Volume 57 (2013): Issue 1 (March 2013)

The purpose of the experiment was to compare apoptosis induced by equine influenza virus (EIV A1 and EIV A2) infection in MDCK, RK13, and NEURO-2A cell lines. Flow cytometry was used to observe two symptoms of apoptosis: phosphatidylserine translocation in plasmalemma (annexin V assay) and the fragmentation of DNA generated by endonuclease activity (TUNEL assayterminal deoxynucleotidyl transferase biotin-dUTP nick end labelling). The differences in the onset of apoptosis in the studied cells was observed. In MDCK cells infected with EIV A1 and A2, a weak signal of the phosphatidylserine translocation was observed but more cells showed the DNA fragmentation. An opposite effect was observed in case of RK 13 cells. NEURO-2A cells displayed a similar number of annexin V and TUNEL positive cells after the infection with EIV A2, while in case of EIV A1 infection, only the early symptoms of apoptosis were noted. Differences between both viral serotypes could originate from functioning of viral proteins responsible for induction or inhibition of apoptosis. The differences between cell types may result from the activation of cellular pro or anti-apoptotic mechanisms.

The paper describes an outbreak of swine influenza, which was the first H1N2 outbreak confirmed in pig farm in Poland. The outbreak occurred in October 2011 in the farrows to finish farm, first in the fattening units, and subsequently in the reproduction and weaning sectors. Samples of the lungs taken from dead gilts were tested by the use of real time and multiplex PCR, sequencing, and virus isolation methods. The amplification of the genetic material extracted from the lungs confirmed the presence of the M1 gene sequence of type A influenza virus. Using multiplex PCR, the bands of 241 bp, typical for HA1 human-like type, and of 791 bp, typical for NA2, were demonstrated. The new isolates of swine influenza virus, named A/swine/Poland/15817/2011, were obtained in embryonated SPF chicken eggs and MDCK cells. Phylogenetic analysis of the HA and NA genes of the isolate revealed that the virus is of human swine linkage closely related to European human-like viruses. On the basis of such results, it can be stated that A/Swine/Poland/15817/2011 is a new swine influenza virus, representing the H1N2 subtype strain, which emerged in Poland.

The study included a sheep flock comprising five genetic groups. The ELISA was applied to perform constant monitoring (every six months) for the infection of ewes with small ruminant lentivirus (SRLV). The research results demonstrated a negative effect of SRLVs infection on lamb rearing that, depending on the genetic group, proved to be lower 1.3%-1.4% compared to the seronegative mothers. At relatively equal fertility (94%-100%) and more differentiated prolificacy (179%-198%) in all the examined groups (except the Suffolk breed), a rearing index was higher in the seronegative animals 6.8%-24.1% compared to the seropositive mothers. The Suffolk breed proved to be the genetic group most susceptible to SRLV infection. A prolificacy of infected ewes was 10% lower, a lamb rearing rate was 13% lower , and a general reproductive performance was 18% lower in comparison to healthy ewes.

The objective of the study was to develop a simple and rapid molecular method for the detection of GPV. Twenty seven goose parvovirus (GPV) isolates collected from geese flocks in Poland were examined. Three pairs of specific primers: two outer primers (F3 and B3), two inner primers (FIP and BIP), and two loop primers (FL and BL) were used to accelerate the reaction. The optimum temperature and time of the reaction were 60°C and 30 min. The sensitivity of the method was 10-times higher than PCR. The method proved to be a sensitive, rapid, and specific assay for detecting GPV.

The outbreak of chlamydiosis in one of the western provinces of Poland, was diagnosed accidentally as a concurrent infection in a commercial laying hen flock during an outbreak of fowl pox. For histological examination, skin and subcutaneous tissue samples from lesions on heads of the birds were collected. Swabs from throat and trachea have been examined by nested PCR, real-time PCR, and partial ompA sequencing. Detailed electron microscopy analysis revealed fowl pox intracytoplasmic inclusions, called Bollinger bodies, and the presence of other intracytoplasmic inclusions; specific for Chlamydia sp. Results of nested PCR confirmed the presence of Chlamydiaceae sp. in two tested samples. Surprisingly, one of the two Chlamydiaceae-positive cases turned out to be infected with a non-classified strain. Results of real-time PCR and sequencing confirmed the presence of a new Chlamydia species that has not been found in Poland to date. Partial sequencing and BLAST analysis of ompA gene sequence confirmed the highest homology to non-classified poultry strains of Chlamydia sp. that were previously detected in Germany and France. The zoonotic potential and the exact taxonomic status of this atypical strain have yet to be defined.

