Introduction: Bovine parainfluenza virus-3 (BPIV3) and bovine respiratory syncytial virus (BRSV) are the cause of respiratory disease in cattle worldwide. With other pathogens, they cause bovine respiratory disease complex (BRDC) in ruminants. The aim of the study was the detection and molecular characterisation of BPIV3 and BRSV from nasal swabs and lung samples of cows in and around the Erzurum region of eastern Turkey. Material and Methods: In total, 155 samples were collected. Of animals used in the study 92 were males and 63 females. The age of the animals was between 9 months and 5 years, mean 1.4 years. Most males were in the fattening period and being raised in open sheds; females were in the lactating period and kept in free stall barns. All samples were tested for the presence of viral genes using RT-PCR. Gene-specific primers in a molecular method (RT-PCR) identified BRSV (fusion gene) and BPIV3 (matrix gene) strains at the genus level. Results: RNA from BRSV and BPIV3 was detected in two (1.29%) and three (1.93%) samples, respectively, one of each of which was sequenced and the sequences were aligned with reference virus strains. Phylogenetic analyses clustered the strains in genotype C/BPIV3 and subgroup III/BRSV. Conclusion: The results indicate that BRSV and BPIV3 contribute to bovine respiratory disease cases in Turkey. This is the first report on their detection and molecular characterisation in ruminants in Turkey.

Introduction: Clostridium perfringens is commonly found in the gastrointestinal tract of animals and humans and continues to cause one of the most prevalent foodborne diseases in man. Material and Methods: A total of 355 samples were examined for the occurrence of C. perfringens: rectal swabs from cattle, sheep, and goats, fresh stool samples from diarrhoea sufferers having been in contact with these animals, irrigation water and soil samples from the husbandry sites, and preharvesting fresh produce from farms irrigated with the sampled water. All samples were collected from Cairo and Giza governorates, Egypt. PCR analysis was carried out with positive isolates using the α-toxin gene. Sequence analysis of the gene of C. perfringens isolates was performed using the neighbour-joining approach. Bootstrap analysis was executed with 1,000 resamplings. Results: 174 C. perfringens strains were isolated with a 49.01% prevalence. The highest prevalence of C. perfringens in apparently healthy animals was found in sheep (65.45%) followed by goats (58%), buffaloes (55%), and cattle (47.1%). Its prevalence in humans being in contact with these animals was 47.5%. The bacterium’s isolation from the soil and irrigation water was achieved in 40% and 31.7% of samples, respectively, posing a risk, particularly when the water and soil contact food in the field, shown by the fresh produce isolation of 40%. A significant relationship between the prevalence of C. perfringens in animal and environmental samples was identified (P < 0.05). A significant relationship was identified neither between animal species and C. perfringens prevalence, nor between the environmental source and C. perfringens prevalence (P > 0.05). All isolates were positive for the α-toxin gene by PCR. The sequence analysis and the phylogenetic relationship of the α-toxin genes from different samples revealed that C. perfringens from faeces of apparently healthy cattle, buffaloes, sheep, and goats is a significant threat in places where it can contaminate the soil and water. In addition, the sequence of C. perfringens from humans suffering from diarrhoea was found in the same cluster with the sequence from cows, goats, and sheep. Conclusion: The role of apparently healthy animals in transmitting C. perfringens to humans, either through being in direct or indirect contact via water or soil in the cultivation of vegetables and fruits, was demonstrated.

Introduction: Traditionally, evolutionary analysis of equine influenza virus (EIV) is based on the HA gene. However, the specificity of the influenza virus enables the classification of viral strains into different phylogenetic groups, depending on the gene being analysed. The aim of the study was to analyse phylogenetic paths of EIV based on M gene with reference to the HA gene. Material and Methods: M gene of Polish isolates has been sequenced and analysed along with all M sequences of EIV available in GenBank database. Phylogenetic analysis was performed using BioEdit, ClustalW, and MEGA7 softwares. Results: The clustering of the strains isolated not only from Asia but also from Europe into one common Asian-like group of EIV was observed. Twelve nucleotide substitutions in the M gene of strains from the Asian-like group were crucial for the evolutionary analysis. We also observed homology in the M gene of the Asian-like and H7N7 strains. Conclusions: M gene specific for the Asian-like group is present in strains recently isolated in Europe and Asia, which were classified previously in the Florida 2 clade based on HA. Therefore, Asian-like group does not seem to be assigned to a specific geographical region. Traces of H7N7 strains in more conservative genes like M of some contemporary EIV strains may indicate the link between the old phylogenetic group and recent H3N8 strains. Analysis of conservative genes may be more useful in tracking the direction of virus evolution than in the genes where the high variability rate may blur the original relationships.

