Volume 60 (2016): Issue 4 (December 2016)

Increased incidence of protothecal mastitis has been recorded in several countries in the past ten years. The main goal of this article is to draw the attention of scientific and professional community to the emerging issue of mammary protothecosis. The article collates currently known facts about infection reservoirs, predisposing factors for the development of mastitis, clinical manifestations of the disease, and potential transmission routes within the herd as well as the measures for control and eradication. We would like to point out that identification of protothecal mastitis on a dairy farm is associated with a range of problems. Early detection of infected animals can be difficult because of predominantly subclinical course of early-stage infection, which easily spreads between cows via the milking system. Spontaneous recovery has not been recorded and infected cows typically develop chronic mastitis with granulomatous infiltration and progressive loss of functional parenchyma of the mammary gland. Substantial economic losses and health damages associated with mammary protothecosis strongly emphasise the need for developing effective prevention strategies aimed at control of the infection.

Introduction: Bovine viral diarrhoea (BVD), caused by the bovine viral diarrhoea virus (BVDV), is one of the most important diseases of cattle worldwide. The purpose of the study was to determine the BVDV infection status in a dairy herd vaccinated against BVD. Before vaccination started in 2008, there had been no prior identification or the removal of the possible source of infection (persistently infected animals). It was expected that vaccination itself would enable the elimination of viral shedders on a long term basis. Material and Methods: Serological screening for antibodies against BVDV with determination for antibodies titres, BVDV antigen, and the presence of the viral genome with phylogenetic analysis of positive samples in the herd were performed, despite the lack of any clinical problems indicating possible presence of BVDV infection. Results: 19 individuals persistently infected with BVDV were identified among calves and heifers but not in adult cattle. All virus shedders were antibody negative and the genotype of isolated virus was BVDV-1b, indicating a single source of infection. The vaccine used in the herd was composed of BVDV-1a strain. In each of the tested cowsheds, antibody titres against BVDV-1b were higher than against BVDV-1a (median values). Conclusion: Despite a long-lasting vaccination programme and relatively high sequence homology of vaccinal and field strains of BVDV (83.6%), it was not possible to avoid transplacental infections of foetuses and the birth of persistently infected calves from vaccinated heifers although the protection against clinical disease was accomplished.

Introduction: Although HEV infection in pigs does not pose a major economic risk to pork production, the risk of zoonotic transmission to humans is an important aspect of public health. HEV genotype 3 infections were reported in developed countries in individuals who had consumed raw meat or meat products from deer, wild boars, or pigs. The aim of the study was the analysis of the occurrence of HEV-specific antibodies among wild boars and domestic pigs in Poland. Material and Methods: A total of 290 samples from wild boars and 143 samples from pigs were tested. The antibodies were tested by ELISA. Results: The presence of anti-HEV IgG was demonstrated in 44.1% of pigs and 31.0% of wild boars. Anti-HEV IgG antibodies were detected in 1.4% of samples from pigs and in 2.1% of samples from wild boars at borderline level. The statistical analysis shows significant differences in the positive results for anti-HEV IgG between the groups of pigs and wild boars (P = 0.0263). Conclusion: Regular surveillance of the occurrence of HEV in swine and wild boars should be performed in the future.

Introduction: Mycoplasma bovis is one of the main pathogens involved in cattle pneumonia. Other mycoplasmas have also been directly implicated in respiratory diseases in cattle. The prevalence of different Mycoplasma spp. in cattle affected by respiratory diseases and molecular characteristics of M. bovis field strains were evaluated. Material and Methods: In total, 713 nasal swabs from 73 cattle herds were tested. The uvrC gene fragment was amplified by PCR and PCR products were sequenced. PCR/DGGE and RAPD were performed. Results: It was found that 39 (5.5%) samples were positive for M. bovis in the PCR and six field strains had point nucleotide mutations. Additionally, the phylogenetic analysis of 20 M. bovis field strains tested with RAPD showed two distinct groups of M. bovis strains sharing only 3.8% similarity. PCR/DGGE analysis demonstrated the presence of bacteria belonging to the Mollicutes class in 79.1% of DNA isolates. The isolates were identified as: Mycoplasma bovirhinis, M. dispar, M. bovis, M. canis, M. arginini, M. canadense, M. bovoculi, M. alkalescens, and Ureaplasma diversum. Conclusion: Different Mycoplasma spp. strains play a crucial role in inducing respiratory diseases in cattle.

