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.

This paper provides an overview of the current knowledge of chlamydiae. These intracellular microorganisms belonging to the Chlamydiaceae family are widely distributed throughout the world. Constant development of culture-independent approaches for characterisation of microbial genomes enables new discoveries in the field of Chlamydia. The number of new taxa is continuously increasing as well as the range of hosts. New species and genotypes are constantly being discovered, particularly new avian and reptilian agents, which are discussed in this article. Interestingly, wild animals are the main hosts for new Chlamydia species including different species of bird, turtle and snake. The availability of next-generation sequencing opens up a new prospect for research and leads to deeper knowledge of these interesting microorganisms about which much is still to discover.

Introduction: Campylobacter jejuni is one of the most frequently reported causes of foodborne bacterial enteric disease worldwide. The main source of these microorganisms is contaminated food, especially of poultry origin. There are several molecular methods for differentiation of Campylobacter isolates at the subgenus level, and one of these is porA-typing based on the sequencing of the major outer-membrane protein (MOMP) encoding gene. The aim of the study was to test the molecular relationship of C. jejuni strains isolated at different points along the poultry food chain and assess the population structure of the isolates. Material and Methods: A total of 451 C. jejuni were used in the study, and a DNA fragment of 630 bp of the MOMP encoding gene was amplified and sequenced. Results: One hundred and ten sequence types were identified, with 69 (62.7%) unique to the isolates' origin and 30 not present in the database. The most prevalent nucleotide variant 1 was detected in 37 (8.2%) strains. These isolates were identified in all poultry sources tested, especially in faeces (15 isolates) but also in poultry carcasses and meat (11 isolates in each). Conclusion: The porA typing method was highly discriminative for C. jejuni of poultry origin since the Simpson's diversity index (D) achieved a value of 0.876, indicating considerable diversity in the bacterial population tested. The method may be further used for epidemiological investigation purposes.

Introduction: The objective of this study was to determine the current profile of bacteria responsible for the infection of the mammary gland and to assess their sensitivity to selected β-lactam antibiotics.

The objective of this study was to investigate the nasal bacterial microbiota of healthy horses and horses infected with equine herpesvirus 1 (EHV-1). The nasal bacterial microbiota of 10 horses infected with EHV-1 and 11 control horses from a farm experiencing an outbreak was characterized using the Illumina MiSeq platform targeting the V4 region of the 16S ribosomal RNA gene. The nasal bacterial microbiota of healthy horses and EHV-1 horses was significantly different in community membership and structure. Horses shedding EHV-1 had lower bacterial richness (P = 0.002), evenness (P = 0.008), and diversity (P = 0.026) than healthy horses. Healthy horses had a higher relative abundance of Firmicutes and Bacteroidetes, but lower Proteobacteria than horses with EHV-1 (P < 0.05). This study provides the basis for generating hypotheses and investigations on the role of bacterial-viral interactions in the health and diseases of adult horses.

The aim of the present study was to characterize the bacterial microbiota of anal sacs in healthy dogs using NGS. Swabs were used to sample the rectum and secretions from each anal sac in 15 healthy dogs. DNA was extracted from swabs and the V4 hypervariable region of the 16S rRNA gene was amplified and sequenced with Illumina MiSeq. Overall, 14 different bacterial phyla were identified in the rectum and in both anal sacs, the 5 main ones being Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Fusobacteria. The rectum had higher microbial diversity and richness than the left and right anal sacs. Community membership and structure significantly differed between the rectum and both anal sacs, but not between the right and the left anal sacs. This study showed that the diversity and richness of the bacterial microbiota of the anal sacs in dogs is greater than what has been reported in previous studies with culture-based methods. In conclusion, the bacterial microbiota of the anal sacs in dogs varies between individuals and differs from the rectal bacterial microbiota.

