Volume 58 (2014): Issue 4 (December 2014)

Chlamydiales, one of the oldest bacterial orders in evolutionary terms, are widespread among animals. Blinding trachoma, a disease caused by Chlamydia trachomatis, was already known in ancient times, whereas modern reports on psittacosis date from 1879. Though these pathogens have long been known and lead to serious health problems both in human and animals, data on Chlamydiales biology has been limited. It is due to their intracellular life style and complex developmental cycle. New molecular biological methods have been recently developed expanding the possibilities of chlamydial research and diagnosis. This paper reviews data concerning avian chlamydiosis, its aetiological agent C. psittaci, newly proposed species isolated from birds, namely C. ibidis sp. nov., C. avium sp. nov., and C. gallinacea sp. nov., and their zoonotic potential.

The aim of this study was to investigate the presence of proviral DNA and colostral antibodies in lambs born to and fed by ewes infected with small ruminant lentiviruses (SRLV). It was demonstrated that all 20 lambs tested 24 h after colostrum ingestion were serologically positive with high antibody titres. These gradually decreased with time, and at week 12 all lambs were seronegative. Twenty percent of lambs tested at the 2nd week postpartum were provirus positive by qPCR as a result of consumption of infected colostrum or in utero infection. When tested at three months of life, 95% of the lambs were provirus positive, probably as a result of horizontal transmission of the virus. Since these animals could play an important role in the early propagation of SRLV to susceptible herdmates, early removal of provirus-positive animals could help to prevent new infections.

The aim of the study was to determine the aetiologic agent causing deaths in two flocks of Pekin ducklings at the age of 12 d in Poland. on the basis of clinical symptoms and pathological changes, viral hepatitis infection was suspected in the birds. During the necropsy, liver sections were collected, from which total cellular RNA was isolated. Then, real-time polymerase chain reaction (RT-PCR) was performed using primers complementary to the pre-S region of the duck hepatitis virus genome. In all liver samples, the presence of a 530 bp PCR product was detected. The RT-PCR demonstrated the presence of genetic material of duck hepatitis virus type 1 (DH type 1) in the examined ducklings.

The article presents data on histopathological studies of the kidneys of cows, which either recovered or died from leptospirosis. Fragments of seven kidneys from slaughtered cows, positive for Leptospira antibodies in the microscopic agglutination test (MAT) (titres of 50 and higher) were used in the study. The MAT was conducted with eight serological groups of Leptospira: Canicola, Grippotyphosa, Hebdomadis, Icterohaemorrhagiae, Pomona, Sejroe, Tarassovi, and Australis. Microscopic changes in all morphological structures of the kidneys were presented. Micromorphological criteria, which can be used for post-mortem diagnosis of leptospirosis were established. They included: serous glomerulonephritis with granular dystrophy of podocytes, necrosis and collapse of the inner layer of Bowman's capsule, partial destruction of capsule and vascular glomeruli, granular and vacuolar degeneration and destruction of tubular epithelial cells, foci of interstitial oedema, and infiltrations predominantly with monocytes and isolated neutrophils. Microscopic changes in the kidneys suggest that the cows died from leptospirosis as a result of toxic shock syndrome.

The aim of this study was to confirm the presence of leptospires from the Sejroe serogroup in aborting seropositive sows and to identify which serovars of this serogroup are responsible for the infection. Serum and urine samples from 20 aborting sows, reared in five farms infected with the Sejroe serogroup, were submitted for examination. Serum samples were examined by the microscopic agglutination test (MAT) and urine samples by polymerase chain reaction (PCR) with primer sets specific for genus Leptospira, species L. borgpetersenii, the Sejroe serogroup, and the Hardjo-bovis genotype. All the examined serum samples showed titres specific to the Sejroe serogroup, ranging from 100 to 6400. PCR indicated the presence of Leptospira genus-specific DNA sequences in all urine samples examined. DNA sequences specific for both L. borgpetersenii and the Sejroe serogroup were found in 12 of these samples. Additionally, the sequences specific only for L. borgpetersenii were found in five urine samples and sequences for the Sejroe serogroup in two samples. The DNA sequence specific for the Hardjo-bovis genotype was not found in the urine samples. PCR confirmed the results of the MAT, displayed the presence of Leptospira sp. DNA in the urine of all the sows examined, proved the affinity of the detected leptospires to the Sejroe serogroup or at least to L. borgpetersenii, and excluded the Hardjo serovar as the reason for the infections described in the sows.

