Volumen 66 (2022): Heft 2 (June 2022)

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.

Antimicrobial resistance is currently one of the major public health threats. In order to prevent its spread, the WHO, OIE and FAO have formed an alliance to promote the study of antibiotic resistance evolution in human, animal and environmental bacteria posing a public health threat; however, the studies performed in wild animals are scarce so far. The main objective of this study was to assess the antibiotic resistance of Enterococcus spp. isolated from wild mammals in Aragón, Spain.

Rectal samples were collected from 103 wild mammals – 70 hunt prey and 33 rescued animals. Isolates were identified by matrix-assisted laser desorption/ionisation–time of flight mass spectrometry and susceptibility tests to 10 antibiotics were also carried out. Statistical analysis was performed (P ≤ 0.05).

A total of 126 isolates of seven different Enterococcus species were recovered. Among them, E faecalis (37.60%), E. casseliflavus (20.63%) and E. faecium (17.46%) were the most prevalent. The antibiotics quinupristin-dalfopristin and ciprofloxacin most frequently lost efficacy against the isolates. Multi-drug resistance was more prevalent in enterococci isolated from the rescued mammals.

This study found resistance widely distributed among enterococci isolated from the studied mammals. This points to the need for additional study of its genetic determinants and investigation of the sources and measures to avoid contributory environmental contamination.

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.

Clinical mastitis (CM) is one of the most common diseases of dairy cows globally, has a complex aetiology and recurs easily. Staphylococcus aureus is a frequently isolated pathogen responsible for bovine mastitis and remains difficult to eradicate.

To characterise the transcriptional profiles of dairy cows infected by S. aureus, we performed an RNA-seq analysis of peripheral blood leukocytes in lactating Chinese Holstein dairy cows with CM and did the same with healthy cows’ samples as controls.

A total of 4,286 genes were detected in the CM cases infected with S. aureus which were differentially expressed compared to the controls, 3,085 of which were upregulated, the remainder being downregulated. Notably, we observed that some differentially expressed genes (DEGs) had strong protein–protein interaction. Of these, six downregulated DEGs (AKR1C4, PTGS2, HNMT, EPHX2, CMBL, and IDH1) were involved in the metabolic pathway, while eight upregulated DEGs (VWF, GP9, MYLK, GP6, F2RL3, ITGB3, GP5, and PRKG1) were associated with the platelet activation pathway.

The transcriptome dataset of CM cases would be a valuable resource for clinical guidance on anti-inflammatory medication and for deeper understanding of the biological processes of CM response to S. aureus infection, and it would enable us to identify specific genes for diagnostic markers and possibly for targeted therapy.

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.

Potential biomarkers for chronic seasonal heat stress in Kagoshima Berkshire pigs reared in the subtropical region were investigated by comparing the biomarker changes in the summer (a period of chronic heat stress) and winter (a thermoneutral period) seasons.

Pigs were allocated to summer- and winter-finishing cohorts, 12 each. The evaluations included assessment of carcass traits and internal organs’ normality carried out at the time of slaughter, and measurement of biomarkers in whole blood: derivatives of reactive oxygen metabolites (d-ROMs) and biological antioxidant potential as markers of oxidative stress, and serum amyloid A and albumin/globulin (A/G) ratio as markers of acute and chronic inflammation, respectively.

The summer-finished pigs reared under subtropical field conditions showed lower carcass quality than the winter-finished pigs, indicating a potential adverse effect of summer temperatures on the swine industry. Marginal changes were observed in d-ROMs and the A/G ratio between the summer- and winter-finishing cohorts.

The results demonstrate that d-ROMs and the A/G ratio could be used as sensitive markers for heat stress under field conditions.

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.

Carvacrol is an essential oil derived from oregano that is used as a natural additive to improve the efficiency of livestock nutrition. Residues of natural additives such as carvacrol should be monitored in food of animal origin to ensure consumer safety. The aim of this study was to appraise the quick, easy, cheap, effective, rugged and safe (QuEChERS) approach coupled with liquid chromatography and mass spectrometry as a means of carvacrol analysis in chicken tissue.

A 5 ± 0.05 g portion of plasma, lung, muscle and liver was mixed for 15 min with 5 mL of 1-butanol and 20 mL of water, then centrifuged. A 0.5 mL volume from the top layer was transferred, then 60 mg of octadecylsilane sorbent, 30 mg of primary and secondary amine and 200 mg of MgSO₄ were added. The extract was mixed and centrifuged. The top layer was filtered and then transferred to an autosampler vial for analysis by high-performance liquid chromatography–tandem mass spectrometry.

