Volumen 57 (2013): Edición 4 (December 2013)

The Forkhead genes (transcription complex Fox) play many important roles in the maintenance and determination of biological processes underlying carbon-based life. The expression of Fox genes occurring as a result of reciprocal interactions at the transcriptional level influences the correct function of the immune system as regards the context of general activity of immunological parameters, as well as exposure to aetiological agents. In the case of model organisms and species, in which the knowledge about genomic sequences is incomplete, understanding the above-mentioned transcriptional complex is still insufficient, despite the existence of numerous scientific publications. It is worth noting that Fox genes exist in amphioxus Branchiostoma lanceolatum and in the majority of species they were not characterised. Among many coding sequences included in the transcriptional complex Forkhead, Foxp3, Foxn1, and Foxj1 genes are of a great importance from immunological point of view, being partially jointly responsible for determining features related to productivity. The paper provides general characterisation of selected Fox genes and is an introduction to the presentation of new data, which can be applied at the level of biological differentiation of the populations of domestic and wild animals.

Between 2008 and 2011, commercial turkey flocks in Poland were examined for the presence of rotaviruses. Ten faecal swabs from each of 207 turkey flocks (turkeys aged one to 19 weeks) were collected in different regions of the country and tested using a PCR assay that targeted the NSP4 gene. The prevalence of rotavirus was 20.3% in the flocks tested. Phylogenetic analysis revealed a clear division into groups dependent on geographical origin of the analysed viruses. All Polish rotaviruses belonged to the European group. However, they were found to be genetically variable based on the sequence analysis. The most frequently identified rotaviruses belonged to RV-1 subgroup and two of them formed a distinct subgroup of RV-2. Rotaviruses were detected in healthy and enteric turkeys. The observed amino acid changes probably did not affect the group affiliation, nor the pathogenecity of the studied rotavirus strains.

The aim of the study was to determine the effect of field strain, serotype 7 (FAdV-7 JN-5/10), of fowl adenovirus infection on the replication of Rispens/CVI988 strain of Marek’s disease virus. Ninety one-day-old SPF chickens were divided into six groups. The chickens from group I were vaccinated against Marek’s disease (MD) and 24 h later infected with the adenovirus; chickens from group II were vaccinated and infected simultaneously; chickens from group III were infected with the adenovirus and 24 h after the infection vaccinated against MD. The chickens from groups IV-VI were: control of infection (IV), control of vaccination (V), and neither vaccinated nor infected (VI). After 3, 7, 14, 21, and 28 d post infection, the number of copies of pp38 gene of Rispens strain and hexon gene of FAdV strain was determined in the bursa of Fabricius and liver using real-time PCR. The results indicated that in all cases the replication of Rispens strain was reduced to about 1.0 log10 - 3.5 log10 in chickens infected with the adenovirus and vaccinated against MD compared with the chickens only vaccinated. Sixty-three one-day-old SPF chickens infected with adenovirus and vaccinated against MD were challenged with vv MD virus field strain. The protection index in this experiment was 55.6%-77.8%.

An epidemiological study was conducted to identify risk factors related to small ruminant lentivirus (SRLV) infection in the central region of Spain. Between October 1998 and October 2000, a total of 194 sheep from 10 flocks and 163 goats from three flocks were tested for SRLV antibodies, resulting in 65.5% and 8.0% of seroprevalence, respectively. The relationship between differences in prevalence of SRLV, geographical location of the flock, and possible factors related to the flock that could enhance transmission were studied. Results of multivariable analysis showed an association between SRLV infection and geographical location of the flock and the rearing system. In addition, the differences in the productivity between infected and non-infected animals were explored. The productivity parameters were measured in 62 sheep and 28 goats. All productivity parameters studied (milk production, number of milking days, and lambing rate) appeared to be reduced in the SRLV-seropositive group in both goats and sheep. Even though, these differences were not statistically significant, it seems that animals infected are less productive than these non-infected. Statistical analyses comparing infected and non-infected sheep showed no statistical relationship between SRLV infection and milk quality.

