Marteiliosis in mussels: a rare disease?

Among 1280 cultured and wild adult Mediterranean mussels, Mytilus galloprovincialis, collected over a 1-year surveillance period from the Slovene Adriatic Sea, 0.3% were histologically positive for the presence of Marteilia spp. The infection was concentrated in winter. Employing the molecular methods of PCR, cloning, DNA restriction and sequencing, only Marteilia refringens type M was detected in all the infected mussels. Although all life-cycle stages of M. refringens severely infected digestive glands, only sporadic disruption of epithelial cells of digestive tubules and focal destruction of digestive tubules were observed in the infected mussels. This was the first detection of M. refringens in M. galloprovincialis from the Slovene Adriatic Sea with the lowest prevalence reported to date. In addition, our results highlight the need for sequencing to complement the established PCR-RFLP analysis for correct parasite typing.

Distribution and amount of cathepsin B in gentamicin-induced acute kidney injury in rats.

The aim of our study was to investigate how the distribution and amount of cathepsin B change during acute kidney injury. The research was done on a rat model of acute kidney injury that was induced by nephrotoxic antibiotic gentamicin. Gentamicin was injected at a dose of 40 mg/kg body weight (the first treated group) and 80 mg/kg body weight (the second treated group) for 14 days. Control groups received injections of physiological saline only. One day after the last injection, animals were euthanized, dissected and kidney samples were taken and fixed in 10% buffered formalin. Tissue sections were stained with haematoxylin and eosin, periodic Acid Schiff (PAS) and Oil-red-O. Immunohistochemistry was used for the demonstration of cathepsin B. Vacuolar degeneration of the proximal convoluted tubules was the most prominent pathologic lesion found in the first treated group, while necrosis prevailed in the second treated group in the same localisation. In both treated groups significantly weaker immunohistochemical reaction for cathepsin B was noticed in the proximal convoluted tubules in comparison to the control groups (P < 0.05). The decrease of positive reaction was the largest in the proximal convoluted tubules of the outer renal cortex. Stronger positive reaction for cathepsin B, although not statistically significant, was found in the proximal straight tubules (P > 0.05), as well. However, more numerous cathepsin B-positive large granules appeared in the proximal straight tubules of the second treated group then in the second control group (P < 0.05). We can conclude that the amount of cathepsin B in the affected proximal convoluted tubules significantly decreases along the increased severity of the histopathological lesions of the proximal convoluted tubuls, the amount of enzyme in the well preserved proximal straight tubules increases and more cathepsin B-positive large granules appear in the cytoplasm.

Skin biopsies predict acute graft-versus-host disease after small bowel transplantation in pigs.

Intestinal transplantation is being increasingly performed to treat patients with irreversible intestinal failure. The major cause of intestinal graft failure is graft-versus-host disease (GVHD) that represents a life-threatening complication after small bowel transplantation (Itx). The purpose of this study was to assess the diagnostic and prognostic value of skin biopsy histological changes for acute GVHD after Itx in pigs. Thirty-four Large White pigs were divided into three groups: Group 1 with Itx only, Group 2 with Itx and donor bone marrow infusion (Itx BM) and Group 3 (control group - before the operation). Animals received tacrolimus-based immunosuppression from day 0 to day 30 postoperatively. Skin and small bowel biopsies were histologically assessed, analysed and classified from grade 1 to 4 on postoperative days 15, 30, 45 and 60. There was a strong correlation between the histological grading values of skin biopsy changes and the histological grading values of small bowel biopsy changes (Kendall's tau_b is 0.855 for the Itx group and 0.730 for the Itx BM group). The significant correlation found between skin and small bowel histological changes suggests the prognostic value of skin biopsies after Itx. In conclusion, our findings emphasise the diagnostic and prognostic value of skin biopsy analysis for acute GVHD after Itx.

Simultaneous Occurrence of Pancreatic Adenocarcinoma and Brunner's Gland Adenoma in a Siberian Tiger (Panthera tigris altaica).

