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  • Author or Editor: Boglárka Sellyei x
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The 16 somatic serotype type strains and 60 field isolates of Pasteurella multocida, representing various avian species and geographic regions in Hungary, were characterised by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the ompH gene with DraI restriction endonuclease. The type strains yielded eight different (I-VIII) profiles. Strains whose PCR fragment was uncut by DraI (profile IV) could be differentiated with HindIII and PvuII restriction endonucleases. Five of the eight PCR-RFLP profiles (I, III, V, VI and VII) were detected among the field strains. Only a correlation of limited strength was found between the classical somatic serotypes and the PCR-RFLP profiles. However, the results confirmed that molecular methods could confidently distinguish serotype A:1 strains from the other serotypes. Moreover, the specific relationship between somatic serotypes and PCR-RFLP types among isolates from turkey raises the possibility of the existence of host-specific clones within the P. multocida population.

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A total of 146 Pasteurella multocida strains isolated from swine in Hungary in the last 20 years were examined. Biochemical characterisation and PCR-based techniques were used to determine species, subspecies, biovar, capsule type and presence of the toxA gene. Eighty-seven percent of the isolates belonged to P. multocida ssp. multocida , and 98% of these had biovar 3 or were trehalose-or lactose-fermenting or ornithine decarboxylase negative variants of that. Ten percent of the strains were P. multocida ssp. septica , and within this group 80% of the strains showed sorbitol-negative biovars (5, 6 and 7). The rest of the strains (20%) were lactose positive. Only 3% of the porcine isolates were P. multocida ssp. gallicida and 3 out of the 4 strains belonged to the dulcitol-fermenting biovar 8. Using a capsule-specific multiplex PCR, 60% of the strains belonged to capsule type D, 38% to capsule type A, and only 1 isolate had capsule type F. In contrast with data published in the literature, only 3% of capsule type D isolates carried the toxA gene, while this ratio was 41% for the type A strains. A remarkable regional distribution of toxA gene positive strains was observed. All but two isolates were found in swine herds located in the Transdanubian region, separated from other parts of Hungary by the river Danube.

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During a general annual fish health survey in natural waters and ponds, epitheliocystis infections were recorded in fingerlings of two cyprinid fish species, the cultured common carp and the wild gibel carp. Benign and heavy infections were equally observed without mortality. In addition to the general health inspection of fish, histopathological examinations of infected gills and molecular biological investigations of separated epitheliocysts were performed. Epitheliocysts were formed both in the interlamellar epithelial cells and in the lamella-free multilayered epithelium of the gill filaments. At the early stage of infection darkstaining inclusion bodies densely stuffed with some pathogenic agents were located at the centre of the cell, while in a progressive stage of the process inclusion bodies within the host cells were disseminated in the cytoplasm and stained pale. Molecular studies demonstrated three different agents related to Neochlamydia, Protochlamydia and Piscichlamydia based on sequence analysis of short regions of the 16S rRNA gene. Among them, Piscichlamydia is a primary fish pathogen, while Neochlamydia and Protochlamydia mostly infect free-living amoebae but have adapted thoroughly to fish.

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Authors: Boglárka Sellyei, Zsuzsanna Rónai, Szilárd Jánosi and László Makrai

Bovine respiratory disease (BRD) is the leading cause of significant economic losses in the intensive beef industry worldwide. Beside numerous risk factors Pasteurella multocida, which is regarded as a secondary pathogen, may play a role in the development of the disease. Previous studies of strains from swine pneumonia revealed that there are a few clones associated with clinical disease, suggesting that some strains may be more virulent than others. This linkage may be true in the BRD, however composition of P. multocida populations in the herds are slightly characterized. Thus, we decided to perform phenotypic and genotypic characterisation of strains isolated from calves with respiratory infection at 31 different herds in Hungary. The results demonstrated the presence of two dominant strain types. At the identical taxonomic background (P. multocida subsp. multocida) with slight phenotypic variability they could be separated by trehalose fermentation capacity, α-glucosidase activity and molecular fingerprint patterns of ERIC- and M13-PCR. Independent prevalence and geographical origin of the strain types may refer to their significance in the illness, but their comparison with strains isolated from healthy individuals is taken into consideration.