A total of 319 Pasteurella multocida (Pm) strains isolated from pigs in Poland were examined. Phenotypic characterisation included: biochemical tests (to determine species, subspecies, and biovar), capsular typing, and antimicrobial susceptibility. Genotypic characterisation included detection of the toxA gene by PCR. All tested Pm strains were classified as Pm subsp. multocida: 87.2% biovar 3, 10.7%-2 and 0.9%-12. One strain was classified as biovar 1. Three strains of Pm did not suit any of the biovars. Using capsular typing methods, 77% of Pm strains isolated from nasal swabs belonged to type D and 33% to type A. Among Pm strains isolated from internal organs, 59.5% belonged to type A and 40.5% to type D. All the isolates showed a high susceptibility to β-lactams: ampicillin and amoxicilin with clavulonic acid (97.8%), penicillin (86.7%), doxicilline (100%), oxytetracycline (97.8%), and tetracycline (93.2%). It was found that all strains were susceptible to norfloxacin, 97.8% to enrofloxacin, and 95.6% to SxT. 24.4% and 15.6% of the strains were resistant to linco-spectin and tiamulin, respectively. The presence of toxA gene was confirmed by PCR in 20.8% of the strains isolated from nasal swabs and 29.1% of isolates from internal organs.

Changes in selected blood lymphocyte subpopulations in dogs administered with a soluble parasitic antigen (SPA) derived from a supernatant of 18S RNA-A and 18S RNA-B Babesia canis cell culture were investigated. The studies included 20 dogs divided into three groups: group I (n=8) - comprised of dogs receiving SPA twice, at 3 week intervals; group II (n=5) - nonvaccinated control dogs, and group III (n=7) - dogs vaccinated twice with a commercial B. canis vaccine. Cytometric analysis revealed that vaccination with SPA derived from B. canis culture had similar effects to the vaccination with a commercial vaccine. The vaccination lowered the percentage of T lymphocytes (CD3+), T helper cells (CD4+), cytotoxic/supressor T cells (CD8+), B lymphocytes (CD21+), and MHC II lymphocytes in the blood in comparison to non-vaccinated dogs. Statistical analysis of the results demonstrated that mean values of the tested parameters at each stage of the study were similar in groups I and III and significantly higher in group II. The lowered level of the lymphocyte subpopulations in groups I and III persisted during the whole period of the study. The results presented that SPA has immunosuppressive effect in the first period after being administrated.

The study included 40 strains of Malassezia pachydermatis obtained in pure culture from external ear canal of dogs and the reference strain. Protein fractions were received by mechanical disruption of the fungal cells. Denaturing protein electrophoresis was performed according to Laemmli method. More than 90% of the all tested strains were characterised by the presence of the following protein fractions: 83.0; 77.0; 59.0; 55.0; 48.0; 38.0; 35.0; 28.0, and 27.0 kDa. In other regions of electrophoregrams, a relatively high differentiation was observed. The strains isolated from animals with otitis externa were characterised by the presence of the following protein fractions: 75.0; 61.0; 52.0; 36.0; 19.5; 16.0; 13.8, and 13.0 kDa. These fractions were absent in the commensal strains. The use of statistical analysis confirmed the obtained results and allowed to generate dendrogram grouping potentially pathogenic and commensal strains on two different branches. Such results may indicate significant differences between protein profiles of strains of M. pachydermatis isolated from healthy and diseased animals.

