Volume 57 (2013): Issue 2 (June 2013)

The article describes the recent data dealing with the fatty acid content in cow, goat, and sheep milk. A large body of evidence demonstrates that fatty acid profile in goat and sheep milk was similar to that of cow milk. Palmitic acid was the most abundant in milk. Goat milk had the highest C6:0, C8:0, and C10:0 content. Sheep milk was the richest source of conjugated linoleic acid and α-linolenic acid. Ewe’s milk had lower value of n-6/n-3 then goat and cow milk.

The study describes construction of five recombinant very virulent (vv) and very virulent plus (vv+) strains lacking meq and viral telomerase (vTR). Deletion of both copies of meq and vTR was achieved by Red E/T recombination in GS1783 E. coli cells. The constructed bacterial artificial chromosome (BAC) clones reconstituted in chicken embryo fibroblasts were examined by immunofluorescence assay to compare the features of recombinant strains with wild-type viruses. The results demonstrated that recombinant BAC strains caused slightly reduced cytophatic effect and decreased level of the fluorescence obtained from the monoclonal antibody in comparison to the parental viruses. Generation of recombinant BAC clones may provide more detailed information on the function of Marek's disease virus oncogenes and the potential use of recombinants for the preparation of the new vaccine against Marek’s disease.

The aim of the study was to evaluate the status of bovine viral diarrhoea virus (BVDV) infection in selected dairy herds in Poland with the use of commercial enzyme linked immonosorbent assay for the detection of specific antibodies (BVDV-Ab ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) for the detection of viral RNA, using bulk tank milk (BTM) samples. Two hundred and thirty-one samples of BTM were collected from 99 dairy herds in Poland. The herds were divided into four different classes according to the Swedish system of classification. The results showed that 70.7% of herds were BVDV antibodypositive. High levels of antibodies in 52.85 % (37 herds in class 3) of all antibody positive herds indicated acute BVDV infection. Thirty five samples with the highest antibody levels were tested by RT-PCR and five of them were positive for viral RNA. Dairy herds in Poland have high levels of antibodies against BVDV in BTM. Since no vaccination was implemented in the herds tested, high seroprevalence of BVDV antibodies in cattle indicates the widespread of BVDV infection in Polish cattle.

Fluorescence polarisation assay (FPA) was evaluated as a potential tool improving specificity of serological diagnosis of brucellosis in cattle and pigs. The evaluation was performed by comparing the results of FPA with the results of rose Bengal test (RBT), serum agglutination test (SAT), complement fixation test, and indirect ELISA when problematic sera, regarded as false positive, were tested. Four hundred and seventy-five sera, including 201 porcine and 274 bovine samples, reacting positively in at least one classical serological assay were used. Only six bovine sera were FPA positive (two positive in SAT and RBT and four positive in SAT only). Different situation was observed when porcine sera were examined. Out of 201 sera, 109 were also positive in FPA. The studies confirmed that in cattle FPA enables to reduce highly the number of false positive reactions for brucellosis. On the other hand, in pigs, the method is a little more specific in comparison to other methods applied.

The aim of the study was to assess selected methods of storage and preservation of horse faecal samples on the detectability of eggs and survival rate of larvae of strongylid nematodes. Physical and chemical factors were found to have a significant effect on the egg detectability and survival of the larvae. The comparison of control and variously preserved samples revealed that each storage method affected the reliability of the results to some extent. Short-lasting, three-day freezing resulted in a decrease of detectability from 100% to 94.1%, with an equally significant decrease in the mean number of eggs per one gram of faeces, from 1,238.97 to 983.82. Extending the freezing period from 3 to 14 d had a highly significant effect on the prevalence, which was then 85.3%, while the EPG (eggs per gram) value decreased more than twice, compared to control samples, and was 503.68. Thirty-day freezing had no further effect on the parasitological indices, which did not differ significantly from those obtained after a fortnight of freezing. Storing samples in 4% formalin and 40% ethanol had a significantly more negative effect on the detectability of strongylid eggs. The prevalence in alcohol-preserved samples was 91.2% and the EPG was 247.79; the values for formalin-preserved samples were 88.2% and 153.68. The way of preserving faecal samples was also found to affect the viability of strongylid larvae.

