the SLT probes (VTEC-AEEC isolates). Isolates hybridizing with the eae and the SLT1 probes were the most frequent: 56 isolates (ie, 93% of all VTEC-AEEC). Members of the second class (10 isolates) failed to hybridize with either SLT probe (non-VTEC-AEEC isolates).(ABSTRACT TRUNCATED AT 250 WORDS)

Nine CNS bovine herpesvirus type 1 (BHV-1) isolates, recovered from bovine brain samples submitted to the Texas Veterinary Medical Diagnostic Laboratories from 1974-1989, were compared by analyzing their DNA restriction endonuclease (RE) fragment migration pattern. Seven had pattern similar to that of the respiratory BHV-1 Cooper strain. The remaining 2 isolates, however, had variant patterns, similar to that of each other, but completely different from patients for the other 7. The RE patterns of these 2 variants were similar to published RE patterns for 2 encephalitic or neuropathogenic BHV-1 strains--the Australian N-569 strain and the Argentine A-663 strain. One of the Texas encephalitic variants (No. 30326) was isolated from the CNS of a calf that died during an epizootic of encephalitis in 1974. The other, designated TX-89, was isolated in 1989 from the CNS of a 7-month-old feedlot steer with acute fatal encephalitis. Microscopic lesions of encephalitis with neuronal degeneration and intranuclear inclusions were observed for 3 of the 9 isolates, the 2 variant isolates (No. 30326 and TX-89), and a respiratory isolate. The remaining 6 CNS isolates, all respiratory subtypes, were recovered from cattle that did not have clinical CNS disease or gross or microscopic CNS lesions; in 5 of these cattle, virus was recovered from at least 1 other organ (lungs) besides the CNS. We conclude that the CNS of calves can be naturally infected with 2 distinct BHV-1 subtypes, the respiratory and the encephalitic, and that the encephalitic subtype (subtype 3 or BHV-1.3) has been present in Texas cattle since at least 1974.

Three available differential stains, Camco-Quik, Diff-Quik, and Wright-Giesma were compared for detection of intraerythrocytic Anaplasma marginale in bovine blood smears. In samples where < 1% to more than 51% of the RBC were infected, statistical analysis of the data indicated no significant difference in the detection of A marginale with Camco-Quik or Diff-Quik stains. However, a significantly lower percentage of infected RBC were detected when blood smears were stained with the Wright-Giemsa stain, compared with the other 2 methods.

Results of a prospective serologic and virologic study of ruminant livestock in Central America and the Caribbean islands revealed bluetongue virus (BTV) to be enzootic in the 9 countries participating in the study. Bluetongue virus serotypes 1, 3, 6, and 12 were isolated from sentinel animals. To the authors' knowledge, these are the first isolations of BTV from the region studied and the first isolations of these serotypes in the Western Hemisphere. Clinical disease attributable to BTV infection was not observed in sentinel animals. The incidence pattern, with respect to age and geographic location, was determined. The need to evaluate the epizootiologic features or arthropod-borne viruses (arboviruses) on a regional ecologic basis is stressed.

To establish whether Mycobacterium paratuberculosis could be cultured from Dulbecco phosphate-buffered saline solution (DPBSS) and to test 3 sampling methods, DPBSS supplemented with 2% fetal bovine serum was inoculated with M paratuberculosis at concentrations of 10(4), 10(3), 10(2), 10(1), and 10(0) colony-forming units/ml. The inoculated media was sampled after mixing, after centrifugation, and after centrifugation and decontamination with 0.75% hexadecylpyridinium chloride. The samples were inoculated onto 3 slants of Herrolds egg yolk medium supplemented with sodium pyruvate and mycobactin J and 1 slant without mycobactin J. Mycobacterium paratuberculosis was isolated following all 3 sampling methods for all concentrations. Treatment with hexadecylpyridinium chloride decreased the number of colonies isolated. To test the efficacy of a 10-step wash procedure for removing M paratuberculosis from bovine ova, washed zona pellucida intact bovine ova were incubated in DPBSS supplemented with 2% fetal bovine serum containing concentrations of 10(4), 10(3), 10(2), 10(1), and 10(0) colony-forming units of M paratuberculosis/ml for 12 hours at 22 C. Ten zona pellucida intact ova were removed from each concentration and washed by passing through 10 changes of DPBSS supplemented with 15% fetal bovine serum. The media from each wash step was inoculated onto slants of Herrolds egg yolk medium. The ova were included with the tenth wash step. Mycobacterium paratuberculosis was isolated from 1 of 10 tenth-wash steps at the 10(4) concentration and 5 of 10 tenth-wash steps at 10(3).

