Volume 61 (2017): Issue 4 (December 2017)

Infectious diseases of swine, particularly zoonoses, have had a significant influence on nutritional safety and availability of pig meat as high-energy protein product since the time that pigs were domesticated back in the 7^th century BC. The main sources of swine infectious diseases include the so-called primary sources (direct infection, i.e. through contact with infected and sick animals) and secondary sources (contaminated meat products, slaughter products, and vectors, including ticks). At present, the most serious epidemiological and economic threat to swine breeding in Europe is African swine fever (ASF). This disease, originally coming from Africa, is incurable and causes death of infected pigs and wild boars during 7−10 days after infection. Among the various factors that influence the spread of ASF, important role is played by ticks from the genus Ornithodoros, mainly from the species Ornithodoros moubata. Research on the ASF indicates that other species of ticks can also transmit the virus to healthy pigs in laboratory conditions. Sylvatic and domestic cycles of ASF virus transmission, which have been described so far, require further studies and updating in order to point the potential new vectors in the Caucasus and Eastern Europe affected by the ASF. Effective methods of control and biosecurity may significantly slow down the spread of ASF, which undoubtedly is a major threat to world pig production and international swine trade.

Introduction: The genomes of nine H5 subtypes of low pathogenic avian influenza virus (LPAIV) strains identified in wild birds in Poland between 2010 and 2015 were sequenced, and their phylogenetic relationship was determined.

Material and Methods: AIV genome segments were amplified by RT-PCR and the PCR products were sequenced using Sanger method. Phylogenetic trees were generated in MEGA6 software and digital genotyping approach was used to visualise the relationship between analysed strains and other AIVs.

Results: High genetic diversity was found in the analysed strains as multiple subgroups were identified in phylogenetic trees. In the HA tree, Polish strains clustered in two distinct subclades. High diversity was found for PB2, PB1, PA and NP, since 5-8 sublineages could be distinguished. Each strain had a different gene constellation, although relationship of as much as six out of eight gene segments was observed between two isolates. A relationship with poultry isolates was found for at least one segment of each Polish strain.

Conclusion: The genome configuration of tested strains indicates extensive reassortment, although the preference for specific gene constellation could be noticed. A significant relationship with isolates of poultry origin underlines the need for constant monitoring of the AIV gene pool circulating in the natural reservoir.

Introduction: The reverse transcription polymerase chain reaction (RT-PCR) is one of the most extensively used methods for identification of animals infected with bluetongue virus (BTV). There are several RT-PCR protocols published and several real-time RT-PCR (rtRT-PCR) commercial kits available on the market. Because Poland faced BTV-14 infection in 2012, different protocols were implemented in the country to confirm the RT-PCR results positive for this virus. The article presents a comparative study of several RT-PCR protocols and discusses their diagnostic reliability and applicability.

Material and Methods: Six rtRT-PCR/RT-PCR protocols were compared for the laboratory diagnostic of fourteen BTV-14 isolates circulating in Poland in 2012–2014.

Results: All 14 isolates were positive in the protocols of Shaw et al. (18), a commercial LSI NS3 kit, and Eschbaumer et al. (5). Four out of fourteen BTV-14 isolates gave positive results in Hoffmann’s 2 and 6 protocols and none of the 14 isolates yielded positive results in Maan et al. (8) method. Phylogenetic study of a short fragment of 450 nt of BTV segment 2 (258–696 positions) revealed 100% identity within Polish variants and with Russian and Spanish isolates.

Conclusion: The paper points to the possible false negative results in the diagnosis of BTV infections depending on the protocol used.

Introduction: Aujeszky’s disease virus (ADV) infects a wide range of animals, including members of the Suidae family, i.e. domestic and wild pigs, as well as wild boar. Since wild boar are a potential ADV reservoir and a source of infection for domestic pigs, the aim of the study was to evaluate ADV antibody prevalence in the Polish wild boar population, during the years 2011 to 2014.