The aim of the study was to isolate and identify staphylococci from the intestinal samples of 24 trouts from East Slovakian waters. Moreover, their relation to antimicrobials was tested. The count of staphylococci in the trouts reached in average 4.0 x 101 colony forming units per gram. Twenty-two strains were identified by validated species-specific oligonucleotide array targeting the manganese-dependent superoxide dismutase-sodA gene. The identified strains were allotted to five species (Staphylococcus warneri, S. haemolyticus, S. epidermidis, S. hominis, S. pasteuri) clustered to three groups according to 16S rRNA sequences (S. epidermidis group, S. haemolyticus group, S. warneri group). These species belong to coagulase-negative staphylococci. All strains were sensitive to eight antibiotics out of 14 tested; the majority of strains were also sensitive to the remaining six antibiotics with the inhibitory zones from 13 to 41 mm. The strains were also sensitive at least to three enterocins of nine tested. Strains SW24/2, SHo 19/2, SHo20/1, SP19/1 were sensitive to eight of nine enterocins. All strains were sensitive to Ent A, P=EK13, and Ent EM41 with activity 100-6400 AU/mL. Strains SHo19/2 and SP19/1 were sensitive to Ent 2019 with activity up to 25600 AU/mL.

In the study, antimicrobial resistance and plasmid profile of Vibrio alginolyticus strains isolated from diseased European sea bass (Dicentrarchus labrax, L.) collected from a commercial farm located on the Agean Region coast of Turkey, were investigated. The agent was identified by classical biochemical tests and the API 20NE system. Bacterial strains were tested for resistance to different antimicrobials by disc agar diffusion method and minimal inhibitory concentration (MIC) at two different incubation temperatures (22 ± 2ºC and 28 ± 2ºC for 24 to 28 h), and screened for plasmid DNA by agarose gel electrophoresis. It was found that 15 strains were susceptible to kanamycin but they revealed resistance to ampicillin, bacitracin and streptomycin at 22ºC. The strains were resistant to bacitracin and streptomycin but 14 strains were found to be intermediately resistant. One strain was resistant to ampicillin at 28ºC; two strains showed sensitivity to kanamycin, whereas 12 strains were intermediately resistant, and one strain was resistant to the same antibiotic at 28ºC. The strains demonstrared susceptibility to low concentrations of chloramphenicol, kanamycin, and trimethoprim (MICs of ≤ 8 μg/mL) at 22ºC. The examined strains were found to be susceptible to chloramphenicol (MICs of ≤8 μg/mL) and trimethoprim (MICs of ≤8 μg/ mL) at 28ºC. The strains harboured 2-3 plasmids, with sizes ranging from 68 to 126 kb.

A total of 135 L. monocytogenes strains isolated from slaughtered cattle and beef meat were tested by the pulsed field gel electrophoresis (PFGE). The AscI restriction analysis revealed a genetic heterogeneity among investigated isolates since 31, 9, and 35 profiles were distinguished among hide, carcass, and meat strains, respectively. The PFGE profiles of the isolates were also analysed in relation to serotypes, virulence genes, and antimicrobial resistance. It was shown that strains displaying the same PFGE type were of the same serotype while correlation between pulsotype and antimicrobial resistance was poor. The obtained results suggest that a cross-contamination between bovine hides and carcasses may occur during the slaughter process. Moreover, identification of identical PFGE types among L. monocytogenes found during a study period may suggest a common source of contamination or presence of persistent strains able to survive for a long time. These results emphasise the importance of molecular subtyping methods, including PFGE, in monitoring and tracking pathogen contamination along food chain.