Shedding time of bovine respiratory syncytial virus (BRSV) and bovine parainfluenza virus 3 (BPIV3) in calves vaccinated intranasally with modified live Rispoval RS-PI3 vaccine was determined. Blood and nasal swabs were collected on selected days before and after vaccination. Antibodies against BRSV and BPIV3 were tested by Respiratory ELISA Pentakit and the viral RNA was detected by RT-PCR. Twenty eight days after administration of the vaccine, a marked increase of specific antibody titres to BRSV and BPIV3 was detected in vaccinated calves. All animals were RT-PCR positive both for BRSV and BPIV3. Both viruses were excreted with nasal discharges within 8 d after vaccination but the course of shedding in individual calves was variable.

The aim of the study was to evaluate the susceptibility of algae of the genus Prototheca to antifungal and antibacterial antibiotics. The study involved 27 isolates of Prototheca zopfii obtained from milk of mastitis-affected cows kept in a detached cowshed in the North-Eastern part of Poland. Analysis of P. zopfii susceptibility has demonstrated low effectiveness of both antifungal and antibacterial antibiotics. All algae isolated from milk were resistant to clotrimazole, fluconazole, econazole, flucytosine, cefoperazone, cephalexin, enrofloxacin, lincomycin, and oxytetracycline (100% of resistant isolates), as well as miconazole (92.6% of resistant isolates). Nystatin, ketoconazole, and amphotericin B showed the highest activity amongst the antifungal antibiotics (88.9% and 0.0%, 51.9% and 22.2%, 0.0% and 48.1% of susceptible and intermediate susceptible isolates, respectively). In the group of antibacterial antibiotics, the high activity against P. zopfii was observed only in the case of gentamicin, kanamycin (96.3% and 92.6% of susceptible isolates, respectively), and polymyxin B (59.3% of susceptible and 33.3% of intermediate susceptible isolates).

Systemic mycobacteriosis was diagnosed in a group of ornamental fish. Although a large number of acid-fast bacterial rods were identified in the kidneys, liver, and muscles of each fish, no granulomas were observed in internal organs. Mycobacterium peregrinum was identified using the GenoType Mycobacterium CM assay. This study illustrates a considerable risk of atypical mycobacteriosis in humans.

The paper presents the results of genotypic differentiation and antimicrobial susceptibility of Y. enterocolitica O:9 isolates that originated from cows and pigs positive in serological reactions for brucellosis, and also from the animals, which were serologically negative. The genetic relationship between Y. enterocolitica O:9 isolates originating from different sources was determined by the use of ERIC-PCR, and resulted in detection of 6 to 13 DNA amplicons of different size. The clonal analysis was based on dendrogram created by Unweighted Pair Group Method with arithmetic mean and Jaccard’s coefficient, which enabled to divide Y. enterocolitica O:9 isolates into 16 different clonal groups. Among all Y. enterocolitica O:9, MIC value was >32 mg/L for the ampicillin, ≤0.008 mg/L for ciprofloxacin, ≤8 mg/L for sulphametoxazole, ≤2 mg/L for colistin, and ≤1 mg/L for tetracycline. The wide range of MIC for ceftazidime (≤0.25-2 mg/L) and cefotaxime (≤0.06-1 mg/L) among Y. enterocolitica O:9 isolates was also observed. No significant differences were observed between MIC values of Y. enterocolitica O:9 isolates originating from animals serologically positive for brucellosis, and the isolates from cows and pigs, which provided serologically negative reactions.

The effect of three different field isolates of Mycoplasma bovis on selected immunological parameters in experimentally infected calves was studied. Calves were kept separately in 4 experimental groups, and animals of 3 groups were infected intratracheally with one of the three selected isolates of M. bovis. The control group was inoculated intra-tracheally with sterile physiological saline. Nasal swabs and blood samples were collected just before the calf inoculation, then daily for seven days, and then weekly for another three weeks. The presence of M. bovis antigen, M. bovis antibodies, total protein, gamma globulins, IgA, IgM, IgG, acute phase proteins (haptoglobulin and serum amyloid A), as well as interferon-γ and interleukin-4 concentrations were determined. M. bovis was detected intermittently during the study in the infected groups from day 1, whilst the control group remained free of the pathogen. M. bovis antibodies were detected in some of infected animals in the second, third, and fourth week after infection. The stimulation and/or immunological suppression varied between the M. bovis isolates used for the inoculation. All M. bovis isolates induced a rise of APP and gamma globulin concentrations in infected calves. However, in this study the mucosal immune response appeared to be down-regulated, which was expressed with a general lack of IgA stimulation.