Material and Methods: Wild boar blood samples were collected during three consecutive hunting seasons; i.e. 2011/2012, 2012/2013, and 2013/2014, and tested for ADV antibodies by ELISA.

Results: ADV antibodies were detected in samples from all tested voivodships. The average seroprevalence reached 32.2%. Seroprevalence, over the examined hunting seasons, was 27.4% in 2011/2012, 32.4% in 2012/2013, and 35.5% in 2013/2014. The highest percentage of seroreagents was detected in four voivodships, situated along the western border of Poland, i.e. Zachodnio-Pomorskie (ZP), Lubuskie (LB), Dolnośląskie (DS), and Opolskie (OP). This area is positively correlated with the highest density of the wild boar population and the highest wild boar hunting bag.

Conclusion: The results of this study confirm that the wild boar population may still pose a threat to domestic pigs, which is of special importance at the final stage of Aujeszky’s disease eradication programme in Poland.

Introduction: The aim of the research was to investigate the antiviral and immunoregulatory effects of saikosaponin A, saikosaponin D, Panax notoginseng saponins, notoginsenoside R1, and anemoside B4 saponins commonly found in Chinese herbal medicines.

Material and Methods: control mice were challenged intramuscularly (im) with 0.2 mL of porcine circovirus 2 (PCV2) solution containing 10⁷ TCID₅₀ of the virus/mL. Mice of high-, middle-, and low-dose saponin groups were initially challenged im with 0.2 mL of PCV2 solution and three days later treated intraperitoneally (ip) with one of five saponins at one of three doses (10, 5, or 1 mg/kg b.w.). In the drug control group, mice were dosed ip with 10 mg/kg b.w. of a given saponin, and mice in a blank control group were administered the same volume of normal saline.

Results: The results revealed that the saponins could reduce the incidence and severity of PCV2-induced immunopathological damage, e.g. body temperature elevation, weight loss, anaemia, and internal organ swelling. In addition, it was seen that the saponins could affect the immunoglobulin levels and protein absorption.

Conclusion: The data suggested that the saponins might effectively regulate immune responses.

Introduction: The study was conducted to investigate the prevalence and genetic diversity of Chlamydia spp. in poultry in Poland and estimate possible transmission to humans.

Material and Methods: Molecular diagnostic methods followed by sequencing and strain isolation were used on cloacal/faecal swabs collected from 182 apparently healthy poultry flocks including chickens, turkeys, ducks, and geese. Serum samples obtained from people exposed (study group) and non-exposed (control group) to birds were tested by complement fixation test to acquire data on Chlamydia spp. antibody level.

Results: Overall, 15.9% of the tested flocks were Chlamydiaceae-positive and three Chlamydia spp. were identified. Predominant chlamydial agent found was C. gallinacea occurring in 65.5% of all positive poultry flocks and in 73.0% of positive chicken flocks. The sequences from four chicken flocks were assigned to C. abortus, whereas C. psittaci was confirmed in one duck and one goose flock. The analysis of ompA variable domains revealed at least nine genetic variants of C. gallinacea. Chlamydial antibodies were detected in 19.2% of human serum samples in the study group in comparison with 10.8% in the controls.

Conclusion: The obtained results confirm that chlamydiae are common among chicken flocks in Poland with C. gallinacea as a dominant species. Moreover, the presence of C. abortus in chickens is reported here for the first time. Further investigation should focus on possible zoonotic transmission of C. gallinacea and C. abortus as well as potential pathogenic effects on birds’ health and poultry production.

Introduction: Colibacillosis – the most common disease of poultry, is caused mainly by avian pathogenic Escherichia coli (APEC). However, thus far, no pattern to the molecular basis of the pathogenicity of these bacteria has been established beyond dispute. In this study, genomes of APEC were investigated to ascribe importance and explore the distribution of 16 genes recognised as their virulence factors.

Material and Methods: A total of 14 pathogenic for poultry E. coli strains were isolated, and their DNA was sequenced, assembled de novo, and annotated. Amino acid sequences from these bacteria and an additional 16 freely available APEC amino acid sequences were analysed with the DIFFIND tool to define their virulence factors.