The effect that topical administration of cyclosporine would have on the number and type of microorganisms isolated from the corneal surface of dogs with keratoconjunctivitis sicca was studied. Schirmer tear tests were performed on and corneal swab specimens were collected from 61 eyes of 31 dogs with keratoconjunctivitis sicca prior to and after 3, 6, and 12 months of treatment with cyclosporine. In eyes that responded to cyclosporine treatment (Schirmer tear test value increased by greater than or equal to 5 mm/min, compared with pretreatment value), the percentage of eyes from which bacteria were isolated after 3, 6, and 12 months of treatment was significantly (P < 0.001) less than the percentage from which bacteria were isolated prior to treatment. However, among eyes that did not respond to treatment, we did not detect a significant change over time in prevalence of bacteria or type of bacteria isolated. The percentage of eyes from which fungi were isolated decreased during treatment; however, the small number of eyes in which fungal culture results were initially positive precluded demonstration of a significant change. For all eyes, we did not detect any significant differences over time in the frequency with which specific bacterial genera were isolated, with the exception of beta-hemolytic Streptococcus spp. Opportunistic corneal infections were not detected even though none of the dogs received antibiotics. An increase in production of tears, which contain anti-infection proteins, was believed to be the primary factor responsible for the decrease in the percentage of eyes from which microorganisms could be isolated.

Fiberoptic bronchoscopy was performed in pigs to assess bacterial contamination of bronchoalveolar lavage fluids (BALF) obtained by use of the method and to determine the aerobic bacterial species in bronchoalveolar airways of healthy pigs. Bacterial contamination of BALF caused by insertion of the bronchoscope was evaluated, using a chromogenic bacterial tracer strain, and was found to be 0.22% of total colony-forming units (CFU), with range between 0 and 1.6%. A total of 164 pulmonary-healthy pigs from 6 closed herds were selected. The BALF obtained from these pigs were examined bacteriologically. Bacteria could not be isolated from 10.4% of all BALF; 5.5% of the BALF samples yielded pure cultures; and 84.1% yielded mixed aerobic bacterial growth. In BALF from 29.2% of the pigs, less than or equal to 5 X 10(2) CFU of bacteria/ml were isolated. The total number of bacteria in BALF from 50% of the pigs varied between 5 X 10(2) and 10(3) CFU/ml; 10.4% of BALF samples contained between 10(3) CFU/ml and 5 X 103 CFU/ml. More than 1 bacterial species were isolated from a single lung lavage of 84.1% of the pigs. Up to 6 species were isolated from a single BALF sample. A total of 443 bacterial isolates were differentiated into 25 bacterial genera and species. Samples of BALF yielded staphylococci (67.6%: Staphylococcus hyicus from 13.4% of the samples and S aureus from 2.4%), alpha-hemolytic streptococci (49.4%), Escherichia coli (42.1%), non-hemolytic streptococci (26.2%), Klebsiella spp (18.3%), micrococci (12.8%), and Coryneformes (11.0%). Other bacterial species were found, but less frequently. In our study, BALF from all pigs yielded < 5 X 103 CFU/ ml. Thus, low numbers of bacteria known to be facultative pathogens were isolated from BALF without causing detectable pneumonia.

The prevalence of mycoplasmal and ureaplasmal recovery from tracheobronchial lavage specimens and prevalence of mycoplasmal recovery from pharyngeal swab specimens from cats with (28) or without (18) pulmonary disease were determined. Mycoplasmas were recovered from tracheobronchial lavage specimens in 21% of cats with pulmonary disease, but in no cats without pulmonary disease; this difference is significant (P = 0.04). Mycoplasmal recovery from tracheobronchial lavage specimens was not significantly associated with concurrent Pasteurella spp isolation, septic inflammation, or bronchitis. Ureaplasmas were only isolated from a tracheobronchial lavage specimen in cat with pulmonary disease and in no cats without pulmonary disease. Similar mycoplasmal recovery rates were found for pharyngeal swab specimens from cats with (39%) or without (35%) pulmonary disease. Seemingly, mycoplasmas are part of the normal pharyngeal flora in approximately a third of the feline population, but mycoplasmas are not normal inhabitants of the lower respiratory tract in cats. It is unknown whether mycoplasmas isolated from tracheobronchial lavage specimens in cats with pulmonary disease are primary pathogens or opportunistic invaders. Seemingly, ureaplasmas are seldom associated with pulmonary disease in cats, and are not normal inhabitants of the trachea and bronchi of cats.