The aim of the study was to optimise selected PCR methods for identification of T. solium, and to compare their effectiveness and usefulness. The investigation concerned three PCR methods described earlier: PCR I (specific to oncospherespecific protein Tso31 gene), PCR II (specific to large subunit rRNA gene), and PCR III (cytochrome c oxidases ubunit 1 gene). Each of them needed optimisation in connection with some changes in the procedures. Among the examined procedures, PCR I was found to be the most useful, requiring the least corrections during optimisation - only a higher concentration of polymerase was necessary. Testing an optimised PCR II method showed strong unspecific reactions with E. granulosus and T. saginata. This method was not considered diagnostically useful in distinguishing T. solium. PCR III method yielded products only when annealing temperature was lowered by 2 C. Under such conditions, there were no unspecific reactions with three others Taenidae parasites; however, annealing at a temperature only 1oC lower generated a distinct unspecific PCR product from T. saginata DNA. Therefore, this method was of limited usefulness. Comparison of the effectiveness of the two selected methods (PCR I and III) in detection of T. solium in successive DNA dilutions showed a large difference between them: in the same DNA sample, PCR I showed positive results in a sample diluted 1:3200, while PCR III failed at dilutions greater than 1:50. The results showed that among the three different methods used in the investigations, the most specific and effective for identification of T. solium was PCR I.

The aim of the study was a preliminary determination of occurrence of extended spectrum β-lactamases (ESBL)- and AmpC-producing Escherichia coli (E.coli) in raw meat samples collected from slaughter-houses located in different regions of Poland. A total of 141 samples were tested, comprising 78 pork samples, 44 beef samples, and 19 chicken meat samples. Isolated and identified E. coli strains were examined for the ESBL and/or AmpC β-lactamases production by the use of four disc diffusion and minimum inhibitory concentration tests. All strains positive in one or both tests were examined by PCR for the presence of the bla_CTX, bla_TEM, bla_SHV, and bla_{CMY-2 group} genes. During the study, 154 E. coli strains were isolated from 95 samples. Among these, 18 (11.7%) strains were identified in phenotypic tests as ESBL-producing and seven (4.5%) strains as AmpC-positive. The presence of the genes encoding selected ESBL-s (TEM, CTX, SHV) was identified in 14 of the strains recognised as ESBLpositive in phenotypic tests. All AmpC-positive isolates showed the presence of the CMY-2 group encoding genes. One of these strains had also the CTX-M and TEM genes, and four of them expressed the TEM marker.

Between 2003 and 2012, 1413 samples of kidneys, liver, and muscles from swine, cattle, sheep, horses, chickens, turkeys, geese, ducks, and fish were examined for the presence of ochratoxin A. The examination was performed in the framework of “The National Residue Control Programme for Chemical, Biological, and Drug Residue in Animal Tissues and in the Food of Animal Origin”. The mycotoxin was determined by liquid chromatography with fluorescence detection after immunoaffinity column clean up. The limit of quantification was 0.2 μg/kg. Ochratoxin A was found only in swine kidney samples (n = 1092). It was detected in 28.8% of the kidney samples at the concentrations from 0.2 to 29.2 μg/kg. The most of the samples (25.5%) contained OTA at the concentration ranging from 0.2 to 5 μg/kg, which is below the provisional action level for OTA in kidneys, established in Poland at the concentration of 5 μg/kg. Furthermore, 24 (2.2 ) samples had mycotoxin concentrations between 5 and 10 μg/kg and 13 (1.2 ) samples above 10 μg/kg.