A method for simultaneous determination of 13 organophosphorus pesticide residues in milk samples has been developed and validated. The method is based on the extraction of the sample with acetone and petroleum ether, cleanup by gel permeation chromatography, and solid phase extraction, and determination by gas chromatography with flame photometric detection. The recovery, investigated by analysing blank milk samples spiked with azinphos, chlorfenvinphos, chlorpyrifos, chlorpyrifos-methyl, diazinon, fenitrothion, methacrifos, methidathion, parathion-methyl, pyrazophos, and triazophos at concentrations of 10, 20, and 30 μg/kg, and with parathion and pirimiphos-methyl at concentrations of 25, 50, and 75 μg/kg, ranged from 73.1% to 106.2%. Performance characteristics, such as repeatability and within-laboratory reproducibility expressed as coefficient of variation were less than 20%. Measurement uncertainty was lower than 22% for all validated organophosphorus pesticides. The limits of quantification were 10 μg/kg for all compounds and allowed determination of residues at, or even below, the maximum residue limits set by the European Union. The satisfactory z-score results of international proficiency tests confirm good accuracy, reproducibility, and reliability of the developed method.

For the measurement of tulathromycin distribution in swine plasma an accurate and reliable analytical method was developed. The extraction was performed with oxalic acid buffer (pH=4.0). Plasma samples were cleaned up by solid phase extraction procedure using polymeric cartriges. Chromatographic separation was achieved on a C 18 analytical column using mobile phase consisting of acetonitrile, 0.1% formic acid in gradient mode. Detection was carried out by liquid chromatography tandem mass spectrometry. Azithromycin was used as internal standard. The method has been successfully validated. The recovery from spiked samples ranged from 94% to 110%. The limit of detection was 2 ng/mL and the limit of quantification was 5 ng/mL. The method was developed to investigate the pharmacokinetics of tulathromycin in swine plasma. Applicability of the method was tested with plasma from swine administered with a single dose of tulathromycin.

The occurrence and concentrations of organochlorine pesticides and polychlorinated biphenyls (PCBs) were determined in 141 adipose tissue samples of wild boars, roe deer, and red deer from different regions of the country, collected in 2010/2011. The determinations of HCH isomers (α-, β-, and γ-HCH), HCB, DDTs (p,p’-DDT, o,p’-DDT, p,p’-DDE, and p,p’-DDD), and PCBs (six indicator PCB congeners 28, 52, 101, 138, 153, and 180) were carried out using the capillary gas chromatography. The mean concentrations of DDTs expressed on the fat basis ranged from 0.241 mg/kg (wild boar) to 0.032 mg/kg (red deer), and for PCBs from 0.015 mg/kg (wild boar) to 0.010 mg/kg (roe deer). The mean contribution of p,p’-DDE to DDTs and PCB 153, PCB 138, and PCB 180 to PCBs exceeded 80% and 90% respectively, in all game animal samples studied. Higher contents of chlorinated hydrocarbons were detected in game animals originating from industrial regions.

The objective of the study was to determine expression of gene of insulin receptor (INSR) in adipose tissue of postpartum dairy cows fed diets containing different amounts of energy at the antepartum period. Healthy pregnant dairy cows (n=45) on 21^st d of the antepartum were divided into three groups differing in diet composition, namely: control group fed a normal diet, high energy group fed a high energy diet, and low energy group fed a low energy diet. Twenty-one days after parturition, INSR gene expression in adipose tissue was determined by internally controlled reverse transcriptase PCR. The level of INSR mRNA in adipose tissues of cows fed the high energy diet was substantially lower than that in cows fed normal or low energy diets. A relatively higher level of INSR mRNA in the adipose tissue of cows fed low energy diet may be beneficial for gluconeogenesis and lipogenesis, which can relieve an energy negative balance. Reduced level of INSR mRNA in adipose tissue of cows fed high energy diet indicates that the response to insulin has significantly decreased.

