Leishmaniasis is a life-threatening zoonosis of which dogs are the major reservoir and sandflies are the vectors. Until now, the prevalence of canine leishmaniasis (CanL) in the Slovenian dog population was unknown. Epidemiological data, eye swabs and blood samples were taken from 465 dogs born in Slovenia and older than one year. Commercial ELISA kits and real-time PCR were used. For ELISA-positive samples, an immunofluorescence antibody test (IFAT) was performed. Descriptive statistics were used to characterise the samples. The one-sample nonparametric chi-square test was used to test whether the categories of a variable were equally distributed. A 59.9% proportion of the recruited dogs had travelled to endemic regions and 62.1% of them had not been protected by insect repellents. Skin symptoms that might be CanL-related were described in 109 of the dogs’ histories (23.4%), inappetence and/or weight loss in 25 (5.4%), and anaemia, intermittent fever, and/or lymphadenopathy in 19 (4.1%). At the time of recruitment, all dogs were asymptomatic. All samples were PCR negative, nine (1.9%) were ELISA positive, but none were IFAT positive. Five of the nine ELISA-positive dogs were non-travellers. We conclude that the seroprevalence of canine leishmaniasis of 1.9 % in the autochthonous Slovenian dog population may pose a risk of endemic spread of the disease.

Horses (Equus caballus) are susceptible to tick-borne diseases. Two of them, Lyme borreliosis due to Borrelia burgdorferi and granulocytic anaplasmosis due to Anaplasma phagocytophilum were investigated in Algerian horses. The diseases have been less extensively studied in horses and results pertinent to Algeria have not been published. Blood samples were obtained from 128 horses. IgG antibodies directed against Anaplasma phagocytophilum and Borrelia burgdorferi were detected by an indirect immunofluorescence antibody test (IFAT) and ELISA. The potential effects of age, gender, breed, and health status on seropositivity were also evaluated. Using IFAT, 28 (21.8%) and 25 (19.5%) animals were positive for B. burgdorferi and A. phagocytophilum, respectively. Using ELISA, 19 (14.8%) and 33 (25.9%) animals were positive for these bacteria. The study shows that horses in Algeria are exposed or co-exposed to tick-transmitted zoonotic bacterial species.

Introduction: Numerous mutations in the bovine tumour necrosis factor receptor type two (TNF-RII) gene have been identified, but their biological consequences remain poorly understood. The aim of this study was to determine whether polymorphism in the analysed loci of the bovine TNF-RII gene is linked with the size of cell subpopulations naturally infected with bovine leukaemia virus (BLV) which serve important immune functions in the host. Material and Methods: Samples originated from 78 cows. Polymorphisms in the studied gene were determined by PCR-RFLP and DNA sequencing by capillary electrophoresis. BLV infection was diagnosed by the immunofluorescence (IMF) technique and nested PCR. Cell subpopulations were immunophenotyped with IMF. Results: Similar and non-significant differences in the average percentages of TNFα+, IgM+TNFα+, and CD11b+TNFα+ cells infected with BLV were noted in individuals with various genotypes in the polymorphic sites g.-1646T > G and g.16534T > C of the TNF-RII gene, and significant differences in the percentages of these subpopulations were observed between selected microsatellite genotypes (g.16512CA(n)). Conclusion: STR polymorphism and the number of CA dinucleotide repeats in intron 1 of the TNF-RII gene influence the frequency of TNF+, CD11b+TNF+, and IgM+TNF+ subpopulations naturally infected with BLV. Polymorphism in the gene’s other two sites do not affect the size of these cell subpopulations.