The study was conducted to detect Brucella sp. and Leptospira sp. in blood samples of dogs in Isfahan and Shahrekord province in Iran. A total of 94 blood samples were collected from dogs of different breed, age, sex, and dogs’ type (stray or nonstray). The samples were examined using conventional polymerase chain reaction (PCR). Fourteen (14.89%) dogs were positive for Brucella sp. and 18 (19.15%). dogs for Leptospira sp. There were no significant differences between the prevalence of the pathogens, provinces, sex, and age groups (P > 0.05). However, there was a statistically significant difference in prevalence of Brucella sp. and Leptospira sp. between stray and non-stray dogs (P < 0.0001; χ2 = 30.3767). The study also demonstrated that PCR was successfully used for the first time in Iran for the detection of Brucella sp. and Leptospira sp. in blood samples of dogs. Therefore, we recommend the PCR as a supplementary method with other commonly recognised methods (e.g. serological methods) for the diagnosis of subclinical infections with the microorganisms. Strict measures for the control of stray dogs are also highly recommended.

The aim of the study was to develop and validate a real-time PCR method, using a TaqMan probe, for quantification of Mycoplasma suis in porcine blood. No PCR signals with closely related non-haemotrophic mycoplasmas were obtained. The detection limit of PCR for plasmid combined with blood DNA was determined to be 103/reaction (5 μL of DNA) (1.2x10⁵ target copies in 1 mL of blood). The linearity of real-time PCR (near 1) indicates its use as a quantitative method. Real-time and quantitative PCR were sensitive and specific for the detection and quantification of M. suis in the blood of animals with acute and chronic form of eperythrozoonosis. Developed quantitative PCR cannot be used to detect carrier animals with a small amount of M. suis in their blood. The validity of real-time PCR used in the studies was confirmed by the low inter- and intra-assay coefficients of variation. This fact confirms the applicability of the assay in other laboratories.

The paper concerns molecular study on pathogenicity markers of fourteen Y. enterocolitica O:9 isolated from pigs in which initially positive serological reactions for brucellosis were observed (n = 41), healthy pigs, which were brucellosis-negative (n = 258), and wild boars serologically negative for brucellosis (n = 209). PCR identification proved that all isolates were ail, ystA- and myfA-positive. The plasmid encoding yadA marker was detected in nine isolates that originated from pigs serologically positive or negative for brucellosis, and from one isolate of wild boar origin. Furthermore, none of the examined isolates was ystB-positive. Results of the investigations indicate that the Y. enterocolitica O:9 isolates from pigs or wild boars, regardless of whether they were serologically positive or negative for brucellosis, may also be potentially pathogenic for humans, due to the presence of chromosomal and plasmid encoded molecular markers.

The aim of the study was to estimate the prevalence of gastrointestinal parasites in raccoons with particular regard to zoonotic parasites. Fifty-five raccoons, hunted or found dead on roads, were examined. The small and large intestines were collected from all raccoons and, additionally, the stomach was collected from 43 animals. The samples were examined with the use of sedimentation and counting technique. The intestines and stomach were examined separately. Samples of raccoon faeces were collected from their environment localised in Słubice district, Lubuskie province (Poland). The samples were collected once a month in 2012. In total, 154 faecal samples were obtained and examined with the use of McMaster flotation technique. The following parasites were detected in the intestinal and stomach contents: tapeworms Mesocestoides sp. (67.3%), Echinostomatidae flukes (34.5%), and nematodes Capillaria sp. (25.5%). Moreover, Acanthocephala were found in the intestines of three raccoons. The highest intensity of infection were observed in case of Mesocestoides sp. Mesocestoides sp. and Echinostomatidae were found statistically more often in the intestines than in the stomach. In the case of these two parasites, there was positive correlation between the intensity of infection in the intestines and the presence of the same parasites in the stomach. Moreover, significantly higher prevalence and intensity of Mesocestoides sp. in males than in females were also observed. Faecal samples contained Baylisascaris procyonis eggs (mean 60 epg). These eggs were found in three samples collected in November and December. Furthermore, in some faecal samples eggs of flukes, Capillaria sp., Mesocystoides sp., and coccidian oocysts were found. It is one of rare reports concerning Baylisascaris procyonis in Poland confirming the presence of this dangerous parasite in Polish raccoon population.