The aim of the study was to evaluate the immune effects of genetically modified (GM), insect resistant corn (MON810) expressing toxin protein of Bacillus thuringiensis, and glyphosate-tolerant soybean meal (Roundup Ready MON-40-30-2), which are used as the feed mixture components in domestic animals. The study was conducted on 60 pigs (36 fatteners and 24 sows), 20 calves, 40 broilers, and 40 laying hens. Each species was divided into four basic nutritional groups: group I (control) - conventional feed, group II - feed consisted of GM soybean meal and non-modified corn, group III - non-modified soybean meal and GM corn, group IV - GM soybean meal and GM corn. Moreover, in the experiment on fatteners two additional groups were formed: group V - animals fed both conventional soybean meal and bruised grain, and group VI - GM soybean meal and conventional bruised grain. The results of study did not reveal any significant effect of feed mixtures containing GM components on the immune response in all animals regardless of their species and technological producing groups.

β-glucan (Macrogard^®) was administrated to enhance the immunity and growth of Cyprinus carpio fry. The whole body homogenate of fish sampled with one week intervals during 2^nd - 6^th weeks post hatching was assayed for the total protein content, lysozyme activity, α-2-macroglobulin content, classical complement pathway activation, and weight gain. After the 3^rd week of experimental feeding, the total protein content and the classical complement activity of fish fed β-glucan supplemented diet were higher than controls. Significantly higher lysozyme activity and α-2-macroglobulin levels were noted in group fed β-glucan at the 2^nd and 3^rd week of diet application. It was demonstrated that β-glucan enriched feeding increased the immunity of common carp at the earliest stages of their development.

The objective of the study was to assess ratio of oxidation to antioxidation in the blood of horses in selected equine diseases. The study was conducted on 47 Polish half-breed horses (36 mares, six stallions, and five geldings) aged 2 to 16 years. Nineteen mares were diagnosed with endometritis, seven horses with symptoms of colic, and six with upper respiratory tract infection. The remaining clinically healthy horses (n=15) served as control. The following parameters were measured: oxygen metabolism in neutrophils using a chemiluminescence (CL) method, total antioxidant status (TAS) of blood plasma, as well as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase activity in whole blood. The results demonstrate increased CL of neutrophils in mares with endometritis, as well as in horses with symptoms of colic. This finding was associated with decreased values of SOD, GPx, and TAS (chiefly in mares with endometritis), which suggests oxidative stress. These results underscore the importance of a broader use of oxidant/antioxidant status assessment in studies of the pathogenesis and pathomechanisms of equine diseases.

The aim of the research was to obtain “native” referential values for the indicators of the red and white blood cell systems and platelet indicators in the clinically healthy foals of purebred Arabs and Polish cold-blooded breed, in the period from birth till weaning. The morphological examination was performed ex tempore with help of the automatic haematological analyser. The obtained results demonstrate that the majority of haematological indicators of the red blood cell and white blood cell systems of warm-blooded foals, especially in the first 2-3 months of their life, are statistically higher. It can be assumed that within the indicators of the white blood cell system, the statistically important differences between the groups occur rarely, as do mutual (interfactor) correlative connections or the trends comparable for both groups.

The group of 36 warm-blooded half-bred horses (18 stallions and 18 mares) and their riders (20 men and 16 women), who ended three-day-events, were selected for the study. The horses were aged 4 to 6 years, while the riders were 19 to 34-year-old. The saliva samples were collected after each phase of the competitions. The cortisol concentration was determined using an immunoassay method. The following factors were considered: type of competition, horse sex, and rider gender. In horses, the statistically important correlation was found between the results obtained for the dressage and cross-country, for the cross-country and show jumping, and for the dressage and show jumping. An analogous comparison for the riders suggests a statistically significant correlation between the data obtained for the cross-country and show jumping. Comparing the data of horses and their riders, a significant correlation coefficient was found for the cross-country group of woman and the dressage group of men. In conclusion, the salivary cortisol level in individual horses in each phase of three-day-event was found to be repetitive. Therefore, the salivary cortisol test is demonstrated to be a useful method to evaluate the horse response to each type of competition during three-day-events.