The aim of the study was to determine serum free amino acid concentration in male dogs suffering from benign and malignant perianal tumours. Serum concentrations of cysteic acid, glutamic acid, glutamine, aspartic acid, glycine, γ-aminobutyric acid, tryptophan, methionine, arginine, taurine, threonine, tyrosine, alanine, valine, isoleucine, and leucine were significantly changed in dogs suffering from benign and/or malignant tumours when compared to the control group (P<0.05). Serum concentration of serine, phenylalanine, lysine and histidine was not influenced in dogs with neoplastic disease (P>0.05). The evaluation of serum free amino acid concentration, has shown that threonine, glutamic acid, alanine, valine, isoleucine, leucine, γ-aminobutyric acid, and glycine have the highest diagnostic and prognostic value in dogs suffering from benign and malignant perianal tumours. Highly diagnostic and prognostic value in relation to benign tumour growth was also found while evaluating branched chain and acidic groups of amino acids. Serine, phenylalanine, lysine, and histidine, as well as alkaline amino acids were proved not to have diagnostic and prognostic value in dogs with the tumours. Thus, the evaluation of free amino acid concentration may serve diagnostic purposes and help in tumour malignancy differentiation. The elaborated experimental model may be used in further studies on neoplastic disease development and monitoring of applied treatment efficiency.

The study aimed at the analysis of prevalence frequency and localisation of tumours in domestic animals. The research material comprised 4,212 tumours developed in dogs, cats, horses, and exotic animals, isolated during surgery, autopsy or biopsy, performed for the purpose of histopathological diagnosis. The most numerous group involved canine tumours, including 3,585 cases (85.1%), followed by tumours in cats (532 cases, 12.6%), ferrets (34 cases, 0.81%), rats (19 cases, 0.45%), horses (15 cases, 0.36%), and rabbits (14 cases, 0.33%). A significant increase in incidence of tumours was noted, as compared to studies performed in the same region of Poland in 1957-1995 and 2000-2004 or in 2005-2008. This was particularly evident in exotic animals, in which 80 cases (1.9%) were detected in 2009-2011 but only 20 cases (1.2%) in 2005-2008. The most frequent localisation of neoplasia involved the skin, subcutaneous tissue, and mammary gland.

Efficacies of hCG and GnRH with respect to follicular diameter in Cosynch protocol were investigated. Lactating cows (n=119) received injections of GnRH and PGF2 7 d apart. Norgestomet ear implant was inserted at GnRH administration and removed prior to PGF2 injections. Presence of the corpus luteum (CL) at GnRH and PGF2 injections, and diameter of preovulatory follicle [small (SPF) <8 mm or large (LPF) ≥8 mm] at PGF2 were determined with transrectal ultrasonography (USG). The animals were randomly treated with GnRH (0.01 mg; GPG, n=62) or hCG (1,500 IU; GPH, n=57) at timed artificial insemination (TAI) 56 h after injection of PGF2α. Pregnancies were diagnosed with USG 32-35 d after TAI. Pregnancies per TAI (P/TAI) were significantly (P<0.05) lower in GPH cows (28.1%; 16/57) compared to those in GPG animals (46.8%; 29/62). P/TAI with respect to follicular size did not differ between GPG and GPH cows, although P/TAI was numerically higher in LPF (30.0%; 15/50) than in SPF (14.3%; 1/7) in GPH cows. P/TAI in cows without CL at PGF2 was 2.5 (1.0-6.7) times higher (P<0.01) in GPG (20.0%; 3/15) compared to GPH (10.5%; 2/19). In conclusion, use of hCG in milieu of the second GnRH in Cosynch+progesterone insert protocol did not have any advantage in lactating cows.

The aim of the study was to assess the influence of the oestrous cycle phase on neutrophil secretory activity and to extrapolate it to susceptibility to uterine infections. The obtained results indicate that the highest enzyme release seen in the late follicular phase (elastase release was 42.18 ±3.11% of maximal release, myeloperoxidase was 45.0 ±5.12%, and alkaline phosphatase was 44.75 ±9.0%) was related to the level of 17β-oestradiol in plasma. Similarly, a free radical generation was also the most enhanced during this phase. Significantly lower values were obtained from sows during the luteal phase in regard to both enzyme release (36.62 ±3.58% for elastase, 27.87 ±8.7% for myeloperoxidase, and 22.12 ±2.4% for alkaline phosphatase), and that of free radicals (2.28 1.6 μM/106 cells for nitric oxide and 2.47 0.6 nM/106 cells for superoxide).

