Yersiniosis is a zoonosis causing gastroenteritis, diarrhoea, and occasionally reactive arthritis and septicaemia. Cases are often linked to meat consumption and the most common aetiological agent is the Gram-negative bacilliform Yersinia enterocolitica bacterium. The occurrence of Yersinia spp. among wild animals has mostly been studied in wild boar, but it has seldom been in other species. A total of 1,868 faecal samples from animals found dead or hunted were collected between 2015 and 2018 in the Valle d’Aosta region of the northwestern Italian Alps. Alpine ibex faecal samples were collected during a health monitoring program in 2018. Bacteria were isolated via PCR and confirmed as Y. enterocolitica biochemically. Strain antimicrobial susceptibility was tested by Kirby–Bauer disc diffusion, and the presence of virulence factors and antimicrobial resistance genes was investigated using whole-genome sequencing. Yersinia enterocolitica strains of biotype 1A were detected in six faecal samples from red deer (0.93%), roe deer (0.49%) and red foxes (0.7%). Strains found in beech martens (3.57%) and Alpine ibex (2.77%) belonged to biotypes 1B and 5, respectively and harboured the pYPTS01 plasmid that had only been detected in Y. pseudotuberculosis PB1/+. All the isolates were resistant to ampicillin and erythromycin. The biovar 1A strains exhibited different virulence factors and behaved like non-pathogenic commensals. The strain from an Alpine ibex also harboured the self-transmissible pYE854 plasmid that can mobilise itself and the pYPTS01 plasmid to other strains. The beech marten could be considered a sentinel animal for Y. enterocolitica. Phenotypic resistance may account for the ability of all the strains to resist β-lactams.

Multi-class and multi-residue analyses are very complex procedures because of the physico-chemical properties of veterinary drug residues and other contaminants. The purpose of the study was to develop an analytical method for the sensitive determination of 69 analytes in bovine milk by liquid chromatography electrospray ionisation–tandem mass spectrometry. Antimicrobial, anabolic hormone, lactone, β-agonist, mycotoxin and pesticide residues were analysed in 120 raw milk samples from different dairy farms in North Macedonia. Stable isotopically labelled internal standards were used to facilitate effective quantification of the analytes. The linear regression coefficients were higher than 0.99, the limits of detection ranged from 0.0036 to 47.94 μg/L, and the limits of quantification ranged from 0.053 to 59.43 μg/L. The decision limit values ranged from 0.062 to 211.32 μg/L and the detection capability from 0.080 to 233.71 μg/L. Average recoveries of the analytes spiked in raw milk were in the range of 70.83% to 109%, intra-day coefficient of variation (CV) values from 2.41% to 22.29%, and inter-day CV values from 3.48% to 23.91%. The method was successfully applied in the testing of bovine milk samples. In five samples residues were detected. They were sulfadimethoxine (in two samples), enrofloxacin, tetracycline and oxytetracycline and were at concentrations below the EU maximum residue limit. The method is useful for routine testing for this group of chemical hazards in bovine milk.

It has been suggested that coagulase-negative staphylococci can serve as reservoirs of virulence genes for other bacteria. This study assessed the presence of such genes in selected isolates recovered from meat of the giant African snail (Achatina achatina). Virulence genes were detected using a polymerase chain reaction targeting specific primers. Two representative isolates were identified using 16S rRNA gene sequencing. The results showed that the staphylococcal enterotoxin A gene (sea) was present in five out of the eight isolates studied. The isolates expressed resistance mainly to three antibiotics: chloramphenicol, norfloxacin and cloxacillin in descending order of incidence. Most importantly, the Staphylococcus sciuri isolate NEDU 181, in addition to being resistant to the three aforementioned antibiotics, also harboured the sea gene. Our findings demonstrate, for the first time, the presence of toxigenic and antibiotic-resistant coagulase-negative Staphylococcus spp. in commercially-available fresh snail meat. With staphylococcal enterotoxin A known to survive cooking temperature, this presents a food safety concern.