A total of 2668 swabs from poultry (n = 2166), pig (n = 311), and cattle (n = 191) carcasses were collected in slaughterhouses all over Poland and tested for the presence of Campylobacter. It was found that 1319 (49.4%) of them were contaminated with these bacteria. The percentages of the positive samples were different in each year of the study and the highest proportion of Campylobacter contaminated samples occurred in 2009, when 64.1% of investigated carcasses were positive. On the other hand, the lowest prevalence of Campylobacter was observed in 2013, in the last year of the survey. In all kind of carcass samples both C. jejuni and C. coli were identified, although the pork meat was more contaminated with C. coli (75.3% of positive samples) than with C. jejuni (24.7%), whereas poultry was nearly equally positive for C. jejuni and C. coli (50.6% and 49.4% respectively). The analysis of seasonal contamination of the carcasses revealed that more positive results were found during the second half of year than between January and June. The prevalence of Campylobacter showed that in all provinces, except one (Pomorskie), the mean percentage of the positive samples was above 40%. The most contaminated samples were identified in Lubelskie (69.3%) and Zachodniopomorskie (66.3%) regions. The obtained results showed that slaughtered animals in Poland, especially broilers, were often contaminated with Campylobacter, either C. jejuni or C. coli.

The aim of the study was to investigate if the enterotoxigenic strains of S. aureus isolated from raw milk are able to produce staphylococcal enterotoxins (SEs) A - E. A total of 168 of S. aureus isolates from raw milk collected in the south - east region of Poland (Lubelskie Province) were tested for SE production by the ELFA, while multiplex PCR was applied for detection of enterotoxin genes (sea, seb, sec, sed, see). It was found that 20 (11.9%) out of 168 strains were positive for one or more classical SE markers and 19 of them produced a detectable level of enterotoxins. The results obtained by mPCR and ELFA were in agreement, when the presence of A, B, and D toxin types was tested; whereas SEC was not found by the ELFA although the S. aureus was positive for the respective gene. The results of the two methods showed that mPCR identified one more strain potentially producing enterotoxin than the ELFA, which may suggest that the enterotoxigenic S. aureus are not always able to express the toxin protein.

Eighty-nine isolates of coagulase-negative staphylococci (CoNS) of eight species from subclinical bovine mastitis were screened for the phenotypic and genotypic methicilline-resistance. In addition, all methicillin-resistant (MR) isolates indicating the mecA gene were examined by PCR for the antimicrobial susceptibility patterns, and staphylococcal cassette chromosome mec (SCCmec) types were also determined by multiplex PCR. A total of 21 (23.6%) CoNS isolates were found to be resistant to oxacillin in broth microdilution assay. All isolates phenotypically resistant to oxacillin did not have the mecA gene, which was only found in 14.6% (13) of the isolates. Most MR-CoNS isolates were highly resistant to erythromycin (92.3%), fusidic acid (84.6%), penicillin (76.9%), and rifampycin (61.5%), and susceptible to mupirocin (100%), tetracycline (100%), vancomycin (100%), clindamycin (92.3%), and sulfamethoxazole-trimethoprim (69.2%). In conclusion, a high rate of antimicrobial resistance among MR-CoNS isolated from food producing animals emphasises the need for periodic surveillance of their resistance.

In the present study, 25 Escherichia coli strains isolated from beef, pork, and poultry meat, and producing extendedspectrum β-lactamases (ESBL) (18 strains) or AmpC- cephalosporinases (7 strains) were tested for antimicrobial resistance using the minimum inhibitory concentration method with 16 antimicrobial agents. All examined strains were resistant to ampicillin and the first-generation cephalosporins. Variable resistance to the third-generation cephalosporins (40%-100% among ESBLproducing strains and 0-72% among AmpC-producing strains) was noted. Less than 30% of examined strains were resistant to ciprofloxacin. All isolates were susceptible to the fourth-generation cephalosporins, cephalosporins connected with inhibitors of β-lactamases, carbapenems, and gentamycin