Spleen cells from a calf immunized with bovine herpesvirus-1 (BHV-1) were fused with the nonsecreting murine cell line SP2/0. Several bovine-murine hybridomas secreting bovine immunoglobulins were stabilized. Of these, 9 hybridomas secreted bovine monoclonal antibodies that specifically bound to BHV-1 in a radioimmunoassay. Two of these monoclonal antibodies reacted specifically with BHV-1 in an indirect fluorescent antibody test and immunoprecipitated a BHV-1 glycoprotein with molecular mass of 97 kilodaltons.

Five virus strains with identical antigenic properties were isolated from 3 RBC suspensions obtained from 2 healthy sentinel calves and from 2 pools of Culicoides oxystoma in cultures of a hamster lung cell line (HmLu-1). The virus was tentatively named Chuzan virus. The Chuzan virus was classified as a new member of the Palyam subgroup of the genus Orbivirus on the basis of its physicochemical, morphologic, and antigenic properties.

Vaccination of calves with a Pasteurella haemolytica serotype 1 antigen preparation elicited a serotype-specific inhibition of nasal colonization by P haemolytica under field conditions. Inhibition was evidenced by a low frequency of nasal colonizations and by relatively few P haemolytica serotype 1 organisms isolated from vaccinated calves. The study comprised 3 field trials, each on a separate year, and included 480 calves.

Kidneys were cultured for leptospires and blood was collected for leptospiral serologic examination from 306 cattle slaughtered in Florida during June, July, and August 1980. Leptospires were found in 82(36%) of 226 otherwise bacterial-free cultures. Forty-five isolates were identified as Leptospira interrogans serovar hardjo, 3 as serovar balcanica, and 3 as serovar pomona. Leptospiral antibodies were found in 218 (71%) of the cattle. Serum titers to hardjo were found in 162 cattle (53%) and to pomona in 28 (9%). Low titers to tarassovi were found in 14 cattle (4.5%).

Beginning in 1973, all available laboratory and field strains of bluetongue virus (BTV) from the United States were serotyped. Of the viral strains serotyped, 27 were collected from 1953 through 1972; 173 were collected from 1973 through 1977. Although 20 BTV serotypes have been found worldwide, only BTV serotypes 10, 11, 13, and 17 have been found in the United States. Since 1973, serotypes 11 and 17 have been the prevalent serotypes. Samples were collected over a 24-year period in the United States and represent a wide geographic area and diverse host sources (sheep, cattle, wild ruminants, and insect vectors). The collection was not a statistical sampling.

Strains of Salmonella typhimurium (n = 175) isolated from animals and birds in northern Japan were differentiated into 5 biovars (1, 2, 7, 10, and untypeable) by 6 kinds of fermentation tests (inositol, bitter-xylose, rhamnose, xylose, Stern's glycerol, and trehalose) of Brandis' scheme, and were subdivided into 9 primary and 38 full biovars by a new biotyping method, using additional biochemical reactions. The full biovar classified by the new biotyping method was considered to be a marker for assessing the widespread outbreaks of infection with S typhimurium. In particular, strains of biovars 25hi and 27hi were characteristically found in pigeons, quail, and fantails, and were thought to be of avian origin.

Pathogenicity of 17 Campylobacter isolates for pregnant guinea pigs was investigated. Of 14 isolates, 12 (86%) produced rates of abortion ranging from 13% to 87%. Two isolates did not produce abortion. Reference strains of C fetus subsp venerealis produced abortion in 60% to 87% and C fetus subsp fetus produced abortion in 60% of the guinea pigs. Inoculated organisms were recovered from uterus, blood, liver, kidney, spleen, and gallbladder of the guinea pigs at rates as high as 83% for 2 ovine isolates and as low as 13% for 2 bovine and 1 human isolates. Most isolations were from the uterus. Two avian isolates were not recovered. Within the C jejuni and C coli group, the ovine and the human isolates appear to be more pathogenic. Swine, bovine, and avian isolates were less pathogenic. Seemingly, the pregnant guinea pig was a suitable and practical model for evaluating the pathogenicity of Campylobacter organisms, regardless of their host of origin.