The study was carried out on live bivalve molluscs available on Polish market. Samples of the molluscs (n = 124) were collected from warehouses and markets. Six different species of molluscs (mussels, oysters, vongole, scallops, Japanese clams, and razor clams) were used for the determination of saxitoxine (responsible for paralytic shellfish poisoning, PSP) by ELISA. The maximum concentration of PSP toxins (756.69 μg/kg of meat) was found in scallops. The majority of tested mussels were free from the PSP toxins or contained biotoxins bellow the permitted level (800 μg/kg). The analysis of toxicological status of raw bivalve molluscs available on Polish market indicated that they are safe for consumers.

The aim of the study was to evaluate the contamination of animal muscle, liver, and milk with lead, cadmium, mercury, and arsenic. Determination of the elements was carried out using several techniques of atomic absorption spectrometry. Between 2008 and 2012, samples of muscles and liver from 1305 cattle, 2345 pigs, 758 horses (only muscles), 1721 poultry (chickens, turkeys, geese, ducks), and 736 samples of raw milk were collected. Only 48 (0.7%) samples exceeded the maximum acceptable levels of the elements, especially lead and cadmium. In the case of lead, the highest number of samples exceeding the legal limits was found in muscles of pigs (6), where the maximum value reached 0.376 mg/kg. For cadmium, the highest number of samples (22) with values exceeding legal limits was found in muscles of horses. The cadmium content in muscles of horses, at both the mean (0.052 mg/kg) and median (0.023 mg/kg), was in order of magnitude higher than that observed in cattle and pigs. Small percentage of samples with values exceeding the maximum levels of toxic elements in food of animal origin indicates a low risk for the consumers’ health.

The study was conducted at all regional veterinary diagnostic laboratories. Feed materials were examined for Salmonella prevalence and contamination by Enterobacteriaceae, aerobic mesophilic bacteria, total plate count, fungi, Clostridium sp., and Bacillus cereus. Assays were done following international and Polish standards used in food and feed microbiology. Salmonella sp. were most often detected in oil seeds. In most of the examined feed ingredients, the number of Enterobacteriaceae did not exceed 10 cfu/g. The contamination by aerobic bacteria ranged most often from 101to 107 cfu/g, and the highest mycological contamination was noted in cereal grains (10⁸ cfu/g). The results showed that microbial contamination of feed materials in regard to Enterobacteriaceae, fungi, and total plate counts declined over the past years.

A fast and reliable method of liquid chromatography and ultraviolet detection of sulfaguanidine, sulfadiazine, sulfamethazine, sulfamethizole, and sulfamethoxazole in feedingstuffs was described. The method involves THE procedure of preparation of spiked samples, and extraction of sulphonamides from the matrix using a mixture of methanol and acetonitrile, followed by drying the extract and dissolving it in a phosphate buffer. The analysis uses octadecyl (C18) analytical column with UV detection at λ = 260 nm and a gradient programme of mobile phase composition. The analytical procedure has been successfully adopted and validated for quantitative determination of the sulfonamides in feedingstuff samples. Validation included sensitivity, specificity, linearity, repeatability, and intra-laboratory reproducibility. The mean recovery of sulfonamides was 84%, within the working range of 200-2000 mg/kg. Direct, simple sample preparation and HPLC-UV analysis allow the method to be successfully included in the scope of routine analyses. The presented results could be an answer to a need of simple and easy method for sulfonamide determination applicable in medicated feedingstuffs analysis.

The aim of this paper was to present the results of comparative evaluation of the usefulness of PCR method for the detection and identification of bovine DNA in feeds. In the validation study, the limit of detection for PCR was determined as 0.05% for bovine meat and bone meal (MBM). Among 132 feed samples, bovine DNA was detected in eight (6.06%) samples. In the next stage of the study, sediment and flotate from the investigated samples were examined with PCR. Out of 132 sediment and flotate samples, bovine DNA was detected in eight (6.06%) and nine (6.82%) samples, respectively. On the basis of the results obtained with the use of the PCR, it is possible to state that the molecular biology methods can, at present, be used as supplementary tools for detection and identification of bovine MBM.

