Volume 60 (2016): Issue 3 (September 2016)

Introduction: In the present study apoptosis was investigated in the cornu ammonis and cerebellum of 10 dogs naturally infected with rabies virus. Diagnosis of rabies was based on the results of fluorescent antibody staining and experimental inoculation.

Material and Methods: The paraffin tissue sections were stained with haematoxylin and eosin, avidin-biotin complex peroxidase (ABC-P), and terminal deoxynucleotidyl transferase biotin-dUTP nick end-labelling (TUNEL) methods.

Results: Histopathological examination revealed encephalomyelitis of varying severity and the presence of Negri bodies. Dense rabies antigens were determined in the motor neurons with ABC-P method. On the other hand, Bcl-2 protein and Bax protein gave positive reaction in seven and five cases, respectively. TUNEL staining demonstrated very marked apoptotic changes in the nuclei of neurons localised deep in the substantia alba of the cerebellum. Similar changes were also determined in perivascular mononuclear cells and glia cells within the substantia alba. No apoptopic changes were found in the motor neurons of the cornu ammonis.

Conclusion: The absence of apoptotic changes in the neurons was considered to be the consequence of the necrotic changes that developed in these neurons.

Introduction: Aujeszky’s disease (AD), most often related to infection of domestic and feral swine, may also concern other mammals, including dogs. The disease in carnivores, related to consumption of raw meat or offal contaminated with AD virus, is manifested by severe neurological disorders and inevitably leads to animal’s death.

Material and Methods: Karelian bear dog was euthanised due to nervous symptoms that started two days after participation in wild boar hunting. After exclusion of rabies the dog’s carcass was subjected to standard necropsy. Tissue samples were collected for histological examination. Samples of the brain were tested for ADV by real-time PCR and virus isolation. Samples of the liver were collected for toxicological examination.

Results: The presence of ADV was confirmed by real-time PCR and virus isolation. Toxicological examination revealed anticoagulant poisoning. This is the first case of Aujeszky’s disease (AD) in a hunting dog in Poland after exposure to ADV from offal of wild boar.

Conclusion: This infection should be taken into consideration in differential diagnosis of syndromes of neurological disorders in dogs. Since AD is found in both domestic pigs and wild boar in Poland, special care must be taken to prevent spread of infection to other species.

Introduction: Orf virus (ORFV) is a prototype Parapoxvirus species in the Poxviridae family that causes serious zoonotic infectious disease. Goat skin fibroblast (GSF) cells are the major host targets of ORFV. Interleukin 8 (IL-8) and tumour necrosis factor (TNF)-α are known to play a vital role in immune response during viral infections. However, the manner of variation over time of their level of expression in GSF cells remains unclear.

Material and Methods: In this study, quantitative enzyme-linked immunosorbent assay chips were used to detect changes in the levels of these cytokines expressed and secreted in GSF cells after ORFV infection.

Results: Results showed that the expression of IL-8, TNF-α, and decorin was upregulated in the cell lysates, and that secreted decorin and IL-8 were significantly increased in cell supernatant.

Conclusion: The results provided possible approaches to elucidation of how ORFV infection initiates host cell immune response.

The aim of the study was to assess the changes of blood parameters in 12 three-week-old Polish Merino sheep subjected to experimental jaagsiekte sheep retrovirus (JSRV) infection.

Material and Methods: Haematological (WBC with leukocyte subpopulations: GRA, LYM, MID, and RBC, MCV, MCH, MCHC, HGB, HCT, PLT, and MPV) and biochemical blood parameters (acid/base balance, cation/anion content, and gasometry) were determined in blood samples collected one month after JSRV infection, then at four-week intervals for five consecutive months.

Results: A decrease in RBC, HCT, MCV, PLT, MPV, and LYM values in comparison with controls was found in the last month of observation. On the other hand, at the same time, an increase in HGB, MCH, MCHC, WBC, MID, and GRA indices was observed. Moreover, at the end of experiment blood gasometric indices such as pCO₂, HCO₃, and tCO₂, and Na and K ion concentrations were higher in the affected lambs than in the healthy animals. The pH values of the challenged animals exhibited less alkaline character than in the case of controls, which was associated with a decrease in O₂% saturation. However, the majority of differences between JSRV inoculated and control groups was not statistically significant.

