Introduction: The study objective was to develop and evaluate a new TaqMan multiplex real-time PCR method for Salmonella, L. monocytogenes, and verotoxigenic Escherichia coli (VTEC) detection in slaughtered animal carcasses.Material and Methods: The procedure included an enrichment step, DNA extraction, and two multiplex real-time PCRs. The first PCR detected the invA and hly genes of Salmonella and L. monocytogenes respectively, the second the vtx1, vtx2, and eae genes of VTEC.Results: The validation of this method resulted in 100% relative sensitivity, specificity, and accuracy as compared to the reference ISO methods. The limit of detection per swab sample was established at 1 cfu for Salmonella and L. monocytogenes and 2 cfu for VTEC. The authors analysed 265 slaughterhouse-collected swabs from cattle, pig, and poultry carcasses. Among 125 from cattle, 51 were positive for VTEC, 29 for Salmonella, and 1 for L. monocytogenes. Among swabs from pig carcasses (n = 95), three, two, and one sample were positive for these pathogens respectively. None of the microorganisms tested for was identified in 45 samples of poultry origin.Conclusion: The obtained results showed that the method developed can rapidly identify the main bacterial pathogens that may contaminate carcasses of food-producing animals.

Introduction: Mycoplasma bovis is one of the main pathogens involved in cattle pneumonia. Other mycoplasmas have also been directly implicated in respiratory diseases in cattle. The prevalence of different Mycoplasma spp. in cattle affected by respiratory diseases and molecular characteristics of M. bovis field strains were evaluated. Material and Methods: In total, 713 nasal swabs from 73 cattle herds were tested. The uvrC gene fragment was amplified by PCR and PCR products were sequenced. PCR/DGGE and RAPD were performed. Results: It was found that 39 (5.5%) samples were positive for M. bovis in the PCR and six field strains had point nucleotide mutations. Additionally, the phylogenetic analysis of 20 M. bovis field strains tested with RAPD showed two distinct groups of M. bovis strains sharing only 3.8% similarity. PCR/DGGE analysis demonstrated the presence of bacteria belonging to the Mollicutes class in 79.1% of DNA isolates. The isolates were identified as: Mycoplasma bovirhinis, M. dispar, M. bovis, M. canis, M. arginini, M. canadense, M. bovoculi, M. alkalescens, and Ureaplasma diversum. Conclusion: Different Mycoplasma spp. strains play a crucial role in inducing respiratory diseases in cattle.

Introduction: In the present study, some of the commercial fish farms located in the Black Sea region of Turkey, were screened for bacteria between 2012 and 2014.Material and Methods: The bacterial agents isolated from fish were identified by classical biochemical tests and the rapid diagnostic tests (API 20 E and API 20 Strep). All strains were further identified by sequencing of the 16S rRNA genes. The strains were also investigated for resistance to different antimicrobials by the disc diffusion method. Antibiotic resistance genes, including tetracycline (B), β-lactam (ampC, blaTEM, blaPSE), florfenicol (floR), erythromycine (ereA, ereB), sulphonamide (sulI, sulII), and trimethoprim (dhfr1) genes, were determined by the PCR method.Results:Vibrio anguillarum, Vibrio fluvialis, Photobacterium damselae subsp. piscicida, Pseudomonas luteola, Lactococcus garvieae, Streptococcus iniae, Aeromonas hydrophila, and Yersinia ruckeri were isolated from marine and freshwater cultured fish. According to the results of disc diffusion, all isolates were sensitive to florfenicol, trimethoprim+sulfamethoxazole, oxitetracycline, and enrofloxacin, and resistant to lincomycin, penicillin G, and amoxicillin. Also, sulI, sulII, and floR resistance genes were detected in the bacteria.Conclusion: The results of the study open up the opportunity to perform further investigations which could determine the possible role of ARGs in fish pathogens.

Introduction: Chlamydia psittaci is a gram-negative obligate intracellular pathogen of birds. Poultry infections lead to economic losses and can be transmitted to humans. No vaccine is available and the bacterium-host cell interaction is not completely understood. Replicating bacteria cause pneumonia, but C. psittaci can also be non-replicating and persistent inside the cytoplasm of avian cells. RT-qPCR provides insight into the molecular pathogenesis of both active replicating and persistent Chlamydia psittaci in birds, but requires identification of stably expressed reference genes to avoid biases. Material and Methods: We investigated the expression stability of 10 C. psittaci candidate reference genes for gene expression analysis during normal growth and penicillin-induced persistence. C. psittaci Cal10 was cultured in HeLa229 and RNA was extracted. The expression level of each candidate was examined by RT-qPCR and Cq values were analysed using geNorm. Results: The genes tyrS, gidA, radA, and 16S rRNA ranked among the most stably expressed. The final selected reference genes differed according to the bacterial growth status (normal growth versus persistent status), and the time points selected during the duration of the normal chlamydial developmental cycle. Conclusion: The study data show the importance of systematic validation of reference genes to confirm their stability within the strains and under the conditions selected.