A total of 56 Salmonella isolates from animals were tested for susceptibility to 12 antimicrobial agents by the standard disk diffusion and mirodilution methods. All isolates were susceptible to gentamicin, tobramycin, amikacin, chloramphenicol, crimethoprim and sulfonamide, colistin, and nitrofurantoin. Isolates were most frequently resistant to tetracycline (50%) and kanamycin (36%). A low prevalence of resistance to ampicillin (16%), carbenicillin (16%), and cephalothin (4%) was found. The differences in susceptibility results between the two methods were minor.

Fifty-one coagulase-positive staphylococcal isolates from canine urinary calculi or from the urine of dogs with documented urolithiasis, and 17 coagulase-positive staphylococcal isolates from human beings and cattle were identified by a commercially available tray micromethod, as well as by conventional methods. Canine isolates had previously been classified as Staphylococcus aureus on the basis of a positive tube coagulase test. After 5 hours' incubation, the tray method identified all 51 canine urolithiasis isolates as S intermedius, rather than S aureus. All human and bovine isolates were identified as S aureus. Conventional methods supported these findings.

Trigeminal ganglia collected from 56 adult dairy cattle at slaughter were maintained in explant culture. Bovine herpesvirus-1 was isolated from six ganglia after periods of 7 to 24 days' maintenance in vitro. Antibody against bovine herpesvirus-1, indicating exposure to that virus, was found in 45.4% of the sample population from which ganglia were collected.

Thirty calves (3 to 4 months old) were exposed conjunctivally to a pathogenic strain of Brucella abortus. Calves were euthanatized and necropsied at postexposure hours 2 and 4, and at postexposure days (PED) 1, 4, 7, 14, 21, 42, and 49. Selected ocular, pharyngeal, and lymphoid tissues were cultured bacteriologically for brucellae to determine organism distribution. Brucella abortus organisms initially localized in the third eyelids, bulbar conjunctivae, and parotid lymph nodes and were detected in these structures until PED 42, 21, and 49 respectively. In calves euthanatized at PED 7, organisms were in other cranial lymph nodes (mandibular and retropharyngeal), and in calves euthanatized at PED 21, organisms were isolated from peripheral lymphoid tissues. Brucellae were not isolated from mesenteric and bronchial lymph nodes and from the spleen until PED 21. The pattern of isolation indicated that conjunctival exposure probably resulted in entrance of brucellae into the host via ocular tissues.

Bovine embryos were exposed to bovine viral diarrhea (BVD) virus in vitro. An uptake of BVD virus by the embryos could not be detected by several assay systems. A significant decrease in the titer of BVD virus was found to occur when the virus was incubated in saline solution + 5% goat serum or minimal essential medium + 5% goat serum for 24 hours at 37 C. Since there was significant inactivation of the BVD virus during the incubation period, lack of viral infectivity of the embryos may have been due to adverse effects of the experimental environmental conditions on the virus or the embryos or upon viral-embryo interaction.

Members of the genus Salmonella isolated from chickens, turkeys, cattle, and swine and submitted to the National Veterinary Services Laboratories for serotyping during October 1981 through September 1982 were tested for sensitivity to 12 antibacterial drugs. A total of 3,500 isolates was tested. A high rate of drug resistance was observed. Three cultures were resistant to each of the drugs and 30% were resistant to each drug except chloramphenicol, cephalothin, and gentamicin. Multiple resistance patterns were observed for 80% of the cultures with an even higher percentage in cultures from swine.