Results: The DIFFIND tool enabled quick, reliable, and convenient assessment of the differences between compared amino acid sequences from bacterial genomes. The presence of 16 protein sequences indicated as pathogenicity factors in poultry resulted in the generation of a heatmap which categorises genomes in terms of the existence and similarity of the analysed protein sequences.

Conclusion: The proposed method of detection of virulence factors using the capabilities of the DIFFIND tool may be useful in the analysis of similarities of E. coli and other sequences deriving from bacteria. Phylogenetic analysis resulted in reliable segregation of 30 APEC strains into five main clusters containing various virulence associated genes (VAGs).

Introduction: The aim of the study was the application and comparison of real-time PCR methods based on the fluorescence of SYBR Green I intercalating dye and TaqMan probes for the detection of the 23S rDNA gene of Listeria spp. and the hlyA gene of Listeria monocytogenes in biological samples of the liver, brain, and blood.

Material and Methods: Five strains of L. monocytogenes and single strains of each species L. ivanovii, L. innocua, L. grayi, L. welshimeri, and L. seeligeri were used for the experiments. Additionally, five strains of other species of bacteria were used for evaluation of the specificity of tests. In the first stage of the study SYBR Green I real-time PCRs, one allowing detection of the 23S rDNA gene and two based on the amplification the hlyA gene, were performed. In the next part, three TaqMan probe-based real-time PCRs allowing confirmation of belonging to Listeria spp. and L. monocytogenes were conducted.

Results: The observation of amplification curves in real-time PCRs enabled the detection of both genes. A high regression coefficient of 0.99 was found for all reactions. Specific amplification products were obtained for the 23S rDNA and hlyA genes which confirm their belonging to Listeria spp. and L. monocytogenes, respectively. Other microbial species did not reveal real-time PCR products.

Conclusion: Both real-time PCR methods for the detection of Listeria spp. and L. monocytogenes in biological samples demonstrated a significant sensitivity and high specificity.

Introduction:Mycoplasma bovis is a well-known cause of various disorders in cattle, such as pneumonia, arthritis, mastitis kerato-conjunctivitis, pharyngitis, laryngitis, otitis media, meningitis, and reproductive disorders. There are no commercial vaccines against M. bovis in Europe, therefore, experimental ones are still under investigation. The aim of this study was to evaluate the effect of experimental M. bovis vaccine, containing the Polish field M. bovis strain as well as saponin and lysozyme dimer adjuvants, on the T- and B-cell response in calves.

Material and Methods: The study was carried out on 12 calves divided into two equal groups: experimental and control. The experimental group was subcutaneously injected with the vaccine composed of the field M. bovis strain as well as saponin and lysozyme dimer as adjuvants, whereas the control one received phosphate buffered saline (PBS). The blood samples were collected prior to the study (day 0), then in 24 h intervals up to day 7 and then each 7 days until day 84 post immunisation. The T- and B-cell response as CD2⁺ (T-cells), CD4⁺ (T-helper cells), CD8⁺ (T-cytotoxic cells), and WC4⁺ (B-cells) markers was analysed using flow cytometry.

Results: In response to the immunisation, the general stimulation of T-cell was observed, the most seen in an increase in CD8⁺ subpopulation. Similarly, a visible rise in the percentage of WC4⁺ cells was registered in the vaccinated calves when compared to the control animals.

Conclusion: This study demonstrated that the novel experimental M. bovis vaccine containing saponin and lysozyme dimer effectively stimulated the cell-mediated immunity in the calves.

Introduction:Mycoplasma gallisepticum is considered the most pathogenic and economically significant avian Mycoplasma spp. for the worldwide poultry industry. The aim of this study was to develop a novel and sensitive real-time loop-mediated isothermal amplification (LAMP) assay based on the amplification of its mgc2 gene sequence for its rapid molecular detection in poultry.