Introduction: Several Mycoplasma species can cause severe diseases in ruminant hosts, some of which are the diseases listed by the World Organisation for Animal Health (OIE). The role of the Cervidae family in carrying and transmitting ruminant mycoplasma infections in Poland is unknown. Material and Methods: Antibody and antigen detection tests for the main mycoplasma species that can affect wild ruminants were performed on 237 samples (serum, nasal swab, bronchoalveolar lavage, and lung) collected from 161 animals during 2011-2014. The samples were obtained from a cull of healthy population of deer which included: 96 red deer (Cervus elaphus elaphus), 19 fallow deer (Dama dama), and 46 roe deer (Capreolus capreolus). Results: Serological screening tests revealed positive reactions to Mycoplasma bovis in one sample and to Mycoplasma capricolum subsp. capripneumoniae in three samples; however, these three samples were negative by immunoblotting. Other antibody and antigen detection tests demonstrated negative results. Conclusion: Currently wild cervids in Poland do not play a significant role in transmitting mycoplasma infections to domestic animals, but they remain a potential risk.

Introduction: Chlamydia psittaci is a gram-negative obligate intracellular pathogen of birds. Poultry infections lead to economic losses and can be transmitted to humans. No vaccine is available and the bacterium-host cell interaction is not completely understood. Replicating bacteria cause pneumonia, but C. psittaci can also be non-replicating and persistent inside the cytoplasm of avian cells. RT-qPCR provides insight into the molecular pathogenesis of both active replicating and persistent Chlamydia psittaci in birds, but requires identification of stably expressed reference genes to avoid biases. Material and Methods: We investigated the expression stability of 10 C. psittaci candidate reference genes for gene expression analysis during normal growth and penicillin-induced persistence. C. psittaci Cal10 was cultured in HeLa229 and RNA was extracted. The expression level of each candidate was examined by RT-qPCR and Cq values were analysed using geNorm. Results: The genes tyrS, gidA, radA, and 16S rRNA ranked among the most stably expressed. The final selected reference genes differed according to the bacterial growth status (normal growth versus persistent status), and the time points selected during the duration of the normal chlamydial developmental cycle. Conclusion: The study data show the importance of systematic validation of reference genes to confirm their stability within the strains and under the conditions selected.

Introduction: The aim of the study was to optimise and compare two multiplex PCR assays for the detection of Listeria spp. and Listeria monocytogenes in biological samples including the liver, brain, and blood. Material and Methods: Three strains of L. monocytogenes and single strains of each of the species: L. ivanovii, L. innocua, L. grayi, L. welshimeri, and L. seeligeri were used. Additionally, five other species of bacterium were used to evaluate the specificity of the tests. Results: Specific amplification products were obtained for both multiplex PCR assays, which confirmed the tested strains as Listeria spp. and L. monocytogenes, respectively. Isolates of other species did not yield PCR products. Conclusion: Both multiplex PCR assays proved to be significantly sensitive and highly-specific methods for the detection of Listeria strains.