The effect of feed mixture supplementation with MgCl2 on alimentary tract microflora, haematological markers, and selected parameters of reproductive performance of sows was investigated. The animals were divided into control group (K) and two experimental groups (E1 and E2). The sows from the experimental groups were receiving 1 g of MgCl2 6H20/100 kg b.w./day, which constituted 120 mg of pure magnesium, administered in a small portion of a feed mixture. The sows from group E1 were receiving MgCl2 every day for 30 d, whereas sows from group E2 for 60 d. A significant decrease in the count of E. coli, Providencia sp., and Proteus sp. was noted in faeces of the swine of both experimental groups. The study has also demonstrated a reduced number of lymphocytes and an increased number of granulocytes in blood of the sows receiving MgCl2. Feeding of pregnant sows with the addition of magnesium salt decreased the mortality rate of newborn piglets, increased the survival rate of piglets until 21 d of their life, and increased body weight values of litters originating from experimental sows, compared to the litters of control sows.

A total of 142 chicken blood samples were collected and a specific primer set was used to amplify a fragment of growth hormone locus using PCR. PCR products were digested with SacI and MspI restriction endonucleases. The amplified fragment digested with SacI enzyme revealed two “+” (wild type) and “-” (normal type) alleles with the frequency of 0.898 and 0.102, respectively. The amplified fragment digested with MspI enzyme revealed three A, B and C alleles with the frequency of 0.599, 0.102, and 0.299, respectively. Frequencies of +/+, +/- and -/- were 0.817, 0.162, and 0.021, respectively, and those of AA, AB, AC, BB, BC, and CC were 0.338, 0.113, 0.409, 0.007, 0.070, and 0.063, respectively, in the studied population. The results of 2 and likelihood ratio tests showed that this population was at Hardy-Weinberg equilibrium with respect to the marker locus. Marker-trait association analysis revealed statistically significant differences between “SacI-RFLP” genotypes for egg production and rate of laying eggs. The relationship between the molecular marker and these traits can be useful to improve the chicken breeding programmes.

The effect of spraying lysozyme solutions of varying activity on microbiological stability and organoleptic features of chicken legs with skin was investigated. Lysozyme was applied at concentrations ranging from 3,000 to 48,000 U/mL. The effect of storage time at 4oC on the total aerobic bacterial count, coli titre, occurrence of enterococci, anaerobic spore forming bacilli, and pathogenic staphylococci was analysed along with the examination of sensory quality attributes. The investigations showed that the addition of lysozyme resulted in a considerable inhibition of growth of the initial aerobic bacterial counts and a limitation of disadvantageous organoleptic changes during cold storage of the legs. The lysozyme solution with the activity of 48,000 U/mL caused a 20-fold reduction in the initial aerobic bacterial count. Sensory examination showed that samples subjected to the action of lysozyme and stored for 120 h under cold storage conditions did not differ qualitatively from fresh legs. The obtained results revealed that lysozyme might be an effective agent extending shelf-life of portioned poultry meat.

Electrocardiographic examinations were performed on 45 Silesian breed horses (group K, 3-16-year-old, 16 stallions, 29 mares) and on 25 Polish Primitive breed horses (group P, 1.5-19-year-old, two stallions, six geldings, 17 mares). Einthoven and chest leads were used. P-wave dispersion was electronically evaluated. There was no correlation between P-wave dispersion and other ECG parameters, age, sex, body mass, or month of pregnancy. The average value of P-wave dispersion for all horses was 30.77 ms (SD 3.92 ms), however in particular groups it was: in group P - 31.89 ms (SD 3.07 ms), and in group K - 30.15 ms (SD 4.27 ms). The maximum value of P-wave dispersion in the studied population was 40 ms, in groups P and K - 37,6 ms and 40 ms, respectively. The physiological value of P-wave dispersion for the group of horses was below 45 ms.