Material and Methods: Blood samples from 300 broiler and layer chickens were screened using a rapid serum agglutination (RSA) test. A real-time LAMP reaction was conducted with seropositive swab samples at 60°C for 90 min in an ESEQuant tube scanner using 6-carboxyfluorescein as the reporting dye.

Results: The sensitivity of the developed assay was 10 fg/μL of DNA. The assay was found 100% specific, showing no cross-reactivity with other avian Mycoplasma species. The proportion found of the positive samples by the real-time LAMP was 58%. In comparison, the RSA was found to detect 52% of positive cases.

Conclusion: The mgc2 real-time LAMP emerged as a more sensitive and accurate method for molecular detection of M. gallisepticum than RSA. Robustness and precision give it applicability as a potential field diagnostic tool for M. gallisepticum control. The study will be beneficial in reducing economic losses that M. gallisepticum inflicts on the poultry industry. This is the first reported development of a real-time LAMP assay based on the amplification of the mgc2 gene sequence using an ESEQuant tube scanner for galline M. gallisepticum detection.

Introduction: Ornamental fish can suffer from different bacterial diseases. Among them the most prevalent are infections caused by Aeromonas, Shewanella, Citrobacter, Plesiomonas, Edwardsiella, and Pseudomonas. But there is a broad spectrum of rarely identified bacteria which may be causative agents of diseases. The aim of the study was to determine the species of bacteria pathogenic for fish which are prevalent in aquariums.

Material and Methods: Bacteria were isolated from infected ornamental fish from pet shops and private aquariums in the Lublin region in 2015 and classified to species using MALDI-TOF MS.

Results: A total of 182 isolates from ornamental fish were identified. The most frequent bacteria found in diseased fish were Aeromonas veronii (30.8% of total number of strains), A. hydrophila (18.7%), Shewanella putrefaciens (7.1%), Citrobacter freundii (7.1%), Pseudomonas spp. (7.1%), Shewanella baltica (4.9%), and Plesiomonas shigelloides (3.3%).

Conclusion: Isolated bacterial species are facultative pathogens for fish and humans and may be isolated from fish without apparent symptoms of the disease.

Introduction: Due to their immunostimulatory properties TLR ligands are used prophylactically to protect against a variety of viral and bacterial pathogens in mammals. Knowledge of the molecular and functional aspects of TLRs is essential for a better understanding of the immune system and resistance to diseases in birds. For that reason, this study attempted to determine the impact of TLR21 stimulation by its synthetic ligand (CpG ODN, class B) on the chicken immune system.

Material and Methods: Sixty embryonated chicken eggs were randomly allocated into three groups (control and two experimental groups). On day 18 of embryonic development, chickens in one experimental group were administered in ovo a low dose of CpG ODN and the birds of the second experimental group were given a high dose of the ligand. Spleens were collected at 1, 2, 5, and 10 days post-hatching (dph) for analysis of IFN-α, IFN-β, IFN-γ, IL-6, and IL-10 expression using qRT-PCR.

Results: Significant differences were observed in mRNA expression levels of all the measured cytokines associated with the modulation and regulation of the immune response at different time points.

Conclusion: The obtained data clearly demonstrate that immune response induction takes place after in ovo administration of class B CpG ODN, and that the ligand has the ability to induce cytokine responses in neonatal chicken spleen.

Introduction: The aim of study was to estimate the prevalence and intensity of intestinal parasite infections in pigs in Poland and evaluate the influence of factors related to the production system on the infection intensity.

Material and Methods: A total of 70 pig farms of all Polish provinces, differing in the herd size and production system, were selected for the study. Fresh faecal samples were collected from all age groups: suckling piglets, weaners, fatteners, and lactating sows. Moreover, data were obtained regarding the size of the herd, the use of paddock and all-in/all-out system, the presence of diarrhoea, and the type of flooring.

