Introduction: The aim of the study was to test the utility of Flinders Technology Associates filter paper (FTA® Cards) for molecular detection and storage of avian influenza virus (AIV). Material and Methods: There were two strains of AIV used in the study: low pathogenicity H7N1 and high pathogenicity H5N1 subtypes. Detection of viral material was conducted using molecular RT-PCR and rRT- PCR method. Results: The infectivity of LPAIV/H7N1 and HPAIV/H5N1 was completely inactivated within 1 h and 24 h after adsorption to FTA® Cards at room temperature, respectively. Viruses stored on FTA® Cards had detection limit approximately 1 log₁₀ lower than live viruses. Viral RNA of both strains were detectable on the cards by rRT-PCR for a minimum of 150 d, irrespectively of storage temperatures (room temperature, -20ºC). RNA was also detected in all samples obtained from SPF chickens experimentally infected with HPAI/H5N1 on 3ʳᵈ and 4ᵗʰ day post-infection (p.i.). Conclusion: FTA® Cards enable safe and effective alternative transport of samples for molecular diagnosis of AIV.

Introduction: The aim of this study was to use TaqMan real-time PCR technique to investigate Jaagsiekte sheep retrovirus (JSRV) proviral DNA in whole blood samples of sheep, and compare the results to those of histopathological examinations. Material and Methods: Eighty blood samples from clinically healthy sheep were randomly collected before the animals were slaughtered. Ten tissue samples from each lung and associated caudal mediastinal lymph node were taken. Results: Fifteen (18.75%) blood samples were found to contain proviral DNA, and 11 (13.75%) corresponding lung samples showed microscopic changes consistent with ovine pulmonary adenocarcinoma. None of the samples displayed metastases to the caudal mediastinal lymph nodes. The prominent pattern of neoplastic nodules consisted of acinar (alveolar) form. Conclusion: The results indicated the higher sensitivity of real-time PCR compared to histopathological examinations in detection of ovine pulmonary adenocarcinoma.

Introduction: The main problem in poultry farming is the difficulty in producing food of animal origin without using antibacterial agents. Because most antibacterial compounds are dispensed in water, some water supply systems can be contaminated by antibiotics which are then administered to the animals unintentionally. This can lead to unexpected increases in antibiotic residues in food of animal origin. The aim of the present study was to determine whether the constant exposure of chicken broilers to enrofloxacin affects the withdrawal time of a therapeutic doxycycline that is intentionally administered to the chickens.Material and Methods: The concentrations of doxycycline, enrofloxacin, and ciprofloxacin were determined by LC-MS/MS in muscles and liver of the chickens.Results: Doxycycline residue concentrations in the chicken tissues from the group that received trace amounts of enrofloxacin were nearly 50% greater than those of the group that received only doxycycline.Conclusion: These results indicated that constant exposure to enrofloxacin in trace amounts significantly influences the residual doxycycline concentration in chicken tissues.

Introduction: Glycolic changes which occur post-mortem have an impact on the physical and sensory features of beef, which in turn determine the successive processes and influence such beef quality traits as colour, tenderness, and cooling loss. The aim of this study was evaluation of the post-mortem changes in bovine meat during aging, quantitative analysis of glycogen and lactic acid, as well as examination of their impact on technological and sensory quality of selected muscles from Holstein-Friesian × Limousin breed carcasses.Material and Methods: The study included three muscles of different metabolic qualities and sarcomere length: m. semitendinosus, m. longissimus dorsi, and m. psoas major, collected from nine bull carcasses aged 24 ±2 months.Results: Significant correlations were found between the volume of cooling loss on individual days of aging and the pH value of muscle tissue, lactic acid and glycogen content, as well as beef lightness. However, no significant dependency between the volume of glycogen and the intensity of red and yellow colours was detected.Conclusion: The colorimetric analysis of glycogen and lactic acid can be an effective tool in predicting the quality of beef.

Introduction: Leptospirosis affects a wide range of mammals, humans, and even a few poikilothermic animal species. In Pakistan, serological studies of equine leptospirosis have reported a prevalence of over 40%, but no study has ever been conducted towards molecular detection of Leptospira in horses. Material and Methods: Blood samples from 128 horses were screened using ELISA and 41 positive samples were examined for the presence of leptospiral DNA using specific primers for 16S rRNA gene. Results: Out of 41 tested samples, 20 samples were found to be PCR-positive, revealing a fragment of 306 bp after gel electrophoresis. Sequencing and phylogenetic analysis of positive samples revealed circulation of pathogenic Leptospira spp. in Pakistani horses. No evidence of circulation of intermediate species was found in this study. Conclusion: This study reports the first molecular evidence of equine leptospirosis in Pakistan and lays ground for further research in this area. It also confirms the efficiency of 16S rRNA for the diagnosis of equine leptospirosis.

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: 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: The aim of the study was to investigate changes in the serum levels of adiponectin and TNF-α, as well as insulin sensitivity, and to elucidate the possible relationship among the parameters and negative energy balance during the periparturient period of dairy cows.Material and Methods: Thirty primiparous Holstein dairy cows were selected for the study. Blood samples were collected from each cow seven days before the expected calving date, on the calving day, and 7, 14, and 21 days after calving. Blood non-esterified fatty acids (NEFA), β-hydroxybutyric acid (BHBA), glucose, insulin, adiponectin, and TNF- α levels were measured. Revised Quantitative Insulin Sensitivity Check Index (rQUICKI) was calculated using data on NEFA, insulin, and glucose concentrations.Results: When compared to prepartum levels, serum concentration of adiponectin significantly increased on day 21 postpartum. The rQUICKI increased and NEFA levels decreased on day 7 after parturition. Insulin and glucose levels decreased on days 7, 14, and 21 postpartum when compared with prepartum levels. BHBA levels decreased on day 21 and TNF- α concentration also decreased on days 7, 14, and 21 postpartum. Adiponectin levels positively correlated with NEFA during the preparturient period. Negative correlation was detected between adiponectin and rQUICKI on calving day and on 14ᵗʰ day after parturition. TNF- α concentration positively correlated with glucose levels on day 7 prepartum and on 21ˢᵗ day postpartum and with rQUICKI on 21ˢᵗ day postpartum. Negative correlation was detected between adiponectin level and insulin sensitivity.Conclusion: Based on the results of the study, we concluded that adiponectin could possibly increase insulin sensitivity when blood NEFA concentrations are elevated.

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.

Increased incidence of protothecal mastitis has been recorded in several countries in the past ten years. The main goal of this article is to draw the attention of scientific and professional community to the emerging issue of mammary protothecosis. The article collates currently known facts about infection reservoirs, predisposing factors for the development of mastitis, clinical manifestations of the disease, and potential transmission routes within the herd as well as the measures for control and eradication. We would like to point out that identification of protothecal mastitis on a dairy farm is associated with a range of problems. Early detection of infected animals can be difficult because of predominantly subclinical course of early-stage infection, which easily spreads between cows via the milking system. Spontaneous recovery has not been recorded and infected cows typically develop chronic mastitis with granulomatous infiltration and progressive loss of functional parenchyma of the mammary gland. Substantial economic losses and health damages associated with mammary protothecosis strongly emphasise the need for developing effective prevention strategies aimed at control of the infection.