For the purpose of quantitative determination of doxycycline (DC) residues in tissues, a sensitive liquid chromatography - tandem mass spectrometry (LC-MS/MS) method was developed. The method was used to determine DC residues in chicken tissues (breast and thigh muscle, liver and kidney) after oral administration with drinking water to five-weak-old broiler chickens. The DC was administered for five consecutive days at a therapeutic dose of 10 mg/kg b.w. once a day. The tissues were collected after 6 h, 24 h, 7 d, and 8 d. The method was validated and the decision limit was established for muscle - 109.2 μg/kg, for liver - 326.1 μg/kg, and for kidney - 634.0 μg/kg. The detection limit was 2 μg/kg and the limit of quantification was 5 μg/kg. In a short period after ceasing the treatment, the detected concentrations of DC were much higher than the established maximum residue limit values. The highest residue concentrations of DC were observed in the kidney, followed by the liver and muscle. The lowest concentration of DC was determined in tight muscle.

A multiresidue method for the determination of seven nitroimidazoles and their hydroxy metabolites in milk was developed. Milk samples were extracted with acetonitrile and cleaned up on strong cation-exchange solid phase extraction cartridges. Evaporated to dryness first, the extracts obtained were then reconstituted in 0.1% formic acid and injected onto a liquid chromatography-tandem mass spectrometer (LC-MS/MS). The separation of analytes was achieved on gradient mode using a C18 column and a mobile phase consisting of 0.1% formic acid in acetonitrile and 0.1% formic acid in water. Multiple reaction monitoring mode was set for the MS/MS with positive electrospray ionisation. For quantification, the isotope dilution method was used with isotopically labeled analogues of the target analytes. The method was evaluated entirely in accordance with EU Commission Decision 2002/657/EC and all validation criteria were in the required ranges. The method can easily detect and confirm metronidazole, dimetridazole, ronidazole, ipronidazole, and their hydroxy metabolites below the recommended concentration level of 3 μg/kg. The decision limits and detection capabilities ranged from 0.11 μg/kg to 0.22 μg/kg and from 0.19 μg/kg to 0.37 μg/kg respectively. The overall recoveries were between 96.6% and 105.2% with a good coefficient of variation, less than 8.7% under within-laboratory reproducibility conditions.

Samples for analysis were collected from 10 areas, including the major Polish rivers and lakes, with different sources of environmental pollution (industrial, municipal, and farming). The materials was taken from the lakes of Mazury, located in a non-industrialised region, from the Brda River, an area impacted by pig farms, from the lakes of Lipczyno Wielkie/Pomerania, from the Wkra River, an area impacted by poultry farms, from the Dunajec River at the Roznowski Reservoir, from the Vistula River at Cracow and Warsaw, from the Odra River at Wroclaw and the Warta River estuary, and also from Rybnik Power Station Reservoir. Concentrations of Pb, Cd, Hg, and As were analysed in 397 fish muscle and 128 sediment samples using an atomic absorption spectrometry technique. The analytical procedures were covered by a quality assurance programme. It was demonstrated that the average concentrations of lead, cadmium, and arsenic in fish were in the low hundredths and thousandths of a mg/kg and never exceeded permitted limits established for food. Higher values of these elements were found in fish from bodies of water located in the zone of influence of large urban agglomerations, especially the Cracow region. High concentrations of lead and cadmium were also found in Vistula River sediments near Cracow, where the maximum values were 134.10 mg/kg and 21.24 mg/kg dry weight for lead and cadmium respectively. The average concentration of mercury in a predatory fish muscle (0.179 mg/kg) was almost twice as high as in the omnivorous fish (0.103 mg/kg). Only a single fish sample exceeded the maximum limit for this metal (0.50 mg/kg) and did not present a risk to consumers’ health.

The aim of the study was to determine the content of chlorinated hydrocarbon residues in fat of different assortment of rainbow trout (Oncorhynchus mykiss). The research material consisted of 108 fish from six different producers. Thirty-six fish were analysed as fresh, 36 fish as frozen after six months storage, and 36 fish as traditionally smoked. Chromatographic determination of γ-HCH, DDT, DDD, and DDE was performed with an Agilent Technologies 6890N. The presence of the compounds was detected in all tested fat samples. The content of γ-HCH was five-fold higher in frozen fish (average 2.11 ng/g of fat) than in fresh and smoked fish (0.42 ng/g of fat and 0.43 ng/g of fat, respectively). Total DDT (ΣDDT) content was found higher after refrigerated storage but did not exceed acceptable levels, indicating that the fat in the meat of rainbow trout is an attractive nutritional compound with a low concentration of tested chemical pollutants.

