Volume 66 (2016): Issue 3 (September 2016)

The Institute of Virology, Vaccines and Sera Torlak was established in 1927, while the first vaccine was produced in the Institute in 1930. Vaccines production implies using experimental animals, including mice, in in-process controls. The laboratory mice which have been in use in Torlak Institute from the very beginning belong to Swiss albino outbred stock. This stock, which has been in use for more than 80 years contains a large number of mice maintained at all times, was recently named Intor:Swiss. Biological characteristics of Intor:Swiss stock, are presented in this paper for the first time. Taking into account the presented characteristics, the Institute Torlak’s Swiss mice are suitable for use in pharmaceutical studies, vaccine development research and basic research, as well as in toxicological studies. The publication of data on the Intor:Swiss mice represents a contribution to the international scientific community, since it offers the possibility for obtaining an additional outbred mouse stock for research.

The effect of the probiotic strain Lactobacillus plantarum LS/07 on intestinal bacterial enzyme activities – β-glucuronidase (β-GLUCUR), β-galactosidase (β-GAL), and β-glucosidase (β-GLU) in the prevention of cancer, atherosclerosis and dysbiosis was investigated. Male Sprague-Dawley rats were randomly divided into 12 experimental groups: C (control group), AT (atherosclerotic group), CC (carcinogenic group), and then each group in combination with antibiotics and probiotics individually and each group in double combination on antibiotic and probiotic. In the control group the β-glucuronidase activity did not change throughout the experiment. High fat diet in the atherosclerotic group significantly increased the activity of β-glucuronidase (p<0.001) and β-glucosidase (p<0.01). Azoxymethane application in the carcinogenic group significantly increased β-glucuronidase (p<0.01), but reduced β-glucosidase (p<0.01). Daily application of probiotics individually and in double combination with antibiotics increased the activity of β-galactosidase, and β-glucosidase, and positively decreased the level of β-glucuronidase. In the control antibiotic group β-glucuronidase was significantly increased (p<0.05), and β-glucosidase decreased (p<0.01) which can be caused by a change of microflora in favor of coliform bacteria. These finding indicate the positive effects of probiotic Lactobacillus plantarum LS/07 which allows its use in disease prevention in human and veterinary medicine.

For the purpose of precise antibiotic susceptibility testing it is necessary to clearly distinguish Pseudomonas and Stenotrophomonas genera, considering acquired resistance of Pseudomonas species, as well as the intrinsic resistance of Stenotrophomonas species. This is why in the identification of the 51 isolates originated from fish, the following methods were used: standard PCR, 16S rRNA gene sequencing, and MALDI-TOF.

The results of the standard PCR test, 16S rRNA gene sequencing and MALDI-TOF analysis confirmed 35 strains to belong to the Pseudomonas genus. Standard PCR test and VITEK MS device confirmed that 10 strains belong to Stenotrophomonas maltophilia species.

Three strains were positive in both standard PCR tests for Pseudomonas and Stenotrpohomonas. 16S rRNA gene sequencing identified these 3 strains to be 99% Pseudomonas sp. and 99% Stenotrophomonas sp. VITEK MS first identified these three strains as 99% Stenotrophomonas, and in the repeated identification it identified them as 99% Pseudomonas. MALDI TOF/TOF 4800 Plus device identified these strains as Stenotrophomonas.

Three strains were negative in both standard PCR tests for Pseudomonas and Stenotrpohomonas. 16S rRNA gene sequencing identified these 3 strains to be 99% Pseudomonas sp. and 99% Stenotrophomonas sp. VITEK MS first identified these three strains as 99% Stenotrophomonas, and in the repeated identification it identified them as 99% Pseudomonas. MALDI TOF/TOF 4800 Plus device identified these strains as Stenotrophomonas.

Although modern test methods that have very high specificity (PCR, 16S rRNA gene sequencing, MALDI TOF) were used in this study, precise differentiation between Pseudomonas and Stenotrophomonas species for 6 isolates could not be reached using the above mentioned methods.

The aim of the present study was to: 1) check whether it would be possible to detect cows susceptible to mastitis at an early stage of their utilization based on selected genotypes and basic production traits in the first three lactations using ensemble data mining methods (boosted classification tress – BT and random forest – RF), 2) find out whether the inclusion of additional production variables for subsequent lactations will improve detection performance of the models, 3) identify the most significant predictors of susceptibility to mastitis, and 4) compare the results obtained by using BT and RF with those for the more traditional generalized linear model (GLZ). A total of 801 records for Polish Holstein-Friesian Black-and-White cows were analyzed. The maximum sensitivity, specificity and accuracy of the test set were 72.13%, 39.73%, 55.90% (BT), 86.89%, 17.81%, 59.49% (RF) and 90.16%, 8.22%, 58.97% (GLZ), respectively. Inclusion of additional variables did not have a significant effect on the model performance. The most significant predictors of susceptibility to mastitis were: milk yield, days in milk, sire’s rank, percentage of Holstein-Friesian genes, whereas calving season and genotypes (lactoferrin, tumor necrosis factor alpha, lysozyme and defensins) were ranked much lower. The applied models (both data mining ones and GLZ) showed low accuracy in detecting cows susceptible to mastitis and therefore some other more discriminating predictors should be used in future research.