The operational method (V-cut pelvic ostectomy) was applied on seven dogs. Pubic symphysis was cut in an inverted “V” fashion and a bone gap occurred between pelvic halves. The gap was reduced by contracting both sides of the pelvis. A new pubic symphysis developed. Results were evaluated with radiographs and different clinical and orthopedic tests. Radiographic measures between mutual parts of pelvic bone showed a marked increase in Norberg angles and diminishing in Bardens, Barlow, and Ortolani orthopedic tests after operations. Principal conclusion: V-cut pelvic ostectomy was found to be a successful operation technique in treatment of canine hip dysplasia.

Radiographic examination of the distal radius and ulna of dogs showed changes in metaphyseal areas of both bones characteristic of hypertrophic osteodystrophy (HOD). The serum of the patients showed increased activity of alkaline phosphatase (ALKP) and elevated concentrations of Ca and P. Oral application of vitamin C and injection of sodium salt of hyaluronic acid in the bolus caused a reduction in swelling, disappearance of pain symptoms around the joints, and increased appetite. Radiographic examinations performed on the 60^th d of treatment revealed the withdrawal of the changes characteristic for HOD. The chosen biochemical parameters tested during the treatment showed a decrease in comparison to the values before treatment, namely: ALKP by 27.7%, Ca by 22.2%, P by 12.3%, and Mg by 2.4%. Clinical condition of the animals was defined as good or very good. The use of hyaluronic acid and vitamin C in the treatment of HOD in the first stage of the disease proved to be highly successful, resulting in the inhibition of the disease course.

The presence of pamidronate during local use of bisphosphonates (BP)-enriched bone cement was determined. The question was whether pamidronate implanted into the bone cement is eluted. The study was performed on 10 probes of BP-enriched bone cement located in 0.9% NaCl. The probes were incubated for 3 and 6 weeks. Ion-pair HPLC was used for the detection of pamidronate. Then, capillary electrophoresis was applied for quantitative analysis of pamidronate in the 3^rd and 6^th week after incubation. The presence of pamidronate, eluted from BP-enriched bone cement into 0.9% NaCl solution 3 and 6 weeks after incubation, was demonstrated. These results may explain the changes in the level of cytokine RANKL and bone turnover marker osteoprotegrin in rats’ serum treated with BP-enriched bone cement 3 and 6 weeks after surgery. The possibility of effective local use of BP-enriched bone cement in veterinary medicine was underlined. The results, and the former conducted research, point out that the clinical applications of BP-enriched bone cement in vivo may have some validity in the future.

The electrocardiographic (ECG) exam has been routinely used in veterinary cardiology with reference values available for the right lateral recumbent position. Many situations require the exam to be performed in the standing position. The study presents that the quality of the ECG recording is comparable for the standing and right lateral recumbent positions, as well as significant differences of the P, S and T waves recorded in two positions.

The influence of docosahexaenoic (DHA) acid obtained from a new generation of eggs on ventricular electrical function of 24 pigs with experimentally induced tachycardiomyopathy was described. Two groups were formed, 12 pigs had experimentally evoked tachycardiomyopathy and were getting standard feed (control group TIC). Twelve pigs with tachycardiomyopathy received feed with an addition of a dietary supplement containing phospholipids isolated from new generation of eggs at a dose of 1,000 mg of DHA/animal/24 h (experimental group TIC). Electrophysiological study was carried out from an external programmer immediately after implantation of the pacemaker. All the tests were carried out in general anaesthesia. After 8 weeks of fast ventricular pacing at 170 bpm in pigs receiving phospholipids obtained from eggs, a statistically significant shortening of ventricular refraction time was observed during sinus rhythm and also during the ventricular pacing of 130 bpm, and 150 bpm in the group of pigs fed standard feed. The ventricular refraction time in the sinus rhythm was significantly longer after 12 weeks of fast pacing and in the pacing at 130 bpm and 150 bpm it was significantly longer after 8 weeks of fast pacing. Phospholipids containing high percentage of n-3 polyunsaturated fatty acids (PUFA) obtained from new generation of eggs may contribute to the shortening ventricular refraction period after its oral administration. The n-3 PUFA obtained from a new generation of hen eggs may be an alternative to fish oil source of DHA and other polyunsaturated fatty acids.