Introduction: Leptospirosis affects a wide range of mammals, humans, and even a few poikilothermic animal species. In Pakistan, serological studies of equine leptospirosis have reported a prevalence of over 40%, but no study has ever been conducted towards molecular detection of Leptospira in horses. Material and Methods: Blood samples from 128 horses were screened using ELISA and 41 positive samples were examined for the presence of leptospiral DNA using specific primers for 16S rRNA gene. Results: Out of 41 tested samples, 20 samples were found to be PCR-positive, revealing a fragment of 306 bp after gel electrophoresis. Sequencing and phylogenetic analysis of positive samples revealed circulation of pathogenic Leptospira spp. in Pakistani horses. No evidence of circulation of intermediate species was found in this study. Conclusion: This study reports the first molecular evidence of equine leptospirosis in Pakistan and lays ground for further research in this area. It also confirms the efficiency of 16S rRNA for the diagnosis of equine leptospirosis.

Introduction: The aim of this study was to carry out a genetic analysis of Babesia canis isolates detected in dogs in eastern Poland and to study the correlation of the protozoa variant with a specific geographical region. Material and Methods: PCR was used to identify strains of B. canis from naturally infected animals (240 dogs from four provinces: Mazowieckie, Lublin, Podlasie, and Podkarpacie) by amplifying and sequencing a fragment of the 18S rRNA gene. Results: Sequencing the PCR products led to the identification of four variants of B. canis. Two previously described protozoa variants (18S rRNA-A and 18S rRNA-B) were observed in all provinces. Additionally, in the Mazowieckie and Lublin provinces a B. canis variant which contributed to the development of acute or atypical babesiosis was observed. The fourth variant of B. canis was detected only in dogs from the Lublin province, and the course of the disease was subclinical in all dogs infected with this variant. Conclusion: These results indicate the appearance of a new fourth B. canis genotype in Poland and confirm that it is still necessary to study the relationships between the genetic structure of protozoa, geographical distribution of the parasites, and clinical course of the disease.

In recent years, Shewanella putrefaciens, commonly known as a halophilic bacteria, has been associated with serious health disorders in freshwater fish. Therefore, it has been described as a new aetiological agent of the disease, named shewanellosis. S. putrefaciens is a heterogeneous group of microorganisms, belonging to the Alteromonadaceae family. Based on different criteria, three biovars and biogroups as well as four genomic groups have been distinguished. The first infections of S. putrefaciens in fish were reported in rabbitfish (Siganus rivulatus) and European sea bass (Dicentrarchus labrax L.). Outbreaks in farmed fish were reported in Poland for the first time in 2004. The disease causes skin disorders and haemorrhages in internal organs. It should be noted that S. putrefaciens could also be associated with different infections in humans, such as skin and tissue infections, bacteraemia, otitis. Investigations on pathogenic mechanisms of S. putrefaciens infections are very limited. Enzymatic activity, cytotoxin secretion, adhesion ability, lipopolysaccharide (LPS), and the presence of siderophores are potential virulence factors of S. putrefaciens. Antimicrobial resistance of S. putrefaciens is different and depends on the isolates. In general, these bacteria are sensitive to antimicrobial drugs commonly used in aquaculture.

Introduction: The giant liver fluke, Fascioloides magna, has spread across Europe over the years posing a serious threat to the Polish cervid population. Material and Methods: Macroscopic and histopathological studies of the liver of 22 roe deer (Capreolus capreolus), 10 red deer (Cervus elaphus), and 6 fallow deer (Dama dama) were performed. Species determination of the recovered liver flukes and eggs was performed by PCR protocol amplifying fragments of ribosomal DNA (ITS2), according to a standard method. Results: The presence of F. magna was confirmed in three (13.6%) roe deer, seven (70.0%) red deer, and two (33.3%) fallow deer. The fluke eggs were found only in the stools of five red deer and one fallow deer. Conclusion: This study presents detailed pathological and histopathological changes in the liver of wild Polish cervids, including roe deer, which were subjected to such study for the first time. The hepatic lesions typical for different stages of liver cirrhosis varied depending on the host species and stage of the disease.