In this study, determination of enterococcus species that were isolated from mastitic milk samples, investigation of their susceptibilities to antibiotics and identification of the existence of resistance genes in resistant strains were conducted. The specimens consist of 600 mastitic milk samples that were collected from 242 cows. Isolation of enterococcus was carried out in selective media and 94 (15.6%) Enterococcus spp. were isolated. A total of 94 species of Enterococci were identified using both sequencing and polymerase chain reaction (PCR). Enterococcus spp. isolates belong to 5 different species (E. faecalis, E. faecium, E. durans, E. hirae, E. mundtii) in sequence analysis and 4 different species (E. faecalis, E. faecium, E. durans, E. hirae) were identify by PCR method with specific primers. Analyzing 94 enterococcus strains by antibiotic sensitiveness test a high rate of resistance to tetracycline in 77 (81.9%) isolates was shown. The tet resistance genes were identified as follows: 54 were tetM positive, 23 were tetK positive and 17 were positive on tetM and tetK. Resistance to erythromycin was established in 27 (28.7%) isolates (25 ermB) while the chloramphenicol resistance gene was found in 10 (10.7%) of isolates and the cat gene was identified in nine samples and one isolate was resistant to vancomycin (1.06%) with the VanA gene confirmed. In conclusion, it was shown that E. faecalis has the biggest role in enterococcus originated mastitis and these strains were found to be mostly resistant to tetracycline. One vancomycine resistant isolate that had the VanA gene was also determined.

The presence of pseudorabies virus (PrV), porcine parvovirus (PPV) and porcine circovirus 2 (PCV2) was examined in sixty samples (spleen and lymph nodes) and thirty samples of sacral ganglia collected from non-vaccinated swine by virus isolation and polymerase chain reaction (PCR). Using PCR method PrV was detected in three samples, PPV in seven samples and six samples were found positive for PCV2. The phylogenetic analysis of the nucleotide sequences of three PrV isolates identified in this study showed high similarity and significant clustering within the PrV genotype I strains such as Kaplan and Bartha isolated from pigs in Hungary, strain Becker isolated in USA and strain Kolchis isolated in Greece. The nucleotide sequences of two PPV isolates showed high level of similarity with the strain Challenge isolated from pigs in UK, strain Kresse isolated in USA and strains 77 and LZ isolated in China. The phylogenetic analysis of the nucleotide sequences of two PCV2 isolates showed high level of similarity and significant clustering within genotype PCV2b strains such as NIVS-3, NIVS-5 and NIVS-6 isolated in Serbia, strain 3959 isolated in Austria, strain PM165 isolated from pigs in Brasil, and strain XT2008 isolated in China. The results of our study present the molecular characterization of PrV, PPV and PCV2 identified in swine in Republic of Montenegro. Besides that, these results confirmed that PCR is a very useful method for rapid detection of these viruses in subclinically infected swine.

The aim of this study was to examine the effect of added sunflower, linseed or soybean meal to a standard pig fattening diet on the chemical composition, fatty acid profiles of meat and backfat, and on the oxidative stability of backfat from pigs. The content of saturated (SFA) and monounsaturated fatty acids (MUFA) was significantly lower (P<0.01), while content of polyunsaturated fatty acids (PUFA) was significantly higher (P<0.01) in the diet with added linseed. The feeding study was conducted on 30 pigs, with groups of 10 pigs fed one of the three different diets for 46 days before slaughter. There were no differences in the chemical composition (protein, water, fat, minerals) of meat from pigs fed the different diets. The content of SFA and MUFA was significantly higher, while the average PUFA content was significantly lower (P<0.01) in meat and backfat of pigs fed diet with added linseed. The content of n-6 fatty acids was significantly lower and n-3 fatty acids significantly higher, with more desirable n-6/n-3 ratio, in meat and backfat of pigs fed diet with added linseed. Malondialdehyde in the backfat of pigs fed diet with added linseed was significantly lower than that in the other two diet groups after the tissue was stored frozen at −20°C for 3, 6, 9 and 12 months.

Lambs were divided into 3 groups of 6 animals each. For 35 days lambs were fed a diet including 2% rapeseed oil, 1% fish oil and 0.1% carnosic acid (the control group) or two experimental diets supplemented with 0.35 mg ∙ kg⁻¹ Se as selenized-yeast (SeY) (the SeY diet) or selenate (the selenate diet). Muscles (Musculus longissimus dorsi (MLD) and Musculus biceps femoris (MBF)), ruminal fluids and microbiota were collected from each lamb. SeY supplementation most effectively stimulated the accumulation of straight-chain volatile fatty acids (VFAs), iso-branched-chain VFAs, CO₂ and CH₄ in the ruminal fluid. The contents of CO₂, CH₄ and VFAs including straight-chain VFAs with the exception of iso-branched-chain VFAs were most effectively reduced by the selenite diet. The control diet most efficiently increased the concentration sums of odd-saturated fatty acids (odd-SFAs) and iso-SFAs in microbiota. The SeY diet most efficiently reduced acetic acid to propionic acid ratio in the ruminal fluid. The selenate diet improved animal performance by reducing ruminal concentrations of CH₄ and CO₂. The SeY diet and especially the selenate diet reduced the biohydrogenation to C18:0 when compared with the control diet. The selenate diet more efficiently reduced the concentration sums of all SFAs (ΣSFAs) and all fatty acids (ΣFAs) in MLD and MBF than the SeY diet, which most effectively increased the concentrations of ΣSFAs and ΣFAs in MLD and MBF. The selenate diet most effectively increased the body mass gain of lambs.