We describe a case of pancreatic adenocarcinoma and Brunner's gland adenoma in an 18-year-old male Siberian tiger (Panthera tigris altaica) from the Ljubljana Zoo. The tiger was humanely destroyed due to weakness and progressive weight loss. Necropsy examination revealed a large, grey, predominantly necrotic mass replacing the major part of the pancreatic body. Microscopically, the mass was unencapsulated, poorly demarcated, highly cellular and composed of highly pleomorphic, cuboidal to tall columnar cells with basal, round or oval, moderately anisokaryotic nuclei with prominent nucleoli and moderate to large amounts of eosinophilic cytoplasm. The tumour was diagnosed as pancreatic tubular adenocarcinoma with infiltration into the duodenum and mesentery. There were tumour emboli in mesenteric blood vessels and hepatic metastases. The non-affected part of the pancreas exhibited severe chronic pancreatitis. In addition, one firm white neoplastic nodule was observed in the duodenal wall. The nodule was set in the tunica muscularis and was unencapsulated, well demarcated and highly cellular, and consisted of a closely packed layer of normal Brunner's glands and a centrally positioned group of irregularly branched tubules with small amounts of debris in the lumen. The neoplastic nodule was diagnosed as Brunner's gland adenoma. The present case is, to the best of our knowledge, the first report of concurrent pancreatic adenocarcinoma and Brunner's gland adenoma, most probably induced by chronic pancreatitis, either in man or animals.

Ivermectin pharmacokinetics in lactating sheep.

Ivermectin (IVM) concentrations in plasma and milk were studied in six Istrian Pramenka dairy sheep after a single subcutaneous dose of 0.2 mg/kg b.w. of IVM in the early lactation period to describe IVM disposition in milk and to evaluate the transfer of IVM residues via milk to suckling lambs. Large inter-animal in concentration variability of IVM in both matrices was observed. The highest overall concentration was found in the same animal: 21.7 microg/l of H(2)B(1a) in plasma on the second day and 44.9 microg/kg of H(2)B(1a) in milk on the first day after the drug was administered. The mean time in which IVM concentrations fell below the limit of detection for the whole ewe group was 22 and 23 days for plasma and milk, respectively. Time course of IVM concentration in milk was following the time course of IVM concentration in plasma, with an overall mean+/-S.D. of milk/plasma ratio of 1.67+/-0.50 for the first 7 days of the experiment. A mean of 0.7% of the dose was excreted through milk. Individual pharmacokinetic parameters were determined by fitting a one-compartment model to the milk and plasma concentration-time profiles. Mean t(max), c(max), t(1/2k(e)) and AUC values for plasma data were: 1.70+/-0.65 days, 11.88+/-6.96 microg/l, 2.85+/-1.97 days and 63.99+/-28.34 microg day/l, respectively, and for milk: 1.28+/-1.07 days, 22.67+/-18.27 microg/l, 3.56+/-2.01 days and 114.60+/-60.41 microg day/l, respectively. The highest level of concentration in suckling lamb plasma, 0.36 microg/l of H(2)B(1a), was slightly above the limit of determination. The mean lamb to ewe ratio of areas under the plasma concentration-time curve for the first 5 days was 0.02. On the basis of obtained results, it can therefore be claimed that indirect IVM exposure of the suckling lambs via milk was negligible.

Defence reaction in dental pulp after pulp capping and partial pulpectomy in dogs.

The dental pulp was capped indirectly or directly, or partial vital pulpectomy was performed on the 12 functionally most important teeth of 24 beagle dogs. For pulp capping, calcium hydroxide was used, followed by zinc phosphate as a lining, and the preparation was restored with amalgam or composite material. Histological sections were prepared and examined for degree and type of pulp inflammation (hyperaemia, pulpitis, necrosis or gangrene). Degenerative changes of dental pulp (vacuolation, calcification, amyloid or hyaline changes) were also determined. For indirect pulp capping the width of predentine in crown and root dental pulp was measured, and for direct pulp capping and partial pulpectomy (vital pulpotomy) the width and quality of the dentinal bridge were graded. The radical method of partial vital pulpectomy of the coronal part of the dental pulp in dogs produced better quality and continuity of the dentinal bridge than the less radical method of direct pulp capping. This was particularly obvious in small single root teeth such as incisors.

