Introduction: Mycoplasma synoviae (MS) is a chicken pathogen of major economic importance. Material and Methods: Between 2010 and 2016, 906 commercial layer chicken flocks in Poland were examined for MS, and the phylogenetic relationship among the strains was established. Regionally dispersed samples were collected and tested with the use of real-time PCR to detect the 16S–23S intergenic spacer region. Positive samples were also tested with LAMP and conventional PCR to detect the vlhA gene. Results: MS genetic material was detected in 265 (29%) of the tested flocks by real-time PCR, in 227 by the LAMP method and in 202 (22%) by conventional PCR. The by-year percentage of positive samples began at 34% in 2010, rose to 44% in 2012, and declined to 29% in 2016. A phylogenetic analysis of Polish M. synoviae strains using a partial sequence of the vlhA gene showed nine genotypes (A–I), the most frequently occurring being F and C. Pathogenic Polish MS field isolates (n = 27) collected from chickens with clinical signs of infection were grouped for their characteristic symptoms: respiratory for genotypes C, E, F, and I (n = 13), EAA and a drop in laying for genotypes F, E, and C (n = 12), and synovitis for genotype A (n = 2). Conclusion: These data showed the country’s isolate diversity. The high prevalence suggests the need to introduce appropriate control programmes. This is the first report of molecular epidemiological data on M. synoviae infection in layer chickens in Poland.

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 development of Jembrana disease vaccine is an important effort to prevent losses in the Bali cattle industry in Indonesia. This study aims to prepare a Jembrana DNA vaccine encoding the transmembrane portion of the envelope protein in pEGFP-C1 and test the success of its delivery in culture cells using a chitosan-DNA complex. Material and Methods: Cloning of the DNA vaccine was successfully performed on E. coli DH5α and confirmed by colony PCR, restriction analysis and sequencing. The plasmids were prepared as a chitosan complex using the complex coacervation method and physicochemically characterised using a particle size analyser. A transfection assay was performed in HeLa cells with 4 h exposure, and mRNA expression was assessed at 24 h post transfection. Results: With a 1:2 (wt./wt.) ratio of DNA and chitosan, the complexes have a mean diameter of 236 nm, zeta potential value of + 17.9 mV, and showed no high toxicity potential in the HeLa cells. This complex successfully delivered the DNA into cells, as shown by the presence of a specific RT-PCR product (336 bp). However, the real-time PCR analysis showed that the delivery with chitosan complex resulted in lower target mRNA expression when compared with a commercial transfecting agent. Conclusion: pEGFP-env-tm JDV as a candidate vaccine can be delivered as the chitosan-DNA complex and be expressed at the transcription level in vitro. This initial study will be used for further improvement and evaluation in vivo.

Introduction: Canine transmissible venereal tumour (CTVT) is a sexually transmitted tumour affecting dogs worldwide, imposing a financial burden on dog owners. A stable culture cell line in continuous passages for >18 months has only been achieved once. The present study investigated a stable CTVT cell line isolated from a bitch and its potential as a vaccine. Material and Methods: A biopsy from a 2-year-old mongrel bitch with CTVT was obtained for histopathological confirmation and isolation of tumour cells. The isolated cells were cultured to passage 55 and characterised by flow cytometry, with karyotyping by GTG-banding and by PCR detection of myc S-2 and LINE AS1. The isolated CTVT cell line was also used as a preventive vaccine in a canine model. Results: Histopathological analysis of the isolated tumour cells revealed typical CTVT characteristics. Constant proliferation and stable morphological characteristics were observed during culture. Phenotypic analysis determined the expression of HLA-DR⁺, CD5.1⁺, CD14⁺, CD45⁺, CD83⁺, CD163⁺, and Ly-6G-Ly-6C⁺. GTG-banding revealed a mean of 57 chromosomes in the karyotype with several complex chromosomal rearrangements. LINE-c-myc insertion in the isolated CTVT cell line at 550 bp was not detected. However, a 340-bp band was amplified. Isolated CTVT cell line inoculation at a concentration of 1×10⁸ did not induce tumour growth in bitches, nor did a challenge with primary CTVT cells. Conclusion: The present study successfully identified and isolated a stable CTVT cell line that may be useful in CTVT prevention.

