Volume 65 (2015): Issue 3 (September 2015)

The transition from intra- to extra-uterine life is one of the greatest physiological challenges that occur in the life of animals. Immediately after birth, newborn calves have to adapt to new environmental and feeding conditions. Namely, at birth a break of the thermal balance occurs, since calves abruptly pass from a 38.8°C temperature in utero to an environmental temperature that is generally lower than 20°C. Additionally, at birth, the energy intake shifts from a continuous parenteral supply of nutrients (mainly glucose) to discontinuous colostrum and milk intake with lactose and fat as the main energy sources. Therefore, the most important issues related to metabolic changes during the transition from intra- to extra-uterine life are related to maintaining the homoeothermic conditions and control of energy metabolism. Those metabolic adaptations are under control of the endocrine system that is relatively mature at birth, but still requires morphological and functional changes after birth. Key hormones whose concentrations are significantly changed around birth and are involved in an adequate adaptation of calves to extra-uterine life are those related to stress at birth (cortisol and cathecholamines), glucoregulatory processes (insulin and glucagon), thermogenesis (thyroid hormones) and growth (IGF axis).

Experimental models have strongly contributed to the comprehension of the processes of peritoneal damage that take place during peritoneal dialysis treatment in human patients. A variety of peritoneal dialysis models have been developed, mostly using rats and rabbits.

In this study we present the successful development of a custom-made improvised peritoneal catheter for an experimental non-uremic rabbit model of peritoneal dialysis.

A detailed description of the surgical technique of peritoneal catheter implantation, care and removal is provided.

This innovative approach to constructing a peritoneal catheter in rabbit animal model of peritoneal dialysis is easy, reproducible and inexpensive. The surgical technique applied provided adequate tissue samples for both light and electron microscopy.

DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked (DBY or Ddx3y) is a candidate gene for male-specific antigen. The DBY gene detected in capacitated mouse sperm codes putative ATP-dependent RNA helicase. The objective was to produce anti-predicted DBY multi-epitope fusion protein antibody, which could be used to determine male specificity of DBY. Epitope prediction is to aid the design of molecules that can mimic structure and function of a genuine epitope, is a useful tool in protein molecule design. This study predicted the DBY epitopes, prepared rabbit poloclonal antibody against DBY multi-epitope fusion protein, then investigated its immunoreactivity. The fusion protein used as the antigen consisted of three regions of DBY with greatest divergence from other family members, cloned together in-frame (with a His tag to facilitate purification). The resulting antibody recognized both the DBY1-2-3 fusion protein and an endogenous DBY protein of the same size. Furthermore, DBY protein was present (Western blot) in testis, male mouse splenocytes and brain, whereas a weaker band was present in the female brain and splenocytes, and finally, ovary produced only a barely visible protein band. Optical density of DBY protein was higher for males versus corresponding tissues from females. Finally, positive signals of DBY1-2-3 antibody were present on only ~60% of mature murine sperm (based on immunofluorescent staining and flow cytometry), in accordance with the expected proportion of Y-bearing sperm. We hypothesized that our antibodies recognized a specific epitope present in subpopulations of mouse sperm. Therefore, we concluded that anti-DBY1-2-3 antibody could be an alternative way of producing antibodies to DBY protein. Furthermore, this novel DBY antibody against a multi-epitope artificial antigen has potential for both investigating male-specific binding of DBY and as a new method of sex selection.