Sensitive and selective methods for the screening (GC-MS) and confirmatory analysis (GC-MS/MS) of 17α- and 17β- trenbolone in bovine urine were developed. In the first stage of the analysis, the enzymatic hydrolysis of trenbolone metabolites with glucuronidase AS-HP in acetate buffer (pH 5.2) solution was carried out. Free compounds were extracted from urine with diethyl ether. For the purification of the extract solid phase, extraction with C18 and NH2 columns was applied. The evaporated extract was subjected to two derivatisation steps; the first with MSTFA/I2 solution and second with MSTFA. The separation of the analytes on HP-5 ms capillary column was conducted. The methods were validated according to the Commission Decision 2002/657/EC. For GC-MS method, CCα and CCβ were 0.21-0.36 μg L⁻¹ for 17α- trenbolone and 0.20-0.34 μg L⁻¹ for 17β- trenbolone, while for GC-MS/MS method the values were lower and amounted to 0.15-0.25 μg L⁻¹ for 17α- trenbolone and 0.20-0.34 μg L⁻¹ for 17β-trenbolone. Method recoveries in spiked samples ranged from 86%-111% with standard deviation lower than 25% for both detection techniques.

A multiresidue method for determination and quantification of Fusarium mycotoxins: deoxynivalenol, zearalenone, T-2 toxin, HT-2 toxin, and metabolite of Aspergillus and Penicillium species - ochratoxin A in feeds was described. The method was based on the simultaneous extraction of selected mycotoxins from matrix, followed by liquid chromatography coupled with tandem mass spectrometry using a hybrid triple quadrupole - linear ion trap mass spectrometer with the multiple reaction monitoring in both positive- and negative-ion modes. The method was validated in accordance with the Commision Decision 2002/657/EC requirements. The mean recoveries of mycotoxins from spiked feed samples ranged from 74.6% to 113.9%, whereas limit of detection and quantification ranged from 0.72 to 12.4 μg/kg and 1.86 to 31.7 μg/kg, respectively.

The aim of the study was to assess the activity of lysosomal enzymes: aminopeptidases, including alanine aminopeptidase (AlaAP), leucine aminopeptidase (LeuAP), arginine aminopeptidase (ArgAP), and glycosidases, such as β-galactosidase (BGAL), β-glucuronidase (BGRD), β-glucosidase (BGLU), N-acetyl-β-hexosaminidase (HEX), α-glucosidase (AGLU) and α-mannosidase (MAN) in the liver of ostriches (n = 80) fed diet supplemented with linseed (4% and 8%) and rapeseed (5% and 10%), with low and high level of vitamin E. (40 and 100 mg). The results indicate that higher level of vitamin E or 4% linseed supplementation in ostrich diet generally increase the activity of glycosidase enzymes and decrease the activity of aminopeptidases in the liver. The 8% linseed and rapeseeds feeding in decreased the activity of AlaAP, LeuAP, and ArgAP and increased only the activity of BGLU.

To select appropriate diet for hamsters used in the uterotrophic and Hershberger assays two rodent diets were compared: Murigran (Agropol, Poland) and Altromin 7010 (Altromin Spezialfutter GmbH&Co., Germany). The contents of bioactive compounds in feeds were evaluated by liquid chromatography, and their oestrogenic activity by yeast enhanced green fluorescent protein assay. In opposition to Altromin, Murigran contained high amounts (μg/kg) of genistein (765 600) and daidzein (132 000), and the oestrogenic activity of these compounds, expressed as 17β-oestradiol equivalent concentration (EEQ), was found to be 9.54 μg EEQ/kg. In in vivo study, Murigran induced a high degree of oestrogenisation in immature hamsters, and females failed to exhibit a normal uterine response to recommended dose of a model oestrogen agonist 17α-ethinyloestradiol. There was no influence of the diet on the weight of five accessory sex organs (ASO): ventral prostate, seminal vesicles with coagulating glands, levator ani bulbocavernosus muscles, Cowper`s glands, and glans penis of control males. However, the impact on ASO response to model androgen agonist, testosterone propionate was observed. The obtained results provide the evidence that phytooestrogen-rich feed modulates the oestrogenic and androgenic response to chemicals.