Results: Parasite eggs or oocysts were detected in 57 of the 70 examined pig farms. Oesphagostomum spp. eggs were found in the largest number of farms (68.6%). Moreover, coccidia (42.9%), Ascaris suum (28.6%), Trichuris suis (21.4%), and Strongyloides spp. (11.4%) were detected. The highest prevalence of coccidia and Strongyloides spp. was found in suckling piglets, A. suum and T. suis in fatteners, and Oesphagostomum spp. in sows. Higher prevalence of parasites was detected in small farms than in medium and large farms, except the prevalence of coccidia, which was the highest in medium farms. Simultaneous infection with several parasites was more often detected than with one parasite. Odds ratio of parasites occurrence was higher in farms with paddock and litter floor and in farms which do not use all-in/all-out system.

Conclusion: Relatively high prevalence of intestinal parasites was found in pigs in Poland. Moreover, specific distribution of parasites in different age groups and farms of different size was observed. Influence of breeding factors on parasite prevalence was identified.

Introduction: Plate diffusion methods play an important role in the monitoring system for antimicrobial agents in raw materials and foodstuffs of animal origin. The aim of this work was to select a Yersinia spp. strain for the plate diffusion method based on sensitivity to a fluoroquinolone, namely flumequine. Another aim was to determine the optimal conditions of the method with the selected strain of Yersinia ruckeri CCM 8467 and to determine the detection capability (CCβ) of this method for residues of selected fluoroquinolones in milk.

Material and Methods: Optimum method conditions were set: cell concentration in the test agar at the level of 9.10⁵–10⁶ CFU/mL, discs with a diameter of 12.7 mm, Antimicrobial Inhibitor Test Agar with a pH of 6.0, and incubation at 30°C for at least 18 h and up to 24 h.

Results: With respect to the maximum residue limit (MRL), the Y. ruckeri plate method demonstrated the lowest sensitivity to flumequine. The CCβ of the method for flumequine was in the concentration of 100 μg/L⁻¹ (twice the MRL). The study also confirmed that the method exhibits very good sensitivity to the other tested fluoroquinolones, which were marbofloxacin (30 μg/L, 0.4 MRL), ciprofloxacin (10 μg/L, 0.1 MRL), and enrofloxacin (20 μg/L, 0.2 MRL), but lower sensitivity to danofloxacin (42 μg/L, 1.4 MRL).

Conclusion: The method with the CCM 8467 strain of Y. ruckeri showed a higher sensitivity to flumequine than the method with the ATCC 11303 strain of E. coli.

Introduction: Polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (DL-PCBs) belong to a well-known group of pollutants. Present in feedstuffs, they bioaccumulate in tissues of food-producing animals. Food is the source of over 90% of human PCDD/Fs and DL-PCBs intake. Thus, feed control is one of the pillars of the EU strategy and a mean of reducing human exposure. The article presents AhR based reporter gene bioassay method for PCDD/Fs and DL-PCBs analysis in feed and its validation results.

Material and Methods: Analytes were extracted from samples with fat. Subsequently, fat and other interferences were removed from extract using sulphuric acid modified silica. Extract was further cleaned and PCDD/Fs separated from DL-PCBs using carbon column. Contaminants detection was performed using H1L6.1c3 cell line, which produces luciferase in response to AhR ligands present in extract.

Results: Performance characteristics (repeatability, reproducibility, and apparent recovery) fulfil the requirements of Regulation 2017/771/EU. The positive correlation between bioassay and reference HRGC-HRMS method was confirmed. Moreover, the role of screening method used in connection with the confirmatory HRGC-HRMS method in providing feed and food safety has been discussed.

Conclusion: Bioassay is a useful method for dioxin and DL-PCBs analysis, allowing cost reduction of monitoring programmes with minimal risk of false negative results.

Introduction: Ochratoxin A (OTA) is a toxic metabolite mainly produced by Aspergillus spp. and Penicillum spp. fungi. Research on the contamination of cereals, complete feeds, and tissues with this mycotoxin has indicated that it can be a toxicological problem impacting animal health and food safety in temperate climes. OTA contamination mainly besets the global pig industry, necessitating the monitoring of feeds and animal tissues. The aim of the study was to present the results of the official monitoring of OTA in animal tissues and feeds in Poland in 2014–2016 and determine the possible correlation between the presence of OTA in different types of samples.