Thrombocytes in vertebrates other than mammals, inter alia in fish, are analogues of platelets in mammals. In Osteichthyes, these cells take part in haemostatic processes, including aggregation and release reactions in cases of blood vessel damage, and in the immune response development as well. This paper discusses the development of thrombocytes in Osteichthyes, taking into account the need to make changes to the concept of grouping progenitor cells as suggested in the literature. The following pages present the morphological and cytochemical properties of thrombocytes as well as their defence functions, and also point out differences between thrombocytes in fish and platelets in mammals. The paper further highlights the level of thrombocytes’ immune activity observed in fish and based on an increased proportion of these cells in response to antigenic stimulation, on morphological shifts towards forms characteristic of dendritic cells after antigenic stimulation and on the presence of surface structures and cytokines released through, inter alia, gene expression of TLR receptors, MHC class II protein-coding genes and pro-inflammatory cytokines. The study also points out the need to recognise thrombocytes in Osteichthyes as specialised immune cells conditioning non-specific immune mechanisms and playing an important role in affecting adaptive immune mechanisms.

Introduction: Peroxiredoxin 6 (Prdx6) is a bifunctional protein with glutathione peroxidase activity and phospholipase A2 activity. Previous studies have shown a significant positive correlation between the intracellular survival ability of Brucella and Prdx6. Here, the Prdx6 enzyme with a single activity was constructed to facilitate study of the relationship between the single function of Prdx6 and Brucella infection. Material and Methods: The target open reading frame (ORF) DNAs of Prdx6 with a single active centre were prepared using gene splicing by overlap extension PCR (SOE-PCR), and the recombinant eukaryotic expression plasmids inserted by Prdx6 with the single activity centre were constructed and transfected into murine Raw264.7 macrophages. The glutathione peroxidase activity and phospholipase A2 activity of the constructed Prdx6 were examined. Results: The core centres (Ser³² and Cys⁴⁷) of Prdx6 were successfully mutated by changing the 94ᵗʰ nucleotide from T to G and the 140ᵗʰ nucleotide from G to C in the two enzyme activity cores, respectively. The constructed recombinant plasmids of Prdx6 with the single active centre were transfected into murine macrophages showing the expected single functional enzyme activity, which MJ33 or mercaptosuccinate inhibitors were able to inhibit. Conclusion: The constructed mutants of Prdx6 with the single activity cores will be a benefit to further study of the biological function of Prdx6 with different enzyme activity.

Introduction: The study sought to characterise antimicrobial resistance among coagulase-negative Staphylococcus (CNS) species recovered from broiler chickens and turkeys in Poland including the presence of 12 antimicrobial resistance genes and five classical genes of staphylococcal enterotoxins. Material and Methods: A panel of 11 antimicrobial disks evaluated the phenotypic sensitivity of the tested strains to antibiotics. Five multiplex PCR assays were performed using primer pairs for specific detection of antibiotic resistance genes and staphylococcal enterotoxin A to E genes. Results: Selected antimicrobial agent susceptibility testing revealed 100% of such in in vitro conditions to cefoxitin among strains of Staphylococcus sciuri and S. chromogenes. The blaZ (for ß-lactam) and mecA (for methicillin resistance) genes were in 58.3% and 27.5% of strains, respectively. Among genes resistant to tetracyclines, tetK was most frequent. Fewer (CNS) strains showed genes resistant to macrolides, lincosamides, and florfenicol/chloramphenicol. Multiplex PCR for classical enterotoxins (A-E) detected the see gene in two S. hominis strains, while the seb gene producing enterotoxin B was found in one strain of S. epidermidis. Conclusion: CNS strains of Staphylococcus isolated from poultry were either phenotypically or genotypically multidrug resistant. Testing for the presence of the five classical enterotoxin genes showed that CNS strains, as in the case of S. aureus strains, can be a source of food intoxications.

