High-throughput sequencing (HTS) identifies random viral fragments in environmental samples metagenomically. High reliability gains it broad application in virus evolution, host-virus interaction, and pathogenicity studies. Deep sequencing of field samples with content of host genetic material and bacteria often produces insufficient data for metagenomics and must be preceded by target enrichment. The main goal of the study was the evaluation of HTS for complete genome sequencing of field-case rabies viruses (RABVs). The material was 23 RABVs isolated mainly from red foxes and one European bat lyssavirus-1 isolate propagated in neuroblastoma cells. Three methods of RNA isolation were tested for the direct metagenomics and RABV-enriched approaches. Deep sequencing was performed with a MiSeq sequencer (Illumina) and reagent v3 kit. Bioinformatics data were evaluated by Kraken and Centrifuge software and de novo assembly was done with metaSPAdes. Testing RNA extraction procedures revealed the deep sequencing scope superiority of the combined TRIzol/column method. This HTS methodology made it possible to obtain complete genomes of all the RABV isolates collected in the field. Significantly greater rates of RABV genome coverages (over 5,900) were obtained with RABV enrichment. Direct metagenomic studies sequenced the full length of 6 out of 16 RABV isolates with a medium coverage between 1 and 71. Direct metagenomics gives the most realistic illustration of the field sample microbiome, but with low coverage. For deep characterisation of viruses, e.g. for spatial and temporal phylogeography during outbreaks, target enrichment is recommended as it covers sequences much more completely.

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.

Since 2007, African swine fever (ASF) has posed a serious threat to the European swine industry. In Poland, the numbers of reported outbreaks in pigs and affected areas grow every year. In 2018, the disease was noted in Western Europe, in Belgium specifically, where several hundred infected wild boars have been detected so far. In 2018, the virus unexpectedly emerged in pig holdings in eastern China, northern Mongolia, Vietnam, and Cambodia, causing worldwide concern about its further spread. Since there is still no vaccine available, the only approach to control the disease is biosecurity. Identification of potential sources of the virus is extremely important in light of its phenomenal survivability. The review summarises the current knowledge about ASFV survivability and resistance to environmental conditions, and discusses the role of indirect contact in spreading the disease.

The Shewanella putrefaciens group are ubiquitous microorganisms recently isolated from different freshwater fish species and causing serious health disorders. The purpose of the study was to characterise isolates of the S. putrefaciens group with special emphasis on elucidating serological diversity and determining putative virulence factors. Isolates collected from freshwater fish (n = 44) and reference strains were used. The identification of bacteria was carried out using biochemical kits and 16S rRNA sequencing. Polyclonal antibodies were prepared against the S. putrefaciens group. The bacterium’s susceptibility to antimicrobial agents, its enzymatic properties, and its adhesion ability to fish cell lines were also tested. Finally, selected isolates were used in challenge experiments in common carp and rainbow trout. Excluding six isolates undeterminable for species, the bacteria were classified to three species: S. putrefaciens, S. xiamenensis, and S. oneidensis, and showed some phenotypic diversity. Fourteen serological variants of the S. putrefaciens group were determined with the newly developed serotyping scheme. Serodiversity may play an important role in the virulence of particular isolates. Further, S. putrefaciens group members adhere to epithelial cells and produce enzymes which may contribute to their virulence. Challenge tests confirmed the pathogenicity of the S. putrefaciens group for fish.