The results of antimicrobial susceptibility testing on 262 strains of Pasteurella multocida and 141 strains of Pasteurella haemolytica isolated from cattle and swine from 1971 to 1974 were analyzed for patterns of resistance to streptomycin, penicillin, tetracycline, and chloramphenicol, using a modified Kirby-Bauer procedure. Resistance was recorded for 80.5% of the isolants of P multocida and 92.2% of those of P haemolytica. Resistance to streptomycin was most frequent, followed by resistance to penicillin and tetracycline. Most cultures of P multocida and P haemolytica were susceptible to chloramphenicol. There were 9 patterns of resistance with the aforementioned antibiotics. The combinations, streptomycin and penicillin and streptomycin and tetracycline, each accounted for approximately 10% of the resistance patterns of P multocida. Approximately half of the 14 isolants of P haemolytica were resistant to the combination of streptomycin, penicillin, and tetracycline. These observations underscore the need for antimicrobial susceptibility testing of clinical isolants of P multocida and P haemolytica.

A mature, rumen-cannulated steer fed Coastal Bermuda grass hay for 8 weeks was adapted to a high-grain ration by stepwise increases in grain over a period of 4 weeks. The grain rations had concentrate-to-roughage ratios of 40:60, 70:30, and 85:15 and were fed for 10, 7, and 11 days, respectively. Numerical estimates of lactate-utilizing bacteria in ruminal fluid of the steer the last 3 days each ration was fed were made by colony counts. Lactate-utilizing bacteria were identified as Megasphaera elsdenii, Peptococcus asaccharolyticus, and Selenomonas ruminantium. Maximal numbers of lactate-utilizing bacteria were observed in the ruminal fluid of the steer during feeding the concentrate-to-roughage ration of 70:30.

An indirect ELISA for the detection of bovine immunoglobulin to bovine herpesvirus type 4 (BHV-4) was developed. Three methods of antigen preparation were compared. They included (1) BHV-4-infected Madin Darby bovine kidney (MDBK) cells treated with glycine and then frozen and thawed, (2) BHV-4-infected MDBK cells treated with glycine and then sonicated, and (3) BHV-4-infected MDBK cells treated with a detergent. The antigen preparation that gave the highest reactivity was the first method. We obtained serum samples from 178 cattle in the field and assayed the serum by ELISA and the complement-fixation (CF) test. Eighty-six percent (153) of the serum samples were positive by ELISA, and 70% (124) were positive by the CF test. The ELISA had a higher degree of sensitivity than did the CF test. Also, the ELISA was specific, and the prevalence of BHV-4-infection was more common in beef and dairy herds than previously recognized.

A strain of Pasteurella haemolytica isolated from the lungs of feedlot cattle with pneumonia was shown to be resistant to ampicillin, tetracycline, and streptomycin. Two small, nontransmissible plasmids were shown to be responsible, for tetracycline resistance, a 5.7 X 10(6) dalton plasmid, and for streptomycin resistance, a 5.5 X 10(6) dalton plasmid. Ampicillin resistance passed by transformation to Escherichia coli could not be shown conclusively to be on a plasmid. Ampicillin resistance was shown to be mediated by means of a beta-lactamase with high activity against cephaloridine.

Samples from the rumen and cecum of cattle and sheep were cultured to determine changes in microbial populations resulting from overfeeding with grain. Before the animals were overfed, the predominant organisms from both sides were those that grew anaerobically on a relatively nonselective ruminal fluid medium and would not grow on selective mediums designed to culture lactobacilli, streptococci, coliforms, or Clostridium perfringens. By 24 hours after overfeeding, lactic acid bacteria had increased in numbers so that they were the most numerous organisms in both the rumen and the cecum. The concentrations of coliforms and C perfringens also increased after overfeeding and were generally higher in the cecum than in the rumen.

A sequential extraction procedure was used to provide 3 endotoxin fractions from Pasteurella haemolytica with distinct biological and solubility properties. After acetone dessication, extraction with phenol, chloroform, and petroleum ether (2:5:8) provided a fraction designated rough lipopolysaccharide (LPS). Subsequent extraction of the cells with 45% phenol at 68 C yielded a fraction designated smooth LPS, which was further divided into smooth precipitate and smooth supernatant, based on sedimentation at 105,000 x g for 4 hours. Yields of the 3 fractions were 1.5%, 3%, and 5.5% of the dry weight of the cells. The polysaccharide moieties of the rough LPS amd smooth precipitate fractions were obtained by partial acid hydrolysis followed by chloroform extraction. Biological activities of all 5 fractions were compared with activities of standard LPS fractions from Serratia marcescens and Salmonella typhimurium. Results of chicken embryo lethality, the local Shwartzman's phenomenon, nonspecific resistance enhancement ot challenge exposure by S typhimurium pyrogenicity, and the Limulus amebocyte lysate assay were reported.