Conclusion: The observed changes in the examined blood parameters can be considered as prodromal symptoms in the preclinical phase of adenocarcinoma development associated with JSRV infection.

Introduction: Porcine pleuropneumonia inflicts important economic losses on most commercial herds. Detection of subclinical or chronic infection in animals still remains a challenge, as isolation and identification of A. pleuropneumoniae serotypes is difficult and quantification of the bacteria on agar plates is often almost impossible. The aim of the study was to develop and evaluate a serotype-specific quantitative TaqMan probe-based PCR for detection of serotype 2 in pig lungs, tonsils, and nasal swabs.

Material and Methods: The primers were designed from the capsular polysaccharide biosynthesis genes of A. pleuropneumoniae serotype 2. PCR specificity and sensitivity were evaluated using reference strains and several other bacterial species commonly isolated from pigs.

Results: The real-time qPCR for detection of A. pleuropneumoniae serotype 2 was highly specific and gave no false positives with other serotypes or different bacterial species of pig origin. The detection limit for pure culture was 1.2 × 10⁴ CFU/mL, for lung tissue and nasal swabs it was 1.2 × 10⁵ CFU/mL, and for tonsils - 1.2 × 10⁵ CFU/mL.

Conclusion: The method can be used to serotype A. pleuropneumoniae isolates obtained during cultivation and to detect and identify A. pleuropneumoniae serotype 2 directly in nasal swabs and tonsil scrapings obtained from live pigs or lung tissue and tonsils collected post-mortem.

Introduction: Recently in Europe an increase in the population of red deer (Cervus elaphus), roe deer (Capreolus capreolus), and fallow deer (Dama dama) has been observed. Research on the prevalence of Leptospira infections in Polish cervids has been performed for the first time.

Material and Methods: During 2014/2015 hunting season, 147 blood samples from red deer, roe deer, and fallow deer were collected. The animals originated from different geographical regions across Poland. Serum samples were tested by microscopic agglutination test (MAT) for the presence of specific antibodies to the following Leptospira serovars: Icterohaemorrhagiae, Grippotyphosa, Sejroe, Tarassovi, Pomona, Canicola, Bratislava, Hardjo, Ballum, Zanoni, Hebdomadis, and Poi.

Results: Serum antibody titres specific to Grippotyphosa, Pomona, and Zanoni serovars were found; none of the sera were positive for any of the other serovars. Out of 147 serum samples only 7 were positive, which gave an overall prevalence of 4.8% in the tested animal population.

Conclusion: The low Leptospira antibody titres along with the low number of positive serum samples in deer indicate that these animals may not act as significant reservoirs of Leptospira for either humans or animals in Poland.

Introduction: The present study aimed to investigate the effect of Enterococcus faecium EF55 on chickens, as well as its influence on proliferative activity of epithelial intestinal cells after infection with Salmonella enterica serovar Enteritidis phage type 4 (SE PT4). Moreover, the length and area of duodenal and jejunal villi of the birds were examined.

Material and Methods: A pool of 80 birds was divided randomly into four groups. Probiotic group (EF) and Salmonella + probiotic group (EFSE) received E. faecium EF55 (10⁹ CFU – 3 g per group/day) during 22 d. Salmonella group (SE) and EFSE group were infected with Salmonella Enteritidis PT4 (10⁸ CFU in 0.2 mL PBS) in a single dose per os on day four of the experiment, whereas control birds (C group) received only 0.2 mL of PBS on that day. Samples were taken on the 4^th and 18^th day post infection.

Results: Supplementation of feed with E. faecium EF 55 confirmed its selective antibacterial activity against SE PT4. The chickens infected with SE PT4 and fed E. faecium EF55 supplemented diet showed increased proliferative activity of enterocytes in the jejunum in both samplings. Applied probiotic strain demonstrated positive impact on intestinal morphometry in the jejunum of both non-infected groups and in Salmonella-infected chickens. In the latter group, the beneficial effect of E. faecium EF 55 was manifested by more efficient tissue turnover in the jejunum.