Material and Methods: The presence of ochratoxin A was determined using accepted procedures based on liquid chromatography with fluorescence detection after immunoaffinity column clean-up. Determination of OTA was afforded in the range of 0.3 μg/kg to 300 μg/kg in complete feeds and from 0.2 μg/kg to 150 μg/kg in the kidneys, liver, and muscles.

Results: Over the three year span, about 23.5% of the animal tissues samples were contaminated by ochratoxin A. In the 2014 survey, 10% of the sample tissues contained 5–10 μg/kg (only one sample above 10 μg/kg), and in 2015 and 2016, 24% of samples showed levels above the limit of quantification 0.2 μg/kg, while none of the samples exceeded the established provisional action level of 5 μg/kg for animal tissues. The animal feed analysis showed that 9% was contaminated with ochratoxin A above the limit of quantification of 0.3μg/kg. In 2% of feed samples the OTA concentration was greater than 50 μg/kg.

Conclusion: The results confirm the appropriacy of OTA contamination monitoring and help to increase food safety.

Introduction: The effect of intrauterine administration of Momordica charantia L. (MC) extract on oxidative changes and pregnancy rate in infertile cows was investigated.

Material and Methods: Endometrial smear specimens were taken from 40 cows with fertility problems for cytological examination, and the cows were randomly divided into two groups: group I (n = 20) was subjected to intrauterine administration of 40 mL (0.25 g/mL) of MC extract, group II (n = 20) was subjected to intrauterine administration of 40 mL of pure olive oil. Blood samples were taken starting from the day of administration of MC extract or olive oil (day 0) and then for three weeks at weekly intervals (days 7, 14, 21). Blood serum samples were evaluated for total antioxidant capacity (TAS), total oxidant level (TOS), oxidative stress index (OSI), lipid hydroperoxide (LOOH), and nitric oxide (NO) levels. In addition, on the 14^th day following treatment, two doses of PGF2α were administrated to all cows at 14-day intervals. Following the second PGF2α administration, insemination and GnRH administration was performed at the 60^th h after PGF2α treatment. Smear samples were stained with Giemsa and immunohistochemically to determine cytological changes and inflammatory status.

Results: According to cytological findings, subclinical endometritis was a prevalent disorder in cows with infertility problem (82.5%; 33/40). Additionally, 60.6% (20/33) of the cows with subclinical endometritis had acute inflammation, whereas remaining 13 cows had chronic endometritis. Of the cows with subclinical endometritis, 50% (8/16) and 35% (6/17) became pregnant in group I and II, respectively (P > 0.05). Although the oxidative stress parameters showed similarities between both groups (P > 0.05), there was a significant difference (P < 0.001) between the groups in terms of mean NO and LOOH levels (NO – 31.20 ± 11.38 vs 44.53 ± 11.50 μmol/L and LOOH – 1.22 ± 0.37 vs 1.89 ± 0.36 μmol/L).

Conclusion: The obtained results indicated that MC administration, especially in the presence of active inflammation, may improve the pregnancy rate by positive reduction of oxidative changes.

Introduction: The purpose of the current study was to evaluate the blood glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) levels under seasonal variations in dairy cows during transition period, and to assess the relationship between chosen reproductive parameters, GSH-Px, and MDA.

Material and Methods: Holstein cows calving in January were assigned into winter group (n = 42), while cows calving in August were assigned into summer group (n = 42). Blood samples were collected from the jugular vein 21, 14, and 7 days before calving, at calving (0 day), and 7, 14, and 21 days after calving. Reproductive parameters obtained from farm records were evaluated.

