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.

Introduction: This article presents the analysis of the correlation between the category and health status of calves and the results of their rearing and levels of selected blood parameters.Material and Methods: The study included 105 Polish Holstein-Friesian and beef (Limousine, Charolaise and Hereford) crossbred calves. Young bulls were purchased at the age of two to four weeks. The animals underwent quarantine, were dehorned, and 46 young bulls were castrated. The germ horns were removed by burning out. Castration was carried out with a bloodless method using a rubber band. The calves were kept in groups and fed a milk replacer administered via teats from automated milk-feeding stations. After the period of milk feeding, the calves were fed grass silage ad libitum and a concentrate at 2.5 kg/animal/day. The calves were weighed every two weeks. Blood for analyses was sampled at 43 d of age.Results: After the rearing period finished at the age of six months, young bulls and steers had similar body weights (176.17 and 176.55 kg) and approximate average daily weight gains from birth (0.756 and 0.767 g/day). The healthy calves at six months of age weighed 180.47 kg, whereas the animals which at least once suffered from some diseases during rearing were lighter by approx. 30 kg (P ≤ 0.01). A statistically significant (P ≤ 0.01) difference was found for the count of red blood cells and white blood cells. In comparison with healthy individuals, the diseased animals had less RBC (8.33 and 9.42 10¹²/L respectively) and more WBC (27.03 and 12.26 10⁹/L respectively).Conclusion: Castration of young bulls did not have any impact on the results of rearing and health status of the calves. The magnitude of the analysed parameters depended on the health status of the calves. Thus RBC and WBC parameters may be used to predict the health status of calves during rearing.

Introduction: Avian pathogenic Escherichia coli (APEC) is a leading cause of extraintestinal infection and heavy economic losses. Imparting immunity after vaccination with live attenuated strain vaccination is an ideal strategy for infection control. This study considers an FtsK knockout mutant strain as a candidate. Material and Methods: An FtsK knockout mutant of APEC strain E058 was constructed and the pathogenicity of the mutant and wild-type strains was further evaluated in chickens. Results: The 50% lethal doses of each strain for one-day-old specific-pathogen-free (SPF) chickens challenged experimentally via trachea were 10⁵.⁵ and 10⁷.⁰ colony-forming units (CFU) respectively. Chickens challenged with the wild-type strain exhibited typical signs and lesions of avian colibacillosis, while those inoculated with the mutant strain showed mild pericarditis and pulmonary congestion. The growth rate of the FtsK mutant strain was much slower than the wild-type strain in the heart, spleen, liver, and lung of infected chickens. Conclusion: These results indicated that the APEC FtsK mutant can be attenuated for chickens, and that this mutant has the potential for the development of an APEC vaccine.

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 the study was to explore the effect of enrofloxacin on production of selected cytokines by porcine peripheral blood mononuclear cells (PBMCs).Material and Methods: Twenty pigs (10 control and 10 experimental) were used in this research. Pigs from experimental group received enrofloxacin at therapeutic doses. Blood samples were collected before, during, and after treatment with enrofloxacin. PBMCs were incubated with or without lipopolysaccharide (LPS). Production of IL-6, IL-10, INF-γ, and TNF-α were determined by ELISA.Results: Administration of enrofloxacin to healthy pigs for 5 d induced a transient reduction of the PBMCs response to LPS in terms of IL-6 and TNF-α secretion. The concentration of IL-6 returned to the day 0 level shortly after treatment, while TNF-α production remained reduced for 10 d after the treatment. The production of IL-10 was not affected by enrofloxacin. The level of IFN-γ was below the detection limit of the tests.Conclusion: The results indicate that enrofloxacin administered in vivo in therapeutic doses has an immunomodulatory effect through its capacity to inhibit secretion of IL-6 and TNF-α by porcine PBMC stimulated by LPS.