Geographically separate United States isolates of Anaplasma marginale were differentiated, using polyclonal bovine antiserum and microfluorometry. Both isolate-common and restricted antigen-specific antibodies were apparent in sera of splenectomized calves after resolution of infection, as judged by the capability of the antisera to recognize heterologous isolate antigens to a lesser extent than homologous antigens. Furthermore, absorption of the antisera with homologous antigens removed homologous and heterologous reactions, whereas heterologous absorptions resulted in the capability to discriminate the tailed or appendage-associated isolates (Virginia and Washington) from the tail-less isolate (Florida).

Typing of R factors by genetic properties was done with Salmonella typhimurium and Escherichia coli isolated from calves on a feedlot where epizootics of clinical or subclinical calf salmonellosis had repeatedly occurred during 5 years. Forty-nine R factors from S typhimurium were fi- (no fertility inhibition) and spp- (no restriction against phage lambda vir). Twenty-three (46.9%) of them belonged to compatibility group Ialpha and the remainder were nontypable. Fourteen R factors from E coli belonged to different genetic types: fi+ (11=78.6%) and fi- (3=21.4%); spp+ (1=7.1%) and spp- (13=92.9%); compatibility groups FII (5=35.7%), N (1=7.1%), and nontypable (8=57.2%). In contrast to the R factors of S typhimurium, 9 (64.3%) of the 14 R factors of E coli carried resistance against aminobenzyl penicillin with or without kanamycin resistance. The compatibility groups of R factors of S typhimurium seemed to be useful as a subsidiary epizootiologic marker in this feedlot.

From Aug 1, 1979 to Aug 15, 1980, a total of 624 samples were collected from the vaginas, cervices, and uteri of 208 dairy cows 30 to 37 days after parturition. An additional 90 samples were collected from the reproductive tracts from 30 of the 208 cows. The 30 cows had aborted or were repeat breeders. Samples from cows that had aborted were collected within a week after clinical abortion or immediately after rectal palpation revealed subclinical abortion. All samples were tested for the presence of cytopathogenic viruses. All samples were negative.

Heparinized blood and serum samples were obtained from 1,295 ruminants in herds or flocks with bluetongue virus (BTV) infection in 4 western states. Submissions were from herds or flocks with clinical bluetongue (BT), as well as from animals on premises with no history of BT disease. Insects, including Culicoides variipennis, were collected in areas enzootic for BT disease. Viral isolations were in 10-day-old embryonating chicken eggs that were then adapted to Vero cells for serotyping. Sera were tested from group-specific antibody to BTV by the micro agar gel precipitin (AGP) test. Viral isolations were from cattle (81), sheep (122), goats (9), antelope (2), and C varipennis (5). There were 7 isolates of serotype 120, 114 of serotype 11, 42 of serotype 13, and 56 of serotype 17. In herds or flocks from which BTV was isolated, 51% of cattle, 56% of sheep, 21% of goats, and 52% of antelope had AGP antibodies. Virus was isolated from 43% of the cattle and 23% of the sheep that had no demonstrable evidence of AGP antibodies. Viral isolations were seasonal, occurring from August until December. Approximately 30% of the herds or flocks from which virus was isolated had more than one serotype of virus causing infection.

Brucella abortus isolates (n = 134) from 38 dairy herds in Vermont, Florida, and Canada exhibited 2 growth patterns on medium containing 5 U of penicillin/ml. The B abortus isolates from 7 herds in Vermont were always susceptible to the antibiotic at that concentration. Eleven of 12 isolates from 1 herd in Florida and 25 isolates (biotypes 1 and 4) from 30 Canadian herds grew at greater than or equal to 5 U of penicillin/ml, as determined using a gradient plate method. Penicillin susceptibility may be of value in identifying the source of infections in cattle.