Introduction: The objective of this study was to determine the current profile of bacteria responsible for the infection of the mammary gland and to assess their sensitivity to selected β-lactam antibiotics.

Material and Methods: The study was conducted on 119 (n = 119) dairy cows of the Polish Black-White breed aged 4 to 10 years with inflammation of the mammary gland. The cows came from different farms: smallholder farms and large dairy cattle farms in the Lublin and Bialystok Provinces. Before the process of collection of milk samples, the teats were cleaned and immersed in a liquid disinfectant. The first streams were collected into containers which were subsequently utilised. Afterwards, 2-4 mL of milk or secretions was milked into sterile disposable tubes. The milk samples were collected into plastic bottles and kept in a cooler with ice until transportation to the laboratory. Tests of resistance to β-lactam antibiotics were performed by disc diffusion.

Results: Contagious and environmental bacteria were isolated from all dairy barns. In the group of contagious bacteria, the presence of typical pathogens responsible for the mammary gland infections, i.e. Staph. aureus, Str. agalactiae, and C. bovis, was detected. A relatively broad group of the isolates was formed by environmental bacteria responsible for inflammation of the mammary gland: Str. dysgalactiae, Str. uberis, Staph. chromogenes, Staph. hyicus, Staph. warneri, and E. coli. Among the environmental organisms, streptococci constituted the largest percentage (23%), followed by staphylococci (13.2%), and E. coli (8.8%). The largest group of infectious pathogens comprised Str. agalactiae (29.6%) and Staph. aureus (20.8%).

Conclusion: Our investigation of the current profile of the isolates responsible for mastitis in the Lublin and Bialystok Provinces showed that environmental bacteria are the major cause of the disease. In view of the substantially varying degrees of sensitivity of the microorganisms isolated from cases of mastitis to β-lactam antibiotics, each therapeutic treatment should be preceded by susceptibility testing.

Introduction: The aim of the study was to determine the prevalence of intestinal helminths in red foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) in the Augustów Primeval Forest (north-eastern Poland), with particular regard to zoonotic parasites.

Material and Methods: Intestines from 53 raccoon dogs and 66 red foxes were examined with the use of sedimentation and counting technique (SCT). Samples of faeces from 51 red foxes and 50 raccoon dogs were examined with the use of flotation method.

Results: Parasitic helminths were found by SCT in 98.5% of red foxes and 96.2% of raccoon dogs. Both species were infected with: Alaria alata (93.9% and 94.3%, respectively), hookworms (68.2% and 83.0%), Apophallus spp. (7.6% and 15.1%), Mesocestoides spp. (57.6% and 24.5%), Taenia spp. (40.9% and 1.9%), and Toxocara/Toxascaris nematodes (33.3% 15.1%). Echinococcus multilocularis was detected only in red foxes (6.1%), but trematodes Echinostomatidae and nematodes Molineus spp. only in raccoon dogs (18.9% and 41.5%, respectively). Additionally, Capillaria spp. eggs were detected by flotation method in 78.4% of foxes and 20.0% of raccoon dogs.

Conclusion: The study showed a very high percentage of red foxes and raccoon dogs infected with intestinal helminths in the Augustów Primeval Forest. Moreover, dangerous zoonotic parasites also were found, which should be taken into consideration in the assessment of infection risk for humans in this region.

In recent years a growing demand for ratite meat, including ostrich, emu, and rhea has been observed all over the world. However, consumers as well as the meat industry still have limited and scattered knowledge about this type of meat, especially in the case of emu and rhea. Thus, the aim of the present review is to provide information on technological and nutritional properties of ostrich, emu, and rhea meat, including carcass composition and yields, physicochemical characteristics, and nutritive value. Carcass yields and composition among ratites are comparable, with the exception of higher content of fat in emu. Ostrich, emu, and rhea meat is darker than beef and ratite meat acidification is closer to beef than to poultry. Ratite meat can be recognised as a dietetic product mainly because of its low level of fat, high content of polyunsaturated fatty acids (PUFA), favourable n6/n3 ratio, and high iron content in comparison with beef and chicken meat. Ratite meat is also rich in selenium, copper, vitamin B, and biologically active peptides such as creatine (emu) and anserine (ostrich), and has low content of sodium (ostrich). The abundance of bioactive compounds e.g. PUFA, makes ratite meat highly susceptible to oxidation and requires research concerning elaboration of innovative, intelligent packaging system for protection of nutritional and technological properties of this meat.

