A novel subgroup among genotypes of equine arteritis virus: genetic comparison of 40 strains

The authors determined partial nucleic sequences of the variable regions of open-reading frame (ORF5) from 151 nucleotide to 668 nucleotide and deduced amino acid sequences of 518 nucleotide respectively of 20 equine arteritis virus (EAV) isolates. About 19 Hungarian and one Austrian EAV strains were subjected to sequence analysis, the further data of 20 EAV strains: six North American and 14 European were obtained from the GenBank. Comparative sequence analysis of the Hungarian EAV strains indicated that among the three variable regions the first has been affected mostly by point mutations. Genetic comparison of the Hungarian strains with other EAV isolates from western Europe and North America (including the Bucyrus reference strain) has been performed on the aforementioned genome region. Besides the already known genetic subgroups of EAV; phylogenetic analysis revealed a novel subgroup comprising mainly Hungarian strains. Compared with the Bucyrus virus, the overall sequence divergencies of the examined Hungarian strains ranged from 81.47 to 90.73% at nucleotide and from 84.88 to 91.86% at amino acid level. Epizootiological studies have shown that the significant part of the EAV strains having been existed in Hungary before and in 2000 belong to this unique cluster (II.D) which was not indicated in former phylogenetic studies. After 2000 new EAV strains emerged in Hungary, one of them causing abortions or neonatal death. The previously dominant 'Hungarian' EAV genotypes were replaced by these new strains belonging to North American and European subgroups (I.A, I.B, II.A, II.B). The anamnesis of these cases revealed connections with persistent virus shedder stallions, those were imported to the country after 2000 or have been infected abroad. One of these Hungarian stallions became the source of abortion storms in Hungarian studs.     

The pharmacokinetics of vedaprofen and its enantiomers in dogs after single and multiple dosing

Vedaprofen is a chiral nonsteroidal anti-inflammatory drug that has been developed as a gel formulation for oral administration to dogs and horses. The pharmacokinetics of vedaprofen and its enantiomers were studied in beagle dogs after single (intravenous solution and oral gel) and multiple (oral gel) dosing at a dosage of 0.5 mg/kg body weight. Plasma concentrations of vedaprofen and its enantiomers were analysed by HPLC. The plasma protein binding of vedaprofen was studied by ultrafiltration. The absorption of vedaprofen was rapid (tmax 0.63 +/- 0.14 h) and almost complete after oral administration (bioavailability 86 +/- 7%). The terminal half-lives after intravenous and oral administration, 16.8 +/- 2.2 and 12.7 +/- 1.7 h respectively, were of the same order of magnitude. Enantioselective analysis showed that the R(-) enantiomer predominated in plasma. The change in the plasma time course of the plasma R(-)/S(+) enantiomer concentration ratio over time was similar after single intravenous and oral dosing, with R(-)/S(+) ratios in the AUC of 1.7 +/- 0.5 and 1.9 +/- 0.2 respectively. Plasma protein binding of vedaprofen and its enantiomers was high (> 99.5%). Vedaprofen is absorbed rapidly from the gastrointestinal tract, has a high bioavailability and does not accumulate in plasma in dogs following repeated oral administration.     

Lymphocytes of dogs immunised with purified excreted-secreted antigens of Leishmania infantum co-incubated with Leishmania infected macrophages produce IFN gamma resulting in nitric oxide-mediated amastigote apoptosis

The role of nitric oxide (NO) in the anti-leishmanial activity has been confirmed both in vitro and in vivo. Recently, we demonstrated that NO-mediated apoptosis-like amastigote death pathway is an important and highly regulated mechanism used for the clearance of Leishmania within infected murine macrophages stimulated to produce NO endogenously. To further characterize these important effector mechanisms in dog, a natural host-reservoir of L. infantum/L. chagasi, we have developed an ex vivo infection model of canine macrophages. Exposure of L. infantum-infected macrophages to autologous peripheral lymphocytes derived from dogs immunised with purified excreted-secreted antigens of L. infantum promastigotes (LiESAp) formulated with muramyl dipeptide (MDP) as adjuvant resulted in a significant leishmanicidal effect due to interferon (IFN)-gamma dependent macrophage activation. Concomitant accumulation of NO(3)(-)/NO(2)(-) in supernatants of co-cultured cells and in situ staining of parasites with terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and YOPRO-1 showed that NO-mediated apoptosis of intracellular L. infantum amastigotes is occurring in canine macrophages as previously observed in mouse models. Monitoring these parameters in dogs after immunisation and before experimental challenge can represent a useful and easy way to rapidly evaluate vaccine candidates against canine visceral leishmaniasis.     