Introduction: Significant improvement of sperm motility within one month effected by oral supplementation of selenium and vitamin E was described in four infertile male dogs which failed to conceive in their last three matings with different bitches. Material and Methods: The dogs (a Golden Retriever, an English Cocker Spaniel, and two Tibetan Mastiffs) were supplemented daily with selenium (Se) (0.6 mg/kg organic Se yeast) and vitamin E (vit. E) (5 mg/kg) per os for 60 days. Semen was collected on days 0, 30, 60, and 90. The sperm concentration and motility parameters were evaluated by the CASA system, sperm morphology was explored by Diff-Quick staining, and live and dead spermatozoa were differentiated by eosin/nigrosin staining. The concentrations of Se and vit. E were measured in peripheral blood serum on semen collection days. Results: Before administration, the concentrations of Se in blood plasma were low (86.0–165.0 µg/L). After 30 days of treatment there was an observable improvement in total and progressive sperm motility and kinematic parameters (VAP, VSK, VCL, ALH, BCF, and RAPID). The percentages of live and normal morphology sperm cells were also higher. There was also an observable increase in Se and vitamin E concentrations in blood serum. Bitches were successfully mated and delivered four to six puppies. Conclusion: Supplementation with Se and vit. E improved rapid sperm motility and restored fertility in infertile dogs with low Se status.

Introduction: Quercetin is a polyphenolic flavonoid which has been used in traditional Chinese medicine as a natural therapeutic agent with a broad spectrum of activities (antioxidant, anticancer, neuroprotective, anti-inflammatory, antiviral and antibacterial). The aim of this study was to develop and validate a rapid and simple ultra-high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) method for the determination of quercetin in milk. Material and Methods: Sample preparation was based on a liquid-liquid extraction with 0.5% formic acid in acetonitrile. The chromatographic separation was performed on a ZORBAX SB-C18 column with methanol and 0.5% formic acid as a mobile phase. Results: The procedure was successfully validated. The mean recovery of the analyte was 98%, with the corresponding intra- and inter-day variation less than 10% and 15%, respectively, and the repeatability and reproducibility were in the range of 3%–7.2% and 6.1%–12%, respectively. The lowest level of quantification was 1.0 μg/kg. Conclusion: The proposed method was successfully applied in evaluating the pharmacokinetics of quercetin in milk obtained from dairy cows with clinical mastitis after intramammary administration.

Introduction: Pacemaker implantation is the only effective symptomatic treatment for life-threatening bradyarrhythmias. Major complications observed after implantation of cardiac pacemakers include lead dislocation, loss of pulse generator function, and inadequate stimulation. The aim of this retrospective single-centre study was to analyse the indications for pacemaker implantation and the incidence and types of complications associated with this procedure in dogs treated for symptomatic bradyarrhythmia. Material and Methods: The retrospective analysis included 31 dogs with symptomatic bradyarrhythmia, implanted with permanent cardiac pacemakers in 1992–2017. The list of analysed variables included patient age, breed, sex, indication for pacemaker implantation, comorbidities, and the incidence of procedure-related complications along with the type thereof. Results: The most common indication for pacemaker implantation was 3ʳᵈ degree AVB, followed by SSS, advanced 2ⁿᵈ degree AVB, and PAS. Pacemaker implantation was associated with a 35% overall complication rate and 6.45% periprocedural mortality. There were no significant differences in terms of procedure-related complications with regard to age, sex, breed, indications for pacemaker implantation, or comorbidities. Conclusions: Cardiac pacing is the only effective treatment of symptomatic bradycardia, but as an invasive procedure, may pose a risk of various complications, including death.