Ochratoxin A (OTA) is a mycotoxin notably produced by Aspergillus and Penicillium spp. Bacillus subtilis fermentation extract (BSFE) contains specific enzymes which hydrolyse OTA. This study evaluated the efficiency of BSFE in ameliorating the immunotoxic and nephrotoxic effects of OTA in broiler chickens. Day-old broiler chicks were divided equally into four groups of ten: control, OTA (0.5 mg/kg feed), BSFE product (1 mL/L water) and OTA + BSFE at the same concentrations. The chicks were vaccinated against avian influenza, Newcastle disease, and infectious bronchitis, and lymphoproliferation was induced in all birds by phytohaemagglutinin-P (PHA-P). Serum samples were taken before sacrifice and organ tissue samples were taken after, in which renal function biomarkers were assayed and the presence of OTA residue was evaluated by high-performance thin-layer chromatography. Protein markers of apoptosis were determined by qPCR, and tissue lesions were examined histopathologically. Exposure to OTA significantly decreased the antibody response to the vaccines and the lymphoproliferative response to PHA-P, and significantly elevated the renal function indicators: serum urea, uric acid and creatinine. It also induced oxidative stress (reduced catalase activity and glutathione concentration), lipid peroxidation (increased malondialdehyde content), apoptosis (increased Bax and Caspase-3 and decreased Bcl-2 gene levels) and pathological lesions in kidney, bursa of Fabricius, spleen and thymus tissue. Residues of OTA were detected in the serum and tissue. BSFE mitigated most of these toxic effects. BSFE counters OTA-induced immunotoxicity and nephrotoxicity because of its content of carboxypeptidase and protease enzymes.

African swine fever virus (ASFV) causes one of the most dangerous diseases of pigs and wild boar – African swine fever (ASF). Since its second introduction into Europe (in 2007), the disease has been spreading consistently, and now ASF-free European countries are at risk. Complex interactions between the host’s immune system and the virus have long prevented the development of a safe vaccine against ASF. This study analysed the possibility of neutralisation of the ASFV in vitro by sera collected from ASF-survivor animals. Two pig and three wild boar serum samples were collected from previously selected potential ASF survivors. All sera presented high antibody titres (>5 log₁₀/mL). Primary alveolar macrophages were cultured in growth medium containing 10% and 20% concentrations of selected sera and infected with a haemadsorbing ASFV strain (Pol18_28298_O111, genotype II). The progress of infection was investigated under a light microscope by observing the cytopathic effect (CPE) and the haemadsorption phenomenon. Growth kinetics were investigated using a real-time PCR assay. Haemadsorption inhibition was detected in the presence of almost all selected sera; however, the inhibition of virus replication in vitro was excluded. In all samples, a CPE and decreasing quantification cycle values of the viral DNA were found. Anti-ASFV antibodies alone are not able to inhibit virus replication. Interactions between the humoral and cellular immune response which effectively combat the disease are implicated in an ASF-survivor’s organism.

Porcine circovirus 4 (PCV4) was first discovered in 2019 in a herd of pigs with porcine respiratory disease, dermatitis and nephropathy syndrome in Hunan Province, China. It has subsequently been detected in other provinces and in South Korea. In consideration of the potential of the virus to cause an epidemic, rapid, sensitive, and specific detection of PCV4 is needed, as is the facilitation of further epidemiological research through elucidation of the whole genome of PCV4. This study had those two aims. Fifty-five blood samples, two pig tissue samples, nine saliva swabs and one semen sample which all originated from Sichuan province pig farms were analysed. The virus’ genome of 1,770 bp was synthesised artificially based on a Chinese reference strain and primers and probes for the ORF2 gene were designed. Then, the amplified target fragment was cloned into the pMD19-T vector and a series of diluted recombinant plasmids were used to generate a standard curve. An optimised real-time TaqMan PCR method was established. The results of this study showed that the established method is specific for PCV4 but not for other viruses, and has amplification efficiency of 99.6%, a regression squared value (R²) of 1.000 and a detection limit of 2.2×10 DNA copies. This method was shown to be analytically specific and sensitive with a low intra- and inter-assay coefficient of variation (<1.67 %). Of a total of 67 clinical samples tested using the established method, three were shown to be positive (4%). This study confirms the existence of PCV4 in Sichuan and provides a promising alternative tool for rapid detection of PCV4.

