Severe asynapsis in spermatocytes of interspecific hybrids of the silver fox (Vulpes vulpes) and the blue fox (Alopex lagopus) leads to pachytene I arrest as a result of sustained H2AXγ phosphorylation.

Infertility is frequently associated with meiotic anomalies which can result in the production of chromosomally abnormal gametes or be concomitant with meiotic arrest. We investigated whether spermatocytes of male interspecific hybrids of the red fox (Vulpes vulpes) and the arctic fox (Alopex lagopus) presented alterations in chromosomal synapses and meiotic checkpoint signalling. Using the immunofluorescence technique with SP1 and SP3 proteins, bivalent structures and their deviations (multivalents, univalents and not fully conjugated bivalents) were analyzed on meiotic preparations. This technique allowed the localization of frequent foci of phosphorylated histones H2AHγ (Ser 139) to the meiotic block in late pachytene. These results indicate a disruption of meiotic division in male fox hybrids, which leads to a high percentage of apoptotic cells in the gonads of these animals and, consequently, sterility.

Comparative analysis of cat bone marrow and adipose tissue cell cultures.

Cell culture transplantation is very promising in the treatment of various diseases. Cells obtained from a number of sources have been analysed to provide a basis for further studies in the area of regenerative medicine. The objective of the study was to compare morphological and phenotypic changes in cat adipose tissue and bone marrow cell cultures from the first to fifth passages. Adipose tissue and bone marrow were used to obtain cell cultures (coming from 3 cats) using standard methods with own modification. Phenotype changes were monitored by CD-marker identification and CD pan-keratin. The cytogenetic analysis was performed on 50 metaphase plates of cell cultures from the first to fifth passage. Cytogenetic assays showed that the adipose tissue cell culture (ATCC) at all passages was more stable than the bone marrow cell culture (BMCC).

Algorithm for establishing the time of death of a dog based on temperature measurements in selected sites of the body during the early post-mortem period.

Post-mortem measurements were made of the body temperature of dogs. The aim of the study was to evaluate and verify a reliable mathematical model that can be used to establish the time elapsed since the death of a dog during the initial post-mortem period at room temperature, using the eye (vitreous body), internal organs (heart, liver, kidney and lung), and rectum as sites for temperature measurement. The measurements were performed at six points in the body using an electronic thermometer in conjunction with a temperature probe. The method of temperature measurement is simple and does not cause perceptible macroscopic changes or disfigure the carcass. Multiple regression analysis was shown to be suitable for estimating the time elapsed from death to the discovery of the body for a period up to 12h post-mortem. The proposed multiple regression equation using body weight and the temperature at a specific site reduces manipulation of the carcass to a minimum and thus reduces error in establishing the time of death. The multiple regression model makes it possible to precisely estimate the time elapsed since the death of the animal.

Effects of maternal treatment with ß-hydroxy-ß-metylbutyrate and 2-oxoglutaric acid on femur development in offspring of minks of the standard dark brown type.

The aim of the study was to evaluate the effect of the diet, mother type and sex of the offspring on the mechanical and geometric parameters of long bones as well as bone tissue density in minks. Primiparous and multiparous dams were supplemented with ß-hydroxy ß-methylbutyrate (a metabolite of leucine, at the daily dosage of 0.02 g/kg of body weight) and/or 2-oxoglutaric acid (a precursor of glutamine, at the daily dosage of 0.4 g/kg of body weight) during gestation. The diet did not influence bone tissue density and the length of the humerus. An increase in the length of the femur was noted in male offspring delivered by multiparous dams. The diet resulted in an increase in the weight of the humerus in males from multiparous dams and a decrease in offspring from primiparous dams. Heavier femora were noted in male offspring delivered by both types of dams. The maximum elastic strength of the humerus was higher in the offspring delivered by multiparous than primiparous dams, irrespective of the offspring sex. The diet resulted in reduction in the ultimate strength of the femur in the male offspring delivered by primiparous dams. Only females born by multiparous dams, irrespective of the diet, showed a significant increase in the cross-sectional area of the humerus, while a significant decline was noted in males delivered by multiparous dams and in all the offspring delivered by primiparous dams. An increase in the cross-sectional area of the femur was noted in the offspring delivered by multiparous dams, while reduction was observed in the offspring delivered by primiparous dams. These results have shown for the first time that the presence of ß-hydroxy-ß-methylbutyrate or 2-oxoglutaric acid in the diet of pregnant primiparous or multiparous dams unambiguously affects the geometry and mechanical properties of offspring's long bones.

