Volume 64 (2020): Issue 1 (March 2020)

Classical swine fever virus (CSFV) causes an economically important and highly contagious disease of pigs, leading to economic losses around the world. Attenuated live vaccines with CSFV antigens have played an important role in the prevention and control of the disease. Porcine kidney 15 (PK15) cells have been widely used for the propagation of CSFV, but this cell line is not efficient or homogeneously susceptible to viral infection.

To achieve a homogeneous PK15 cell line which enabled high titre replication of CSFV, we used the limiting dilution cell cloning method.

We developed two cell clones, PK15-1A6 and PK15-3B1, which respectively have high- and low-permissive phenotypes to CSFV infection. The PK15-1A6, PK15-3B1, and PK15 parent cells showed different characteristics in cell proliferation rate, susceptibility to CSFV infection, and CSFV production. The mean virus titres per millilitre reflected by TCID₅₀ values in PK15-1A6, PK15-3B1, and PK15 parent cells were 106.85, 103.63, and 104.74, respectively.

The PK15-1A6 cell clone is more permissive to CSFV infection than the PK15 parent cells. The screened high-permissive cells will be useful for CSFV propagation and vaccine development in vitro, and facilitate research on the pathogenicity of CSFV.

A research project is underway aiming to develop a field diagnostic tool for six important viruses of the pig sector, namely: African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), porcine parvovirus (PPV), porcine circovirus (PCV2), and classical swine fever virus (CSFV).

To obtain a preliminary sounding of the interest in this type of instrument among its potential operators, a questionnaire was drawn up and submitted to three categories of stakeholders: farmers, veterinarians, and others (including scientific and technical staff working on animal farms). Four countries participated: Italy, Greece, Hungary, and Poland.

In total, 83 replies were collected and analysed in a breakdown by stakeholder type and pertinence, where the areas were the importance of the main diseases within the different countries, diagnostic tool operational issues, and economic issues.

The main end-users of this kind of instrument are expected to be private veterinarians and pig producers. The infectious agents seeming to be most interesting to diagnose with the instrument are PRRSV, SIV, PPV, and PCV2. The most decisive parameters which have been selected by the stakeholders are sensitivity, cost, simplicity, and time required to obtain results. The economic issue analysis showed that the majority of those who would prefer to buy rather than rent the device are willing to pay up to €3,000 for a diagnostic field tool.

Porcine epidemic diarrhoea virus (PEDV) infection causes watery diarrhoea, vomiting, anorexia, and weight loss, especially among neonatal piglets, inflicting on them morbidity and mortality potentially reaching 90%–100%. Despite it being known that certain mammalian cell phases are arrested by PEDV, the mechanisms have not been elucidated, and PEDV pathogenesis is poorly understood. This study determined the effect of an epidemic PEDV strain on cell cycle progression.

We observed the effect of the PEDV SHpd/2012 strain on an infected Vero cell cycle through flow cytometry and Western blot, investigating the interrelationships of cell-cycle arrest, the DNA damage–signalling pathway caused by PEDV and the phosphorylation levels of the key molecules Chk.2 and H2A.X involved upstream and downstream in this pathway.

PEDV induced Vero cell-cycle arrest at the G1/G0 phase. The phosphorylation levels of Chk.2 and H2A.X increased with the prolongation of PEDV infection, and no significant cell-cycle arrest was observed after treatment with ATM or Chk.2 inhibitors. The proliferation of PEDV was also inhibited by treatment with ATM or Chk.2 inhibitors.

PEDV-induced cell-cycle arrest is associated with activation of DNA damage–signalling pathways. Our findings elucidate the molecular basis of PEDV replication and provide evidence to support further evaluation of PEDV pathogenesis.

Marek’s disease virus (MDV) can cause malignant T-cell lymphomas and immunosuppression in chickens. Macrophage migration inhibitory factor (MIF) not only plays a critical role in inhibiting T-cell responses, but also contributes to multiple aspects of tumour progression. The aim of this study was to reveal the potential role of MIF in the pathogenesis of MDV infection.

MIF gene expression levels were measured by using real-time PCR. Expression was assayed at different times in chicken embryo fibroblast (CEF) cells and tissue samples of SPF chickens infected with different MDV strains and fold change was calculated by the 2^–△△CT method.