Contagious agalactia (CA) is a disease affecting small ruminants with worldwide distribution and caused by several mycoplasmas, especially M. agalactiae. The main option for systematic diagnosis under monitoring control programmes is the enzyme-linked immunosorbent assay (ELISA) test. This study was designed to appraise the performance of two commercial indirect ELISA tests using M. agalactiae p48 protein and one using total protein, for antibody detection in small ruminants after natural infection with different M. agalactiae strains. We carried out the test evaluation using sera of confirmed M. agalactiae-positive goats with clinical signs. In addition, test agreement was assessed by kappa between the three commercial ELISA tests. All three ELISA tests showed high validity scores (Youden’s J: 72.9–84%). The sensitivity values for the P48 protein-based tests were 76.9% and 84.6%, and was 79% for the total protein-based test. The specificity of all tests was 100%. In addition, between the total protein-based ELISA test and the other two ELISA tests based on the P48 protein, the agreement was substantial (kappa: 0.762–0.763) and the agreement between the latter two tests was almost perfect (kappa: 0.93). The validity parameters for all tests allowed their application for diagnostic purposes in lactating goats excreting M. agalactiae in milk and presenting clinical signs. The agreements show that any of these ELISA tests could be equally well used for diagnosis in programmes against CA.

Trypanosomosis is an important disease of dromedary camels caused by the pathogenic protozoan Trypanosoma evansi. This study aimed to compare three different tests for its diagnosis in this species: conventional microscopy, the card agglutination test for trypanosomosis/T. evansi (CATT/T. evansi) and real-time PCR. Whole blood and serum samples collected from 77 dromedary camels of Abu Dhabi, United Arab Emirates, were analysed with the test methods stated. Statistical analysis was done using McNemar’s chi-squared test, and Cohen’s kappa index (κ) was calculated. We obtained results with positivity of 18% (14/77) by microscopy, 22% by CATT (17/77) and 60% (46/77) by real-time PCR, with the chain reaction detecting at a respectively three- and two-fold greater rate than the other techniques. Analysis of the data revealed a relative sensitivity of 30.4% and 37.0% for microscopy and CATT, respectively, compared to real-time PCR. The difference between the real-time PCR’s sensitivity and those of the other methods was statistically significant, with X² values of 30.03 and 20.1, respectively (df = 1 and P = 0.05 in both cases). Agreement of microscopy results with those of with CATT was good (κ = 0.72; 95% CI = 0.62–0.82). Cohen’s kappa index showed fair agreement of real-time PCR with microscopy (κ = 0.26; 95% CI = 0.16–0.36) whereas it was in poor agreement with CATT (κ = 0.09; 95% CI = 0.02–0.15). Real-time PCR was found to be more sensitive than microscopy and CATT.

The aim of the study was to present cases of botulism in animals found in Poland in 2019–2021. The analytical laboratory diagnosis and difficulties that occurred in the interpretation of the results are described. From 2019 to 2021, samples of serum, intestinal content, liver, spleen, kidney, faeces, wet feed, dry feed, ensilage, water and mixed samples of internal organs associated with 10 suspected animal botulism cases were sent to the National Veterinary Research Institute. Samples were analysed using a mouse bioassay and culture methods in combination with ntnh and bont gene detection. Among the ten putative botulism cases, only four (40%) were confirmed in the laboratory on the basis of the detection of botulinum toxin (BoNT) or the ntnh or bont genes. The remaining six (60%) were determined as probable despite observable characteristic clinical signs. The diagnosis of botulism in animals is a very difficult task, made so by the heterogeneity of Clostridium botulinum strains and possible loss of toxinogenicity during laboratory processing or the potential degradation of toxins. Laboratory diagnosis is a complex and problematic process which should utilise different prescribed methods for specific types of sample.

Fasciola hepatica is a trematode infecting ruminants worldwide and occasionally affecting other animal species, including humans. It causes significant economic losses. Geographic distribution and patterns of infection must be considered before control and management measures are developed for this parasite. DNA molecular markers are useful for the identification of flukes and elucidation of their genetic evolution. Therefore, the population structure of F. hepatica was studied using this method in sheep in Xinjiang, China. The molecular characteristics, genetic relationships within the population and dispersal patterns of F. hepatica isolates were analysed based on the cox1 and nad1 genes. The population structure of F. hepatica from three regions of Xinjiang was explored and a neutrality test was conducted. The cox1 and nad1 genes have 21 and 42 variable sites, respectively, which can be classified into 34 and 33 haplotypes. Median-joining network and phylogenetic tree analyses showed that there was no significant variation in F. hepatica isolates between the three geographical regions. Analysis of variance revealed that the genetic variation of F. hepatica was mainly present within the populations. The neutrality test indicated that the populations were relatively stable but the Hami population may have undergone short-term expansion. This study revealed for the first time the molecular characteristics, genetic diversity and dispersal patterns of F. hepatica isolates from sheep in Xinjiang, thus providing new insights into the genetic variation and haplotype diversity of F. hepatica from indigenous sheep.