Immunophenotypic characteristics and karyotype analysis of bone marrow-derived mesenchymal stem cells of rabbits during in vitro cultivation.

The aim of this study was to establish the immunophenotypic profile and karyotypic stability of bone marrow mesenchymal stem cells (MSCs) of rabbits at the early passages in vitro following the application of different methods of dissociation of cellular material. MSCs were obtained from the femur bone marrow of three clinically healthy rabbits under general anaesthesia. Bone marrow aspirate was seeded in Petri dishes and cultured in a CO2 incubator with 5% CO2 at 37.0oC using a standard procedure. Immunohistochemical detection of nuclear proteins, cytoskeletal proteins and cell adhesion were performed by immunohistochemical analysis and karyotype analysis of MSCs following the enzyme and chelating methods of dissociation of the cell monolayer. The results of the immunophenotypic analysis of rabbit bone marrow MSCs showed that at the first, seventh, twelfth, and eighteenth passages these cells express markers of mesenchymal, muscle, epithelial and nerve cells. The choice of the enzyme or chelating method of dissociation of a culture of rabbit mesenchymal stem cells affects their cytogenetic variability. Dissociation of the MSCs monolayer with ethylenediaminetetraacetic acid produces a cell culture with fewer quantitative and qualitative changes in the chromosome apparatus as compared to the enzyme method. Rabbit MSCs express markers of mesenchymal (vimentin, actin), muscle, epithelial and nerve (E-cadherin, N-cadherin) cells that are essential for differentiation of these cells. The chelating method of dissociation of a culture of rabbit mesenchymal stem cells, using ethylenediaminetetraacetic acid during cultivation, is more advantageous than the enzyme method of dissociation because it leads to less cytogenetic variability.

High molecular polymorphism of the hypervariable region in the VP2 gene of Aleutian mink disease virus.

Parvoviruses exhibit extreme genetic plasticity. The VP2 protein, containing a hypervariable region, is of particular importance. A single nucleotide change in this part of the genome and its effect on the amino acid sequence may significantly affect the range of infected hosts, tropism for specific tissues, or virulence. The high polymorphism in the hypervariable region can be exploited for phylogenetic analysis. The aim of this study was to analyze the polymorphism of the VP2 hypervariable region in isolates of the Aleutian mink disease virus (AMDV) infecting Polish mink farms and to determine the phylogenetic relationships between the Polish isolates and genetic variants of the pathogen occurring in other countries. The study compares farms from two regions of Poland. The isolates contained five changes in the amino acid sequence, which had not previously been recorded in the NCBI database. There were 21 changes noted between the genotypes obtained and the sequence of the reference strain [GenBank NC_001662.1], of which 8 were in the hypervariable region. The isolates identified in our study exhibit a high degree of similarity within the farms, but between farms there is considerable variation in the amino acid sequence of the VP2 protein fragment. Because variants characteristic for farms were obtained, it will be possible to trace the movement of the virus between farms, and in the longer term to use the characteristic sequences as a marker of the origin of infected animals.

Flow cytometric evaluation of sperm apoptosis in semen of silver foxes in the breeding period.

The objective of the study was to evaluate cytometrically the percentage of apoptotic and necrotic spermatozoa in fresh semen of silver foxes in the breeding season. In males F3 and F4 with high percentages of early apoptotic (A+Pi-), late apoptotic (A+Pi+) and necrotic (A-Pi+) spermatozoa as well as 56-65% of living spermatozoa (A-Pi-) with progressive motility, the semen was characterised by reduced fertility. In males F1 and F2 with spermatozoa showing the motility and viability of 89-90% and high percentages of living cells that do not bind Annexin V and propidium iodide, the semen was assessed as valuable and useful for artificial insemination. Amongst 16 females of group I and II inseminated with semen from F1 and F2 males, 15 (93.75%) had multi-cub litters - on average 6.1 and 4.8, respectively. In contrast, amongst 16 females of group III and IV inseminated with semen from F3 and F4 males, only 10 (62.5%) had litters with few cubs (on average 2.6 in group III and 2.1 in group IV). Our findings explicitly indicate that semen of farm male foxes should be evaluated before the breeding season, as one of the causes of reproduction failures is likely to be a high percentage of apoptotic and necrotic spermatozoa. Thanks to flow cytometry, fresh ejaculates can be speedily evaluated and their usefulness for artificial insemination determined.