The expression of MIF was significantly downregulated (p < 0.05 and FC > 2) in CEF cells infected with the very virulent MDV RB1B strain at 48 h post infection (hpi) and in the skin and spleen at 14 days post infection (dpi). The reduction of MIF expression was also found in CEF cells infected by reticuloendotheliosis virus (REV), avian leukosis virus subgroup J (ALV-J), and MDV vaccine strain CVI988 or in HD11 cells stimulated with TLR2, 3, 4, and 7 ligands. Interestingly, MIF expression decreased continuously from 7 to 28 dpi in the thymus after RB1B virus infection while it increased after CVI988 virus infection. Upregulated expression of MIF was found in CEF infected with RB1B at 96 hpi and in the spleen and skin at 21 and 28 dpi.

The present study revealed the different expression pattern of MIF in response to MDV infection and indicated that MIF level may be associated with MDV pathogenesis.

Sheep pulmonary adenomatosis (ovine pulmonary adenomatosis, OPA, Jaagsiekte) is a chronic contagious bronchoalveolar carcinoma caused by the Jaagsiekte sheep retrovirus. Since effective treatment and a vaccination procedure are not currently possible, control and eradication of the disease is difficult. It leads to serious economic losses around the world, therefore studies are currently underway in order to design control and eradication programmes. In this study, levels and changes in selected tumour markers (carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 125, CA 19-9, CA 15-3, and alphafetoprotein (AFP)-3) and their diagnostic significance were investigated.

A total of 30 sheep were used. Clinical examinations were performed and blood samples were obtained before slaughter from all animals with presumed OPA. Blood samples with positive OPA results by macroscopic and histopathological examination were included in the study as the experimental group and numbered 20. Sheep totalling 10 had negative OPA results and provided control samples.

CEA levels were similar in both groups, and the differences were statistically insignificant (P > 0.05). CA 125, CA 19-9, CA 15-3, and AFP-3 levels were higher in the OPA group than the control group and with statistical significance (P < 0.05). In all OPA animals, CA 125 levels were higher than 1 U/mL.

serum CAs and AFP levels increase significantly in adenomatous sheep. These tumour markers are thought to facilitate the diagnosis of OPA.

Since 2009, Poland has been recognised as a country officially free of bovine tuberculosis (bTB), although in each year of the last five there were from 8 to 18 outbreaks of the disease. In 2008–2016, the largest number of cattle infected with bovine mycobacteria were eliminated in the Masovian Province (the central region of Poland) and the largest number of outbreaks of this zoonosis were recorded in this area. The close proximity of farms where bTB was found led to the suspicion that tuberculosis could have been transmitted between the affected herds. The aim of the study was the molecular characterisation of the pertinent M. bovis/caprae strains and determination of the epidemiological relationship of various bTB outbreaks.

The material for microbiological tests came from 119 cattle (Bos taurus) from nine herds located in five provinces, neighbouring the Masovian Province.

Laboratory tests of tissue material gave results confirming tuberculosis in 54 (45%) animals. All strains belonged to the Mycobacterium bovis species. A two-step analysis of genetic affinity allowed 50 strains to be identified as phylogenetically closely related and separated between three genetic clusters consisting of 2 to 27 strains.

Based on the results of genotyping, bTB outbreaks were found in three herds, and three transmission chains were identified among these herds.

Bovine tuberculosis, caused by M. bovis, is endemic in Mexico and has had a big impact on public health. Jalisco is considered to be an important dairy region in the country, accounting for approximately 19% of the total milk production. Within Jalisco, the region of Altos Sur holds the largest proportion of the cattle inventory of the state.

To determine the frequency of bovine tuberculosis in Altos Sur, Jalisco, as well as M. bovis genetic diversity, sampling of tissue (lymph nodes, lungs, and liver) from Holstein cattle was performed in four abattoirs belonging to three municipalities of this region (Tepatitlán de Morelos, San Miguel el Alto, and Arandas). Spoligotyping and whole-genome sequencing were carried out to assess the genetic relationships of M. bovis strains circulating in this area, as well as a comparison to isolates from other places in Mexico.