Immunohistochemical expression of urokinase plasminogen activator (uPA) was studied in 37 canine malignant mammary tumours to define the relationship between their histopathological type and grade. In 29 (78.4%) cases, expression of uPA by neoplastic cells was more than 10% and in 34 samples (91.9%) uPA expression by stromal cells (fibroblasts) was more than 10%. The uPA was expressed in epithelial and myoepithelial cells of carcinomas and carcinosarcomas and mesenchymal population of carcinosarcoma, chondrosarcoma, and carcinomas arising in benign tumours. The intensity and percentage of expression of uPA by stromal cells was associated with their histological grade (P < 0.05). However, no significant relationship was detected between uPA expression by neoplastic cells (epithelial, myoepithelial, and mesenchymal cell) and histological grade. Increased expression of uPA by tumour stroma was associated with poor prognostic factors. Stromal expression of uPA could be a prognostic indicator for canine mammary tumours.

The objective of this study was the morphological and structural characterisation of glioblastoma multiforme grown in ovo. Glioma cells U87 and U118 were implanted in the chorioallantoic membrane (CAM) of chicken egg. After 10 days of incubation, tumours were resected for further analyses. Culturing two types of glioblastoma tumours from U87 and U118 cell lines has shown a number of differences in their morphology, histology, and ultrastructure. CAM assay proved to be a useful tool for studying glioblastoma growth. The model provides an excellent alternative to current rodent models and could serve as a preclinical screening assay for anticancer molecules. It might increase the speed and efficacy of the development of new drugs for the treatment of glioblastoma.

The cytotoxic potential of fluoroquinolones (enrofloxacin, ciprofloxacin, difloxacin, sarafloxacin, danofloxacin, norfloxacin and marbofloxacin) was investigated using mouse fibroblasts Balb/c 3T3 and human hepatoma HepG2 cell lines. The cells were exposed for 24, 48, and 72 h to drugs at eight concentrations ranged from 0.78 to 100 μg/mL. Four independent cytotoxicity assays were applied, in which various endpoints were assessed: mitochondrial activity - MTT reduction, lysosomal activity - neutral red uptake, total protein content, and cellular membrane integrity - lactate dehydrogenase release. Mean effective cytotoxic concentrations (EC₅₀) calculated at different time points from concentration-response curves ranged from 10 to 100 μg/mL. The most affected endpoint in both cell lines was mitochondrial activity. The EC_{50-MTT-72 h} <10 μg/mL was found for difloxacin, marbofloxacin (fibroblasts), sarafloxacin, and norfloxacin (HepG2). The data shows that cytotoxicity of the fluoroquinolones appears after longer exposure of both cell cultures to these compounds.

A total of 1229 samples, including 406 bovine hides and 406 corresponding carcasses at the slaughter level, as well as 417 beef meat from local supermarkets, were tested for the presence of Salmonella sp. Eighteen (1.5%) samples were positive for the target microorganism, and the highest prevalence (2.2%) was found in meat, followed by carcasses (1.2%) and hides (1.0%). Among the isolated strains, Salmonella enterica serotypes Enteritidis (six isolates) and Schleissheim (six strains), followed by Dublin (four contaminated samples) were the most predominant. The antimicrobial resistance analysis against nine antimicrobials with the MIC technique revealed that most isolates were sensitive to all antibacterial agents. However, one S. Typhimurium of carcass origin was multidrug resistant, and displayed the resistance to four antimicrobials, i.e. ampicillin, streptomycin, tetracycline, and sulphametoxazole. Furthermore, one S. Enteritidis (from carcasses), two S. Dublin (of beef origin), and one S. London (from meat) strains were resistant to sulphametoxazole. The restriction enzyme analysis with XbaI resulted in eight different PFGE types. The obtained results suggest that cattle may be an underestimated source of pathogenic Salmonella for consumers.