Phylogenetic analysis of canine parvovirus CPV-2 strains and its variants isolated in Poland.

Canine parvovirus disease appeared in the world and in Europe during the second half of the 1970s. Over the course of 40 years the original CPV-2 strains mutated and variants 2a, 2b and 2c appeared. Their appearance is connected with specific amino acid changes, mainly in the capsid protein VP2. Strains isolated by the authors were adapted for in vitro cell culture. Phylogenetic analysis revealed differences between strains isolated in Poland in 1982-1985 and in 1995-2009. Strains from the 1980s were shown to belong to variant CPV-2a (11 strains) and variant 2b (2 strains), while no fundamental differences were found among the genetic profiles of the strains from 1995-2009, which were classified as belonging to variant 2c.

Analysis of occurrence of virulence genes among Yersinia enterocolitica isolates belonging to different biotypes and serotypes.

The 150 Y enterocolitica strains isolated from humans and from pigs belonged to biotypes 4 (68.7%), 1A (18.7%) and 2 (4%), or were biochemically untypeable (8.6%). Biotype 4 was comprised of Y. enterocolitica strains representing serotype O:3, within biotype 1A the strains either belonged to serotypes O:5 and O:6 or were untypeable, and biotype 2 was represented by the strains of serotype O:9. The strains which were biochemically untypeable belonged to serotypes O:5, O:6 and O:3. Among the strains tested there also were those of an unidentified biotype and serotype. Nearly all the strains of biotype 1A represented genotype ystB+myfA+, and few belonged to genotype ystB+. The presence of the ystB gene in the strains of biotype 1A and only occasional occurrence of the gene in the other biotypes makes ystB a distinguishing marker of biotype 1A. The strains of genotype ystA+ail+myfA+yadA+ predominated in biotype 4 (serotype O:3). The strains of biotype 2 (serotype O:9) represented genotype ystA+ail+myfA+, and the plasmid yadA gene was detected in some of them. Within the group of biochemically untypeable strains ystB- and myfA-specific PCR products were mainly obtained. The genotypes determined for the tested biotypes and serotypes of Y. enterocolitica, based upon the selected genes of virulence, can be applied as distinguishing markers and indicators of the potential virulence of Y. enterocolitica strains, excluding bioserotyping.

Multiresistant Salmonella enterica serovar 4,[5],12:i:- in Europe: a new pandemic strain?

A marked increase in the prevalence of S. enterica serovar 4,[5],12:i:- with resistance to ampicillin, streptomycin, sulphonamides and tetracyclines (R-type ASSuT) has been noted in food-borne infections and in pigs/pig meat in several European countries in the last ten years. One hundred and sixteen strains of S. enterica serovar 4,[5],12:i:- from humans, pigs and pig meat isolated in England and Wales, France, Germany, Italy, Poland, Spain and the Netherlands were further subtyped by phage typing, pulsed-field gel electrophoresis and multilocus variable number tandem repeat analysis to investigate the genetic relationship among strains. PCR was performed to identify the fljB flagellar gene and the genes encoding resistance to ampicillin, streptomycin, sulphonamides and tetracyclines. Class 1 and 2 integrase genes were also sought. Results indicate that genetically related serovar 4,[5],12:i:- strains of definitive phage types DT193 and DT120 with ampicillin, streptomycin, sulphonamide and tetracycline resistance encoded by blaTEM, strA-strB, sul2 and tet(B) have emerged in several European countries, with pigs the likely reservoir of infection. Control measures are urgently needed to reduce spread of infection to humans via the food chain and thereby prevent the possible pandemic spread of serovar 4,[5],12:i:- of R-type ASSuT as occurred with S. Typhimurium DT104 during the 1990s.

Contents of zinc, copper, chromium and manganese in silver foxes according to their age and mineral supplementation.