A growing interest in enzymatic growth promoters prompted the authors of the study to investigate the effects of a stimulating enzymatic complex on ejaculate characteristics of boars. The enzymatic complex comprising five proteases (proteinases - endopeptidases) and two peptidases (exopeptidases) was obtained by fermentation from Streptomyces fradiae. This complex was added to boar diets for 3 months at the initial doses of 90 (group E1) and 120 (group E2) g/ton of feed in the first week, followed by 40 and 60 g/ton, respectively, in the following weeks. Evaluation was based on the assessment of key ejaculate characteristics (volume, sperm concentration, total sperm count, primary and secondary sperm defects). The enzymatic complex improved the microscopic and macroscopic characteristics of boar ejaculate by increasing sperm concentrations and total sperm counts, and decreasing the volume of the analysed ejaculates without significant changes in the spermiograms of primary and secondary defects.

The resazurin reduction test (RRT) was subjected to spectrophotometric analysis to evaluate the quality of canine semen. Twenty four samples of canine semen were analysed. The absorption peaks for resazurin and resorufin were determined at 615 and 580 nm, respectively. The RRT ratio (RRT_sperm-the ratio for samples containing spermatozoa, RRT_plasma-the ratio for samples containing seminal plasma) was calculated by dividing the absorbance at 580 nm by the absorbance at 615 nm. Spearman’s correlation test was used to determine the significance of correlations between the analysed sperm parameters and the results of the resazurin reduction assay. The RRT ratio was highly correlated with sperm motility (r=0.68, P<0.01), progressive sperm motility (r=0.61, P<0.01), the subpopulation of cells with rapid velocity (r=0.72, P<0.01), and the subpopulation of cells with medium velocity (r= -0.54, P<0.05). A negative correlation was observed between the reducing capacity of seminal plasma vs. sperm with plasma membrane integrity (r= -0.60, P<0.01) and sperm with normal morphology (r= -0.58, P<0.01). The RRT test can be used as an additional tool for evaluation of the quality of canine semen.

The aim of the study was to analyse densitometric and geometric parameters of the compact bone and the trabecular diaphysis of the femur of ostriches with the use of computed tomography. The method has been applied in ostriches for the first time. The study was conducted on the femur of 14-month-old ostriches of both sexes. The obtained results were analysed statistically using one-way ANOVA, with Bonferroni correction for multiple-group comparisons. It was stated that the mineral content (BMC) in the middle of the diaphysis of the femur was significantly higher in males (465.52 mg/mm) than in females (417.67 mg/mm). Similar tendencies were observed for the total bone area (TOT_ A), trabecular area (TRAB_ A), cortical area (CRT_ A), and strength strain index (SSI). The average area of the CRT_A for both sexes was 568.62 mm² and was lower than the TRAB_A - 884 mm², at P≤0.05. Moreover, it was found that strength strain index (SSI) of the diaphysis depended on the TOT_A and the TRAB_A. Both features positively correlated with SSI. Pearson’s correlation coefficient for both features was r = 0.97, at P≤0.01. Further research concerning the dynamics of changes of densitometric and geometric parameters occurring with age and involving both sexes is recommended, as there are many locomotion problems in ostriches, especially older ones, used intensively on farms for many years.