Toxocara canis and Toxocara cati are roundworms of dogs and cats. The purpose of this study was to investigate the infection caused by these ascarids in cats and dogs, using microscopic and molecular analysis methods. Adult ascarids were gathered from the faeces of dogs and cats in Van province, in 2015–2016. Existing keys and PCR sequencing of the ITS-2 fragment were used to identify the morphological features of the parasite species. It was observed that out of 20 adult ascarids, 17 and 3 were found to be Toxocara canis and Toxocara cati, respectively. The ITS-2 gene region was amplified by PCR to perform molecular analysis. Genotyping indicated that the dogs and cats were infected with T. canis and T. cati, respectively, and none had Toxascaris leonina. To the best of our knowledge, this is the first report on the molecular characteristics of adult ascaridoid nematodes from cats and dogs in Turkey. The molecular approaches established in this study enable molecular identification and genetic structure studies of the ascaridoids.

Introduction: The genomes of nine H5 subtypes of low pathogenic avian influenza virus (LPAIV) strains identified in wild birds in Poland between 2010 and 2015 were sequenced, and their phylogenetic relationship was determined. Material and Methods: AIV genome segments were amplified by RT-PCR and the PCR products were sequenced using Sanger method. Phylogenetic trees were generated in MEGA6 software and digital genotyping approach was used to visualise the relationship between analysed strains and other AIVs. Results: High genetic diversity was found in the analysed strains as multiple subgroups were identified in phylogenetic trees. In the HA tree, Polish strains clustered in two distinct subclades. High diversity was found for PB2, PB1, PA and NP, since 5-8 sublineages could be distinguished. Each strain had a different gene constellation, although relationship of as much as six out of eight gene segments was observed between two isolates. A relationship with poultry isolates was found for at least one segment of each Polish strain. Conclusion: The genome configuration of tested strains indicates extensive reassortment, although the preference for specific gene constellation could be noticed. A significant relationship with isolates of poultry origin underlines the need for constant monitoring of the AIV gene pool circulating in the natural reservoir.

Introduction: Due to their immunostimulatory properties TLR ligands are used prophylactically to protect against a variety of viral and bacterial pathogens in mammals. Knowledge of the molecular and functional aspects of TLRs is essential for a better understanding of the immune system and resistance to diseases in birds. For that reason, this study attempted to determine the impact of TLR21 stimulation by its synthetic ligand (CpG ODN, class B) on the chicken immune system. Material and Methods: Sixty embryonated chicken eggs were randomly allocated into three groups (control and two experimental groups). On day 18 of embryonic development, chickens in one experimental group were administered in ovo a low dose of CpG ODN and the birds of the second experimental group were given a high dose of the ligand. Spleens were collected at 1, 2, 5, and 10 days post-hatching (dph) for analysis of IFN-α, IFN-β, IFN-γ, IL-6, and IL-10 expression using qRT-PCR. Results: Significant differences were observed in mRNA expression levels of all the measured cytokines associated with the modulation and regulation of the immune response at different time points. Conclusion: The obtained data clearly demonstrate that immune response induction takes place after in ovo administration of class B CpG ODN, and that the ligand has the ability to induce cytokine responses in neonatal chicken spleen.

Introduction: The aim of the study was to determine microbiota in the cloacal samples of European herring gulls (Larus argentatus) and to compare a variety of genes encoding antimicrobial resistance in cultivable and non-cultivable bacteria. Material and Methods: Cloacal samples from European herring gulls were collected from a Kaunas city dump. Cultivable microbiota were isolated, their microbial susceptibility was tested, and genes encoding antimicrobial resistance were detected. Additionally, a metagenomic study was performed using Next-Generation Sequencing (NGS). Results: In total, 697 different operational taxonomic units at genus level were detected; however, only 63 taxonomic units were detected at the amount of ≥0.1% of the total number of DNA copies. Catellicoccus marimammalium was found to have the highest prevalence. The bacterial amount of other genera was up to 5% with the most highly prevalent being Psychrobacter (4.7%), Helicobacter (4.5%), unclassified Enterococcaceae (3.2%), Pseudomonas (2.9%), and Brachyspira (2.6%). Conclusions: C. marimammalium are predominant microbiota in the cloacal samples of Larus argentatus. This species of gulls is a reservoir of bacteria carrying a wide-spectrum of genes encoding antimicrobial resistance. The same genes were detected in both cultivable microbiota and in the total DNA of the samples.

Introduction: A real-time RT-PCR method for identification and quantification of porcine epidemic diarrhoea virus (PEDV) strains in China was developed.Material and Methods: Based on the conserved sequence of the PEDV nucleocapsid (N) gene, a primer pair and probe were designed to establish a TaqMan-MGB real-time RT-PCR assay for quantitative detection of the virus. The sequence was cloned into the pMD18-T vector and a series of diluted recombinant plasmids were used to generate a standard curve with an R2 value of 0.999.Results: The developed quantitative PCR assay detected viral titres as low as 0.1 TCID₅₀ with high specificity and no cross-reaction with other porcine viruses (PoRV, TGEV, PRRSV, or CSFV). The intra-batch and inter-batch coefficients of variation were both less than 1%, which indicated good reproducibility. Thirty clinical diarrhoea samples obtained from pigs in Shanghai and Fujian were analysed using this quantitative PCR assay. Out of these samples, 93.3% were found to be PEDV positive.Conclusion: This approach is suitable for clinical sample identification and pathogenesis studies.

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.