Determination of tetracyclines in bovine and porcine muscle by high-performance liquid chromatography using solid-phase extraction.

A method is presented for the determination of the three tetracyclines oxytetracycline, tetracycline and chlortetracycline in muscle, spiked at 100 ng/g, using high-performance liquid chromatography (HPLC). The concentration and extraction steps are carried out using Waters Environmental Sep-Pak cartridges. The principal steps involve homogenizing the sample in EDTA-McIlvaine buffer followed by centrifugation and precipitation of the supernatant using trichloroacetic acid. After further filtration and concentration on a Sep-Pak cartridge, the sample is eluted and analysed by HPLC with UV detection and confirmation by diode-array. The column used is a Nova-Pak C18 (4 microns) cartridge (10 cm x 8 mm I.D.). A phosphate-citrate-acetonitrile buffer, utilizing ion suppression, is the mobile phase. The analytes are detectable at levels down to 10 ng/g. The analyte identity can be confirmed at 20 ng/g by the use of diode-array detection and spectral library comparison.

High-performance liquid chromatography of sulphonamides extracted from bovine and porcine muscle by solid-phase dispersion.

A method has been developed for the analysis of sulphonamides in bovine and porcine muscle, based on solid-phase dispersion. Muscle tissue was blended with pre-washed C18 coated silica (55-105 microns), and the resulting homogeneous solid packed into a polypropylene syringe barrel. Fatty material was washed from the sample using hexane, and the sulphonamide analytes eluted with dichloromethane. The collected fraction was dried under nitrogen and reconstituted in 20% methanol in 0.01 M sodium acetate/acetic acid (pH 5) buffer. After sonication and filtration, the sample was analysed by high-performance liquid chromatography on a C18 column using UV diode array detection. Individual sulphonamides could be detected down to 0.01 ppm, whilst analyte identity could be confirmed by diode array spectrum down to 0.02 ppm.

Comparison of the Johne's absorbed EIA and the complement-fixation test for the diagnosis of Johne's disease in cattle.

A commercially available absorbed ELISA for the diagnosis of Johne's disease (JD) (paratuberculosis) in cattle, the Johne's Absorbed EIA, was compared with the conventional complement-fixation test (CFT) used in Australia. Stored plasma from 3 Victorian dairy herds with a history of JD, sera from specimens submitted from animals showing clinical signs of JD and sera from the US National Repository for Paratuberculosis Specimens were used to determine the sensitivity of each test. The EIA detected 48.8% of 43 Australian animals with subclinical JD, while the CFT detected only 12 (21.4%) of 56 subclinically affected cattle. Of 150 subclinically infected US cattle, the EIA detected 47.3% and the CFT detected 52.0%. The EIA detected 59.7% of animals which at the time of sampling were shedding Mycobacterium paratuberculosis in their faeces, but showed no clinical signs of JD, while the CFT detected 57.3%. The EIA correctly identified 88.2% of 136 histologically confirmed clinical cases, and the CFT detected 83.4%. The specificity of each test was determined by testing sera collected at slaughter from animals residing in a known JD-free area of Australia, and from samples from the US National Repository of Paratuberculosis Specimens collected from certified-free herds in Wisconsin. The EIA was found to have a specificity of 99.8% when 998 Australian animals were used as the test population, and 99.0% when 196 US animals were used. The specificity of the CFT using Australian samples was 96.9% and 95.2% using American samples.

Genetic homogeneity of Actinobacillus seminis isolates.

Isolates of Actinobacillus seminis from clinical cases and reference sources had markedly similar Bam H1 restriction endonuclease profiles but were readily distinguishable from the Bam H1 profiles of the Histophilus-Haemophilus group as well as from A lignieresii. For epidemiological purposes this lack of interstrain variation in Bam H1 profiles makes restriction endonuclease analysis of isolates of A seminis unsuitable.

A role for DNA characterisation in ovine footrot.

Bacteroides nodosus involved in several outbreaks of ovine footrot over a number of years were subjected to DNA restriction endonuclease analysis. Individual isolates were found to have characteristic Bam HI profiles which permitted their accurate identification and differentiation from other isolates. Bam HI profiles of B. nodosus isolates were used in epidemiological investigations involving consecutive outbreaks of footrot on individual and neighbouring farms. The relationship of given isolates to a common source could be established by this means. Restriction endonuclease analysis provides an additional epidemiological tool in ovine footrot investigations as it accurately identifies interstrain differences in a manner not possible by conventional bacteriological and serological means.

Restriction endonuclease analysis of Brucella abortus.

The restriction endonuclease profiles of bacterial DNA from Brucella abortus isolates were evaluated. It was not possible to distinguish between vaccine strain 19 and virulent (biotype 1 and biotype 2) strains of B abortus. Restriction endonuclease analysis is therefore not a suitable epidemiological tool in bovine brucellosis investigations. The genetic homogeneity of the Brucella genus was reinforced by these findings.

Characterisation of Histophilus ovis and related organisms by restriction endonuclease analysis.