The study was conducted on 20 clinically healthy stallions, aged 3-4 years. The evaluation of the degree of adaptation of stallions to exercise loads in the course of a 100-d training programme was conducted three times: (I) - on the 1st d of training, (II) - on the 46th d of training, and (III) - on the 97th d of training. Rectal temperature (T) and number of heart and respiratory rates were measured. Blood was sampled before exercise (1), after exercise (2), and 30 min after rest (3). The following indices were determined in the blood: concentration of haemoglobin, haematocrit level, number of red blood cells, white blood cells, lymphocytes, neutrophils, eosinophils, basophils, monocytes, and platelets. Post-exertion changes in the evaluated indicators showed moderate intensity to the applied loads with respect to functional potential of tested horses. The effect of the standard 100-d training was to stimulate the body's adaptation to functional tasks, which was evidenced, among others, by lower variability of post-exertion T and the number of red blood cells. The highest T increase was found at the beginning of the programme (about 2.1%), whereas during the subsequent studies (II and III) it was significantly (P<0.05) lower (0.98% and 0.84%, respectively). The variability in red blood cell count in the course of training showed a downward trend (16.4%, 13.7%, and 11.0%, respectively). The results confirm the relatively low exercise loads and good health of young stallions during the 100-d performance test.

The aim of the study was to evaluate the effect of β-endorphin on oxygen activity of polimorphonuclear neutrophils and to investigate the total antioxidant status of blood serum in rats subjected to chronic treatment with methimazole. The study was carried out on 15 male Wistar rats weighing 270-290 g, divided into two groups. The animals from experimental group were administered a 0.05% drinking water solution of methimazole. The oxygen activity of peripheral blood neutrophils was examined using a modified RBA test. The extra-receptor effect of β-endorphin on the respiratory burst of neutrophils was evaluated. The incubation of neutrophils in the presence of phorbol myristate acetate (PMA) in the control group caused a nearly threefold increase in the oxygen activity of the cells compared to the control. Slightly higher values were obtained after addition of β-endorphin at the concentration of 10-6. Lower concentrations of this opioid (10-8 and 10-10) also stimulated oxygen activity but at a lower level than PMA. Naloxone did not affect the oxygen activity only when using β-endorphin in the concentration of 10-6. Naloxone and β-endorphin in the concentrations of 10-8 and 10-10 evoked a linear decrease in the oxygen activity of neutrophils, below the values obtained for PMA. The obtained results suggest that methimazole significantly modulates the generation of reactive oxygen species under the influence of β-endorphin, which stimulates the respiratory burst via the receptor pathway and inhibits it via the extra-cellular pathway. Methimazole also stimulates the anti-free radical defence.

The toxic effect of various doses of aflatoxin B1 on renal function was studied. Experiments were conducted on randomly chosen Wistar rats. The animals were divided into four groups. Group I received 8% alcohol intragastrically. The other groups received aflatoxin B1 in various doses. The effect of the aflatoxin on renal cells was analysed by means of determination of oxidoreductive balance and development of free radicals. The activity of antioxidative enzymes in renal tissue has decreased with an increase in the dose of aflatoxin B1. Disturbance of oxidation balance in the kidneys confirm a toxic effect of aflatoxin B1 on these organs

The aim of this study was to develop and examine a model of apoptosis and necrosis of hepatocytes induced by a damaging factor - adriamycin, correlating time after its administration with cell death type, and to investigate the localisation within the liver acinus of hepatocytes dying in these two ways. The results obtained in the present and previous studies were compared in order to make a map of cell death localisation in the liver acinus, showing the effect of time in action and dose of adriamycin. The experiment was performed on 32 female Wistar rats, divided into four groups: I and II - experimental, and III and IV - control. Adriamycin (3 mg/kg b.w.) was administered intraperitoneally to rats in groups I and II, and the rats were decapitated after four (group I) and eight (group II) weeks. Animals in control groups III and IV were given 0.5 mL of 0.9% NaCl solution, and decapitated after four and eight weeks respectively. Sections of the liver were examined with a three-stage immunohistochemical method. This method allowed to examine hepatocytes qualitatively and quantitatively for the presence of proteins involved in three types of apoptosis: induced by the mitochondrial pathway (caspase 3, 9), the intrinsic pathway related to endoplasmic reticulum stress (caspase 3, 12), and the extrinsic pathway (caspase 3, 8). One of the inflammatory markers, caspase 1, was also examined. The zonal localisation of all three types of apoptosis was assessed in the liver tissue. More oxidated hepatocytes indicated only signs of the internal mitochondrial pathway, whereas less oxidated hepatocytes induced the internal reticular pathway and the external apoptotic pathway. The period between adriamycin administration and hepatic cell investigation was a main factor of the process. A longer period post insult resulted in a more pronounced effect of the activation of apoptosis. Sections explored eight weeks after treatment with different doses of the drug (3 and 5 mg/kg in the previous study) showed a similar intensity of apoptosis.

