Introduction: In the European Union the use of steroid growth promoters is prohibited under Council Directive 96/22/EC. For effective control of illegal use of natural steroids, highly sensitive analytical methods are required, because sex hormones can be present in very low concentrations in biological samples. The aim of the study was to develop a confirmatory method for the detection of testosterone in bovine serum at ppt level.

Introduction: In the European Union the use of steroid growth promoters is prohibited under Council Directive 96/22/EC. For effective control of illegal use of natural steroids, highly sensitive analytical methods are required, because sex hormones can be present in very low concentrations in biological samples. The aim of the study was to develop a confirmatory method for the detection of testosterone in bovine serum at ppt level. Material and Methods: 17β-testosterone and internal standards of 17β-testosterone-d2 were extracted from serum samples with a mixture of tert-butyl methyl ether/petroleum ether and were directly analysed by an LC/MS/MS on QTRAP 5500 instrument with a TurboIon-Spray source operating in a positive ionisation mode. Chromatographic separation was achieved on the analytical column Inertsil® ODS-3 with an isocratic elution using mobile phase consisting of acetonitrile, methanol, and water. Method validation has been carried out in accordance with the Commission Decision 2002/657/EC. Results: The method was characterised by good recovery (82%) and precision (R.S.D 17 %). Decision limit (CCα) and detection capability (CCβ) was 0.05 μg L⁻¹ and 0.09 μg L⁻¹ respectively. The method met the criteria set out in Commission Decision 2002/657/EC for the purpose of confirmation in terms of retention time and ion ratio in the whole range of its application. Conclusions: The developed method is specific and sensitive, suitable for measuring the natural level of testosterone in blood of cattle and for use in routine control programme for the detection of this hormone in bovine serum.

Introduction: Tuberculosis is a highly infectious disease affecting humans and animals. It is caused by the Mycobacterium tuberculosis complex (MTBC) – Mycobacterium bovis and Mycobacterium caprae, which are aetiological factors of bovine tuberculosis (bTB). In Poland, the bTB eradication programme exists. Animals diagnosed with tuberculosis are in the majority of cases not treated, but removed from their herd and then sanitary slaughtered.

Introduction: Tuberculosis is a highly infectious disease affecting humans and animals. It is caused by the Mycobacterium tuberculosis complex (MTBC) – Mycobacterium bovis and Mycobacterium caprae, which are aetiological factors of bovine tuberculosis (bTB). In Poland, the bTB eradication programme exists. Animals diagnosed with tuberculosis are in the majority of cases not treated, but removed from their herd and then sanitary slaughtered. Material and Methods: In total, 134 MTBC strains isolated from cattle in Poland were subjected to microbiological analysis. The resistance phenotype was tested for first-line antimycobacterial drugs used in tuberculosis treatment in humans: streptomycin, isoniazid, rifampicin, ethambutol, and pyrazinamide. The strains were isolated from tissues collected post mortem, so the test for drug resistance fulfilled only epidemiological criterion. Results: The analysis of drug-resistance of MTBC strains revealed that strains classified as M. bovis were susceptible to 4 antimycobacterial drugs: isoniazid, rifampicin, streptomycin, and ethambutol, and resistant to pyrazynamide. The strains classified as M. caprae were sensitive to all tested drugs. Conclusion: The results indicate that despite enormously dynamic changes in mycobacterial phenotype, Polish strains of MTBC isolated from cattle have not acquired environmental resistance. The strains classified as M. bovis are characterised by natural resistance to pyrazinamide, which is typical for this species.

Introduction: Vascular cell adhesion molecule 1 (VCAM-1) is a member of Ig superfamily. The aim of this study was to prepare highly specific polyclonal antibodies against bovine VCAM-1 and to evaluate the expression of VCAM-1 in the mammary lymph nodes of cows with subclinical mastitis.

Introduction: Vascular cell adhesion molecule 1 (VCAM-1) is a member of Ig superfamily. The aim of this study was to prepare highly specific polyclonal antibodies against bovine VCAM-1 and to evaluate the expression of VCAM-1 in the mammary lymph nodes of cows with subclinical mastitis.Material and Methods: The VCAM-1 gene was cloned from bovine Peyer’s patches and inserted into the pGEX-4T-1 and pET-28a vectors. The recombinant plasmids pGEX-4T-1/VCAM-1 and pET-28a/VCAM-1 were transferred into Escherichia coli BL21 and the recombinant strains were induced by isopropyl-D-thiogalactoside to produce fusion proteins tagged with polyhistidine (His) and glutathione S-transferase (GST), respectively. The expressed fusion proteins His-VCAM-1 and GST-VCAM-1 were identified by SDS-PAGE and Western blot. His-VCAM-1 protein was used as an antigen to immunise Wistar rats and polyclonal antibody serum against VCAM-1 was obtained.Results: The serum titre tested by indirect ELISA was 128,000 using GST-VCAM-1 as the well coating antigen. Western blots indicated that the antibody recognised recombinant VCAM-1 protein as well as endogenous VCAM-1. In addition, using qPCR and Western blot, VCAM-1 mRNA and protein expression levels were measured in dairy cows with subclinical mastitis. It was demonstrated that VCAM-1 levels in the mammary lymph nodes of the cows were significantly higher than those from healthy controls (P < 0.05).Conclusion: These results are to our knowledge the first report that VCAM-1 expression in the mammary lymph nodes is elevated in dairy cows with subclinical mastitis.