Serum, livers and kidneys of 30 silver foxes from one breeding farm were subjected to analysis of the four microelements contents. The samples derived from 3 groups of animals (n = 10) selected according to age and developed reproduction disorders. Cu, Cr and Mn were determined by a graphite furnace AAS whereas Zn by the flame AAS methods. Serum levels of Zn were the least variable (x (n = 30) = 4.72 +/- 2.313 microg x mL(-1)) and the mean of Cu content was 0.26 +/- 0.244, of Cr was 0.029 +/- 0.032 and of Mn was 0.074 +/- 0.085 microg x mL(-1). The livers and kidneys contained respectively: 159.9 +/- 23.66 and 74.25 +/- 14.44 microg g(-1) of Zn; 34.03 +/- 12.43 and 13.66 +/- 1.67 microg g(-1) of Cu as well as 6.28 +/- 0.97 and 2.60 +/- 0.33 microg g(-1) of Mn. The most variable was Cr level achieving 1.00 +/- 1.06 and 1.43 +/- 2.64 microg g(-1) (all results per gram of wet weights). The differences between means and medians within the age groups did not exceed 41%, however chromium was an exception, its values differed more than 2 times. High zinc level found both in the serum and the organs resulted from its concentration in feedstuff being 1040,5 microg g(-1), exceeding the recommended level for farming foxes. Over-supplementation of dietary Zn might suggest connection between subtoxic action of Zn or its interaction with Cu or Cr followed by subsequent effect on reproduction events. Whether nature of Zn effects derived from direct target action or from Zn - Cu or Zn - Cr interactions have to be solved experimentally. Some foxes contained copper and chromium in livers and kidneys at levels exceeded significantly those concidered as physiological ranges for mammals, whereas manganese was within these limits. Thus, manganese was considered to keep homeostasis status of this element in the examined foxes.

Towards an integrated approach to study SNPs and expression of candidate genes associated with milk protein biosynthesis.

MilkProtChip is oligonucleotide microarray allowing bovine genotyping based on single nucleotide polymorphisms (SNPs) in genes influencing milk protein biosynthesis. A total of 71 SNPs in 42 genes were selected as associated with milk protein biosynthesis. Genotyping of about 300 animals of Polish Black-and-White cattle showed that SNPs in acyl-CoA: 1,2-diacylglycerol O-transferase (DGAT1), lactoferrin (LTF), casein kappa (CSN3) and growth hormone receptor (GHR) genes were associated with several milk performance traits. Analysis of correlations between SNPs and milk production traits showed that SNPs in single genes rarely affect the investigated traits. Only 4 of 42 investigated single SNPs had impact on milk production traits while 22 combinations of paired SNPs in these genes had impact. Positive effect SNP combinations in two genes can be a result of additive effect on these SNPs on the same traits or effect of genes interaction. The MilkBovExp chip representing 90 genes encoding transcription factors expressed in the bovine mammary gland and/or involved in mammary gland signaling pathways was designed for further investigation of impact of gene expression and/or its encoded products on milk traits performance.

Molecular typing of Staphylococcus aureus isolated from cows with mastitis in the east of Poland on the basis of polymorphism of genes coding protein A and coagulase.

The aim of this study was to test the diversity of a population of 82 strains of S. aureus isolated from cows with mastitis in the east of Poland. The isolates were typed by analysis of the number of repeats of 24 bp sequences in the X region of protein A (spa) gene and restriction fragment length polymorphism (RFLP) of the coagulase (coa) gene. Twelve different spa types were distinguished. Amplification of region X gave, in 79 cases, one stripe. In a scope of 100-364 bp 10 different products (genotypes) of amplification reaction were defined. For one strain two stripes were obtained and two strains did not contain the spa gene. The most prevalent strains had 10, 11 and 12 repeats of 24 bp sequences, which represented respectively 18%, 30% and 13% of all strains tested. The presence of any strain containing 4 or 9 sequence was not observed. In the case of analysis of the polymorphism of the coagulase gene, 13 different genotypes were identified. The most frequently appearing genotype is genotype C, in which case an amplification product is digested into three DNA fragments: 410, 320 and 160 bp. To this genotype belong 43 strains, which constitute 52% of the examined population. A significant improvement in discriminatory power was observed when results from both genes were analyzed simultaneously. In an analyzed group of 82 strains, 24 genotypes were isolated.