Introduction: The study objective was to develop and evaluate a new TaqMan multiplex real-time PCR method for Salmonella, L. monocytogenes, and verotoxigenic Escherichia coli (VTEC) detection in slaughtered animal carcasses.

Material and Methods: The procedure included an enrichment step, DNA extraction, and two multiplex real-time PCRs. The first PCR detected the invA and hly genes of Salmonella and L. monocytogenes respectively, the second the vtx1, vtx2, and eae genes of VTEC.

Results: The validation of this method resulted in 100% relative sensitivity, specificity, and accuracy as compared to the reference ISO methods. The limit of detection per swab sample was established at 1 cfu for Salmonella and L. monocytogenes and 2 cfu for VTEC. The authors analysed 265 slaughterhouse-collected swabs from cattle, pig, and poultry carcasses. Among 125 from cattle, 51 were positive for VTEC, 29 for Salmonella, and 1 for L. monocytogenes. Among swabs from pig carcasses (n = 95), three, two, and one sample were positive for these pathogens respectively. None of the microorganisms tested for was identified in 45 samples of poultry origin.

Conclusion: The obtained results showed that the method developed can rapidly identify the main bacterial pathogens that may contaminate carcasses of food-producing animals.

Introduction: The main problem in poultry farming is the difficulty in producing food of animal origin without using antibacterial agents. Because most antibacterial compounds are dispensed in water, some water supply systems can be contaminated by antibiotics which are then administered to the animals unintentionally. This can lead to unexpected increases in antibiotic residues in food of animal origin. The aim of the present study was to determine whether the constant exposure of chicken broilers to enrofloxacin affects the withdrawal time of a therapeutic doxycycline that is intentionally administered to the chickens.

Material and Methods: The concentrations of doxycycline, enrofloxacin, and ciprofloxacin were determined by LC-MS/MS in muscles and liver of the chickens.

Results: Doxycycline residue concentrations in the chicken tissues from the group that received trace amounts of enrofloxacin were nearly 50% greater than those of the group that received only doxycycline.

Conclusion: These results indicated that constant exposure to enrofloxacin in trace amounts significantly influences the residual doxycycline concentration in chicken tissues.

Introduction: Glycolic changes which occur post-mortem have an impact on the physical and sensory features of beef, which in turn determine the successive processes and influence such beef quality traits as colour, tenderness, and cooling loss. The aim of this study was evaluation of the post-mortem changes in bovine meat during aging, quantitative analysis of glycogen and lactic acid, as well as examination of their impact on technological and sensory quality of selected muscles from Holstein-Friesian × Limousin breed carcasses.

Material and Methods: The study included three muscles of different metabolic qualities and sarcomere length: m. semitendinosus, m. longissimus dorsi, and m. psoas major, collected from nine bull carcasses aged 24 ±2 months.

Results: Significant correlations were found between the volume of cooling loss on individual days of aging and the pH value of muscle tissue, lactic acid and glycogen content, as well as beef lightness. However, no significant dependency between the volume of glycogen and the intensity of red and yellow colours was detected.

Conclusion: The colorimetric analysis of glycogen and lactic acid can be an effective tool in predicting the quality of beef.

Introduction: To identify novel pathways involved in the pathogenesis of ketosis, an isobaric tag for relative and absolute quantitation/mass spectrometry was used to define differences in protein expression profiles between healthy dairy cows and those with clinical or subclinical ketosis.

Material and Methods: To define the novel pathways of ketosis in cattle, the differences in protein expression were analysed by bioinformatics. Go Ontology and Pathway analysis were carried out for enrich the role and pathway of the different expression proteins between healthy dairy cows and those with clinical or subclinical ketosis.