Introduction: The transition period is the most challenging time for dairy cattle, which is characterised not only by negative energy balance but also by fatty tissue mobilisation.

Introduction: The transition period is the most challenging time for dairy cattle, which is characterised not only by negative energy balance but also by fatty tissue mobilisation.Material and Methods: The efficiency of energy pathways, β-oxidation in WBC and glycolysis in RBC (based on deoxyglucose transmembrane transport) were estimated. Insulin in blood plasma was determined using ELISA.Results: After calving and up to one month after delivery, a significant drop in blood plasma level was noticed, simultaneously with a rise in β-oxidation from 18.93 ±3.64 to 30.32 ±5.28 pmol/min/mg protein in WBC. A strong negative correlation between these two indices (r = −0.68) was found. During the period of transition to lactation an increase in glucose cross-membrane transportation from 41.44 ±4.92 to 50.49 ±6.41 μmol/h/g Hb was observed. A strong positive correlation between glucose transportation in RBC and β-oxidation in WBC (r = 0.71) was noticed. These data are in agreement with results of studies on dairy cows using liver slices from dairy cows in late pregnancy and different stages of lactation, in which changes in gene expression were analysed.Conclusion: It seems that measuring fatty acids oxidation and glycolysis using isolated blood cells may be an adequate and relatively simple method for energy state analysis to estimate the state of dairy cow metabolism and animal health.

Introduction: The purpose of this study was to determine the occurrence of avian reovirus (ARV) infections in wild birds in Poland and attempt to propagate the selected ARV strains in chicken embryo kidney (CEK) cells or chicken SPF embryos. Material and Methods: The study included 192 wild birds representing 32 species, collected between 2014 and 2016. A part of the S4 segment encoding the σNS protein of avian reoviruses (ARVs) isolated from different species of wild birds from that period was amplified. Results: The presence of ARV was demonstrated in 58 (30.2%) wild birds belonging to nine orders. The isolated strains were propagated in chicken embryos by yolk sac inoculation, and CPE was induced in the infected CEK monolayer. Agar gel precipitation showed that two ARV isolates from rock pigeon and mute swan shared a common groupspecific antigen with chicken reovirus S1133. Specific products of predicted size were found in two ARV isolates from the chicken embryo passage and 13 ARVs isolated from CEK cells. Conclusion: The study indicates the high prevalence of ARV among wild birds in Poland and its possible transmission to farmed birds.

Introduction: The purpose of this study was to determine the occurrence of avian reovirus (ARV) infections in wild birds in Poland and attempt to propagate the selected ARV strains in chicken embryo kidney (CEK) cells or chicken SPF embryos. Material and Methods: The study included 192 wild birds representing 32 species, collected between 2014 and 2016. A part of the S4 segment encoding the σNS protein of avian reoviruses (ARVs) isolated from different species of wild birds from that period was amplified. Results: The presence of ARV was demonstrated in 58 (30.2%) wild birds belonging to nine orders. The isolated strains were propagated in chicken embryos by yolk sac inoculation, and CPE was induced in the infected CEK monolayer. Agar gel precipitation showed that two ARV isolates from rock pigeon and mute swan shared a common groupspecific antigen with chicken reovirus S1133. Specific products of predicted size were found in two ARV isolates from the chicken embryo passage and 13 ARVs isolated from CEK cells. Conclusion: The study indicates the high prevalence of ARV among wild birds in Poland and its possible transmission to farmed birds.

Introduction: Data collection on the Salmonella occurrence is crucial in effective implementation of different actions or control programmes aiming to protect consumers’ health and to reduce the level of Salmonella prevalence in farm animals. The goal was to describe Salmonella serovar distribution along the food chain in Poland during 2010–2015 and to identify their epidemiological importance.

Introduction: Data collection on the Salmonella occurrence is crucial in effective implementation of different actions or control programmes aiming to protect consumers’ health and to reduce the level of Salmonella prevalence in farm animals. The goal was to describe Salmonella serovar distribution along the food chain in Poland during 2010–2015 and to identify their epidemiological importance.Material and Methods: Slide agglutination according to White-Kauffmann-Le Minor scheme was used to identify Salmonella serovars of 6,928 isolates originating from animals, food, feeds, and fertilisers.Results: In total, 160 Salmonella serovars were identified. Differences in serovar distribution were observed depending on animal species. Among isolates from hens, S. Enteritidis and S. Infantis were the most prevalent. Serovar pattern in turkeys differed from those in hens, with S. Kentucky, S. Newport, S. Saintpaul being the most prevalent. Monophasic S. Typhimurium was predominant in pigs. Serovars found in food reflected those observed among livestock animals. Nine out of the ten most prevalent serovars in animals and humans were also found in organic fertilisers.Conclusion: Serotyping of large number of isolates from different sources is essential for insight on emerging serovars and trends of Salmonella occurrence. This may increase the value of epidemiological data and result in updating of Salmonella control programmes to target further epidemiologically important serovars in animals and better protection of consumers’ health.

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