The hemagglutinating activity of Pseudomonas aeruginosa strains isolated from humans and animals.

The presence and type of adhesins occurring in Pseudomonas aeruginosa strains were determined by hemagglutination test with a 3% suspension of normal and trypsin-treated human group A erythrocytes, with or without the addition of sugar inhibitors (D-mannose, D-glucose, L-fucose, D-galactose, D-fructose, lactose, N-acetylneuraminic acid, N-acetylglucosamine and N-acetylgalactosamine). This study showed that a low percentage of Pseudomonas aeruginosa strains caused the agglutination of normal erythrocytes. Trypsin treatment of erythrocytes did not affect the hemagglutinating properties, indicating that hemagglutination was not dependent upon a trypsin-sensitive protein on the erythrocytes surface. Most of the studied strains agglutinated RBCs at 37 degrees C. A great variability in the inhibiting activity on studied strains was observed among the carbohydrates tested. These results demonstrated the predominant role of N-acetylglucosamine, N-acetylgalactosamine and N-acetylneuraminic acid for Pseudomonas aeruginosa adhesion to RBCs.

Typing of Yersinia Enterocolitica Isolates by ITS profiling, REP- and ERIC-PCR.

Thirty-five Yersinia enterocolitica strains isolated from humans, pigs and foxes were analysed by genotyping including intergenic transcribed sequence (ITS) profiling, REP- and ERIC-PCR. ERIC-PCR revealed the presence of seven different genotypes. Amplification of the 16S-23S rDNA spacer region by ITS profiling gave similar results with nine different genotypes. REP-PCR was found to be more discriminatory for typing of Y. enterocolitica than ERIC-PCR and ITS profiling. Fifteen different DNA patterns were obtained by this technique. Based on data obtained by three methods it was found that: (i) Y. enterocolitica strains belonging to the same serotype can represent different genotypes and vice versa; (ii) isolates recovered from humans, pigs and foxes exhibit limited heterogeneity and, independent of the origin, one or two prevailing genotypes were always observed; and (iii) many human Y. enterocolitica isolates shared common genotypes with porcine isolates.

The activity of chosen bacteriophages on Yersinia enterocolitica strains.

The aim of the present study was to evaluate the lytic activity of three bacteriophages on Yersinia enterocolitica strains isolated from humans and pigs. The Y. enterocolitica strains tested belonged to 0:3, 0:9 and 0:2 serogroups. The ZD5 phage was obtained from a water sample, but remaining phages were obtained from the lysogenic Y. frederiksenii 7291 and Y. enterocolitica 8684 strains. All the Y. enterocolitica strains tested which belonged to 0:9 serogroup did not show any susceptibility to the bacteriophages used. The bacteriophages tested showed different lytic activity on the Y. enterocolitica 0:3 strains investigated. The phage susceptibility of Y. enterocolitica 0:3 strains revealed 9 different phage patterns. ZD5 phage showed the highest lytic activity, because it produced confluent lysis of the most Y. enterocolitica 0:3 strains tested. The Y. enterocolitica 0:2 strains isolated from pigs showed the similar phage susceptibility. The Y. kristensenii and Y. pseudotuberculosis strains tested were not sensitive to the bacteriophages used.

Adherence of Pseudomonas aeruginosa strains to solid surfaces.

The aim of this study was to evaluate adherence of 59 strains of Pseudomonas aeruginosa to nitric-acid cleansed glass surfaces. There were differences in adherence between the investigated strains. The highest adherence was noticed among human strains (the average percentage was 13.3 +/- 7.51%) and the lowest adherence was determined among swine strains (the average percentage amounted 6 +/- .37%). We conclude that strains of Pseudomonas aeruginosa isolated from humans colonise glass surfaces better than strains isolated from animals.

[Evaluation of hydrophobic properties of Yersinia enterocolitica strains isolated from humans and pigs]. / Ocena hydrofobowych wlasciwosci szczepów Yersinia enterocolitica wyizolowanych od ludzi i trzody chlewnej.