Apparent cross-infection with a single strain of Malassezia pachydermatis on a pig farm.

Twenty-nine isolates of Malassezia pachydermatis were recovered from a single farm of 100 pigs in Croatia. In contrast, 290 farm pigs from other locations (northern parts of Croatia and Slovenia) yielded only two non-lipid dependent isolates of M. pachydermatis using the same swabbing procedure. Ten of the 29 isolates from a single farm had their identity confirmed by karyotyping, and were typed by restriction fragment length polymorphism (RFLP) analysis. All but one of these isolates sub-typed were indistinguishable, one isolate produced a slightly different RFLP profile. Control isolates recovered from dog skin gave RFLP profiles that were easily distinguished from those produced by the pig isolates. These results suggest that a single strain of M. pachydermatis had colonised this pig herd.

An attempt to estimate students' workload.

Following the recent introduction of the European Credit Transfer System (ECTS) into several European university programs, a new interest has developed in determining students' workload. ECTS credits are numerical values describing the student workload required to complete course units; ECTS has the potential to facilitate comparison and create transparency between institutional curricula. ECTS credits are frequently listed alongside institutional credits in course outlines and module summaries. Measuring student workload has been difficult; to a large extent, estimates are based only upon anecdotal and casual information. To gather more systematic information, we asked students at the Veterinary Faculty, University of Ljubljana, to estimate the actual total workload they committed to fulfill their coursework obligations for specific subjects in the veterinary degree program by reporting their attendance at defined contact hours and their estimated time for outside study, including the time required for examinations and other activities. Students also reported the final grades they received for these subjects. The results show that certain courses require much more work than others, independent of credit unit assignment. Generally, the courses with more contact hours tend also to demand more independent work; the best predictor of both actual student workload and student success is the amount of contact time in which they participate. The data failed to show any strong connection between students' total workload and grades they received; rather, they showed some evidence that regular presence at contact hours was the most positive influence on grades. Less frequent presence at lectures tended to indicate less time spent on independent study. It was also found that pre-clinical and clinical courses tended to require more work from students than other, more general subjects. While the present study does not provide conclusive evidence, it does indicate the need for further inquiry into the nature of the relationship between teaching and learning in higher education and for evaluation of the benefits (or otherwise) of more "self-directed" study.

Enzootic nasal adenocarcinoma of sheep in Slovenia.

SUMMARY Clinicopathological and electron microscopical findings of eight cases of enzootic nasal adenocarcinoma of sheep, diagnosed solely in one big flock in Slovenia between years 2001 and 2003 are described. All affected sheep were female, their mean age was 4.5 +/- 1.5 years and they either belonged to the Istrian pramenka breed (five sheep) or were crossbreeds (three sheep). Tumours that arose from the ethmoid area of the nasal cavity were unilateral in six cases (75%) and bilateral in two cases (25%). All tumours were classified as adenocarcinomas by histopathological examination and they displayed either a combination of tubular and papillary growth or less often solely tubular proliferation. No metastases were detected in regional lymph nodes, brain or other organs. Electron microscopical studies performed on the reprocessed paraffin-embedded tissues revealed the presence of the virus-like particles with an average diameter between 70 and 90 nm.

Outbreak of tuberculosis caused by Mycobacterium caprae in a zoological garden.