OBJECTIVE To evaluate optimal isolation of endothelial colony-forming cells (ECFCs) from peripheral blood of horses. SAMPLE Jugular and cephalic venous blood samples from 17 adult horses. PROCEDURES Each blood sample was divided; isolation was performed with whole blood adherence (WBA) and density gradient centrifugation (DGC). Isolated cells were characterized by uptake of 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate–labeled acetylated low-density lipoprotein (DiI-Ac-LDL), vascular tubule formation, and expression of endothelial (CD34, CD105, vascular endothelial growth factor receptor-2, and von Willebrand factor) and hematopoietic (CD14) cell markers by use of indirect immunofluorescence assay (IFA) and flow cytometry. RESULTS Colonies with cobblestone morphology were isolated from 15 of 17 horses. Blood collected from the cephalic vein yielded colonies significantly more often (14/17 horses) than did blood collected from the jugular vein (8/17 horses). Of 14 cephalic blood samples with colonies, 13 were obtained with DGC and 8 with WBA. Of 8 jugular blood samples with colonies, 8 were obtained with DGC and 4 with WBA. Colony frequency (colonies per milliliter of blood) was significantly higher for cephalic blood samples and samples isolated with DGC. Cells formed vascular tubules, had uptake of DiI-Ac-LDL, and expressed endothelial markers by use of IFA and flow cytometry, which confirmed their identity as ECFCs. CONCLUSIONS AND CLINICAL RELEVANCE Maximum yield of ECFCs was obtained for blood samples collected from both the jugular and cephalic veins and use of DGC to isolate cells. Consistent yield of ECFCs from peripheral blood of horses will enable studies to evaluate diagnostic and therapeutic uses.

Adipose-derived stem cells (ADSCs) are multipotent, and can be differentiated into many cell types in vitro. In this study, tissues from pigs were chosen to identify and characterize ADSCs. Primary ADSCs were sub-cultured to passage 28. The surface markers of ADSCs: CD29, CD71, CD73, CD90, and CD166 were detected by reverse-transcription polymerase chain reaction assays and the markers CD29, CD44, CD105, and vimentin were detected by immunofluorescence. Growth curves and the capacity of clone-forming were performed to test the proliferation of ADSCs. Karyotype analysis showed that ADSCs cultured in vitro were genetically stable. To assess the differentiation capacity of the ADSCs, cells were induced to differentiate into osteoblasts, adipocytes, epithelial cells, neural cells, and hepatocyte-like cells. The results suggest that ADSCs from pigs showed similar biological characteristics with those separated from other species, and their multi-lineage differentiation shows potential as an application for cellular therapy in an animal model.

Objective: To estimate the prevalence of perinuclear antineutrophilic cytoplasmic autoantibodies (pANCA) in the serum of healthy Soft Coated Wheaten Terriers (SCWTs) in the United Kingdom and to identify potential risk factors and heritability patterns associated with a positive result for pANCA. Animals: 188 SCWTs (age range, 18 months to 14.3 years). Procedures: Blood samples were obtained from SCWTs in various locations in England. Serum was tested for pANCA by use of an immunofluorescence assay, and total protein and albumin concentrations were determined. Pedigrees were evaluated to identify close relatives that had protein-losing enteropathy (PLE) or protein-losing nephropathy (PLN). Results: 39 of 188 (20.7%) dogs, including young dogs, had positive results for pANCA. Dogs had significantly higher odds of having positive results for pANCA if they had at least 1 littermate that had PLE or PLN (odds ratio, 12.1) or if they had at least 1 full sibling from another litter known to be affected with PLE or PLN (odds ratio, 4.0). Conclusions and Clinical Relevance: This study revealed a high prevalence of pANCA in the serum of a representative sample of healthy SCWTs in the United Kingdom and a significant association between positive results for pANCA and a diagnosis of PLE or PLN in a sibling.