Introduction: Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) belonging to the clonal complex 398 (CC398) emerged recently in livestock as a new type of MRSA, which may cause zoonotic infections. This study presents data on the characterisation of S. aureus isolated from the meat processing plants. Material and Methods: S. aureus was isolated from 90 samples collected in the raw meat warehouse, from devices and surfaces of meat processing plants, and from finished meat products. The isolates were subjected to molecular analysis in order to investigate the presence of enterotoxin genes, the mecA gene, and to verify whether they belong to the clonal complex 398. The genetic relatedness of the isolates was determined using pulsed-field electrophoresis. Likewise, antimicrobial susceptibility was tested. Results: From 21 S. aureus strains isolated, five belonged to the CC398, two of which were recognised as MRSA and three as methicillin-sensitive Staphylococcus aureus (MSSA). The most prevalent enterotoxin genes were seg and sei. Two MRSA CC398 isolates, three MSSA CC398, and one MSSA were classified as multidrug-resistant. Conclusion: The first isolation of MSSA CC398 from beef in Poland indicates contamination of beef by strains belonging to this clonal complex. The occurrence of multidrug-resistant enterotoxigenic S. aureus isolates in the finished meat products constitutes a potential risk for the consumers.

Introduction: Growing consumption of shellfish is associated with an increased risk of food poisoning. The study was carried out on live bivalve molluscs available on the Polish market between 2009 and 2013. Material and Methods: ELISA was used for the determination of the following marine biotoxins: paralytic shellfish poison (PSP), amnaesic shellfish poison (ASP), and diarrhoeic shellfish poison (DSP). The molluscs, of which seven species were examined, were obtained from wholesale companies and markets. Results: Marine biotoxins were detected below the permitted levels in 67.6% of the samples. The maximum amounts of PSP and ASP biotoxins were found in great scallops (532.6 μg/kg and 1.0 mg/kg respectively) and the peak for DSP was in blue mussels (107 μg/kg). Conclusion: The analysis of toxicological status of raw bivalve molluscs available on the market in Poland indicates that they are safe for consumers.

The progressive growth and spread of tumour cells in the form of metastases requires an interaction of healthy host cells, such as endothelial cells, fibroblasts, and other cells of mesenchymal origin with immune cells taking part in innate and adaptive responses within the tumour lesion and entire body. The host cells interact with tumour cells to create a dynamic tumour microenvironment, in which healthy cells can both positively and negatively influence the growth and spread of the tumour. The balance of cellular homeostasis and the effect of substances they secrete on the tumour microenvironment determine whether the tumour has a tendency to grow or disappear, and whether the cells remain within the lesion or are capable of metastasis to other regions of the body. Intercellular interactions also determine the tumour’s susceptibility to radiation or other types of cancer treatment. They may also be a rational explanation for differences in treatment outcomes, in which some metastases regress and others progress in response to the same treatment method.

Introduction: The present study is a comprehensive overview of the natural occurrence of 17β-oestradiol and testosterone in serum of cattle in Poland. Material and Methods: The serum samples (n = 826) were collected from cattle within five years. The samples were examined for the presence of oestradiol and testosterone using ELISA or gas chromatography with mass spectrometry. Results: In 98 samples (24%) 17β-oestradiol was detected above decision limits of applied methods, including five samples over the recommended concentration of 0.1 μg L^-1. Of the serum samples taken from cows (≤18 months of age), 95 and 99 percentiles of the animals had 17β-oestradiol concentration below 0.027 and 0.086 μg L^-1 and of samples from cows over 18 months of age - below 0.059 and 0.125 μg L^-1 respectively. Calculated values for bulls (≤18 months of age) were 0.025 and 0.034 μg L^-1 and for the animals older than 18 months of age - 0.035 and 0.041 μg L^-1. The natural presence of testosterone was detected in 201 serum samples (48.7%). According to the obtained data, 95% and 99% of cows (≤18 months of age) serum samples had testosterone concentration below 0.05 and 0.23 μg L^-1 and the animals over 18 months of age - 0.30 and 0.49 μg L^-1, respectively. For bulls these values did not depend on the age of the animals and were in the ranges of 5 - 6.3 μg L^-1 (95%) and 11.4 - 12.1 μg L^-1 (99%). Conclusion: Our study showed that the threshold values for these hormones in plasma of cattle designated years ago are correct, but they need to be supplemented for animals older than 18 months.