Parasitemia in an immunocompetent horse experimentally challenged with Sarcocystis neurona sporocysts

Equine protozoal myeloencephalitis (EPM) is a serious neurological disease of horses in Americans. Most cases are attributed to infection of the central nervous system with Sarcocystis neurona. Parasitemia has not been demonstrated in immunocompetent horses, but has been documented in one immunocompromised foal. The objective of this study was to isolate viable S. neurona from the blood of immunocompetent horses. Horses used in this study received orally administered S. neurona sporocysts (strain SN 37-R) daily for 112 days at the following doses: 100/day for 28 days, followed by 500/day for 28 days, followed by 1000/day for 56 days. On day 98 of the study, six yearling colts were selected for attempted culture of S. neurona from blood, two testing positive, two testing suspect and two testing negative for antibodies against S. neurona on day 84 of the study. Two 10 ml tubes with EDTA were filled from each horse by jugular venipuncture and the plasma fraction rich in mononuclear cells was pipetted onto confluent equine dermal cell cultures. The cultures were monitored weekly for parasite growth for 12 weeks. Merozoites grown from cultures were harvested and tested using S. neurona-specific PCR with RFLP to confirm species identity. PCR products were sequenced and compared to known strains of S. neurona. After 38 days of in vitro incubation, one cell culture from a horse testing positive for antibodies against S. neurona was positive for parasite growth while the five remaining cultures remained negative for parasite growth for all 12 weeks. The Sarcocystis isolate recovered from cell culture was confirmed to be S. neurona by PCR with RFLP. Gene sequence analysis revealed that the isolate was identical to the challenge strain SN-37R and differed from two known strains UCD1 and MIH1. To our knowledge this is the first report of parasitemia with S. neurona in an immunocompetent horse.     

Enhancement of bone healing using non-glycosylated rhBMP-2 released from a fibrin matrix in dogs and cats

OBJECTIVES: To test a non-glycosylated recombinant human bone morphogenetic protein-2 (ngly-rhBMP-2)/fibrin composite, which has been shown experimentally to enhance healing of bone defects in rodents, in a clinical case series of dogs and cats undergoing treatment for fracture non-unions and arthrodesis. METHODS: A ngly-rhBMP-2/fibrin composite was applied in 41 sites in 38 dogs and cats for which a cancellous bone autograft was indicated, replacing the graft. RESULTS: Bridging of the bone defect with functional bone healing was achieved in 90 per cent of the arthrodesis and fracture nonunions treated in this manner. CLINICAL SIGNIFICANCE: This prospective clinical study demonstrates the beneficial effects of ngly-rhBMP-2 in a specially designed fibrin matrix on the treatment of bone defects, and validates the use of this composite as an alternative to bone autografts in dogs and cats.     

Treatment of 10 dogs with discospondylitis by fluoroscopy-guided percutaneous discectomy

Ten dogs suffering from discospondylitis were treated by percutaneous discectomy and local and systemic antibiotic therapy. With fluoroscopic guidance, a cylinder 5 mm in diameter was removed from the centre of the intervertebral space, yielding a fenestration and decompressing the disc without producing any spinal instability. The causative bacteria were identified in nine of the 10 biopsy specimens, but in only three urine cultures and four blood cultures. In two cases, the antibiotics used initially had to be changed owing to the organisms' antibiotic resistance. The clinical signs of the dogs improved markedly after two to nine days (mean 4.2 days) and had resolved completely after five to 14 days (mean 9.1 days). In all the cases the disease could be classified histologically as either acute or chronic, and the disease was classified as chronic in one dog. No side effects were observed.     

Effects of dietary cation-anion difference on blood cortisol and ACTH levels in reproducing ewes

Formulation of rations to induce a compensated metabolic acidosis in the post-partum cow has proved a useful strategy for prevention of milk fever. Such acidification improves the ability of the animal to maintain calcium homeostasis by promoting the absorption of calcium from the intestine and mobilization of calcium from the bone. In humans, an acidogenic diet results in mild metabolic acidosis in association with a state of cortisol excess and this increase in plasma cortisol may increase bone catabolism. The objective of our experiment was to induce acidification by anionic salt supplementation and to study the effects of anionic supplementation on plasma cortisol and adrenocorticotropic hormone (ACTH) levels in sheep. Twenty-seven twin-bearing sheep were assigned to two experimental groups and a control group, depending on dietary cation-anion difference (DCAD) (+272.6, -88.9 and +164.5 mEq/kg DM respectively). Sheep assigned to each dietary treatment received their respective rations beginning 6 weeks pre-partum and continuing until 12 days post-partum. Anionic diet induced a non-respiratory systemic acidosis in association with a mild increase in plasma cortisol concentration without changes in plasma ACTH levels. Our data suggest that the mild hypercortisolism observed in sheep fed the anionic diet may not be an effector for bone resorption induced by anionic salts. A mild hypercortisolism of this magnitude may lead to osteoporosis but this might require many years of adrenal hypersecretion while anionic salts are only used during the last weeks of pregnancy.     