The aim of the study was to evaluate the hydrophobic properties of Yersinia enterocolitica and to determine the influence of the culture conditions, such as: type of medium, temperature, and duration of the culture on the manifestation of these properties. The subject of the study were 117 of Y. enterocolitica strains isolated from humans and pigs. The ammonium sulphate salt aggregation test according to Lindahl modified method was used to evaluate the hydrophobic properties of Y. enterocolitica strains. Strains of Y. enterocolitica were cultured for 24 h at 25 degrees C on TSA (Difco) medium. During investigation of the influence of the culture conditions the chosen strains were incubated for 24 h and 48 h at 25 degrees C and 37 degrees C on TSA (Difco), LB (Difco), enrichment agar (Biomed), and enrichment agar with 5% sheep blood (Graso). A total of 44.5%, 17.9%, 9.4%, and 28.2% strains of Y. enterocolitica showed very strong hydrophobic properties, strong hydrophobic properties, some hydrophobic properties, and were non-hydrophobic, respectively when strains of Y. enterocolitica were cultured for 24 h at 25 degrees C on TSA medium. A total of 75.5% strains isolated from humans showed very strong hydrophobic properties and 13.5% strains were non-hydrophobic. Among strains isolated from pigs 30% showed very strong hydrophobic properties but 35% were non-hydrophobic. The hydrophobic properties of Y. enterocolitica depended on the temperature, duration of the culture and the type of media. The highest number of strains with very strong hydrophobic properties (89.6%) was obtained after 48 h of the incubation at 37 degrees C on the enrichment agar with 5% sheep blood. The highest number of non-hydrophobic strains of Y. enterocolitica (28.5%) was obtained after 24 h at 25 degrees C on TSA medium.

[Occurrence of beta-lactamase type ESBL and IBL in Pseudomonas aeruginosa rods]. / Wystepowanie beta-laktamaz typu esbl i ibl u paleczek Pseudomonas aeruginosa.

The aim of the study was to determine extended spectrum beta-lactamases (ESBL) and inducible beta-lactamases (IBL) among Pseudomonas aeruginosa strains. A total of 43 strains isolated from humans (6), hospital sink (1), fish (15), cattle (5), swine (5), dog (1), redder (1) fur animals (9) were studied. ESBL-producing strains were detected with double disc diffusion test according to Jarlier et al. (8). Clavulonate and tazobactam were used as the inhibitors of ESBL. Inducible beta-lactamases were determined using double disc method according to Sanders (15). Cefoxitin was the inductor of these beta-lactamases. The susceptibility study was carried out using the disc diffusion method according to NCCLS standards. A total of 8 ESBL (18.6% of all strains) and 31 (72%) IBL producing strains were detected. The obtained results indicate the necessity of monitoring of ESBL- and IBL-producing strains of Pseudomonas aeruginosa.

[Analysis of bacteriocinogenic properties of Yersinia enterocolitica strains]. / Analiza bakteriocynogennych wlasciwosci szczepów Yersinia enterocolitica.

The aim of the study was the investigation of bacteriocinogenic properties of 102 Yersinia enterocolitica strains. The influence of selected factors on the production of bacteriocins by Y. enterocolitica and properties of jersiniacin 44JPSBKOH were also investigated. Bacteriocinogenic properties of Y. enterocolitica strains were tested by using the delayed cross-streaking method. It was found that the production of bacteriocins by Y. enterocolitica depended on the type of media on which the producer and indicator strains were grown. It turned out that some strains of Y. enterocolitica showed bacteriocinogenic properties at 25 degrees C, 30 degrees C and 37 degrees C irrespective of the presence of manganese ions in medium. In the presence of iron ions these strains showed bacteriocinogenic properties only at 25 degrees C. Y. enterocolitica strains which required Mn2+ or Mn7+ ions for bacteriocins production showed this activity only at 25 degrees C but in presence of Fe3+ ions they had no bacteriocinogenic properties. The partially purified jersiniacin 44JPSBKOH is a protein, its molecular weight was estimated to be 40 kDa. Yersiniacin 44JPSBKOH was active in the pH range of 3 to 9. Its bactericidal activity was rapidly lost when heated to 100 degrees C and treated with proteolytic enzymes. Yersiniacin 44JPSBKOH showed bactericidal activity against other Y. enterocolitica strains and some strains of Pseudomonas aeruginosa isolated from humans.