Introduction: Hard antlers of deer are unique bioindicators of environmental metal pollutions, but sampling methods presented in the literature are inconsistent. Due to the specific growth pattern of antlers and their histological structure, sampling methods described in the literature were reviewed, the suitability of using mixed samples of both antler layers as element bioindicators was assessed, and the codified method of antler sampling used for bioindication was described. Material and Methods: Lead, cadmium, mercury, arsenic, copper, zinc, and iron in trabecular and cortical parts of hard antlers of red deer (Cervus elaphus) were determined using different methods of atomic absorption spectrometry (depending on the element). Results: Mean mercury content in trabecular bone (0.010 ±0.018 mg/kg) was 5 times higher than in cortical bone (0.002 ±0.003 mg/kg). Mean iron concentration was approximately 15 times higher in trabecular (239.83 ±130.15 mg/kg) than in cortical bone (16.17 ±16.44 mg/kg). Concentrations of other analysed elements did not differ statistically between antler layers. Conclusion: In mixed antler samples, concentrations of mercury and iron depend on the particular antler layer contents. This therefore warrants caution when comparing results across studies and specification of the sampling methodology of antlers is highly recommended.

The aim of this article was to evaluate the influence and effects of chosen bioaccumulative substances i.e. heavy metals, pesticides, and polychlorinated biphenyls (PCBs) on fish, as well as provide information on time trends and potential threat to human health. Chemical substances which pollute water may affect living organisms in two ways. First of all, large amounts of chemical substances may cause sudden death of a significant part of the population of farmed fish, without symptoms (i.e. during breakdown of factories or industrial sewage leaks). However, more frequently, chemical substances accumulate in tissues of living organisms affecting them chronically. Heavy metals, pesticides, and polychlorinated biphenyls are persistent substances with a long-lasting biodegradation process. In a water environment they usually accumulate in sediments, which makes them resistant to biodegradation processes induced by, e.g., the UV light. These substances enter the fish through direct consumption of contaminated water or by contact with skin and gills. Symptoms of intoxication with heavy metals, pesticides, and PCBs may vary and depend on the concentration and bioavailability of these substances, physicochemical parameters of water, and the fish itself.

Introduction: The aim of the study was to evaluate the effect of administration of therapeutic doses of ceftiofur and tulathromycin on the circulating lymphocyte subpopulations in healthy pigs. Material and Methods: The study was conducted on thirty healthy 7- to 10-week-old pigs, assigned to three groups: the TUL group, injected with tulathromycin (n = 10); the CEF group, injected with ceftiofur (n = 10); and the C group, the control with no antibiotic administration (n = 10). Blood samples were collected before, during, and after treatment with antimicrobials. Lymphocyte subpopulations circulating in the blood were determined by immunostaining and flow cytometry analyses. Results: Following administration of a therapeutic dose of tulathromycin, there were no changes in the lymphocyte subpopulations circulating in blood. In contrast, administration of ceftiofur at the recommended dose decreased the absolute number of CD3+, CD21+, CD4+CD8-, CD4-CD8+, and double positive CD4CD8 cells. Conclusion: Results from the study indicate that ceftiofur possesses the ability to modulate the immune system in healthy pigs by decreasing lymphocyte subpopulations circulating in blood.

