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.     

Feeding-unrelated factors influencing the plasma leptin level in ruminants

The triglyceride content of lipid depots associated with the current feeding level is the primary determinant of leptin gene expression and the circulating leptin level. In laboratory rodents and primates the plasma leptin is influenced also by the age, gender and physiological status (puberty, pregnancy, lactation, postpartum period), and by the health condition such as sepsis due to Gram-negative (GN) bacteria. Some pathologic conditions with intensive cytokine release evoke an increase in plasma leptin, which is thought to depress the subsequent feed intake. However, the effect of these secondary factors may be species-dependent, with still unknown clinical relevance in ruminants. In our ovine and bovine models plasma leptin increased after castration and dexamethasone treatment, decreased after experimental administration of synthetic androgens in castrated rams, but remained unchanged throughout the ovarian cycle and after ovariectomy. The circulating leptin level increased temporarily during synthetic progestin (fluorogestone) treatment in ewes, but similar changes were not seen in progesterone-supplemented ewes and norgestomet-treated cows. In a second trial on dairy cows we wanted to study whether elevated plasma leptin levels are induced by experimental endotoxin mastitis, or by natural outbreak of GN mastitis and puerperal metritis. Experimental endotoxin mastitis resulted in some-hour elevation in cortisol and insulin, with a simultaneous decrease in IGF-I and thyroid hormones. In the first 14 days of lactation GN mastitis induced the same endocrine alterations as the experimental endotoxin challenge, but in natural cases these changes varied within a wider range, and were more protracted and robust. Cows with puerperal metritis had more obvious catabolic changes in the early weeks of lactation, than their healthy counterparts. However, both mastitis and puerperal metritis failed to increase the circulating leptin level, showing that in cows the plasma leptin is not responsible for the anorexia associated with these inflammatory diseases.     

Congenital aganglionosis in a 3-day-old Holstein calf

Necropsy of a 3-day-old Holstein heifer revealed proximal megacolon and distal colorectal hypoplasia. Histologically, the hypoplastic distal colon and rectum lacked submucosal and myenteric ganglia. Clinical history, physical examination, and pathologic findings were consistent with intestinal aganglionosis, a congenital anomaly well documented in humans and foals but not previously reported in cattle.     

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.     

Longterm investigation in an Austrian dairy herd with low prevalence of paratuberculosis detection of antibodies in blood and milk

Paratuberculosis (Johne's disease), which is widely distributed throughout the world, is caused by Mycobacterium avium subspecies paratuberculosis (MAP). Diagnosis of subclinically infected cattle is challenging and is especially problematic in herds with low prevalence of MAP. The aim of this long-term study was the comparison of different diagnostic tests for MAP and specific antibodies in a herd with low prevalence of MAP. Three different commercially available serum-ELISA (Svanovir-ELISA, Svanova, Uppsala, Sweden; IDEXX-ELISA, IDEXX Laboratories, Maine, USA; Pourquier-ELISA, Institut Pourquier, Montpellier, France) and two milk ELISA (Svanovirm-ELISA Svanova, Uppsala, Sweden; Pourquier-ELISA, Institut Pourquier, Montpellier, France) were compared. Apart from these indirect diagnostic tests, two methods for the detection of the etiologic agent (bacteriologic culture and real-time PCR of faecal samples) were performed. In January 2005 the first and in April 2005 the second herd investigation of all animals older than 2 years (n=335) were carried out. Blood, milk and faecal samples were taken. From November 2005 until April 2006 follow up investigations were performed. For this purpose, blood-, milk- and faecal samples were monthly taken from 63 selected animals. The highest number of blood- and milk samples with a detectable antibody-level was found by the Svanovir-ELISA. There was a significant correlation between serum- and milk- Svanovir-ELISA results, whereas the agreement between ELISA and faecal culture/PCR was low. Significant correlations between Svanovir-serum-ELISA results and milk somatic cell counts could be registered. Moreover, there was significant agreement between IDEXX-serum-ELISA results with the age and number of lactations of the cows, as well as the mother's MAP-status.     

Genetic variation within and between G1 and G3 genotypes of Echinococcus granulosus in Italy revealed by multilocus DNA sequencing

Numerous studies have provided evidence that Echinococcus granulosus exists as a complex of different strains, that differ in a wide variety of criteria that have an impact on the epidemiology, pathology and control of cystic hydatid disease (CHD) and, to date, 10 distinct genotypes (G1-G10) have been identified. In Italy, sequence analysis of the mitochondrial cox1 and nad1 genes showed the occurrence of the G1 genotype, the common sheep strain, the G3 genotype, the buffalo strain and of one isolate identified as G2 genotype, the Tasmanian sheep strain. In the present work, we have analysed E. granulosus strains in Italy, by genotyping a large sample of isolates and by checking out the genetic differentiation within and among the G1 and G3 genotypes using an additional mitochondrial gene as marker, the rrnS gene. Sequencing of the rrnS gene revealed a significant genetic differentiation between isolates identified as belonging to the G1 and G3 genotypes, with fixed nucleotide substitutions. This study provides further evidence of the occurrence of the E. granulosus G3 buffalo strain in Italy, a strain previously thought to be confined to the Indian region.     

Evaluation of an antigen capture ELISA for the early diagnosis of Hypoderma lineatum in cattle under field conditions

An antigen capture or sandwich ELISA (sELISA) was evaluated for the diagnosis of Hypoderma lineatum in cattle under field conditions in northwestern Spain. The kinetics of circulating hypodermin C (HyC) and specific antibodies during the course of natural infestation were determined in a group of 10 Frisian calves. In addition, oesophagi and blood samples were taken from 105 cows at a slaughterhouse in order to compare three methods for the diagnosis of H. lineatum: sandwich ELISA for the detection of the antigen HyC (sELISA), indirect ELISA for the detection of antibodies anti-HyC (iELISA) and the detection of first instars (L1) in the oesophagus. In naturally infested cattle, HyC was present in circulation at low levels during the early and late phases of the infestation. However, in the middle phase, coinciding with the presence of L1 in the oesophagus, two peaks of increased HyC concentration were observed. Specific antibodies increased progressively until the first appearance of larvae in warbles on the back. There was no correlation between antigen or antibody levels and the number of grubs in the back. Prevalence of first instars in the oesophagi of slaughtered cows was 21.9% (23/105). The percentage of cattle that were positive for circulating antigen was slightly higher (24.8%), suggesting the recent destruction of migrating larvae in some animals. However, there was no correlation between the number of L1 and HyC levels. With the iELISA, 79% of the animals were positive to Hypoderma, which means that a high percentage of those animals have been exposed to the parasite but they had no apparent current infestation. The sELISA is a good tool to follow larval development within the host; however, the episodic elevation of HyC levels limits the usefulness of this test for the early diagnosis of Hypoderma under field conditions.     

Molecular cloning of DNA from a bovine herpesvirus 1 strain isolated in Hungary

Molecular cloning of the HindIII fragments of bovine herpesvirus 1 (BHV-1) strain HB144, isolated from infectious bovine rhinotracheitis (IBR) in Hungary, and of an infectious pustular vulvovaginitis (IPV) reference strain (K22) is reported. So far 52% of the IBR viral genome and 28% of the IPV viral genome have been cloned. The analysis of differences between the strains is currently in progress.