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.

Introduction: Numerous mutations in the bovine tumour necrosis factor receptor type two (TNF-RII) gene have been identified, but their biological consequences remain poorly understood. The aim of this study was to determine whether polymorphism in the analysed loci of the bovine TNF-RII gene is linked with the size of cell subpopulations naturally infected with bovine leukaemia virus (BLV) which serve important immune functions in the host. Material and Methods: Samples originated from 78 cows. Polymorphisms in the studied gene were determined by PCR-RFLP and DNA sequencing by capillary electrophoresis. BLV infection was diagnosed by the immunofluorescence (IMF) technique and nested PCR. Cell subpopulations were immunophenotyped with IMF. Results: Similar and non-significant differences in the average percentages of TNFα+, IgM+TNFα+, and CD11b+TNFα+ cells infected with BLV were noted in individuals with various genotypes in the polymorphic sites g.-1646T > G and g.16534T > C of the TNF-RII gene, and significant differences in the percentages of these subpopulations were observed between selected microsatellite genotypes (g.16512CA(n)). Conclusion: STR polymorphism and the number of CA dinucleotide repeats in intron 1 of the TNF-RII gene influence the frequency of TNF+, CD11b+TNF+, and IgM+TNF+ subpopulations naturally infected with BLV. Polymorphism in the gene’s other two sites do not affect the size of these cell subpopulations.

Introduction: Laryngeal swab samples collected from three waterfowl slaughterhouses in central Taiwan were cultured and suspected isolates of Riemerella anatipestifer were identified by API 20NE and 16S rDNA PCR. Material and Methods: Serum agglutination was used for serotyping, and antimicrobial susceptibility was tested. Results: Seventy-six R. anatipestifer isolates were detected, and the prevalences in the ducks and geese were 12.3% (46/375) and 8.0% (30/375), respectively. The positive isolation rates were 65.6% for all arriving waterfowl, 76.0% for birds in the holding area, 1.6% for defeathered carcasses, but zero for degummed carcasses. A PCR examination detected R. anatipestifer in the slaughtering area frequently. Serotype B was dominant in both duck (34.8%) and goose (46.7%) isolates, but the wide serotype distribution may very well impede vaccination development. All isolates were resistant to colistin, and 79.7% were resistant to more than three common antibiotics. Conclusion: The results proved that most ducks had encountered antibiotic-resistant R. anatipestifer in rearing, which suggests that the bacterium circulates in asymptomatic waterfowl. It is worth noting that most waterfowl farms were found to harbour R. anatipestifer, and contaminated slaughterhouses are a major risk factor in its spread. Effective prevention and containment measures should be established there to interrupt the transmission chain of R. anatipestifer.

Viral infections are the greatest threat to waterfowl and cause significant economic losses. Diagnosis and differentiation of three goose viruses is difficult in the field and often requires laboratory confirmation. Therefore, the aim of the study was to develop a triplex PCR and optimise its parameters for simultaneous detection of DNA of goose parvovirus (GPV), goose polyomavirus (GHPV), and goose circovirus (GoCV). The DNA of viruses isolated from field cases from the National Veterinary Research Institute’s own collection was used for the study. The primer attachment temperature, the number of reaction cycles, and the Taq DNA polymerase and Mg²⁺ concentrations were optimised. The sensitivity and specificity of this triplex PCR was also determined. Based on the obtained results, triplex PCR parameters were optimised for simultaneous detection of DNA of GPV, GHPV, and GoCV in one sample. The following PCR products of the expected size were obtained: GPV DNA of 806 bp, GoCV DNA of 571 bp, and GHPV DNA of 180 bp. The developed triplex PCR method proved to be useful for simultaneous detection of infections with three waterfowl viruses and will be used in relevant laboratory diagnostics.

Avian pathogenicEscherichia coli (APEC) causes serious colibacillosis and significant economic losses. Data on profiles of virulence factors and antibiotic resistances among APEC strains are crucial to the control of infection. In this study, strains were isolated from eastern China, and the prevalence of virulence factors and distribution of antibiotic resistance were determined. APEC strains were isolated and characterised by PCR for O serogroups, virulence factor genes, antibiotic resistance, and phylogenetic groups. O78 was the most prevalent serogroup and type A was the most frequent phylogenetic group. ThefimH,feoB, andiron genes were the most prevalent among the isolates. All isolates were multiresistant, and all strains were resistant to ampicillin and tetracycline, which are widely used in the poultry industry in China. This study provided important data on the presence of virulence genes and antibiotic resistance profiles of APEC from poultry farms in eastern China.