In the autumn of 2004, tuberculosis caused by Mycobacterium caprae occurred in a zoo in Slovenia. A dromedary camel (Camelus dromedarius) was killed after a history of progressive emaciation. Necropsy findings indicated disseminated tuberculosis, which was confirmed by cultivation of M. caprae. Consequently, a tuberculin skin test was performed in all epidemiologically linked animals and another dromedary camel and six bison (Bison bison) were positive and killed. Mycobacterium caprae was isolated from two bison while M. scrofulaceum and Mycobacterium spp. were found in two other bison, respectively. The second dromedary camel was found to be negative for mycobacteria under both microscopic and culture tests. The isolates were investigated with commercial identification kits, IS6110 PCR, IS6110 restriction fragment length polymorphism analysis, spoligotyping and mycobacterial interspersed repetitive units typing. Genotyping results revealed that the dromedary camel and the two bison were infected by the same M. caprae.

Tolerance of therapeutic doses of abamectin and doramectin in Istrian Pramenka sheep.

Avermectin endectocides are very effective and safe veterinary drugs, when used at recommended doses. Adverse reactions are described in some species and breeds of animals. In this study, the effects of therapeutic doses of abamectin and doramectin on some haematological and biochemical parameters in Istrian Pramenka sheep are discussed. In the pilot trial, we compared selected haematological and blood biochemical parameters of an experimental sheep flock (40 sheep) with the reference values. Then, two groups of 12 sheep (and their suckling lambs) were chosen from the experimental sheep flock. Each group was subdivided into a control (six animals) and treated (six sheep and their six suckling lambs) groups. We compared haematological and biochemical parameters between control and treated group before subcutaneous administration of abamectin or doramectin (0.2 mg/kg b.w.) and on days 15 and 42 after treatment. In addition, animals were observed for neurological signs. We detected some significant differences (P < 0.05) in some haematological and biochemical parameters between control and treated animals, but none of them appeared to be of clinical importance. No neurological symptoms were observed. Therefore, abamectin and doramectin might be well tolerated in Istrian Pramenka sheep.

Morphometric analysis of AgNORs in tubular and papillary parts of canine mammary gland tumors.

OBJECTIVE: To test the value of the silver staining nucleolar organizer regions (AgNORs) technique on canine mammary gland tumors using image analysis and to estimate differences in AgNOR parameters in structurally different parts of canine mammary gland tumors. STUDY DESIGN: Analysis was performed on 13 complex type and 10 simple type malignant canine mammary gland tumors containing tubular and/or papillary structures. Ten normal mammary glands were used as controls. Morphometric analysis was done by a computer-assisted image analysis system and consisted of evaluation of nuclear area, number and area of AgNORs per nuclear area, ratio of nuclei with five or more AgNORs, nuclear perimeter, area fraction between nuclear area and area of AgNORs, and area, equivalent diameter, volume equivalent sphere, perimeter and circularity of a singular AgNOR. RESULTS: Distinct differences were detected between normal and malignant mammary gland tissue for all measured parameters. There were no significant differences between the tubular and papillary parts of the same tumor or between the tubular and papillary parts of complex and simple type tumors. CONCLUSION: Despite the fact that no significant differences were found for AgNOR parameters between papillary and tubular structures of mammary gland tumors, the results of grouping tumors by the number of AgNORs indicate that this might help with classification of canine mammary gland tumors.

Immune suppression of commercial broilers in Croatia, Slovenia, and Bosnia and Herzegovina from 1981 to 1991.

A continuous decline in immune responses to Newcastle disease (ND) vaccine was observed in commercial broiler flocks in Croatia, Slovenia, and Bosnia and Herzegovina beginning in 1982. Floating mean haemagglutination inhibition (HI) titres declined from log(2) 4 in 1983 to a low of log(2) 2.4 in 1986, then were log(2) 2.9 in 1990. Several causes of the decline were discounted, leaving mycotoxins in feed and infection with chicken anaemia virus (CAV) as the two most likely causes. Mycotoxins in feed could not be evaluated retrospectively, but archival tissues were available from Croatia and Slovenia. Tissue sections were examined by in situ hybridization for CAV. Whereas only one chicken from early in the decade was infected, all but one of the chickens from late in the decade were. The increase in CAV detection correlated inversely with ND HI titres. Whereas this correlation does not establish cause and effect, CAV cannot be eliminated as a contributory cause of immune suppression.