Sixteen white Wistar female rats were divided into two equal groups. Experimental group received per os 40 mg/kg b.w. of L-arginine, every other day for 2 weeks and were decapitated after 3 weeks of the experiment. Control rats received in the same manner 2 ml of distilled water and were decapitated after 3 weeks of the experiment. The renal lesions observed under electron microscope were of focal character and concerned only the experimental group. The tubules with necrotic cells were observed among normal tubules or single normal epithelial cells of the tubular wall. The boundaries between epithelial cells of the tubule wall were blurred. The mitochondria indicated abnormal structure. Numerous lysosomes and peroxysomes with dark, homogenous content were observed. The rough endoplasmic reticulum had widened channels and was focally completely destroyed. The nucleus of damaged cells was most commonly located in one of the cell poles; its shape was changed and visibly smaller than the nuclei of normal cells. Condensation and peripherally located chromatin were noticed. The lesions observed were characteristic for apoptotic cells.

The paper presents preliminary results of the implantation of calcium sulfate type Hartform HF1 developed at the Institute of Glass and Ceramics in Warsaw. The study was conducted on 10 New Zealand White rabbits, in which after tibial osteotomy the biomaterial was implanted directly into the fracture site. The animals were divided into two groups based on the method of fracture stabilisation: intramedullary pinning using Kirschner wires in one group and acrylic external fixator in the other. After 12 weeks, bone union was observed in all animals as estimated by clinical and radiological findings. Histological tests revealed resorption of the biomaterial into the bone fracture area. It was concluded that type Hartform 1 calcium sulfate can be used as a biomaterial in veterinary orthopedics. Due to the lack of observable side effects during implantation and resorption, the material can be considered as a valuable ingredient in composite biomaterials containing calcium sulfate.

The aim of the study was to estimate prevalence of tricuspid dysplasia (TD) in dogs with respect to breed, age, sex, clinical signs, and echocardiographic findings and to compare this data with literature. TD was found in 15 dogs (6.5% of congenital cardiac disease) of 215 dogs with congenital heart defects. All dogs had right heart enlargement on thoracic radiographs, echocardiography, and electrocardiography. Doppler echocardiography revealed tricuspid valve regurgitation. Seven dogs presented no clinical symptoms to date. TD took the form of Ebstein anomaly in all Labrador Retrievers, one Boxer, and one German Shepherd dog. TD predominated in males (11 males vs. three females). The body weight of the affected dogs, with the exception of the Miniature Schnauzer, exceeded 20 kg. Two dogs (Boxer and Bull Terrier) had additional congenital cardiac lesions in the form of mitral valve dysplasia. The most affected breeds in the study were the Labrador Retriever and Boxer.

Ten mixed-breed female dogs were used in the study. Abdominal wall lifting was performed with a sterilised cotton strip. Four portal sites were used to complete gasless laparoscopic ovariohysterectomy (GLOHE) procedures. The proper and suspensor ligament, ovarian pedicle, and broad ligament of the uterus, uterine artery and vein were coagulated using bipolar electrocoagulation. After the uterine body was coagulated and cut, the end of the cervix was ligated with a loop suture. Blood samples were obtained before the surgery, immediately after the procedure, and on 1, 3, and 5 d postoperatively for the determination of interleukin-6 (IL-6) and C-reactive protein (CRP). No significant surgical complications occurred. After gasless laparoscopic ovariohysterectomy, a significantly higher serum IL-6 level was found immediately after the surgery and 1 d postoperatively when compared with the level observed after traditional laparoscopic ovariohysterectomy (LOHE). However, no significant differences were observed in CRP level between GLOHE and LOHE. GLOHE proved to be a safe and feasible procedure in bitches.

Nine dogs with Chiari malformation and syringomyelia, which were subjected to low-field magnetic resonance imaging (MRI) test, were described. The results of MRI examinations were presented. The outcomes of pharmacological therapy involving two dogs and surgical treatment of one dog were also described. The applied treatments produced positive short-term outcomes, and they eliminated the clinical symptoms of the disease.