Introduction: The aim of this study was to propose the optimal methodology for stallion semen morphology analysis while taking into consideration the staining method, the microscopic techniques, and the workload generated by a number of samples. Material and Methods: Ejaculates from eight pure-bred Arabian horses were tested microscopically for the incidence of morphological defects in the spermatozoa. Two different staining methods (eosin-nigrosin and eosin-gentian dye), two different techniques of microscopic analysis (1000× and 400× magnifications), and two sample sizes (200 and 500 spermatozoa) were used. Results: Well-formed spermatozoa and those with major and minor defects according to Blom’s classification were identified. The applied staining methods gave similar results and could be used in stallion sperm morphology analysis. However, the eosin-nigrosin method was more recommendable, because it allowed to limit the number of visible artefacts without hindering the identification of protoplasm drops and enables the differentiation of living and dead spermatozoa. Conclusion: The applied microscopic techniques proved to be equally efficacious. Therefore, it is practically possible to opt for the simpler and faster 400x technique of analysing sperm morphology to examine stallion semen. We also found that the number of spermatozoa clearly affects the results of sperm morphology evaluation. Reducing the number of spermatozoa from 500 to 200 causes a decrease in the percentage of spermatozoa identified as normal and an increase in the percentage of spermatozoa determined as morphologically defective.

Introduction: Due to the high heterogeneity of canine lymphoma, the aim of the present study was to test in vitro the chemosensitivity of canine high-grade primary lymphoma cells to various cytostatic drugs commonly used to treat dogs: 4-HO-cyclophosphamide, doxorubicin, dexamethasone, prednisolone, vincristine, etoposide, chlorambucil, lomustine, and cytosine arabinoside. Material and Methods: To determine the cell viability and drug ability to induce apoptosis two different tests were used: an MTT assay and annexin V/propidium iodide staining. Results: Both in vitro tests were found to be useful tools. Significant differences in the sensitivity, depending on the drug type, between B-, T- and mixed/null-type lymphoma cells were found for the majority of the tested drugs. B-type cells were the most sensitive in vitro, whereas T-type cells seemed to be the most resistant. Doxorubicin, chlorambucil, etoposide, and vincristine most strongly reduced the cell viability and induced apoptosis. Conclusion: In vitro assays, such as the MTT test and especially the annexin V/PI assay, may be useful tools for predicting a response to the treatment of high-grade lymphoma in dogs or improving the treatment outcomes in individual animals.

Introduction: Several Mycoplasma species can cause severe diseases in ruminant hosts, some of which are the diseases listed by the World Organisation for Animal Health (OIE). The role of the Cervidae family in carrying and transmitting ruminant mycoplasma infections in Poland is unknown. Material and Methods: Antibody and antigen detection tests for the main mycoplasma species that can affect wild ruminants were performed on 237 samples (serum, nasal swab, bronchoalveolar lavage, and lung) collected from 161 animals during 2011-2014. The samples were obtained from a cull of healthy population of deer which included: 96 red deer (Cervus elaphus elaphus), 19 fallow deer (Dama dama), and 46 roe deer (Capreolus capreolus). Results: Serological screening tests revealed positive reactions to Mycoplasma bovis in one sample and to Mycoplasma capricolum subsp. capripneumoniae in three samples; however, these three samples were negative by immunoblotting. Other antibody and antigen detection tests demonstrated negative results. Conclusion: Currently wild cervids in Poland do not play a significant role in transmitting mycoplasma infections to domestic animals, but they remain a potential risk.

Introduction: The article presents a rapid and simple analytical procedure for determination of four tetracyclines (oxytetracycline, tetracycline, chlortetracycline, and doxycycline) in animal medicated feedingstuffs. Material and Methods: Two-gramme samples were extracted by a Na2EDTA-McIlvaine buffer (pH 4)/methanol mixtures (40/60, v/v). The determination was achieved by liquid chromatography using a Zorbax Eclipse XDB C18 analytical column with mass spectrometer detection (LC-MS). Results: Recoveries of the antibiotics from spiked feed samples ranged from 78.2% to 113.5%. The LOD and LOQ for tetracyclines in feeds ranged from 2.8 to 4.2 and from 4.3 to 5.7 mg/kg, respectively. Conclusion: The method was successfully validated and proved to be efficient, precise, and useful for quantification of tetracyclines in medicated feedingstuffs.

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.