Miklós Rusvai

Comparison of the pathogenicity of QX-like, M41 and 793/B infectious bronchitis strains from different pathological conditions

Five QX-like infectious bronchitis virus (IBV) strains, isolated from different field outbreaks and two reference IBV strains of known serotypes (M41 and 793/B), were used in the present study to investigate and compare their pathogenicity for 1-day-old specific pathogen free chickens. The ability of the strains to inhibit trachea epithelium ciliary activity, to induce immune response, to replicate and to cause histopathological lesions in designated organs was followed by repeated samplings during a period of 42 days post infection. Clear differences in pathogenicity and in organ distribution of the three serotypes were found. Strain 793/B had the least capacity to invade the investigated organs, while it produced a good humoral response as measured by enzyme-linked immunosorbent assay. The QX-like strains generally replicated to higher titres, although differences were found among the five strains in their pathogenicity and affinity for different organs. The Chinese isolate of QX-like virus caused the most severe lesions and induced the highest antibody titres. Severe kidney damage and dilatation of the oviduct were the prominent lesions that could be related to the QX-like IBV strains, although neither marked virus replication nor histopathological lesions were detected in the oviduct.

Phylogenetic Analysis of Acute Bee Paralysis Virus Strains

ABSTRACT Reverse transcription-PCR assays have been established for a quick, sensitive, and specific diagnosis of acute bee paralysis virus (ABPV), a common virus of the honeybee (Apis mellifera), directly from clinical samples. A 3,071-nucleotide fragment of the ABPV genome, which includes the entire capsid polyprotein gene, was amplified from Austrian, German, Polish, and Hungarian ABPV samples and sequenced, and the sequences were compared. The alignment of a smaller fragment with ABPV sequences from the United States and the United Kingdom revealed nucleotide identity rates between 89 and 96%, respectively. Phylogenetic trees which display the molecular relationship between the viruses of different geographic origin were constructed.

Genetic diversity of Hungarian canine distemper virus strains

Abstract To achieve proper diagnosis of dogs based on acute clinical symptoms and poorly preserved field samples taken from animals that died due to canine distemper (CD), a new differential diagnostic test has been developed based on polymerase chain reaction (PCR). In this study, more than 150 samples collected from dogs showing respiratory, gastrointestinal and neurological signs suggesting canine distemper virus (CDV) infection were examined. The samples consisted of urine, blood and nasal swabs collected from clinically ill patients, sent to our laboratory by clinicians from various veterinary clinics throughout Hungary. Various organs collected during the necropsy of dogs with pathological changes that suggested CDV infection were also included. Three distinct PCRs were designed. For diagnostic purposes, a primer pair specific to a 409 bases-long segment within the conservative part of the large polymerase region (L) of the CDV genome was designed. Using this test, out of the 150 analyzed samples, 46 (30.66%) proved to be positive for CDV, indicating that CDV still represents a high risk to the canine population in Hungary. For the phylogenetical analysis, a primer pair that completely encompasses the hemagglutinin (H) gene of the CDV genome was designed. The amplicons of this region were sequenced in both directions using the appropriate primers. Our results indicate that several different CDV genotypes are currently present in Hungary. Nine of the analyzed Hungarian strains turned out to belong to the so-called Arctic group of CDVs, and were most closely related to non-European strains from North America, China and Greenland, as well as to the phocine distemper virus 2 (PDV-2) isolated from Baikal seals (Phoca sibirica). One of the Hungarian strains showed high similarity to other European isolates from Denmark, Germany, Italy and Turkey, as well as to other isolates from geographically more distant regions, such as the USA. Three Hungarian strains seem to join a new cluster that is formed by only a couple of strains, one isolated from a mink in Denmark, and another from a dog in North America. Using a third set of primers, a restriction fragment length polymorphism (RFLP) assay has also been designed for the fast and reliable differentiation of the wild-type CDVs from the vaccine strains.