Effects of dopaminergic drugs on inflammatory bowel disease induced with 2,4-dinitrofluorbenzene in BALB/c mice.

The aim of the study was to demonstrate the effects of dopaminergic drugs on 2,4-dinitrofluorbenzene (DNFB) induced experimental inflammatory bowel disease (IBD) in previously sensitized BALB/c mice. The number and extent of ulcerations and erosions, the intensity of haemorrhages, oedema, and accumulation of neutrophils and eosinophils within colonic lamina propria and submucosa were scored and statistically evaluated. The 180 BALB/c mice, were allocated into three equal groups. The mice in the first experimental group were treated with domperidone (DP), a peripheral dopamine (DA) antagonist. The mice from the second experimental group were treated with bromocriptine (BC), a dopamine agonist. The mice from the control group were treated with an equivalent volume of normal saline in the same manner. Ten animals from each group were killed on days 1, 2, 3, 5 and 10, subsequent to the challenge enema of DNFB solution. Gross and microscopic examination of the colon was performed. Treatment with BC resulted in clinical improvement and decreased mortality rate by 2 of 60 (3%), while domperidone treatment increased mortality rate to 12 of 60 (20%) compared with the controls [4 of 60 (6%)]. The analysis of the microscopic lesions indicated that the beneficial effects of BC were the result of maintenance of vascular integrity.

Subchronic liver injuries caused by microcystins.

The subchronic effects of cyanobacterial lyophilizate (CL) containing microcystins on liver were investigated in female New Zealand rabbits. Sterilised CL containing microcystins was injected i.p. Liver toxicity was assessed by histological examination of liver samples. Non-invasive magnetic resonance imaging (MRI) of liver was also performed in order to assess changes in the homogeneity of liver tissue. Subchronical intoxication with microcystins caused morphological changes of liver tissue that were also detected by use of MRI. Histological analysis showed that changes seen on MRI represent liver injury characterised with fatty infiltration and periportal fibrosis. This demonstrates that subchronic exposure to microcystins can lead to liver degeneration, which can easily be detected in vivo by use of MRI.

Gizzerosine-induced histopathological lesions in broiler chicks.

1. The aim of this study was to investigate pathomorphological changes in broiler chicks fed with different doses of gizzerosine, a substance produced during the heat treatments of fish meal. 2. The experiment was carried out in Ross broiler chicks which were divided into three groups: group A received 100% of non-medicated commercial mash for broiler chicks. During an experimental 5-d period, 50% of commercial mash was replaced with unheated fish meal (0.65 ppm gizzerosine) in group B and in group C with heated fish meal (1.15 ppm gizzerosine). Fourteen chicks from each group were killed every day. Samples of gastrointestinal and lymphoid organs, lung, pancreas, liver, brain and kidney tissue were sampled for histopathological analysis. Organs were embedded in paraffin and stained with hematoxylin-eosin stain and using periodic acid-Schiff reagent (PAS) and Sudan III (frozen sections). 3. Necropsy did not reveal notable differences between treated groups. There were no significant histopathological changes in immunocompetent organs nor in the lungs, the pancreas, the kidney or the brain. Sharply demarcated multiple vacuoles were observed in the myocardium in group C toward the end of the experiment. In group C, the prevalent changes in the gizzard and the proventriculus were slight to severe cuticle erosions and oedema of the lamina propria with or without multiple vacuoles, respectively, towards the end of the experiment. The most prominent changes toward the end of the experiment were dispersed cell vacuolisation in duodenal, jejunual, ileal and caecal lamina propria in group C. 4. In conclusion, it should be emphasised that extra-gizzard gizzerosine-induced lesions are probably not mediated by H2-receptor stimulation, but could be a consequence of cellular hypoxia.