The aim of this study was to investigate the influence of prebiotic inulin diet intervention on the activity of β-glucuronidase and counts of coliforms and lactobacilli in fresh caecal digesta, cytokine levels (IL-6, IL-8) and transcription nuclear factor kappa beta (NFkB) activities in the colon tissue and blood samples of rats with dextran sulphate sodium (DSS) induced acute colitis. Male Sprague-Dawley rats (8 per group) were randomly divided into three groups: Control, Acute colitis and Prebiotic. Colitis was induced using 5% DSS in drinking water for 7d. DSS application significantly increased the activity of β-glucuronidase (p<0.001), increased counts of coliform bacteria and decreased lactobacilli count (p<0.05) in comparison to the control group. Serum and tissue levels of IL-6 and IL-8 as well as tissue NFkB activities showed an increased expression in the acute colitis group. These results correspond to the average value of the disease activity index score (DAI) and revealed the maximum DAI score (6.5) in the acute colitis group. A decrease in the DAI score (4.13) was observed after application of the prebiotic inulin. Inulin diet intervention positively modified the number of microorganims and decreased β-glucuronidase activity. Colon tissue activities of NFkB were significantly suppressed (p<0.001). The synthesis of proinflammatory cytokines IL-6 (p<0.01) in the serum and in the colon tissue, as well as tissue IL-8 (p<0.05) in the prebiotic group were downregulated. These findings indicate that the dietary intake of inulin suppressed the expression of the observed markers, which play an important role in the inflammatory process, which predisposes to the use of inulin in the prevention or treatment of acute colitis in human and veterinary medicine.

Apart from being against the law, illegal waste dumping also poses a threat to human health and to the environment. Solid and decomposing waste is an ideal breeding ground for a number of rodents, insects, and other vermin that pose a health risk through the spread of infectious diseases. The main objective of this study was to survey disease vectors and rodents for the presence of Leishmania sp. from waste sites along the Istrian Peninsula in Slovenia and Croatia.

During the survey five sandfly (Phlebotomus neglectus, P. perniciosus, P. papatasi, P. mascitii, Sergentomyia minuta) and five rodent species were collected (Rattus rattus, Mus musculus, Apodemus agrarius, A. flavicollis and A. sylvaticus).

Sandflies and rodents were screened using a molecular probe to amplify an approximately 120 bp fragment of the kinetoplast DNA (kDNA) minicircle for the detection of Leishmania sp. parasites. Leishmania infantum DNA was detected in the spleen of one juvenile black rat (R. rattus). Despite few published records on Leshmania sp. infection in black rats, the addition of our record highlights the importance of further investigation into the frequency and distribution of such occurrences so that we may better classify the role of rodents as potential reservoirs of leishmaniasis in the Mediterranean basin.

The aim of this study was to investigate the etiology of subclinical mastitis (SCM) in dairy Jersey cows with the use of bacteriological and molecular identification methods. In the study 121 Jersey and 78 hybrid Jersey cows with SCM were observed in the Samsun district of Turkey. A total of 411 California mastitis test (CMT) positive milk samples from these animals were examined bacteriologically. The prevalence of subclinical mastitis was 54.75% and 67.2% in Jerseys and hybrids, respectively. On bacteriological examination, a total of 92 strains were isolated from 411 milk samples. The most prevalent bacteria were Staphylococcus spp. (69.56%). Among them 24 isolates were Staphylococcus aureus (26.08%) the other isolates were Streptococcus dysgalactiae (23.91%), Enterococcus spp. (3.26%) and Streptococcus agalactiae (3.26%). All strains were identified with bacteriological culture methods, as well as by Polymerase Chain Reaction (PCR). Gram-negative bacteria were not isolated. In conclusion, the etiology of SCM in full blood and hybrid Jersey dairy cows in Samsun and the prevalence of bacteria were determined. The relatively high prevalence of SCM indicates the potential need for the consideration of some factors contributing to the formation of mastitis (e.g. management) as well as bacterial agents. The present study and further studies may be useful to develop mastitis vaccines by means of providing true vaccine strain sources.