First detection and dominance of Nosema ceranae in Hungarian honeybee colonies

Microsporidiosis (nosema disease) of the European honeybee ( Apis mellifera L.) is present in bee colonies worldwide. Until recently, Nosema apis had been regarded as the causative agent of the disease, which may have many negative effects on the colony and cause heavy economic losses in apicultures. Another microsporidium species, Nosema ceranae , was reported to infest the Asian honeybee ( Apis ceranae ), but both honeybee species are susceptible to both microsporidia. In the European honeybee N. ceranae was first detected in Spain in the year 2006. As it is difficult to distinguish N. ceranae and N. apis morphologically, a rapid and accurate assay has been developed to differentiate N. apis and N. ceranae based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the partial large subunit ribosomal RNA. The assay was tested on 38 Nosema -infested bee samples, which were collected from geographically distant Hungarian bee colonies representing all regions of the country. Only one sample contained N. apis , and in the other 37 samples N. ceranae was detected, which indicates the dominance of N. ceranae in Hungarian apiaries. This is the first report on the presence of N. ceranae in Hungary.

Naturally occurring parvoviral infection in Hungarian broiler flocks

The major enteric disease (ED) complex in broiler chickens is runting–stunting syndrome and in turkey broilers is poult enteritis mortality syndrome. Viruses from numerous families have been identified in the intestinal tracts of poultry with ED, such as Astroviridae, Coronaviridae, Reoviridae, Rotaviridae, and Parvoviridae. The objective of the present study was to directly demonstrate the presence of the scarcely known chicken parvovirus (ChPV) and turkey parvovirus (TuPV) in Hungarian flocks experiencing clinical signs of ED. ChPV and TuPV infection were demonstrated in 15 chicken flocks and two turkey flocks, in intestinal samples collected between 2008 and 2010. The histopathological investigation revealed enteritis in the duodenum and jejunum, and atrophy of the lymphoid organs. Indirect immunohistochemistry (IHC) suggested the intestinal epithelium of chickens and turkeys as a potential replication site of the virus, similarly to other parvoviruses, while in case of the turkey samples IHC positivity was also observed in the bursa of Fabricius, liver and pancreas. However, no direct connection could be established between the presence of the pathogen in the above-mentioned tissues and the histopathological changes observed in the investigated flocks. The phylogenetic analysis performed on the partial nucleic acid sequence of the NS1 gene revealed an evident clustering tendency of the ChPV and TuPV strains, but also highlighted the potential reciprocal role of these two species in the epidemiology of these viruses. The role of the ChPV and TuPV in the ED is far from understood, but the results of the present study emphasize the fact that in certain, still not fully elucidated conditions, ChPV and TuPV may participate in the emergence of ED in chicken flocks, as suggested by previous experimental infections.

Development of a one-step real-time quantitative PCR assay based on primer-probe energy transfer for the detection of porcine reproductive and respiratory syndrome virus

Abstract A one-step real-time RT-PCR method has been developed for the simultaneous detection of both genotypes of porcine reproductive and respiratory syndrome virus (PRRSV). The assay is based on primer-probe energy transfer, and the most important advantage of this is the relative tolerance towards mutations in the target-probe region. The primers and the probe were designed using an alignment of 235 Type 1 (including all subtypes) and Type 2 PRRSV strains. According to the alignment, multiple degenerations were included in the forward and reverse primers to enable the detection of all PRRSV strains deposited in the GenBank. Specificity was tested using 37 different PRRSV strains and eight other swine pathogen viruses. The detection limit was approximately 10 copies of RNA prepared from the Lelystad virus, a European Subtype 3 virus (Belarus strain Soz-8), and an American vaccine virus (Ingelvac MLV®). One TCID50 was the detection limit in the case of the cell cultured Lelystad virus and an American wild type isolate, respectively. The melting point analysis revealed melting point decrease, but no significant sensitivity and signal loss in the presence of numerous (up to five) target-probe mismatches, indicating the capability of tolerating even more mutations. The method was suitable for the detection and quantitation of phylogenetically divergent strains and can serve as a robust, high throughput tool for molecular diagnosis of the PRRSV.

Identification of CNS neurons involved in the innervation of the epididymis: A viral transneuronal tracing study