The limit of detection was calculated at 0.06 μg g⁻¹ and the limit of quantification was 0.2 μg g⁻¹, with relative standard deviation repeatability and reproducibility below <20%.

The validation results showed that this method could be a good alternative to determination of carvacrol by gas chromatography and is suitable for carvacrol analysis in different matrices.

Pyrrolizidine alkaloids (PAs) and tropane alkaloids (TAs) are natural contaminants of honey and respectively hepatoxic and neurotoxic compounds. Because honey is a popular constituent of the human diet, it is relevant to warrant the safety of the product. For that reason, a method for simultaneous determination of PAs and TAs in honey based on liquid chromatography– mass spectrometry was developed.

The analytical protocol used sulphuric acid extraction and solid-phase extraction purification. The developed procedure was subjected to validation in terms of linearity, selectivity, repeatability, reproducibility, limits of quantification and determination, matrix effect and uncertainty. A total of 29 honey samples were analysed for the determination of PAs and TAs.

All the evaluated validation parameters fulfilled the requirements of European Commission Decision 2002/657/EC. At least one of the monitored alkaloids was determined in 52% of the samples. Among the most abundant alkaloids were echimidine, intermedine and lycopsamine. The total PA concentrations ranged from 2.2 to 147.0 μg kg⁻¹. Contrastingly, none of the monitored TAs was detected in the analysed samples. An assessment of the dietary exposure to PAs from the consumption of the contaminated honeys showed that three of them would pose a risk to consumers, especially if they were children.

A sensitive method suitable for simultaneous determination of PAs and TAs in honey was developed and validated. The analysis of 29 honey samples for PAs and TAs revealed that honey destined for retail could pose a risk to consumers.

This study aimed to determine the profile of immunoglobulins and cortisol concentrations in serum around the periparturient period in sows suffering from postpartum dysgalactia syndrome (PDS) and in healthy sows.

A total of 45 sows with lactation impairment (Group PDS) and 58 clinically healthy sows with a physiological peripartum period (Group H) were subjected to a serological test (ELISA) for measurement of serum immunoglobulins (IgG, IgM, and IgA) and cortisol concentration.

The serum contents of IgG, IgM and IgA had highly similar profiles in PDS-affected sows and healthy ones. A significantly higher concentration of IgG at 28 and 14 days ante partum compared to days 3 and 7 post partum was only observed in Group H. The mean cortisol content remained at a highly similar level throughout the entire experiment in both groups.

The results of the study indicate that lactation impairment such as PDS did not influence the immunoglobulin or cortisol concentration in sow serum.

Dairy cows may infrequently give milk tinged with blood after calving, which is a condition termed haemolactia. Economic losses for dairy farmers are caused by cases of haemolactia because of the condemnation of such milk, potential contamination of good bulk tank milk with haemolactic milk, and need for veterinarian intervention. This study was performed to elucidate the oxidative status of dairy cows with haemolactia during the peripartum period.

Plasma glutathione peroxidase, malondialdehyde (MDA) and superoxide dismutase concentrations along with serum vitamin A, C and E concentrations were determined as indices of oxidative stress. The sampled dairy cows comprised two haemolactic (n = 11 and n = 6) and two non-haemolactic (n = 11 and n = 6) groups.

On the first day when haemolactia was identified in colostrum (at mean 2.1 days after parturition), a significantly increased concentration of plasma MDA was noted in the haemolactic group. During the prepartum period, low levels of serum vitamin E were continuously observed from prepartum week 4 to the parturition day but only in the haemolactic group.

These results demonstrate that continuous low levels of serum vitamin E in the prepartum period may play a pivotal role as a requisite factor in the onset of haemolactia after calving.

Bisphenols, as endocrine disruptors, may cause a wide range of health problems in humans, but so far, not all of them have been confirmed in animals, including pigs. Since animals are also exposed to bisphenols, we hypothesised that these substances may have an effect on uterine contractility in pigs. Therefore, the aim of the study was to investigate the effect of the most-used bisphenol, bisphenol A (BPA), and a selected analogue, bisphenol F (BPF), on the contractile activity of the pig uterus.

The investigation utilised smooth muscles from immature pigs (n = 6), cyclic pigs on days 12–14 of the oestrous cycle (n = 6) or early pregnant pigs on days 12–16 of pregnancy (n = 6). Strips of the myometrium were exposed to BPA and BPF at concentrations of 10⁻¹³–10⁻¹ M. Smooth muscle contractility was determined with equipment for measuring isometric contractions.