Results: Differences were identified in 19 proteins, 16 of which were relatively up-regulated while the remaining 3 were relatively down-regulated. Sorbitol dehydrogenase (SORD) and glyceraldehyde-3-phosphate dehydrogenase (G3PD) were up-regulated in cattle with ketosis. SORD and G3PD promoted glycolysis. These mechanisms lead to pyruvic acid production increase and ketone body accumulation.

Conclusion: The novel pathways of glycolysis provided new evidence for the research of ketosis.

Introduction: Peroxiredoxin 6 (Prdx6) is a bifunctional protein and a unique 1-Cys Prdx of the peroxiredoxin family. The expression and regulation of Prdx6 are implicated in numerous physiological and pathological processes.

Material and Methods: Eight stepwise truncated DNA fragments obtained from the 5′-flank region of the Prdx6 gene were prepared and subcloned into the pSEAP2-Enhancer vectors. To investigate the transcriptional activity of the truncated DNA fragments, the recombinant plasmids were transfected into the COS-1 cells and the transcriptional activity was measured via assaying the expression of the reporter gene of the secreted alkaline phosphatase.

Results: A 3.4 kb 5′-upstream flank region of the Prdx6 gene was cloned and sequenced. The region from −108 nt to −36 nt of the 5′-flanking region of the Prdx6 gene contained basal transcriptional activity.

Conclusion: This result provides the basis for further studies on the gene regulation of the Prdx6-mediated biological processes and on screening for the transacting factors that interact with cis-acting elements of the Prdx6 gene promoter.

Immunohistochemical studies have become an indispensable element of establishing the correct histopathological diagnosis of poorly differentiated lesions, proving particularly suitable, and occasionally indispensable, for diagnosis of poorly differentiated neoplastic tumours. Knowledge of the mechanism of action and normal reaction of individual proteins is required in selection of the antibody pattern for a given tissue and in evaluation of the obtained results. This paper aims to promote the application of immunohistochemical techniques in routine diagnosis, especially in cases of poorly differentiated or undifferentiated tumours.

Introduction: Apocrine sweat gland carcinomas (ASGCs) are malignant neoplasms of dogs and other animals, rarely reported worldwide. The aim of this study was to summarise the occurrence of this cancer in a population of dogs in Poland between 2009 and 2014 with regards to histological features and body location of the tumours, as well as age, sex and breed of the cancer-affected dogs.

Material and Methods: The study involved 40 canine ASGC cases diagnosed in five national veterinary pathology laboratories. The material was processed according to routine histological methods.

Results: Histological types of the tumours involved simple and complex apocrine carcinoma of cystic/papillary (62.5%), solid (15%), and tubular type (12.5%), as well as apocrine ductal carcinoma (10%). The epidemiological analysis revealed peak incidence of the cancer in dogs between 8 and 14 years of age, with the most commonly affected sites being forelimbs and thorax. The highest number of the cancer cases was diagnosed in mixed breed dogs and German Shepherds; no sex predilection was noted.

Conclusion: To the authors’ knowledge, this is the first report recounting the study on canine malignant apocrine sweat gland tumours in Poland providing detailed phenotypical and histological data, which are otherwise rarely described in veterinary literature. This type of cancer appears to be diagnosed more frequently in dogs than in humans. Being an easily accessible material for research, canine ASGCs might serve as a relevant animal model for studies related to pathogenesis of sweat gland tumours.

Introduction: The objective of the study was immunophenotypic and cytogenetic analysis of mesenchymal stem cells from equine bone marrow and foal umbilical cords during in vitro culture.

Material and Methods: The mesenchymal stem cells were obtained from equine bone marrow of three horses and from foal umbilical cords of six foals. The cells were cultured in CO₂ incubators by standard procedure. Quantitative abnormalities of chromosomes, i.e. aneuploidy and polyploidy, and structural aberrations, i.e. breaks in chromosomes and chromatid, were taken into account during the study.