Prevalence was 15.06%, and distribution similar among the three municipalities. The most frequent spoligotypes were SB0673, SB121, and SB0145. Whole-genome sequencing revealed three main clades (I, II, III), but isolates did not show clustering by region.

Phylogenetic analysis suggested ongoing transmission between herds of the different regions, and no unique source of infection was determined. This hinders efforts under the national program for the control and eradication of the disease, so serious attention must be paid to rural regions such as Altos Sur in order to improve its success.

Tularaemia is a zoonotic disease caused by the gram-negative bacterium Francisella tularensis, which is endemic to Ukraine. The aim of this work was to provide screening of different field samples (rodent tails, ticks, pellets, water, and hay) to obtain an actual picture of the tularaemia epizootic situation in the Kharkiv, Dnipropetrovsk, and Mykolaiv oblasts.

Samples were collected using the flag method (for ticks) and break-back traps (for rodents). Also, hay, water and owl pellets were collected for study. The F. tularensis genetic material in samples was detected using a 16S qPCR.

It was found that in Kharkiv oblast, 23% of collected samples were positive for F. tularensis, in Dnipropetrovsk oblast 1.9%, and in Mykolaiv oblast 0.4%.

Among the sample types, 34.7% of ticks, 1.8% of rodents, and 36.4% of pellets were positive for F. tularensis. The most frequent carriers of F. tularensis were the D. reticulatus and I. ricinus ticks (74.2% and 29.3%, respectively, of positive results).

The aim of this study was to present two outbreaks of bovine abortion due to Leptospira infection in cattle herds located in the northern part of Sicily (Italy). The animals were positive for Leptospira interrogans serogroup Sejroe serovar Hardjo in a microscopic agglutination test (MAT).

A total of 23 Charolaise cows (farm A) and 75 Limousine bulls and Cinisara and Modicana cows (farm B) were enrolled in this study. The blood samples were collected from all subjects at the following time points: before a cycle of intramuscular treatment with oxytetracycline dihydrate (T0), after 5–6 weeks from the treatment (T1), and every 10 weeks until seronegativisation (T2 in Farm A and T3 in Farm B). A serological test (MAT) was used for the diagnosis of leptospirosis.

Two samples from farm A (2/23) and 29 samples from farm B (29/75) were positive to Leptospira interrogans, serogroup Sejroe, serovar Hardjo in the MAT. Leptospira spp. DNA was detected by real-time PCR in the urine sample of one positive cow on farm A, and in placenta and brain samples belonging to one aborted foetus on farm B.

It is important to use serological and molecular diagnostic techniques complementarily to identify infected individuals.

The aim of the study was to establish the prevalence of Bartonella spp. in cats in eastern Poland, and to determine the factors associated with the infection.

PCRs were performed to detect Bartonella DNA in the whole blood of 672 cats from four regions in eastern Poland (the Lublin, Podlasie, Masovian, and Subcarpathian provinces). The association between the previously selected variables and the dependent variable (presence of Bartonella DNA) was investigated using a logistic regression model.

The overall prevalence of infection was 40.48%. All PCR positive cats were infected with B. henselae. The living conditions of the animals (free outdoor roaming), mixed breed cats, Subcarpathian region, and absence of tick control were significant risk factors associated with Bartonella infection at a 95% confidence level.

Cats in eastern Poland appear to be at risk of a bartonellosis epizootic. Factors which seem to impact the likelihood of infection in cats and factors which seem not to impact it have been suggested. We advocate additional research into the ways bartonellosis spreads, its geographical scope, and the factors that favour its development.

Leishmaniasis is a zoonotic disease which is caused by protozoan parasites of the genus Leishmania. Canids are the most important reservoir of the parasites; however, limited data are available on the species of Leishmania prevalent in these animals and their impact on human health. The objective of this study was to estimate the seroprevalence of leishmaniasis in dogs from an inter-Andean region of Colombia during July 2016–July 2017, and to describe the clinical and histopathological features of the disease.

A total of 155 dogs were subjected to clinical examination and a serological test for detection of antibodies against Leishmania. Necropsy was carried out on positive animals and tissue samples were processed by routine histopathology.