A simple and sensitive gas chromatography method was developed to determine a group of oestrogens in surface water. In the first stage of analysis, enzymatic hydrolysis of oestrogen metabolites with glucuronidase AS-HP was performed. Free compounds were extracted from 200 mL of water sample on C18 SPE column (6 mL, 1000 mg). The evaporated extract was subjected to derivatisation with a mixture of MSTFA/NH4I/DTT (1000:2:5, v/w/w). The separation of the analytes on HP-5ms capillary column was conducted. The method was validated according to the Commission Decision 2002/657/EC. Recovery in spiked samples ranged from 90% to 120 % with standard deviation lower than 30% for all examined compounds. The decision limit and detection capability of five oestrogens were in the range of 0.3-0.6 ng L^-1 and 0.5-0.9 ng L^-1, respectively. Nineteen water samples collected from different sites of several Polish rivers and lakes were tested for the presence of oestrogens. Some target compounds such as 17α-oestradiol, 17β-oestradiol, oestrone, oestriol, and 17α-ethynyloestradiol were found in trace amounts in the analysed samples. The highest concentration observed for oestradiol reached 23 ng L^-1.

The aim of the study was to test and compare the cytotoxic potential of two synthetic oestrogens: diethylstilboestrol (DES) and ethinyloestradiol (EE₂) and two androgens: testosterone propionate (TP) and trenbolone (TREN) on two cell lines. The fibroblast cell line Balb/c 3T3 and the hepatoma cell line HepG2 were selected. To get more insight into the mode of toxic action, four methods were used, which evaluated different biochemical endpoints: mitochondrial activity (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide reduction assay), lysosomal activity (neutral red uptake assay), total protein content, and lactate dehydrogenase release. Cytotoxicity was assessed after 24, 48, and 72 h exposure to eight concentrations ranging from 0.78 to 100 μg/mL. Concentration- and time- dependent effects were observed. Depending on the line and assay used, half maximal effective concentration after 72 h (EC_50-72h) values ranged as follows: DES 1-13.7 μg/mL (Balb/c 3T3) and 3.7-5.2 μg/mL (HepG2); EE₂ 2.1-14.3 μg/mL (Balb/c 3T3) and 1.8-7.8 μg/mL (HepG2); TP-14.9-17.5 μg/mL (Balb/c 3T3), and 63.9- 100 μg/mL (HepG2); and TREN 11.3-31.4 μg/mL (Balb/c 3T3) and 12.5-59.4 μg/mL (HepG2). The results revealed that oestrogens were more toxic than androgens and the most affected endpoint was mitochondrial activity. In contrast to oestrogens, for which EC_50-72h values were similar in both lines and by all assays used, Balb/c 3T3 cells were more sensitive than HepG2 cells to TP.

A liquid chromatography-ultraviolet detection method for the determination of florfenicol (FF) and thiamphenicol (TAP) in feeds is presented. The method comprises the extraction of analytes from the matrix with a mixture of methanol and acetonitrile, drying of the extract, and its dissolution in phosphate buffer. The analysis was performed with a gradient programme of the mobile phase composed of acetonitrile and buffer (pH = 7.3) on a Zorbax Eclipse Plus C18 (150 × 4.6 mm, 5 μm) analytical column with UV (λ = 220 nm) detection. The analytical procedure has been successfully adopted and validated for quantitative determination of florfenicol and thiamphenicol in feed samples. Sensitivity, specificity, linearity, repeatability, and intralaboratory reproducibility were included in the validation. The mean recovery of amphenicols was 93.5% within the working range of 50-4000 mg/kg. Simultaneous determination of chloramphenicol, which is banned in the feed, was also included within the same procedure of FF and TAP stability studies. Storing the medicated feed at room temperature for up to one month decreased concentration in the investigated drugs even by 45%. These findings are relevant to successful provision of therapy to animals.