Salmonella enterica serotype Enteritidis is an important alimentary pathogen that recently gained special attention due to the ability of a large number of strains to form biofilms. Qualitative testing of biofilm forming ability was performed by observing the morphotype of the colonies on Congo Red agar and by conducting the pellicle test, while quantitative testing was carried out by Cristal violet assay on microtiter plates. A total of 14 isolates of S. Enteritidis were tested for biofilm forming ability, while Salmonella Enteritidis ATTC 13076 was used as the reference strain. Based on the morphotype of colonies cultivated on Congo Red agar at 25°C incubation temperature, among tested isolates three morphotypes were detected – red, dry and rough (rdar), brown, dry and rough (bdar) and smooth and white (saw). Half of the tested isolates demonstrated rdar morphotype. All isolates that showed a specific morphotype at this incubation temperature also formed the corresponding type of pellicle at the air-liquid interface. Additionally, comparing OD (optical density) values obtained by crystal violet test between groups of isolates that represent one of the three detected morphotypes (rdar, bdar and saw), statistically significant differences were detected. Based on OD values obtained by crystal violet test at both applied incubation temperatures, isolates were classified into three categories, regarding their ability to form biofilms: strong, moderate and weak biofilm producers. By comparing the amounts of the biofilms formed after 48h at 25°C and 37°C, statistically significant differences were noted (P<0.05). In this research we presented micrographs and a reconstruction of three-dimensional projections of biofilm developing phases of rdar morphotype isolates, which were obtained using confocal laser scanning microscopy.

The aim of our study was to stablish the possible alternations in fatty acid composition of brain phospholipids in rats on a standard and high-fat diet supplemented with buckwheat leaf and flower mixture (BLF) and subsequent possible beneficial effects of BLF. Four months old male Wistar rats were randomly divided into five experimental groups fed a standard diet, standard diet supplemented with 5% BLF, high-fat diet, high-fat diet with full-period (13 weeks) of 5% BLF supplementation and high-fat diet with partial-period (7 weeks) of 5% BLF supplementation. Gas-liquid chromatography was performed to analyze the fatty acids in hexane lipid extracts of whole rat brains.

Supplementation with BLF did not induce significant changes in fatty acid composition of whole brain phospholipids in rats fed the standard diet. In rats on high-fat diet concomitant (full-period) BLF supplementation increased eicosapentaenoic acid (20:5n-3, EPA), total n-6 and n-6/n-3 ratio, and decreased the percentage of oleic acid (18:1n-9) and estimated activity of Δ-9 desaturase. When BLF application was postponed (partial-period) in the case of developed hyperlipidemia, a decrease of stearic acid (18:0) accompanied with an increased estimated Δ-9 desaturase activity was observed. Regardless of BLF supplementation all high-fat diet-fed groups showed an elevated percentage of linoleic acid (18:2n-6, LA) and a reduced estimated Δ-6 desaturase activity.

BLF contributes to the maintenance of stable fatty acid composition of brain phospholipids and supports normal brain function in high-fat diet rats, with more positive effects when BLF was applied before hyperlipidemia developed. This could be the mode of buckwheat health beneficial effects on the brain.

The aim of this study was to evaluate the effects of 60 days of rose-hip and grapeseed dietary supplementation of a balanced home-cooked diet on serum oxidative stress parameters: ROMs, MDA and FRAP in army service dogs before and after regular physical exercise. The dogs were fed a balanced cooked diet as instructed by army standards until the initial blood sampling in June. Thereon the dogs were randomly allotted to 4 groups according to the dietary regime: dogs maintained on a balanced cooked diet according to army standards, branded dry dog food, cooked diet with added 500 mg rose-hip extract, and cooked diet with added 100 mg grapeseed extract for a 60 day period from June to September after which all 4 groups were fed the standard cooked meal diet. Sampling was performed at the beginning of the experiment (June), 60 days from the start of the treatment (September) and finally 60 days after the end of supplementation (November). Statistical analysis of the results included descriptive statistical parameters: mean (M), standard deviation (SD), and variation coefficient (CV%). In order to test the statistical significance of the differences between treatments a multifactor variance test (ANOVA) was performed for ROM, MDA and FRAP and the combined effects of diet, exercise and time period were observed. The initial (in June) increase in MDA and ROMs after exercise indicates the presence of oxidative stress 30 minutes after exercise. However, the antioxidative effects of rose-hip and grapeseed extracts are not conclusive, as multifactor ANOVA testing of time, diet, and exercise factors did not reveal for MDA statistically significant differences either at 60 days of supplementation nor 60 days after withdrawal of the supplements. Only one distinct exception was recorded for the prolonged antioxidative effects reflected in significantly decreased (p<0.01) ROMs before and after exercise in dogs fed the branded dry food 60 days after the end of such dietary regime (November). FRAP values tend to be higher (p>0.05) after exercise compared to before exercise in all experimental groups in November, indicating on a possible redistribution and upregulation of endogenous antioxidants during the experiment.