Results: The results of cytogenetic analysis of equine bone marrow mesenchymal stem cells at the third and fourth passages indicated that the level of karyotype variability of these cells corresponded to the spontaneous level of karyotype variability typical of the peripheral blood lymphocytes of this species. Equine bone marrow contained several clones of stem cells that differed in the expression of specific nuclear markers characteristic of proliferating cells.

Conclusion: Mesenchymal stem cells from foal umbilical cords during in vitro cultivation are characterised by quantitative abnormalities of the chromosomal apparatus.

Introduction: The aim of this study was to determine the effect of deoxynivalenol (DON), given alone or with bentonite (which eliminates mycotoxicity) in the diet of mink dams throughout mating, pregnancy, and lactation period to pelt harvesting, on the mechanical properties and geometry of their long bones.

Material and Methods: The minks were randomly assigned into two groups: a control group (not supplemented with DON, n = 15) and a group fed naturally DON-contaminated wheat and divided into three sub-groups (each sub-group n = 15), depending on bentonite dose: 0 M – sub-group fed naturally DON-contaminated wheat at a concentration of 3.7 mg kg⁻¹ alone; 2 M – sub-group fed naturally DON-contaminated wheat at a concentration of 3.7 mg kg⁻¹ and bentonite at a concentration of 2 kg 1000 kg⁻¹; 0.5 M – sub-group fed naturally DON-contaminated wheat at a concentration of 3.7 mg kg⁻¹ and bentonite at a concentration of 0.5 kg 1000 kg⁻¹.

Results: The DON treatment reduced the length of the femur compared to the control group and reduced the bone weight dependently on the amount of bentonite supplementation. However, DON treatment reduced the MRWT and CI of the femur, irrespective of the bentonite supplementation, compared to the control. The total BTD and BMC decreased in all DON-treated groups (irrespective of the bentonite supplementation). Furthermore, the densitometric analysis showed that the main changes in BMD and BMC indicated bone loss in the proximal and distal parts of bone covering the trabecular bone; whereas when bentonite was given at the dose of 2 kg 1000 kg⁻¹ an increase in the whole BMD and BMC was observed in the femoral midshaft.

Conclusion: Analysis of the geometrical parameters seems to indicate that endosteal resorption was delayed after bentonite supplementation. The addition of bentonite diminished the DON action on bone homeostasis in the mink dams. Thus bentonite could prevent DON-induced bone loss in a dose-dependent manner.

Introduction: The study aimed to investigate the anti-fertility effect of fennel (Foeniculim vulgare Mill) seed extract in male rats.

Material and Methods: Forty Wistar rats were divided into five equal groups. The control group received distilled water and the experimental groups were orally administered 1 ml of hydro-alcoholic extract of fennel seed in four doses of 35, 70, 140, and 280 mg/kg/b.w. daily for 60 days. After the last gavage, the rats were anaesthetised and the caudal part of the right epididymis was used for sperm counting. After fixation of the testes, microscopic sections were prepared and histological changes were evaluated.

Results: The number of spermatogonia after doses of 140 and 280 mg/kg and Sertoli cells after a dose of 140 mg/kg decreased significantly as compared with the control group (P < 0.05). The number of primary spermatocytes and sperm count decreased significantly in the experimental groups (70, 140, and 280 mg/kg) when compared to the control group (P < 0.05). Furthermore, thickening of the basement membrane, cell apoptosis, and irregular arrangement of the germinal epithelium were observed in the experimental groups.

Conclusion: Hydro-alcoholic fennel seed extract at these doses could reduce reproductivity and has anti-fertility activity in male rats.

There are numerous biomarkers of central and peripheral nervous system damage described in human and veterinary medicine. Many of these are already used as tools in the diagnosis of human neurological disorders, and many are investigated in regard to their use in small and large animal veterinary medicine. The following review presents the current knowledge about the application of cell-type (glial fibrillary acidic protein, neurofilament subunit NF-H, myelin basic protein) and central nervous system specific proteins (S100B, neuron specific enolase, tau protein, alpha II spectrin, ubiquitin carboxy-terminal hydrolase L1, creatine kinase BB) present in the cerebrospinal fluid and/or serum of animals in the diagnosis of central or peripheral nervous system damage in veterinary medicine.