The aim of this study was to identify mutations in the D-loop region of mtDNA in head, neck, and limb tumours in dogs, and determination of their relationship with the process of neoplastic transformation. Blood and tumour tissue samples from 19 dogs with diagnosed sporadic malignant tumours were analysed. DNA extraction, amplification, and sequencing of the mtDNA D-loop, and bioinformatic analyses were performed. Five mutations and 19 polymorphisms were observed in 68.42% of all tumours. Polymorphic variants were noted in 42.86% of the head and neck tumours and in 58.33% of the limb tumours. Mutations were observed in 21.05% of dogs. The mutations were found in 28.57% of the head and neck tumours and in 16.66% of the limb tumours. The mutations were identified in 50% of the studied epithelial cancers. In the mesenchymal tumours, no mutations in the D-loop region were observed. Mitochondrial haplotype A17 was found in over 40% cases of limb tumours. No association between the age, breed, sex, type of tumour, and detected polymorphic variants were observed. Different mutational changes in the D-loop sequences of mtDNA identified in the blood and tumour tissues may indicate a relationship between the type of tumour and individual changes in the D-loop nucleotide sequences of mtDNA.

The study was an attempt to determine the possibilities of using ovocystatin, a component of a new generation product of natural origin, in local therapy of atopic dermatitis in dogs by suppressing pruritus during illness. Chicken egg cystatin was used locally in the interdigital spaces of forelimbs of dogs used in the experiment. The degree of pruritus and clinical changes in the animals were defined using CADESI-03 scale before and after the beginning of the experiment. The results obtained proved that ovocystatin may be used as a substance suppressing pruritus in atopic dermatitis.

The aim of the study was to demonstrate the orthodontic treatment of malocclusions in dogs, a condition which can lead to cranio-mandibular and functional disorders of the stomatognathic system. The treatment involved the use of maxillofacialorthopaedic appliances, which type depended on the type of disorder and the degree of malocclusion. The applied treatment induced changes in the alveolar bone. Throughout the process of the treatment a great attention was paid to regular brushing off the orthodontic appliance using antiseptics for prophylactic prevention of inflammation of gingival tissue and the palate caused by food getting stuck in the spaces between the teeth.

Retrograde neuronal tracing, using fast blue, in combination with a single-labelling immunofluorescence technique, was applied to determine whether somatostatin (SOM) participates in sensory innervating of the porcine adrenal glands in physiological conditions and after adrenalectomy. In control animals, SOM-like immunoreactive neurons comprised 7.0 ± 0.7% of adrenal gland-projecting cells in dorsal root ganglia (DRG) at neuromeres Th6-7 and 6.5 ± 1.2% at neuromeres Th12-14. After adrenalectomy the percentage of SOM-positive DRG cells considerably increased and attained the level of 44.7 ± 2.5% at neuromeres Th6-7 and 36.6 ± 1.7% at neuromeres Th12-14. The obtained results demonstrate that SOM is not only a neuromediator within sensory neurones supplying the porcine adrenal glands, but also suggest the role of this substance during repairing processes within the nervous system after adrenalectomy.

The aim of the study was to investigate the nephrotoxic effects of gentamicin in adult male sheep, and to identify the earliest signs of toxicity and the extent of clinical and biochemical changes. Twenty clinically healthy yearling male Iranian fattailed sheep were injected with gentamicin sulfate at a daily dose of 80 mg/kg for 9-10 d when nephrotoxicosis was induced. Blood samples were collected weekly before and after induction of nephrotoxicosis. Gentamicin-induced nephrotoxicity was characterised by increased creatinine and urea levels in serum, electrolyte imbalances, occurrence of albuminuria, and renal dysfunction. Significant elevation in respiratory and heart rates were observed one week after treatment (P < 0.05). There was a noticeable increase in water consumption, lethargy, and loss of appetite in treated sheep. There were significant correlations between serum creatinine and potassium (P = 0.004, r = 0.759), sodium (P = 0.017, r = 0.501), and urea (P = 0.021, r = 0.617) levels. Additionally, significant negative correlations between serum total protein and albumin and creatinine (P = 0.023, r = -0.484) and urea (P = 0.036, r = -0.381) were found. At necropsy, the kidneys were pale, swollen, and wet on the cut surface, especially perirenal tissues and ureters were oedematous. These findings confirmed the previous reports in other species.