Cell groups of the spinal cord and the brain transsynaptically connected with the epididymis (caput, cauda) were identified by means of the viral transneuronal tracing technique. Pseudorabies virus was injected into the caput or the cauda epididymidis, and after survival times 4 and 5 days, the spinal cord and brain were processed immunocytochemically. Virus-labeled neurons could be detected in the preganglionic sympathetic neurons (lower thoracic and upper lumbar segments) and following virus injection into the cauda epididymidis, also in the sacral parasympathetic nucleus (L6-S1). Virus-infected perikarya were present in several brain stem nuclei (lateral reticular nucleus, gigantocellular and paragigantocellular nucleus, A5 noradrenergic cell group, caudal raphe nuclei, locus coeruleus, Barringtons nucleus, nucleus of the solitary tract, periaqueductal gray) and in the diencephalon (hypothalamic paraventricular nucleus, lateral hypothalamus). At the longer survival time, some telencephalic structures also exhibited virus-labeled neurons. The distribution of infected neurons in the brain was similar after virus injection into the caput or cauda epididymidis; however, earlier onset of infection was observed after inoculation into the cauda. The present findings provide the first morphological data on a multisynaptic circuit of neurons innervating the epididymis and presumably involved in the control of epididymal functions. reserved.

Comparative histopathology and immunohistochemistry of QX-like, Massachusetts and 793/B serotypes of infectious bronchitis virus infection in chickens.

Summary The aim of this study was to compare experimentally the pathogenicity and tissue distribution of the recently emerged QX-like strain of infectious bronchitis virus (IBV) with the widespread M41 and 793/B serotypes of the virus. Histopathological and immunohistochemical methods were employed to define the main sites of virus replication. One-day-old specific pathogen free chickens were inoculated with five different QX-like strains, or with the M41 and 793/B IBV strains and monitored for 42 days post-infection. Tracheal lesions developed in all infected birds, confirming the ability of all of the tested strains to induce respiratory disease. Replication of the isolates in the alimentary tract was detected, but the infection did not cause significant gut lesions. Four of the five QX-like IBV strains induced severe kidney lesions. Dilation of the oviduct with accumulation of serum-like fluid in the lumen of this structure, reported previously from field cases of QX-like IBV infection, was observed following experimental infection with all of the five QX-like strains. Microscopical and immunohistochemical examination of the affected oviducts did not help to elucidate the pathogenesis of this lesion.

Evaluation of microvessel density (MVD) in canine mammary tumours by quantitative immunohistochemistry of the claudin-5 molecule

In our recent investigation, angiogenesis was evaluated and quantified by immunohistochemical evaluation of microvessel density (MVD) using claudin-5 (CLDN-5) as a marker for vascular endothelium in 67 canine mammary gland tumours. Computer image analysis was used to measure the intratumoural MVD. Higher intratumoural MVD was detected in malignant simple neoplasms compared with benign tumours. Furthermore, the results of MVD were correlated with histological grade, higher grades being accompanied by higher MVD. In simple adenomas and grade I tubular-tubulopapillary simple carcinomas the intratumoural microvessels were wide and regular in shape with evident erythrocytes in their lumen. In grade III solid carcinomas the microvessels were smaller, less regular and had irregular shape, often without a distinct lumen, and isolated endothelial cells were frequently present. In the complex carcinomas MVD was low and the intratumoural microvessels were mostly irregular in shape without a distinct lumen. The evaluation of MVD by CLDN-5 immunohistochemistry may give useful additional information on the angiogenic potential of breast cancers in dogs.

Expression of claudin-1, -2, -3, -4, -5 and -7 proteins in benign and malignant canine mammary gland epithelial tumours.

Claudins are tight junction proteins expressed by epithelial and endothelial cells. The present study has evaluated the expression of claudin-1, -2, -3, -4, -5 and -7 in 115 hyperplastic and neoplastic lesions of the canine mammary gland and compared this expression with that of normal mammary epithelium. The lesions studied included lobular hyperplasia (n=15), simple adenoma (n=20), non-infiltrating carcinoma in situ (n=20) and infiltrating carcinomas of histological grades I, II and III (n=20 of each). There was strong expression of claudin-1, -3, -4, -5 and -7 by epithelia within examples of lobular hyperplasia and simple adenoma, and strong expression of claudin-3 and -4 by non-infiltrating carcinomas and all three grades of infiltrating carcinoma. By contrast, there was reduced expression of claudin-5 and -7 by non-infiltrating and infiltrating carcinomas and the expression of these two molecules was inversely correlated with histological grade. Claudin-1 was expressed focally within carcinoma in situ, but this molecule was not detected in any invasive carcinoma. Claudin-2 was weakly expressed within areas of lobular hyperplasia and by simple adenomas, but was not expressed by any carcinoma cells. These results suggest that loss or reduction of expression of claudin-1, -2, -5 and -7 may lead to cellular disorientation, detachment and invasion in canine mammary neoplasia.