The aim of this study was to investigate the influence of marination, on the growth of Salmonella spp. in contaminated broiler breast fillets during storage. In the conducted study, broiler breast fillets were inoculated with a cocktail of different Salmonella strains and afterwards marinated in different solutions of table salt, sodium tripolyphosphate and/or sodium citrate. The total count of Salmonella spp. was examined on the 0, 3^rd, 6^th and 9^th day of storage. Broiler breast fillets salted in 6% solution of table salt were used as the control. Proximate composition and salt content, pH value and a_w value, were determined as the meat quality parameters and parameters which can affect environmental conditions for bacterial growth, as well. Compared to initial contamination, Salmonella spp. count in marinated and salted fillets did not change significantly (p<0.001) until the 3^rd and 6^th day of storage, respectively, but it increased significantly on the 9^th day of storage. Marination of broiler breast fillets in different solutions of table salt, sodium tripolyphosphate and/or sodium citrate had a significant influence(p<0.05; p<0.01) on pH and a_w value, moisture, ash and salt content in marinated broiler meat, but there was no significant influence (p>0.05) on protein and fat content in broiler meat. According to the results obtained it can be concluded that marination of broiler breast fillets in solutions containing table salt, sodium trpolyphosphate and/or citrate, in some way, can prolongate the lag phase of Salmonella spp. growth, where sodium citrate is more effective than sodium tripolyphosphate.

The objective of this study was to assess cell-free fermented culture broth of 9 Lactobacillus plantarum strains as antibiotic alternatives for the inhibition of in vitro growth of enterotoxigenic Escherichia coli (ETEC) and Shiga toxin-producing E. coli (STEC) isolated from postweaning pigs with colibacillosis and edema disease in 2014. A total of 10 ETEC and 5 STEC strains isolated from postweaning pigs were tested in antimicrobial susceptibility tests. ETEC and STEC strains used in this study possessed at least one of fimbrial, enterotoxin, and Shiga-toxin genes when tested by polymerase chain reaction. Among 9 L. plantarum strains tested, 3 strains (Lp 2-05, 2-06, and 1-03) showed inhibitory activity of in vitro growth against 10 ETEC strains (100%) and 7 strains (Lp 6-13, 3-06, 3-05, 7-01, 2-06, 1-03, and 6-05) showed inhibitory activity of in vitro growth against 5 STEC strains (100%). Three strains (Lp 3-05, 2-06, and 1-03) showed inhibitory activity of in vitro growth against 10 ETEC (100%) and 5 STEC (100%) strains. The results of this study show the inhibitory activity of cell-free fermented culture broth of L. plantarum against ETEC and STEC isolated from postweaning pigs with colibacillosis and edema disease.

Rhabdomyosarcoma is a malignant tumor of the skeletal muscles that is occasionally observed in free-living and pet birds. A case of malignant rhabdomyosarcoma in the musculature of the right wing of a captive female cockatiel (Nymphicus hollandicus) was investigated. The clinical aspect and radiographic images suggested an invasive neoplasm. Histologically, the tumor was composed of a patternless array of large primitive polygonal cells or sheets of bizarre cells with giant nuclei and atypical mitoses set in a collagenous stroma. Anaplastic cells were poorly differentiated and exhibited notable cellular and nuclear pleomorphism. Nuclei showed extreme variability in number, size, shape, chromatin distribution and nucleolar size and number. The cross striations were encountered in elongated multinucleated cells known as “strap cells” and in ovoid cells known as “racquet cells” and numerous mitotic figures were also observed. Neoplastic cells were positive for muscle actin and negative for desmin, suggesting the diagnosis of anaplastic rhabdomyosarcoma. It is the first reliable record of a rhabdomyosarcoma in a cockatiel (Nymphicus hollandicus) in Romania.