Altogether 19 dogs were positive in the serological test, establishing a 12% seroprevalence of Leishmania. Clinical examination and necropsy revealed exfoliative and ulcerative dermatitis with haemorrhagic borders on the ears, head, nose, and legs. Histopathology revealed severe multifocal dermatitis with abundant Leishmania amastigotes within the cytoplasm of phagocytic cells, depletion of lymphocytes in lymphoid tissues, interstitial pneumonia, and interstitial nephritis. Tissue samples were positive for Leishmania by PCR.

The macro- and microscopic changes correlated with the presence of Leishmania as established by serological test and PCR.

Quasiamidostomum fulicae (Rudolphi, 1819) Lomakin, 1991, is a species of which the systematic position is still unclear, and it is reported in the literature under many synonyms. In the present study, an attempt has been made at establishing the ultimate systematic position of Quasiamidostomum fulicae against the backdrop of selected Amidostomatinae species.

The parasites were identified based on measurements of external and internal structures. Ecological analysis of Q. fulicae was carried out using the quantitative indices (frequency, prevalence, mean intensity, relative abundance, and dominance index). Statistical analyses (discriminant analysis) were performed on measurement data.

The intestines of 77 coots were examined. They yielded a total of 398 parasites, including 67 identified as Q. fulicae. Both males and females were located in the muscular gizzard. The morphometric analysis of Q. fulicae in this study showed the dimensions of all the internal organs to be in agreement with measurements reported by other authors. The discriminant analysis, used to find the differences between the examined nematode species (Amidostomoides acutum, A. petrovi, A. monodon, Amidostomum anseris, and Quasiamidostomum fulicae), gave highly significant results (P < 0.0001) with respect to both males and females.

The results justify the separation of Q. fulicae from the genus Amidostomum.

Common parasites of the European bison include gastro-intestinal and pulmonary nematodes, liver flukes (Fasciola hepatica), tapeworms, and protozoa of the genus Coccidia. This study compared the extensiveness and intensities of European bison parasitic invasions in three north-eastern Polish forests in different seasons and queried the role of parasitological monitoring in sanitary and hygienic control of feeding places.

Faecal samples were collected in the Białowieża, Knyszyńska, and Borecka Forests between 2014 and 2016, as were some from an area neighbouring the Białowieża Forest outside the Natura 2000 protected area. Parasites were detected in individual samples with the flotation, decanting and Baermann methods.

The eggs of Trichostrongylidae, Aonchotheca sp., Nematodirus sp., Strongyloides spp., Trichuris sp., Moniezia spp., and Fasciola hepatica; the larvae of Dictyocaulus viviparus; and the oocytes of Eimeria spp. were identified. Significant variation in invasion intensity and diversity was seen by origin and season. The relationships were assessed first by univariable tests and next multivariately, when origin and season emerged as the major risk factors for exposure to most of the parasites.

The differences in the level of parasitic infection between the forests did not have implications for its sufficiency to cause clinical symptoms. However, the associations and risk factors found enable the necessary preventive measures to be taken to protect the E. bison from exposure or decrease the risks. Additionally, parasitological monitoring is appropriate as the method of sanitary and hygienic control of European bison winter feeding places. Threats to public health through adventitious invasions by zoonotic factors such as F. hepatica have been identified.

Land application of manure that contains antibiotics and resistant bacteria may facilitate the establishment of an environmental reservoir of antibiotic-resistant microbes, promoting their dissemination into agricultural and natural habitats. The main objective of this study was to search for acquired antibiotic resistance determinants in the gut microbiota of wild boar populations living in natural habitats.

Gastrointestinal samples of free-living wild boars were collected in the Zemplén Mountains in Hungary and were characterised by culture-based, metagenomic, and molecular microbiological methods. Bioinformatic analysis of the faecal microbiome of a hunted wild boar from Japan was used for comparative studies. Also, shotgun metagenomic sequencing data of two untreated sewage wastewater samples from North Pest (Hungary) from 2016 were analysed by bioinformatic methods. Minimum spanning tree diagrams for seven-gene MLST profiles of 104 E. coli strains isolated in Europe from wild boars and domestic pigs were generated in Enterobase.