OBJECTIVE To determine population pharmacokinetics of enrofloxacin in purple sea stars (Pisaster ochraceus) administered an intracoelomic injection of enrofloxacin (5 mg/kg) or immersed in an enrofloxacin solution (5 mg/L) for 6 hours. ANIMALS 28 sea stars of undetermined age and sex. PROCEDURES The study had 2 phases. Twelve sea stars received an intracoelomic injection of enrofloxacin (5 mg/kg) or were immersed in an enrofloxacin solution (5 mg/L) for 6 hours during the injection and immersion phases, respectively. Two untreated sea stars were housed with the treated animals following enrofloxacin administration during both phases. Water vascular system fluid samples were collected from 4 sea stars and all controls at predetermined times during and after enrofloxacin administration. The enrofloxacin concentration in those samples was determined by high-performance liquid chromatography. For each phase, noncompartmental analysis of naïve averaged pooled samples was used to obtain initial parameter estimates; then, population pharmacokinetic analysis was performed that accounted for the sparse sampling technique used. RESULTS Injection phase data were best fit with a 2-compartment model; elimination half-life, peak concentration, area under the curve, and volume of distribution were 42.8 hours, 18.9 μg/mL, 353.8 μg•h/mL, and 0.25 L/kg, respectively. Immersion phase data were best fit with a 1-compartment model; elimination half-life, peak concentration, and area under the curve were 56 hours, 36.3 μg•h/mL, and 0.39 μg/mL, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that the described enrofloxacin administration resulted in water vascular system fluid drug concentrations expected to exceed the minimum inhibitory concentration for many bacterial pathogens.

Bovine anaplasmosis is the disease caused by the bacterium Anaplasma marginale. It can cause production loss and death in cattle and bison. This was a reportable disease in Canada until April 2014. Before then, infected herds were quarantined and culled, removing infected animals. In North America, A. marginale is biologically vectored by hard ticks (Acari: Ixodidae), Dermacentor variabilis and D. andersoni. Biting flies, particularly horse flies (Diptera: Tabanidae), can also act as mechanical vectors. An outbreak of bovine anaplasmosis, consisting of 14 herds, was detected in southern Manitoba in 2008. This outbreak lasted multiple rounds of testing and culling before eradication in 2011, suggesting local maintenance of the pathogen was occurring. We applied novel approaches to examine the vector ecology of this disease in this region. We did not detect A. marginale by screening of 2056 D. variabilis (2011 and 2012) and 520 horse flies (2011) using polymerase chain reaction (PCR).

OBJECTIVE To compare clinical disease and lung lesions in calves experimentally inoculated with Histophilus somni 5 days after metaphylactic administration of tildipirosin or tulathromycin. ANIMALS Twenty-four 3-month-old Holstein and Holstein-crossbreed steers. PROCEDURES Calves were randomly allocated to 3 groups of 8 calves. On day 0, calves in group 1 received tildipirosin (4 mg/kg, SC), calves in group 2 received tulathromycin (2.5 mg/kg, SC), and calves in group 3 received isotonic saline (0.9% NaCl) solution (1 mL/45 kg, SC; control). On day 5, calves were inoculated with 10 mL of a solution containing H somni strain 7735 (1.6 × 109 CFUs/mL, intrabronchially; challenge). Calves were clinically evaluated on days 5 through 8 and euthanized on day 8. The lungs were grossly evaluated for evidence of pneumonia, and bronchial secretion samples underwent bacteriologic culture. RESULTS The mean clinical score for each group was significantly increased 12 hours after challenge, compared with that immediately before challenge, and was significantly lower for tildipirosin-treated calves on days 6, 7, and 8, compared with those for tulathromycin-treated and control calves. The mean percentage of lung consolidation for tildipirosin-treated calves was significantly lower than those for tulathromycin-treated and control calves. Histophilus somni was isolated from the bronchial secretions of some tulathromycin-treated and control calves but was not isolated from tildipirosin-treated calves. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that metaphylactic administration of tildipirosin to calves 5 days prior to H somni challenge prevented subsequent culture of the pathogen from bronchial secretions and was more effective in minimizing clinical disease and lung lesions than was metaphylactic administration of tulathromycin.