BPA caused a significant decrease in contraction amplitude, and frequency and in myometrial tension in all groups examined. BPF caused a decrease in the amplitude and frequency of contractions in all groups and in myometrial tension in the early pregnant group.

The obtained results indicate that both BPA and BPF relaxed the porcine myometrium, but these changes, especially in the amplitude and frequency of contractions, were more evident after BPF treatment. The extent of relaxation is dependent on the physiological status of the animals.

Enhancer of zeste homologue 2 (EZH2) is the human homologue of Drosophila zeste gene enhancer. The aim of this study was to determine the expression of EZH2 in canine mammary carcinomas (CMCs) and its relationship with clinicopathological features.

The expression of EZH2 mRNA and protein in 53 CMC tissue and 8 normal mammary gland tissue samples was measured by quantitative real-time PCR and immunohistochemical staining assay, respectively. The relationship between EZH2 protein expression and clinicopathological features was analysed by χ2 test to further explore the clinical significance of EZH2 in CMCs.

Compared with normal mammary gland tissues, EZH2 mRNA expressions were significantly increased in CMC tissues (P < 0.01). Moreover, normal mammary glands did not express the EZH2 protein but carcinomic glands did, and expression increased in CMCs with high histological grades, especially in histological grade II (P < 0.05). However, EZH2 expression was not related to age, tumour size, or metastasis (P > 0.05). The expression of EZH2 in one type of CMC was not significantly different from the expression in any other type (P > 0.05).

EZH2 is highly expressed in CMCs, indicating that it can be used as a molecular marker for early diagnosis, prognosis, or therapy of CMCs.

Inflammatory mammary carcinoma (IMC) is a rare disease with a poor prognosis and one affecting dogs. Inflammatory breast carcinoma (IBC) is a subtype of malignant breast cancer in humans with a high degree of malignancy and a similarly poor prognosis. Since the clinical symptoms and prognoses of both are similar, canine IMC has been considered as a model of human IBC. In this study, we newly established a stable IMC-derived cell line from a patient at the Yamaguchi University Animal Medical Center in Japan.

The patient was a female toy poodle presenting with an inflamed mammary gland, which was diagnosed as IMC. The cell line was established from a tissue biopsy. Surface antigen marker (CD24 and CD44) expression was determined. Cystine/glutamate antiporter (xCT) expression was determined by Western blotting, flow cytometry and fluorescence immunostaining, and sulfasalazine was administered to ascertain if it suppressed xCT expression. Stem cell marker (Nanog, Sox2, Myc and Klf4) expression and aldehyde dehydrogenase (ALDH) activity were also investigated.

The cultured cells showed xCT, and its suppression showed downregulation of stem cell markers and ALDH activity. Stable cell proliferation was verified.

A new canine IMC-derived cell line was established. In the future, we aim to study the effect of xCT on the maintenance of cancer stem cell properties in canine tumours, and propose a new therapeutic method for the treatment of canine IMC by targeting xCT.

Dogs with chronic kidney disease (CKD) may have alterations in the glomerular filtration barrier, including podocyte loss. Detection of podocyte mRNA in urine could be useful for assessing podocyturia in dogs with kidney disease. The objective of this study was to evaluate the presence of nephrin mRNA (NPHS1) and podocin mRNA (NPHS2) in urine sediments of dogs with naturally occurring CKD and healthy dogs.

Twenty-four dogs, 14 with CKD and 10 as healthy controls, underwent clinical evaluation. The dogs with CKD were divided into two groups, according to the International Renal Interest Society criteria: stage 1 or 2 CKD (n = 5) and stage 3 or 4 CKD (n = 9). Urine was collected by catheterisation or free catch and RNA isolation from the urine sediments was optimised using glycogen as a co-precipitant. Detection of NPHS1 and NPHS2 in the sediment samples was performed using quantitative real-time PCR.

Both types of mRNA were detected in samples from all groups, but the percentages of detection were higher in the group of dogs with stage 1 or 2 CKD and lower in the group of dogs with stage 3 or 4 disease.

Physiological podocyturia was observed in healthy dogs, and the results suggest differential podocyturia in dogs with CKD, according to the stage of the disease, i.e. an increase in podocyturia in dogs at stage 1 or 2 and a reduction in podocyturia in dogs at stage 3 or 4.