Rapid isolation of Brachyspira hyodysenteriae and Brachyspira pilosicoli from pigs

The aim of this study was to compare and evaluate the time required to isolate Brachyspira hyodysenteriae and Brachyspira pilosicoli from porcine faeces. This was done using previously described selective media (spectinomycin) S400, (colistin, vancomycin and spectinomycin) CVS and (spectinomycin, vancomycin, colistin, spiramycin and rifampin with swine faecal extract) BJ, compared with the method based on blood agar modified medium, with spectinomycin and rifampin (BAM-SR), including a pre-treatment step. Fourteen spirochaetal strains were obtained in pure cultures after 5 days (48 h in BAM-SR primary plate and three passages every 24 h in brain heart infusion (BHI) without antibiotics) pre-treating simulated samples in brain heart infusion broth with spectinomycin (400 microg/ml) and rifampin (15 microg/ml), before streaking on the selective BAM-SR medium. Spirochaetes from samples in S400, CVS and BJ, with and without pre-treatment, were obtained in pure cultures only after repeatedly transferring on plates of the same selective medium requiring 15-18 days according to the strain. BAM-SR used after the pre-treatment step showed a detection limit ranging from 3.5 x 10(2) to 6.7 x 10(7) cells/g faeces and was the only method able to support the growth of spirochaetes after 48 h.     

Babesia canis canis in dogs from Hungary: detection by PCR and sequencing

Canine babesiosis in Hungary has always been a severe and frequent disease, attributed to infection with Babesia canis transmitted by Dermacentor reticulatus. Identification of the disease agent has been based merely on size and morphology of the intraerythrocytic parasites and no evidence has been found concerning the subspecies (genotype) of B. canis. Therefore, a molecular survey on natural Babesia infection of dogs in Hungary using PCR and sequence analysis was attempted to clarify the subspecies (genotype) and to obtain information on the occurrence of B. canis. A total of 44 blood samples from dogs showing clinical signs of babesiosis were collected. A piroplasm-specific PCR amplifying the partial 18S rRNA gene yielded an approximately 450 bp PCR product in 39 (88.6%) samples. Thirteen positive samples originated from Budapest and 26 from 21 other locations. Five PCR products were chosen randomly for sequencing. The partial 18S rDNA sequences were submitted to GenBank (accession numbers AY611729; AY611730; AY611731; AY611732 and AY611733). The sequences showed 100% homology to one another or differed by one nucleotide. BLAST search against GenBank revealed the highest similarity (99.8 or 100%) with Babesia canis canis. The implication of these data, for the further study and diagnosis of canine babesiosis is discussed.     

Hyperextension of fetlock joints in German Holstein and German Holstein-Limousin crossbred calves

In two different dairy farms six calves exhibiting bilateral flexion of the fetlock joints in front or rear legs were born in 2000 to 2002. Four of the affected calves from the same farm were crossbred between German Holstein cows with red and white coat colour and a bull of the breed Limousin. The other two affected calves born on another farm were purebred German Holsteins with a black and white coat colour. The tests for BVD virus antigen and antibodies were negative in all affected calves. Three of the calves showed a lower selen and a higher glutamate dehydrogenase concentration in the analyses of blood metabolites. Two crossbred calves showed a degeneration of the liver with a progressive periportal fibrosis in a histological examination. In one calf an edema of astrocytes of the central nervous system was seen. The analysis of the pedigrees revealed for the four crossbred calves the Limousin bull as common ancestor and the mothers of the calves as relatives. For the affected purebred German Holstein calves also a sire was identified as a common ancestor. The pedigrees support inheritance through a monogenic autosomal recessive locus or more recessive gene loci with variable expressivity. However, the analysis could not clarify whether different gene loci are responsible for the congenital anomalies observed in the calves from the two farms and thus, the observed anomalies may be different genetic entities. Obvious environmental reasons were not found.