The banding profiles generated by Bam H1 restriction endonuclease cleavage of bacterial DNA from clinical and reference isolates of Histophilus ovis, Haemophilus somnus and related bacteria were compared. H. ovis, H. somnus and Haemophilus agni isolates were found to have distinct similarities in banding profiles characterised by 10 common bands between 2.0 and 9.6 kilobases (kb). The close taxonomic relationship of these isolates was reinforced by these findings. The reference isolates examined in this study--Actinobacillus lignieresii, Actinobacillus seminis, H. agni, H. somnus, H. ovis, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus parahaemolyticus--could be distinguished from each other on the basis of their characteristic banding profiles. Actinobacillus sp were observed to have more bands between 2 and 23 kb compared with the H. ovis and Haemophilus sp isolates studied. Analysis of isolates from an experimental infection trial illustrated the potential of restriction endonuclease analysis in molecular epidemiological applications. It was possible to demonstrate by this means that the post-challenge isolates had identical banding profiles to the challenge (or infecting) isolate which had a distinctly different banding profile from that of pre-challenge H. ovis isolates. Furthermore, restriction endonuclease analysis of H. ovis isolates obtained from follow-up investigations of a recurrent problem of epididymitis in unmated rams, indicated that the H. ovis isolates implicated in epididymitis, were present as a single strain in a number of sheep over a period of time. This suggested that the mechanism of transmission was by perinatal perputial contamination.

Antimicrobial resistance among Pasteurella spp recovered from Missouri and Iowa cattle with bovine respiratory disease complex.

A retrospective study was conducted to determine the prevalence of antimicrobial resistance among Pasteurella spp recovered from cattle with bovine respiratory disease complex. The study extended from January 1976 through May 1980, and included a review of the necropsy records of 386 beef cattle. Susceptibility or resistance of the Pasteurella isolants was determined by using the standard disk diffusion susceptibility test. Each isolant was tested for susceptibility with 15 different antimicrobial agents. A high prevalence of resistance (greater than 80%) was found when Pasteurella was tested with triple sulfonamides. For P haemolytica isolants, 57% to 70% were resistant to ampicillin (56/97), penicillin (58/101), and streptomycin (70/100); for unidentified Pasteurella spp isolants, 64% to 91% were resistant to ampicillin (83/129), penicillin (89/129), and streptomycin (118/129). For P haemolytica (21/100) and P multocida (34/146) isolants, 21% to 23% were resistant to tetracycline. Most of the P multocida isolants did not show marked antimicrobial resistance to 9 of the 15 drugs tested. However, 58% of the P multocida isolants (84/145) were resistant to streptomycin and 88% of them were resistant to three combined sulfonamides (126/144).

Semiselective medium for isolation of Moraxella bovis from cattle with infectious keratoconjunctivitis.

The incorporation of 2.5 micrograms/ml of cloxacillin into 5% bovine blood agar provided an inexpensive, easily prepared culture medium for the primary isolation of Moraxella bovis from bovine lacrimal and nasal secretions. With this medium, the time required to identify and isolate M. bovis from large numbers of field specimens was substantially reduced, whereas the sensitivity of isolation was increased by 60%.

Antimicrobial susceptibility of Moraxella bovis determined by agar disk diffusion and broth microdilution.

The antimicrobial susceptibility of 84 isolates of Moraxella bovis was evaluated by the standard agar disk diffusion and broth microdilution procedures. All isolates were resistant to cloxacillin by disk diffusion, with 97% of isolates having a minimal inhibitory concentration of greater than or equal to 2 micrograms/ml. Of the hemolytic isolates, 68% were resistant to streptomycin. A high frequency of susceptibility was recorded for all other antimicrobial agents tested. Quantitative data supported the use of sulfonamides, but not tylosin, for parenteral infectious bovine keratoconjunctivitis therapy.

Risk factors related to the prevalence of infectious bovine keratoconjunctivitis.

A cross-sectional epizootiologic study was conducted by mailed questionnaire to determine how the prevalence of infectious bovine keratoconjunctivitis (IBK) related to selected predisposing causes. The prevalence of IBK in Missouri during the summer of 1978 was 4.97 cases/100 cattle, with 45% of respondents reporting cases of IBK. A higher prevalence of IBK was associated with calves and yearlings, Hereford and Hereford-cross cattle, backgrounding cattle operations, and herds with a history of IBK. Vaccination of mature cattle against infectious bovine rhinotracheitis was associated with significantly higher prevalence of IBK in calves. A significantly lower prevalence of IBK was associated with dairy cattle operations, older cattle, and the winter months. There was no association between the prevalence of IBK and locale, nutrient supplementation, pasture management, use of vaccines other than infectious bovine rhinotracheitis, parasite prophylactic measures, or fly control measures.

Effects of Moraxella bovis vaccination schedules on experimentally induced infectious bovine keratoconjunctivitis.

An oil-adjuvant Moraxella bovis bacterin was administered to weanling calves, using different vaccination schedules. Calves were given a booster vaccination after 3 weeks and were challenge exposed 2 weeks later with virulent M bovis recovered from calves with clinical infectious bovine keratoconjunctivitis (IBK). The effects of different routes of vaccination and homologous and heterologous challenge exposure on the incidence, severity, and duration of induced IBK was evaluated. All calves given a placebo developed clinical IBK. Calves vaccinated subcutaneously in the neck had the shortest duration of M bovis infection, the lowest incidence and the shortest duration of acute IBK, and the lowest disease severity score, compared with effects in calves given a placebo or vaccinated subconjunctivally. Calves challenge exposed with the homologous strain of M bovis had more infected eyes, more eyes with acute IBK, longer duration of infection, and a higher severity and duration disease score.