Introduction: The clinical symptoms of portosystemic shunts (PSSs) and hepatic microvascular dysplasia (HMD) – portal vein hypoplasia (PVH) in dogs are similar. PSSs are abnormal vascular connections between the portal vein system and systemic veins. HMD is a very rare developmental vascular anomaly, recognisable during histopathological examination. The study aim was to assess the prevalence of HMD–PVH and hepatocellular and vascular pathologies in the liver. Material and Methods: Liver biopsies from 140 dogs (of different breeds and both sexes) arousing clinical suspicion of PSS were examined histopathologically. Results: An initial PSS diagnosis was confirmed in 125 dogs (89.29%). HMD–PVH was found in 12.32% of dogs, as an isolated disease in 9.29%, especially in Yorkshire terriers, and with extrahepatic PSS in 6.67%. Histopathological analysis of muscles around sublobular veins showed that HMD cases presented hypertrophy or hypertrophy with fibrosis. In 2.17% of all dogs with liver vascular developmental disorders calcification was visible around vessels (without correlation by degenerative changes in those vessels), suggesting prior onset of deep metabolic disorders. Clinical suspicion of PSS was also formed upon quite different pathological processes in young dogs. Conclusion: Histopathological findings diagnosed the type of vascular anomalies (PSS or HMD–PVH) or other pathological changes conclusively, therefore detailed hepatic histopathology is an indispensable component of the clinical diagnostic process.

Introduction: The thyroid and parathyroid glands play a major role in maintaining physiological homeostasis in all vertebrates. Reptiles have plasma concentrations of thyroid hormones far lower than mammals. Low levels of these hormones in reptiles impede thyroid hormone detection with assays designed for the higher levels of mammals. The aim of this study was to explore teaming this with ultrasound imaging of the thyroid to appraise glandular function. Material and Methods: Thyroid function of four pond sliders was evaluated based on the results of T4 analyses and ultrasound. Results: The concentrations of T4 varied considerably between the examined animals from <9 nmol/L to >167.3 nmol/L. Ultrasound examination revealed uniform echogenicity and a smooth outline of the thyroid gland in all animals. Conclusion: Monitoring of thyroid function based on T4 and electrolyte concentrations is helpful in assessing the health and living conditions of reptiles, which is important in veterinary practice but problematic. Ultrasound examinations are useful in diagnosing changes in gland structure, such as tumours and goitres, and a combination of both methods supports comprehensive assessments of the anatomy and function of the thyroid gland.

Recumbency is a frequent symptom occurring throughout lactation. Its cause can be related to the energy or mineral metabolism, or to trauma or infectious diseases. We compared various clinical chemistry parameters between healthy and recumbent cows and between cows with different causes of recumbency and determined if hypocalcaemia manifests in later lactation. Recumbent (n = 32) and healthy (n = 32) German Holstein cows were studied. After clinical examination, a serum sample was taken to measure the concentrations of Mg, Ca, Fe, Na, K, Pi, β-hydroxybutyrate, total bilirubin, non-esterified fatty acids (NEFA), urea, and creatinine as well as activities of alkaline phosphatase, aspartate aminotransferase (AST), creatine kinase (CK), and γ-glutamyl transferase in recumbent cows > 5 d in milk and control cows matched for age, lactation number, and pregnancy stage. In recumbent cows, mean serum concentrations of NEFA, bilirubin, and CK were statistically higher, while those of Fe, K, and Pi were significantly lower. Parameters compared between different recumbency diagnoses showed some descriptive Fe, K, urea, and AST differences, but these were not statistically significant. The results show that only a limited number of parameters have diagnostic besides therapeutic value. Although of minor importance in our study, hypocalcaemia should be considered a cause of recumbency, even outside the typical risk period of parturient paresis.

The characteristics of immune factors in somatic cells from lactating dairy cows and their association with commensal bacteria in normal milk have not been clarified. This study investigated the relationship between the pathogenic bacteria in milk and somatic cell immune factors in healthy lactating cows. In total 44 healthy Holstein cows were studied on one farm. Milk samples were collected aseptically using a cannula and these samples were cultured for detection of bacteria and analysis of mRNA of immune factors expressed by somatic cells. Cows were divided into two groups based on the microbial status of their milk samples: 12 cows showed bacteria in cultures (positive group), and the other 32 cows did not (negative group). The mRNA levels of IL-6, lactotransferrin, and cathelicidin expressed by somatic cells after milking decreased significantly compared to those before milking in both groups (P < 0.05). There were significantly lower mRNA levels of IL-6 and cathelicidin in the positive group compared to those in the negative group before milking. These results suggest that mRNA levels of IL-6 and cathelicidin expressed by the somatic cells may be affected by the presence of bacteria in healthy lactating dairy cows.

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: 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.