Streptococcus suis is an emerging zoonotic pathogen that causes septicemia, meningitis, arthritis, and pneumonia in swine and humans. The present study aimed to characterize the genetic diversity of S. suis serotype 2 isolated from pigs showing signs of illness in Brazil using pulsed-field gel electrophoresis (PFGE), single-enzyme amplified fragment length polymorphism (SE-AFLP), and profiling of virulence-associated markers. A total of 110 isolates were studied, 62.7% of which were isolated from the central nervous system and 19.1% from the respiratory tract. Eight genotypes were obtained from the combination of virulence genes, with 43.6% and 5.5% frequencies for the mrp (+) /epf (+) /sly (+) and mrp (-) /epf (-) /sly (-) genotypes, respectively. The presence of isolates with epf gene variation with higher molecular weight also appears to be a characteristic of Brazilian S. suis serotype 2. The PFGE and SE-AFLP were able to type all isolates and, although they presented a slight tendency to cluster according to state and year of isolation, it was also evident the grouping of different herds in the same PFGE subtype and the existence of isolates originated from the same herd classified into distinct subtypes. No further correlation between the isolation sites and mrp/epf/sly genotypes was observed.

This study was conducted to investigate the high mortality of young lambs in two sheep farms in Pekan, Pahang over a period of 3 years. Samples from postmortem of 1,451 lambs below one year of age by a farm veterinarian were submitted for laboratory diagnosis at the Bacteriology Section of the Regional Veterinary Diagnostic Laboratory in Kuantan. Escherichia coli is the most commonly recorded bacteria with 161 lambs diagnosed in 2013. In 2014 and 2015, there was a decrease in occurrence of E. coli related deaths, with 120 and 75lambs respectively. A total of 25% of the cases showed Escherichia coli positive by culture on blood agar and MacConkey agar, and confirmed by biochemical tests. A total of 21% of the cases were positive for staphylococcus sp, 3% and 6% for Streptococcus sp and Klebsiella pneumonia, respectively. Other bacteria were isolated in 45% of the cases. It was further noted that a total of 285 lambs between the ages of one to four months of age followed by 58 lambs (20%) less than one month old had E.coli isolation. It is also noteworthy that there were 10 lambs with E.coli infection in one to fourteen day-old lambs during the 3-year period from January 2013 to December 2015. This information was collated as a result of routine diagnosis of field cases submitted and with the intention of highlighting the common pathogens causing high mortality in local small ruminant farms so that preventive action may be taken for future farming ventures. E. coli infections or Colibacillosis is an important finding and indicator of poor management including poor nutrition, hygiene and environmental contamination which can reduce animal immunity and render it susceptible to other infections.

The aim of this study was to determine the pharmacokinetics of amikacin and penicillin G sodium when administered in combination as an intravenous regional limb perfusion (IVRLP) to horses. Seven healthy adult horses underwent an IVRLP in the cephalic vein with 2 g of amikacin sulfate and 10 mill IU of penicillin G sodium diluted to 60 mL in 0.9% saline. A pneumatic tourniquet set at 450 mmHg was left in place for 30 min. Synovial fluid was collected from the metacarpophalangeal joint 35 min and 2, 6, 12, and 24 h after infusion of the antimicrobials. Concentrations of amikacin and penicillin in synovial fluid were quantitated by liquid chromatography tandem-mass spectrometry analysis. Therapeutic concentrations of amikacin and penicillin for equine-susceptible pathogens were achieved in the synovial fluid. Maximum synovial concentrations (Cmax) (mean ± SE) for amikacin and penicillin were 132 ± 33 μg/mL and 8474 ± 5710 ng/mL, respectively. Only 3 horses had detectable levels of penicillin at 6 h and 1 at the 12 h sample. The combination of amikacin with penicillin G sodium via IVDLP resulted in reported therapeutic concentrations of both antibiotics in the synovial fluid. The Cmax:MIC (minimum inhibitory concentration) ratio for amikacin was 8:1 and Time > MIC for penicillin was 6 h. At 24 h, the mean concentration of amikacin was still above 4 μg/mL. Terminal elimination rate constants (T1/2 lambdaz) were 13.6 h and 2.8 h for amikacin and penicillin, respectively. The use of IVDLP with penicillin may therefore not be practical as rapid clearance of penicillin from the synovial fluid requires frequent perfusions to maintain acceptable therapeutic concentrations.