"Haemophilus somnus' was isolated from 77% of 31 reproductive tracts of bulls from an Ontario slaughterhouse. Identification of H somnus was based on morphologic and cultural characteristics and on fluorescent antibody and immunodiffusion tests, using antisera prepared against a known pathogenic encephalitic isolate of H somnus. The infection rate and distribution of H somnus within the tract were as follows: preputial orifice--71% preputial cavity--71%; urinary bladder--26%; accessory sex glands--19% and ampulla of ductus deferens--10%. Isolates were not obtained from the testes or epididymides. On 2 occasions, H somnus was isolated in pure culture from the preputial cavity. A higher prevalence of infection was found in young bulls. There were no differences found in infection rates between breeds. Differences in hemolytic activity and minor antigenic variation between isolates indicated that a series of biotypes within the species H somnus may exist. The study indicates that organisms presently identified as H somnus may normally form part of the flora of the bovine prepuce and that dissemination from the male bovine reproductive tract is one possible means of infection in H somnus-associated diseases. The pathogenic significance of genital isolates of H somnus awaits further study.

Colony hybridizations with 4 enterotoxins (STaP, STaH, STb, and LT) and 1 adherence factor (K99) gene probes were done on Escherichia coli isolated from calves. Agreement between the K99 probing and a serologic assay to detect the K99 antigen was 99% for the identification of K99+ and K99- isolates. Ninety-five of the isolates (22%) hybridized with at least 1 enterotoxin gene probe (Ent+ isolates), and 82 (19%), with the K99 gene probe. The majority of Ent+ isolates (85%) reacted with probes for STaP and K99 genes. The STaP gene was present by itself in 4 of the Ent+ isolates (4%) and with the STb gene in 6 of the Ent+ isolates (6%). Five of the Ent+ isolates (5%) carried the STb and LT genes, and none (0%) of the isolates carried the STaH gene. All but 2 of the isolates with the K99 gene also had the STaP gene. Twenty-eight isolates shown to produce STa enterotoxin in previous studies failed to hybridize with any of the enterotoxin gene probes. These 28 isolates were also phenotypically negative when the tests for enterotoxin production were repeated. These isolates probably lost their genes for enterotoxin production during storage in the laboratory.

The serotypes 1 and 2 of Breda virus from feces of experimentally infected gnotobiotic calves were studied with respect to their sedimentation and density properties in sucrose gradients and their structural polypeptides; Berne virus, the proposed prototype of the new family Toroviridae, was included for comparison. After Breda-1 virus had been stored at 4 C for a prolonged period, it showed a heterogeneous sedimentation behavior (480 to 520 Svedberg units [S]) and density (1.18 to 1.21 g/ml) indicative of its poor state of preservation. In contrast, freshly prepared Breda-2 virus sedimented at 350 S and showed a buoyant density of 1.18 g/ml; these values compare well with those of Berne virus (400 S and 1.16 g/ml, respectively). Efficient purification of the Breda viruses could be achieved by a 2-step method, involving pelleting by ultracentrifugation followed by isokinetic and isopyknic sucrose gradient centrifugation. Radioiodinated purified virus showed polypeptides with apparent molecular weights of 105,000, 85,000 37,000, and 20,000; another labeled protein of 65,000 D is of doubtful virus specificity. Mouse immune serum raised against Breda-2 virus recognized the polypeptides of the homologous virus and the 2 highest molecular weight proteins of Breda 1 virus in radioimmune precipitation. The same serum inhibited hemagglutination of the heterologous serotype to a low, but significant, degree and efficiently neutralized the infectivity of Berne virus. These observations are taken as indications that the 105,000- and 85,000-D polypeptides represent surface structures of torovirions, probably peplomeric proteins.

The pulmonary clearance rate of Serratia marcescens was determined in 17 calves exposed to an aerosol from a modified Hendersen apparatus. The mean bovine pulmonary clearance was 50.2 +/- 4.1% at postaerosolization hour (PAH) 1, 72.5 +/- 7.0% at 2 hours, 88.6 +/- 5.2% at 4 hours, 97.3 +/- 2.8% at 8 hours, and 99.9% at PAH 24 and 48. Regional pulmonary mean percent retention (MPR) of S marcescens in 10 calves was determined in dorsal or ventral and in cranial or caudal regions. Dorsal MPR was less than ventral MPR for PAH 1, 2, and 4, whereas cranial MPR was greater than caudal MPR for PAH 1, 2, 4, and 8. Difference in regional airway oxygen tensions was suggested as the most probable reason for differing regional MPR values.