Introduction: Thyroid hormones affect protein turnover, and in the case of hypothyroidism a decrease in protein synthesis and reduced release of certain amino acids from skeletal muscles are observed. Changes in the amino acid system of skeletal muscles may be responsible for the occurrence of muscle disorders. Material and Methods: The study measured the content of selected amino acids in the gastrocnemius muscle of Wistar rats during experimental hypothyroidism induced by oral administration of methimazole at a concentration of 0.05% in drinking water for 90 d. The rats were divided into four groups: E1 (n = 6) - experimental males, E2 (n = 6) - experimental females, C1 (n = 6) - control males, and C2 (n = 6) control females. Results: A statistically significant reduction occurred in leucine, isoleucine, and 1-methylhistidine levels in males, and 1-methylhistidine in females, in comparison to the control groups. Conclusion: The hypothyroidism-induced changes in amino acid content may be responsible for the occurrence of skeletal muscle function disorders.

Introduction: The cytotoxicity of anthelmintic agent, albendazole (ABZ) and its two major metabolites, sulfoxide (ABZSO) and sulfone (ABZ-SO₂), on non-hepatic Balb/c 3T3 line, two hepatoma cell lines (FaO, HepG2), and isolated rat hepatocytes was investigated. Material and Methods: Cell cultures were exposed for 24, 48, and 72 h to eight concentrations of the compounds ranging from 0.05 to 100 μg/mL (ABZ) and from 0.78 to 100 μg/mL (ABZ-SO and ABZ-SO₂). Three different assays were applied in which various biochemical endpoints were assessed: lysosomal activity - neutral red uptake (NRU) assay, proliferation - total protein contents (TPC) assay and lactate dehydrogenase (LDH) leakage assay. Results: The most toxic was albendazole whose EC₅₀ values calculated from the concentration effect curves ranged from 0.2 to 0.5 μg/mL (Balb/c 3T3 ) and from 0.4 to 73.3 μg/mL (HepG2). Rat hepatoma line and isolated rat hepatocytes were less sensitive to the impact of ABZ. Toxic action expressed as EC₅₀ was recorded after 72 h exposure only in LDH release assay at 0.8 μg/mL and 9.7 μg/mL respectively. The toxicity of metabolites was much lower. The most sensitive to ABZ-SO were fibroblasts and EC_50-72h values were similar in all three assays used, i.e. NRU (14.1 μg/mL), TPC (15.8 μg/mL), and LDH (20.9 μg/mL). In the case of ABZ-SO₂ the mean effective concentrations were the highest, and could be reached only in one LDH assay. These values (μg/mL) were as follows: 65.3 (FaO), 65.4 (HepG2), 75.8 (hepatocytes), and 77.4 (Balb/c 3T3). Conclusion: The differences in in vitro toxicity of albendazole depend on metabolic ability of the cellular models. Primary cultured rat hepatocytes represent a valuable tool to study the impact of biotransformation on the cytotoxicity of drugs.

Electrophoretic techniques, used to separate mixtures of electrically charged particles, are widely used in science. One of these techniques, native protein electrophoresis in an agarose gel, is applied in human and veterinary medicine. Changes in the proportions of individual protein fractions correspond to significant changes in the physiology of the body. Although the pattern obtained by electrophoretic separation rarely indicates a specific disease, it provides valuable information for the differential diagnosis. Decades of research on the types of patterns obtained in the case of particular diseases have led to the accumulation of substantial knowledge. The paper presents the available information on this topic. Serum protein electrophoresis is recommended in cases of increased levels of total protein in order to reveal the nature of the process. The basic information which can be obtained from electrophoretic separation includes the immune status of the organism. Both increased antigenic stimulation and immunodeficiency are clearly visible in electropherograms. Moreover, the level of heterogeneity of the corresponding protein fractions can help to distinguish between infectious diseases and cancer - multiple myeloma - the latter producing a homogeneous immunoglobulin fraction. Analysis of other protein fractions helps to detect or confirm an ongoing inflammatory process and provides information regarding liver function. Even when the concentration of total protein is within the reference range, this analysis can be recommended as a basic laboratory test.