Results: In both groups of cows, GSH-Px activity decreased from 21 days before calving to day 0, and it gradually continued to increase until 21 days after calving. GSH-Px activity was higher in winter group compared to summer group during the transition period (P < 0.05). MDA levels in both groups increased over time starting from 21 days before calving to 0 day, but it gradually decreased thereafter. MDA levels were higher in summer group compared to winter group during the transition periods (P < 0.05). Summer group of cows showed higher intervals of calving-to-oestrus, calving-to-conception, and higher insemination index (P < 0.01). Negative correlation was recorded between GSH-Px and MDA during all examination days (P < 0.01). MDA levels correlated with calving to conception interval on day 21 before calving and day 0 (P < 0.01) and insemination index on day 0 and 21 days after calving (P < 0.01). GSH-Px activity was negatively correlated with calving to conception interval on day 21 before calving, day 0, and 21 days (P < 0.01) after calving. Negative correlation on day 21 before calving and day 0 was also determined between GSH-Px and insemination index (P < 0.01).

Conclusion: This study showed that blood oxidant and antioxidant levels have affected the fertility parameters in cows under seasonal variations.

Introduction: The study aimed to clarify the changes in the concentration of inflammatory mediators, proteases, and cartilage degradation biomarkers in the synovial fluid of joints in an equine osteoarthritis model.

Material and Methods: Osteoarthritis was induced in eight Mongolian horses by a sterile intra-articular injection of amphotericin B, which was injected into the left carpal joint in a dose of 2 mL (25 mg/mL). The control group comprised five horses which were injected with an equal dose of sterile physiological saline into the left carpal joint. Synovial fluid was obtained at baseline and every week after injection. Test methods were based on ELISA.

Results: In the course of the osteoarthritis, the concentration of biomarkers in joint synovial fluid showed an increasing trend. IL-1, IL-6, MMP-9, MMP-13, ADAMTS-5, CS846, GAG, HA, CTX-II, and COMP concentrations sharply increased before the onset of significant symptoms of lameness, whereas TNF-α, MMP-2, and MMP-3 concentrations rose sharply after the occurrence of such symptoms.

Conclusion: The results obtained confirm that the concentrations of IL-1, IL-6, MMP-9, MMP-13, ADAMTS-5, CS846, GAG, HA, CTX-II and COMP increase substantially in equine osteoarthritis, which provides a theoretical basis for the rapid diagnosis of the disease.

Introduction: Radioactive iodine (RAI) is commonly used for the treatment of hyperthyroidism caused by Graves’ disease or thyroid nodules. However, information available on the impact of RAI therapy on male gonadal function is scarce. This study aimed to determine any possible damage to testicular tissue and sperm quality caused by RAI therapy, and the radioprotective effect of amifostine against such damage.

Material and Methods: In total, 36 rats were randomly allocated to three groups, including a control group, RAI group (111 MBq Iodine-131), and RAI + amifostine group (111 MBq Iodine-131 and a single dose of 200 mg/kg amifostine). Blood and epididymal sperm samples were taken for hormone analyses and the evaluation of spermatological parameters. The TUNEL assay and haematoxylin-eosin were used to stain testicular tissue samples to detect histological changes and apoptosis.

Results: The groups differed insignificantly for the testicular mass index and spermatozoa concentration. However, spermatozoa motility and percentage of viable spermatozoa were higher in the RAI + amifostine group, compared to the RAI group. Sperm DNA fragmentation and the index of apoptotic germ cells significantly decreased in the amifostine group, in comparison to the radioiodine group. While the testosterone levels showed no significant change, the follicle stimulating hormone (FSH) levels significantly decreased in the RAI + amifostine group.

Conclusion: All histopathological parameters and some spermatological parameters showed that RAI therapy caused statistically significant damage of testicular tissue and this damage was reduced by amifostine.

Introduction: The aim of the study was to assess the influence of α-lipoic acid (ALA) on the morphology of the aorta and liver of rabbits fed high fat diet with addition of oxidised (ORO) and non-oxidised rapeseed oil (N-ORO).