In the ileum of a diarrhoeic boar, a dominant E. coli O112ab:H2 strain with intermediate resistance to gentamicin, tobramycin, and amikacin was identified, displaying sequence type ST388 and harbouring the EAST1 toxin astA gene. Metagenomic analyses of the colon and rectum digesta revealed the presence of the tetQ, tetW, tetO, and mefA antibiotic resistance genes that were also detected in the gut microbiome of four other wild boars from the mountains. Furthermore, the tetQ and cfxA genes were identified in the faecal microbiome of a hunted wild boar from Japan.

The gastrointestinal microbiota of the free-living wild boars examined in this study carried acquired antibiotic resistance determinants that are highly prevalent among domestic livestock populations.

The aim of this study was to determine the content of fatty acids in eggs harvested from two edible subspecies of Polish-bred common garden snail from the Cornu genus, as well as this content in the retail-ready product obtained from these eggs.

Material for the study consisted of eggs from two subspecies of edible snails: the small (Cornu aspersum aspersum), and large (Cornu aspersum maxima) common garden snails. The eggs studied were in two forms, the first of which had undergone initial processing to the half-product stage and the second of which was the final product available on the Polish market under the name “Snail Eggs”. The gas chromatography method was used to determine the content of fatty acids.

More than 75% of the studied fats were saturated fatty acids, dominated by palmitic and stearic acids. The average content of polyunsaturated fatty acids was 0.37%, and it was a combination of two acids: linoleic (C18:2n6c), and its trans isomer (C18:2n6t). No significant differences were found comparing individual fatty acids content between the two species’ eggs as half-products, or between the half-products and the final product.

The fat in raw and processed eggs of common garden snails holds low nutritional value, and the processing did not affect the content of fatty acids.

Enolases are enzymes in the glycolytic pathway, which catalyse the reversible conversion of D-2-phosphoglycerate into phosphoenol pyruvate in the second half of the pathway. In this research, the effects of α-enolase (ENO1) on steroid reproductive-related hormone receptor expression and on hormone synthesis of primary granulosa cells from goose F1 follicles were studied.

Primary granulosa cells from the F1 follicles of eight healthy 8-month-old Zi geese were separated and cultured. An ENO1 interference expression vector was designed, constructed and transfected into primary cultured granulosa cells. The mRNA expression levels of follicle-stimulating hormone receptor (FSHR), luteinising hormone receptor (LHR), oestrogen receptor α (ER α), oestrogen receptor β (ER β), growth hormone receptor (GHR) and insulin-like growth factor binding protein-1 (IGFBP-1) in the cells were evaluated as were the secretion levels of oestradiol, activin, progesterone, testosterone, inhibin and follistatin in cell supernatant.

α-enolase gene silencing reduced the expression of FSHR, LHR, ERα, ERβ, GHR, and IGFBP-1 mRNA, potentiated the secretion of oestrogen, progesterone, testosterone, and follistatin of granulosa cells, and hampered the production of activin and inhibin.

ENO1 can regulate the reactivity of granulosa cells to reproductive hormones and regulate cell growth and development by adjusting their hormone secretion and reproductive hormone receptor expression. The study provided a better understanding of the functional action of ENO1 in the processes of goose ovary development and egg laying.

Five-minute heart-rate variability (HRV) measurement is a useful tool for assessing the autonomic nervous system (ANS) balance in humans, but there are no studies on healthy dogs. The aim of the study was, therefore, to provide the reference ranges in small and medium-sized breeds for short-term HRV time and frequency domain (TFD) analyses.

A total of 79 healthy dogs were included in the study between 2015 and 2019. Grouping by age with the breakpoint at six years and subgrouping by reproductive status and sex was imposed. All the dogs were included after physical and cardiological examinations and blood analyses. The TFD of HRV were analysed from a five-minute-long digital ECG recording after removal of non-sinus complexes.

There were no statistically significant differences in any TFD parameters between age, reproductive status or sex groups. A mild increase in all time domain parameters and the high-frequency (HF) band was observed in older dogs, and the low frequency (LF):HF ratio decreased in these dogs. In males, the time domain parameters and HF band increased slightly.

