Introduction: The aim of the study was to optimise and compare two multiplex PCR assays for the detection of Listeria spp. and Listeria monocytogenes in biological samples including the liver, brain, and blood. Material and Methods: Three strains of L. monocytogenes and single strains of each of the species: L. ivanovii, L. innocua, L. grayi, L. welshimeri, and L. seeligeri were used. Additionally, five other species of bacterium were used to evaluate the specificity of the tests. Results: Specific amplification products were obtained for both multiplex PCR assays, which confirmed the tested strains as Listeria spp. and L. monocytogenes, respectively. Isolates of other species did not yield PCR products. Conclusion: Both multiplex PCR assays proved to be significantly sensitive and highly-specific methods for the detection of Listeria strains.

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: Porcine pleuropneumonia inflicts important economic losses on most commercial herds. Detection of subclinical or chronic infection in animals still remains a challenge, as isolation and identification of A. pleuropneumoniae serotypes is difficult and quantification of the bacteria on agar plates is often almost impossible. The aim of the study was to develop and evaluate a serotype-specific quantitative TaqMan probe-based PCR for detection of serotype 2 in pig lungs, tonsils, and nasal swabs.Material and Methods: The primers were designed from the capsular polysaccharide biosynthesis genes of A. pleuropneumoniae serotype 2. PCR specificity and sensitivity were evaluated using reference strains and several other bacterial species commonly isolated from pigs.Results: The real-time qPCR for detection of A. pleuropneumoniae serotype 2 was highly specific and gave no false positives with other serotypes or different bacterial species of pig origin. The detection limit for pure culture was 1.2 × 10⁴ CFU/mL, for lung tissue and nasal swabs it was 1.2 × 10⁵ CFU/mL, and for tonsils - 1.2 × 10⁵ CFU/mL.Conclusion: The method can be used to serotype A. pleuropneumoniae isolates obtained during cultivation and to detect and identify A. pleuropneumoniae serotype 2 directly in nasal swabs and tonsil scrapings obtained from live pigs or lung tissue and tonsils collected post-mortem.

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 evaluate adherence of methicillin-resistant Staphylococcus pseudintermedius (MRSP) to 5 suture materials commonly used in small animal surgery. SAMPLE 10 epidemiologically unrelated MRSP isolates (obtained from dogs with clinical infections) that had strong biofilm-forming ability and 5 types of suture. PROCEDURES The 5 types of suture evaluated were monofilament polyglecaprone 25, monofilament polydioxanone, triclosan-coated (TC)–monofilament polydioxanone, braided polyglactin 910, and barbed monofilament polydioxanone. Suture segments were incubated in standard suspensions of MRSP for 2 minutes. Segments were then placed in tryptone soy broth and incubated overnight. After incubation, segments were rinsed with PBS solution and sonicated to dislodge adherent bacteria. Resulting suspensions were used to create serial dilutions that were plated, incubated overnight, and counted the following day. Bacterial adherence to 1 segment of each suture type was assessed by use of scanning electron microscopy. RESULTS There was significantly less adherence of MSRP to TC–monofilament polydioxanone than to polyglecaprone 25, polyglactin 910, barbed monofilament polydioxanone, and monofilament polydioxanone. There was significantly less adherence of MSRP to polyglecaprone than to polyglactin 910. CONCLUSIONS AND CLINICAL RELEVANCE Barbed suture had a bacterial adherence profile comparable to that for monofilament suture. Adherence of MRSP was greatest for braided polyglactin 910. Use of TC–monofilament polydioxanone can be considered for patients that are at high risk of developing surgical site infections and for which a surgeon chooses a multifilament suture.

OBJECTIVE To develop and validate a real-time quantitative PCR (qPCR) assay for the detection and quantification of Mycoplasma ovis in goats and investigate the prevalence and risk factors for hemoplasma infection of goats located in Indiana. ANIMALS 362 adult female goats on 61 farms. PROCEDURES Primers were designed for amplification of a fragment of the dnaK gene of M ovis by use of a qPCR assay. Blood samples were collected into EDTA-containing tubes for use in total DNA extraction, blood film evaluation, and determination of PCV. Limit of detection, intra-assay variability, interassay variability, and specificity of the assay were determined. RESULTS Reaction efficiency of the qPCR assay was 94.45% (R2, 0.99; slope, −3.4623), and the assay consistently detected as few as 10 copies of plasmid/reaction. Prevalence of infection in goats on the basis of results for the qPCR assay was 18.0% (95% confidence interval, 14% to 22%), with infected goats ranging from 1 to 14 years old, whereby 61% (95% confidence interval, 47% to 73%) of the farms had at least 1 infected goat. Bacterial load in goats infected with M ovis ranged from 1.05 × 103 target copies/mL of blood to 1.85 × 105 target copies/mL of blood; however, no bacteria were observed on blood films. Production use of a goat was the only risk factor significantly associated with hemoplasma infection. CONCLUSIONS AND CLINICAL RELEVANCE The qPCR assay was more sensitive for detecting hemoplasma infection than was evaluation of a blood film, and production use of a goat was a risk factor for infection.

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 determine effects of in ovo administration of a probiotic on development of the intestinal microbiota of 2 genetic lineages (modern and heritage) of chickens. SAMPLE 10 newly hatched chicks and 40 fertile eggs to determine intestinal microbiota at hatch, 900 fertile eggs to determine effects of probiotic on hatchability, and 1,560 chicks from treated or control eggs. PROCEDURES A probiotic competitive-exclusion product derived from adult microbiota was administered in ovo to fertile eggs of both genetic lineages. Cecal contents and tissues were collected from embryos, newly hatched chicks, and chicks. A PCR assay was used to detect bacteria present within the cecum of newly hatched chicks. Fluorescence in situ hybridization and vitality staining were used to detect viable bacteria within intestines of embryos. The intestinal microbiota was assessed by use of 16S pyrosequencing. RESULTS Microscopic evaluation of embryonic cecal contents and tissues subjected to differential staining techniques revealed viable bacteria in low numbers. Development of the intestinal microbiota of broiler chicks of both genetic lineages was enhanced by in ovo administration of adult microbiota. Although the treatment increased diversity and affected composition of the microbiota of chicks, most bacterial species present in the probiotic were transient colonizers. However, the treatment decreased the abundance of undesirable bacterial species within heritage lineage chicks. CONCLUSIONS AND CLINICAL RELEVANCE In ovo inoculation of a probiotic competitive-exclusion product derived from adult microbiota may be a viable method of managing development of the microbiota and reducing the prevalence of pathogenic bacteria in chickens.