Material and Methods: The study was conducted on male chinchilla rabbits divided into six groups. The control group (C) was fed a breeding standard diet (BSD), group I received BSD with the addition of ALA in the dose of 10 mg/kg b.w., groups II and III received BSD enriched with 10% addition of N-ORO or ORO, whereas rabbits from groups IV and V received BSD with 10% addition of N-ORO or ORO and ALA.

Results: Addition of ORO caused necrosis and steatosis of hepatocytes, as well as atherosclerotic plaques of various intensification in the aorta. In the liver of rabbits from group II (N-ORO) infiltrations of mononuclear cells was observed in the area of liver triads and between liver lobules. The beneficial influence of ALA was demonstrated in rabbits fed a diet containing N-ORO or ORO. In case of ORO, the activity of ALA was not fully effective.

Conclusion: Diet supplementation with ALA counteracts the changes generated in the liver and aorta under increased exposure to higher fat content in diet, in particular thermally treated fats.

Introduction: The purpose of this study was to investigate the therapeutic effect of hydrogen on the therapy of onion poisoned dogs.

Material and Methods: A total of 16 adult beagle dogs were divided into two groups (control and hydrogen) and all were fed dehydrated onion powder at the dose of 10 g/kg for three days. The dogs of the experimental group were given subcutaneous injection of 0.2 mL/kg of hydrogen for 12 days after making the poisoned model successful. Blood samples were collected before feeding onions, one day before injecting hydrogen, and 2 h after the injection of hydrogen on days 1, 3, 5, 7, 9, and 12. Control dogs were not treated with hydrogen.

Results: The levels of leukocyte production, anaemia, red blood cell degeneration which was reflected by the values of Heinz body count, haemolytic ratio, and oxidative products in hydrogen treated group were lower than in control dogs on some days. The capacity of medullary haematopoiesis that was based on reticulocyte counts, and the antioxidation in hydrogen group were higher compared with control group. However, the differences in renal function were not obvious in both groups.

Conclusion: Accordingly, it was concluded that subcutaneous injection of hydrogen could alleviate the symptoms in onion poisoned dogs.

Introduction: Eight microsatellite loci were used to define genetic diversity among five native water buffalo breeds in Pakistan.

Material and Methods: Blood samples (10 mL) from 25 buffaloes of each of the Nili, Ravi, Nili-Ravi, Kundhi, and Azi-Kheli breeds were collected aseptically from the jugular vein into 50 ml Falcon tubes containing 200 μl of 0.5 M EDTA. The phenol-chloroform method was used to extract DNA and the regions were amplified for microsatellite analysis. The eight microsatellite markers ETH10, INRA005, ILSTS029, ILSTS033, ILSTS049, ILSTS052, ETH225, and CSSM66 were analysed.

Results: The effective number of alleles across all loci was as usual lower than the observed values with a mean value of 2.52 alleles per locus. The overall allele frequency varied from 0.0041 for alleles B, I, and J over respective loci ILSTS052, INRA005, and ILSTS029 to 0.80 for allele H over locus ILSTS029. The average observed and expected heterozygosity values across all polymorphic loci in all studied buffalo breeds were 0.43 and 0.53, respectively. The overall value for polymorphic information content of considered microsatellite markers was 0.53, suggesting their appropriateness for genetic diversity analysis in buffalo. The mean Fis value was 0.13 and all loci except ILSTS049 were found significantly deviated from HWE, most likely due to non-random breeding. The five buffalo populations were genetically less diverse as indicated by a small mean Fst value (0.07). The average gene flow (Nm) indicative for population migration was calculated as 3.31. Nei’s original measures of genetic distance (Ds) revealed ancient divergence of the Nili and Azi-Kheli breeds (Ds = 0.1747) and recent divergence of the Nili and Ravi breeds (Ds = 0.0374).

Conclusion: These estimates of genetic diversity were seen to coincide with phenotypic differentiation among the studied buffalo breeds. The present study reports the first microsatellite marker-based genetic diversity analysis in Pakistani buffalo breeds, and might facilitate similar studies in other livestock breeds of Pakistan.