Ischemic acute kidney injury is characterized by renal vasoconstriction, filtration failure, tubular obstruction, tubular backleak and overproduction of angiotensin II and reactive oxygen species. Considering this complexity, the aim of our study was to investigate the effects of angiotensin II type-1 receptor blocker - Losartan and superoxide anion scavenger - Tempol, in a combined treatment on acute kidney injury in postischemic hypertensive rats.

The experiment was performed in anesthetized, adult male spontaneously hypertensive rats. The right kidney was removed and the left renal artery was occluded for 40 minutes. Experimental groups received combined treatment (Losartan + Tempol) or saline in the femoral vein 5 minutes before, during and 175 minutes after clamp removal.

Hemodynamics and biochemical parameters were measured and kidney specimens were collected 24h after reperfusion. Histological examination was performed by optical microscopy.

Combined treatment improves renal haemodynamics parameters which were exacerbated due to acute kidney injury. Acute kidney injury significantly decreased creatinine and urea clearance and increased lipid peroxidation in the plasma. Treatment with Losartan and Tempol induced a significant increase of creatinine and urea clearance. Lipid peroxidation in the plasma decreased and glutathione peroxidase enzyme activity in the erythrocytes increased after Losartan + Tempol treatment. This combined treatment reduced cortico-medullary necrosis and tubular dilatation in the kidney.

Our results indicate that synergism of Losartan and Tempol treatment could have beneficial effects on blood pressure and kidney function, during postischemic acute kidney injury development in experimental hypertension.

Caseous lymphadenitis (CLA), also called pseudotuberculosis, is a chronic and highly infectious disease of sheep and goats, subject to obligatory registration in the European Union. The EU law also applies to special territories, which include the Falkland Islands. On the Falkland Islands 99% of sheep and goats are slaughtered in one slaughterhouse, overseen by veterinary authorities, and approved for export to EU countries (mainly the UK). The export season begins about the middle of January and usually lasts 3-4 months. In the years 2013-2015 were examined 162 317 sheep, including 39 971 lambs, 35 357 yearlings and 86 989 mature rams. During the study lung tissue clippings were collected. During the 2013 season lesions were observed in 8 025 of animals (13.9% CLA). In 2014 lesions were noted in 7716 with CLA accounting for 13.91% and in 2015 lesions were noted in 5743 with CLA accounting for 11.7%. Further analysis of cases of CLA on the Falkland Islands seems interesting, as they are British overseas territories and therefore part of the European Union.

Gastroesophageal intussusception is a rare pathology characterized by an acute or intermittent invagination of the stomach into the lumen caudal to the thoracic esophagus, the pathogenesis is not well understood, although it is likely multifactorial. The mortality is high, associated with a late diagnosis and a rapid deterioration. A 4-month-old male domestic shorthair kitten presented for evaluation of intermittent respiratory distress and abdominal discomfort with two weeks of evolution. Based on clinical history, clinical sings and imaging studies a diagnosis of intermittent gastroesophageal intussusception was established. The reduction of the intussusception was performed with endoscopy maneuvers and with medical and nutritional management for some days, but this failed and finally was reduced with a percutaneous gastropexy tube to prevent recurrence with a good long-term outcome. Gastroesophageal intussusception in cats is associated with megaesophagus or hiatal hernia, and should be considered as a differential diagnosis in patients with chronic vomiting, regurgitation or respiratory disease. Survey and contrasted thoracic radiographs can be used to identify gross anatomical abnormalities, but the definitive diagnosis should be made by esophagogastroscopy with the possibility of reducing the intussusception. The use of percutaneous gastropexy provides other possible option to conventional surgery for the management of this pathology in cats.

Gastric dilatation and volvulus is a life-threatening condition characterized by rapid accumulation of food and gases that cause displacement and distension of the stomach. The large and giant, deep-chested breeds of dogs are at higher risk for developing the gastric dilatation and volvulus. Uncommonly, it can also develop in cats, but it is also described in free-range polar bears.

A case of gastric dilatation and volvulus in a brown bear (Ursus arctos) is described in this paper. This case was characterized by lack of any previous symptoms, sudden death, as well as macroscopic findings during necropsy - twisted distended stomach, congested displaced spleen and necrotic gastric wall. According to the available data this is the first described case report of the gastric dilatation and volvulus in brown bear (Ursus arctos).