The normal ranges for HRV derived from short-term ECG recording in the usual clinical environment now have proposed reference ranges. Our findings suggest that accommodation time, age, sex, or reproductive status do not influence the results derived from these recordings, indicating that this method is reliable for assessing the ANS function in small and medium-sized dog breeds.

The therapeutic effect of subcutaneous embedding and revascularisation on the repair of canine bone defects caused by open fracture was examined.

A total of 12 adult beagle dogs were randomly split into a control group (group C) and a test group (group T). A section of the radius was removed from each dog under general anaesthesia and the deficit supported by an orthopaedic implant. Group T had the section surgically implanted next to the blood vessel–rich saphenous vein and Group C had it cryopreserved at −80°C. After eight weeks, the bone was surgically implanted back into the matching radial deficit. Bone healing was evaluated by gross morphological and X-ray examinations, post-mortem histology, and successive blood measurements of key bone biochemical markers.

At 12 weeks, the bone healing boundary was disappearing more quickly in group T dogs than in their group C counterparts. X-ray and histological examinations showed that the cortical repair of group T subjects was complete and the bony plate arrangement was more regular than that in group C. The levels of bone biochemical markers also proved that the healing state of group T was better.

The results showed that the degree of healing, osteoclast activity, and bone formation status of group T were better than those of group C, proving that the vascularised bone graft had a significantly shorter healing time than the cryopreserved bone graft.

Dobermann dogs are reportedly predisposed to familial glomerulonephropathy. Proteinuria is a hallmark of canine familial glomerular diseases. The identification of glomerular abnormalities in breeds so predisposed is of great importance in improving breeding policy. Therefore, markers that allow the detection and localisation of renal damage are needed. The purpose of this study was to investigate the urinary concentrations of immunoglobulin G (uIgG), retinol-binding protein (uRBP), and Tamm–Horsfall protein (uTHP) in a family of Dobermanns with proteinuria and compare these concentrations with the corresponding values in healthy controls.

Ten dogs of the Dobermann breed with proteinuria (five with a urine protein-to-creatinine ratio (UPC) of 0.5–1 and five with a UPC >1) and twelve healthy dogs were enrolled. An ELISA was performed to measure uIgG, uRBP, and uTHP, and these proteins were quantified in relation to urinary creatinine (uCrea).

uIgG/uCr and uRBP/uCr were significantly higher in the family of Dobermanns than in the healthy dogs. A significant difference in the uTHP/uCr value was found only in dogs with a UPC of >1.

IgG seems to facilitate the diagnosis of primary hereditary glomerulopathy in Dobermanns. Moreover, in affected dogs, proteinuria characterisation seems to be a promising alternative option for the detection and localisation of renal lesions.

Oxidative stress (OS) seems to be an important mediator of cellular injury, from which sepsis can proceed. Studies have demonstrated the protective effect of controlled hypothermia in sepsis. This study aimed to evaluate its effects on OS parameters in rat hepatic and renal tissue septic after caecal ligation and puncture (CLP).

Three groups were appointed (10 rats/group): C (control), SN (sepsis normothermic), and SH (sepsis hypothermic). Ten hours from CLP, the liver and kidneys were harvested and total protein concentration, superoxide dismutase (SOD), glutathione peroxidase (GPx) activities, lipid peroxidation level (malondialdehyde (MDA), carbonylated proteins (2,4-dinitrophenylhydrazine (DNPH), and fatty acid profile were analysed.

Sepsis significantly increased SOD and GPx activities in the liver, regardless of the temperature. In renal tissue, GPx activity increased significantly in normothermic conditions and SOD tended to decrease in hypothermic conditions. MDA and DNPH concentrations increase in both tissues after CLP. Hypothermia significantly lowered MDA in the liver but only changed it insignificantly in the kidneys. The DNPH in the liver and kidneys was significantly lower in hypothermic conditions. The unsaturated-to-saturated fatty acids ratio was significantly lower in sepsis, and the fall in temperature raised this ratio.

Experimentally induced sepsis in rats enhances OS in the liver and kidneys. The effect of hypothermia on OS indices is dependent on the type of tissue.