Objective-To assess the use of CSF testing with an indirect fluorescent antibody test (IFAT) for diagnosis of equine protozoal myeloencephalitis (EPM) caused by Sarcocystis neurona. Sample Population-Test results of 428 serum and 355 CSF samples from 182 naturally exposed, experimentally infected, or vaccinated horses. Procedure-EPM was diagnosed on the basis of histologic examination of the CNS. Probability distributions were fitted to serum IFAT results in the EPM+ and EPM- horses, and correlation between serum and CSF results was modeled. Pairs of serum-CSF titers were generated by simulation, and titer-specific likelihood ratios and post-test probabilities of EPM at various pretest probability values were estimated. Post-test probabilities were compared for use of a serum-CSF test combination, a serum test only, and a CSF test only. Results-Post-test probabilities of EPM increased as IFAT serum and CSF titers increased. Post-test probability differences for use of a serum-CSF combination and a serum test only were less than or equal to 19% in 95% of simulations. The largest increases occurred when serum titers were from 40 to 160 and pre-test probabilities were from 5% to 60%. In all simulations, the difference between pre- and post-test probabilities was greater for a CSF test only, compared with a serum test only. Conclusions and Clinical Relevance-CSF testing after a serum test has limited usefulness in the diagnosis of EPM. A CSF test alone might be used when CSF is required for other procedures. Ruling out other causes of neurologic disease reduces the necessity of additional EPM testing.

Objective--To evaluate the immunologic response of a killed tachyzoite vaccine against Neospora caninum and its effectiveness in preventing fetal loss associated with experimentally induced neosporosis in sheep. Animals--30 Dorset ewes. Procedure--Ewes were randomly allocated to receive vaccination on days 1 and 60 of the study with a killed N caninum tachyzoite preparation in a commercially available adjuvant or a saline-adjuvant mixture. A ram was placed on pasture with the ewes from days 15 to 60. Blood was collected from ewes before primary and booster vaccinations and prior to experimental challenge with N caninum tachyzoite performed on day 90; sera were assessed via Neospora agglutination (NA) and immunofluorescence antibody (IFA) assays. Blood was collected from lambs before they suckled, and sera were tested for antibodies against N caninum. Results--Of the 14 vaccinated ewes that became pregnant, 12 gave birth to live-born lambs; in contrast, 5 of 11 pregnant control ewes gave birth to live-born lambs. Whereas vaccination improved fetal survival in pregnant ewes challenged with N caninum tachyzoites, it did not appear to have any appreciable effect on transmission of N caninum to offspring, as indicated by results of NA and IFA assays. Conclusions and Clinical Relevance--The N caninum tachyzoite vaccine used in this study appeared to provide protection against fetal loss associated with experimentally induced neosporosis in a high proportion of pregnant ewes.

Two cytopathic agents were isolated on porcine alveolar macrophages following inoculation with homogenates of lung tissues from pigs showing respiratory problems. These isolates were identified as porcine reproductive and respiratory syndrome (PRRS) virus isolates by indirect immunofluorescence using a PRRS virus (PRRSV) specific monoclonal antibody (MAb) and were designated as LHVA-92-1 and LHVA-92-2. Immunogold electron microscopy using a porcine PRRS positive serum pool and protein A-gold resulted in an intense labelling of aggregates of viral particles. Dark specific cytoplasmic staining of porcine alveolar macrophages infected with both virus isolates could be observed by immunogold silver staining (IGSS) using the specific MAb. This method proved effective in detecting PRRSV antigens in several ethanol-fixed tissues of piglets intranasally inoculated with the supernatants of macrophages infected with each isolate. Immunogold silver staining was also successfully used for the detection of PRRSV antigens on sections of formalin-fixed paraffin-embedded lung tissues and on frozen sections of lungs. The present results indicate that colloidal gold may be useful for the identification and immunohistochemical detection of PRRSV in tissues.

The 43 monoclonal antibody raised against feline T cells was found to react with a single-chain glycoprotein of Mr 72,000 that is present on most thymocytes, 60% of lymph node cells, 20% of splenocytes, and 45% of blood mononuclear cells. All CD4+ and CD8+ T cells were found to express the 43-reactive determinant, as did a small subpopulation of CD4-/CD8-/IgM- lymphocytes in the periphery. The 43-reactive determinant was not detected on B cells, macrophages, or other types of blood cells. The 43 antigen was phosphorylated in resting and activated T cells. Its expression was upregulated by stimulation with phorbol myristate acetate and with phytohemagglutinin. When added to concanavalin A-stimulated T-cell cultures in low concentrations, the 43 antibody was found to augment mitogenesis. The data indicate that this antibody may identify a CD5 homologue on feline T cells.