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.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) has become the leading cause of health care-associated infections. Treatment is difficult due to the lack of an effective antimicrobial therapy, and mortality is high. This study investigated the occurrence of CRPA in farm animals (buffaloes and cattle), livestock drinking water, and humans in Egypt. A total of 180 samples were examined: 50 faecal each from buffaloes and cattle, 30 of livestock drinking water, and 50 stool from humans. The samples were cultured on cetrimide agar and the plates were incubated aerobically at 37°C for 24 h. The isolates were examined for the presence of the blaKPC, blaOXA₋₄₈, and blaNDM carbapenemase-encoding genes using PCR and investigated for the exotoxin A (toxA) gene. The toxA gene from carbapenem- group resistant isolates was phylogenetically analysed. P. aeruginosa was isolated from buffaloes, cattle, drinking water, and humans, with occurrences of 40%, 34%, 10%, and 20%, respectively. Carbapenem resistance genes were found in 60%, 59%, 67%, and 70% in buffalo, cattle, water and human samples, respectively. The toxA gene was detected in 80% of samples. The phylogenetic analysis showed that cattle and water sequences were in one cluster and more related to each other than to human isolates. Occurrence of CRPA among farm animals, drinking water, and humans was high, reflecting the environmental origin of P. aeruginosa and highlighting contaminated water as a potential transmitter of CRPA to livestock and next to humans.

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.

A comprehensive description is presented of four novel cases ofamorphus globosus (ag) foetuses originating from multiple pregnancies of Polish Holstein cows. Four amorphic foetuses were delivered by three cows. Tissue samples were collected during autopsy, embedded in paraffin, sectioned, and stained with haematoxylin and eosin. Genomic DNA was isolated from tissue samples of abnormal foetuses and from blood leukocytes of their healthy siblings. PCR reactions were used to reveal the presence of Y-linked genes (SRY and AMELY) and an X-linked gene (AMELX). All foetuses were classified to the groupholoacardius amorphous (anideus). Molecular analysis clearly showed that at 17 microsatellite loci, the studied amorphous foetuses had identical genotypes to the viable co-twins. Foetuses had monozygotic origin. Histological analysis showed a low level of development of tissues of meso- and ectodermal origin, as well as features of degrading patterns.

Trypanosoma evansi is enzootic in camels in Egypt, and water buffaloes act as a reservoir for camel infection. Molecular techniques have contributed towards understanding the epidemiology of T. evansi. Trypanosoma evansi was detected in acute and chronic stages of the disease in male and female mice by polymerase chain reaction (PCR) using two primers. Two experiments were conducted. In experiment I, two groups consisting of 26 female and 26 male mice received 10(4) trypanosome by I/P inoculation for each mouse. In experiment II, 42 female and 42 male mice were inoculated I/P with 10² trypanosome/mouse. In addition, five mice were kept as uninfected control for each group. Mice were monitored daily for parasitaemia level during the pre-patent period using the micro-haematocrit centrifugation technique (MHCT) and conventional PCR. The primer pairs, (Trypanosoma brucei) TBR1/2 and TeRoTat1.2 (T. evansi Rode Trypanozoon antigen type [RoTat] 1.2), detected the infection after 24 hours earlier than MHCT in both experiments. The course of infection that was detected by MHCT revealed three waves of parasitaemia in female mice and two waves in male mice in the chronic stage of infection. In addition, PCR was able to detect T. evansi in different organs in the chronic stage (i.e. disappearance of parasite from blood). Application of the two primer sets on blood samples from camels showed that all samples were positive by TBR1/2 primers and only 32 of 44 were positive by TeRoTat1.2 primers. Acutely and chronically Trypanosoma-infected mice were detected by PCR in blood and organs. TBR1/2 primers were more sensitive than TeRoTat1.2 primers in detecting Trypanosoma-infected mice, and more reliable in detecting field-infected camels and excluding carrier animals.