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Sixty-one avian strains of Pasteurella multocida were characterised and compared by biochemical tests, capsular PCR typing and ERIC-PCR. The strains were recovered from various avian species (goose, duck, Muscovy duck, turkey, chicken and pheasant) and represented different geographic locations in Hungary. Forty-two strains (69%) were identified as P. multocida subsp. multocida and 19 strains (31%) as P. multocida subsp. septica . The strains were grouped into 7 different biovars (1, 2, 3, 4, 5, 6 and 7). The most prevalent biovars were 1 (25%), 3 (21%) and 6 (21%). Most of the duck isolates (90%) belonged to biovar 1 or 6. The most frequent capsular type was A (93.5%). Type F represented only a small number (6.5%) of the strains. Other capsular types were not identified. From the 61 isolates 24 different fingerprint patterns were generated by ERIC-PCR assay. Based on cluster analysis the strains could be grouped into four larger and four mini-clusters that showed considerable correlation with the geographical origin and the host species. The results indicate that ERIC-PCR may be a suitable technique for studying the host adaptation of P. multocida and the epidemiology of fowl cholera.

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The prevalence and distribution of piscine circoviruses (CVs) were tested in a routine virus monitoring programme in Lake Balaton, Hungary. A high prevalence of European eel CV (EeCV) was found in the apparently healthy eel population (35.5%). The copy number of the viral DNA in different organs was determined by quantitative real-time PCR. The results suggested that some eel specimens were in active viraemic status despite their asymptomatic condition. Furthermore, a novel, previously undescribed CV was also detected in eel and sichel samples. Full genome characterisation confirmed that the virus represents a novel EeCV species (EeCV-2). The genome contains an integrated eel chromosome-derived fragment, suggesting that the original host of the virus was the eel and it probably emerged subsequently in the sichel by host switching. In some samples, an additional, 1,111-nt-long circular ssDNA was also observed involving a CV-like stem-loop structure and an ORF showing homology to CV capsid protein genes, without any sign of a replication initiator protein sequence.

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Abstract

Two adult barbels (Barbus barbus) with visible skin tumours were subjected to histopathological and molecular examinations. The fish were caught in the River Danube near Budapest. Papillomas were found around their oral cavity, at the operculum and at the pectoral fins, while epidermal hyperplasias were seen on the body surface. Cyprinid herpesvirus 1 (CyHV-1) was detected in the kidney of the specimens by polymerase chain reaction (PCR), and barbel circovirus 1 (BaCV1) was found in all internal organs and in the tissues of the tumours. The whole genome of BaCV1 and three conserved genes from the genome of CyHV-1 were sequenced. Previously, BaCV1 had been reported only once from a mass mortality event among barbel fry. The whole genome sequence of our circovirus shared 99.9% nucleotide identity with that of the formerly reported BaCV1. CyHV-1 is known to infect common carp and coloured carp (Cyprinus carpio), and has been assumed to infect other cyprinid fish species as well. We found the nucleotide sequences of the genes of CyHV-1 to be identical in 98.7% to those of the previous isolates from carp. To the best of our knowledge, this is the first molecular confirmation of the presence of CyHV-1 DNA in cyprinid fish species other than carp.

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Abstract

Two adult barbels (Barbus barbus) with visible skin tumours were subjected to histopathological and molecular examinations. The fish were caught in the River Danube near Budapest. Papillomas were found around their oral cavity, at the operculum and at the pectoral fins, while epidermal hyperplasias were seen on the body surface. Cyprinid herpesvirus 1 (CyHV-1) was detected in the kidney of the specimens by polymerase chain reaction (PCR), and barbel circovirus 1 (BaCV1) was found in all internal organs and in the tissues of the tumours. The whole genome of BaCV1 and three conserved genes from the genome of CyHV-1 were sequenced. Previously, BaCV1 had been reported only once from a mass mortality event among barbel fry. The whole genome sequence of our circovirus shared 99.9% nucleotide identity with that of the formerly reported BaCV1. CyHV-1 is known to infect common carp and coloured carp (Cyprinus carpio), and has been assumed to infect other cyprinid fish species as well. We found the nucleotide sequences of the genes of CyHV-1 to be identical in 98.7% to those of the previous isolates from carp. To the best of our knowledge, this is the first molecular confirmation of the presence of CyHV-1 DNA in cyprinid fish species other than carp.

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Authors: Zsuzsanna Varga, Boglárka Sellyei, Petra Paulus, Melitta Papp, Kálmán Molnár and Csaba Székely

The objective of this study was to survey the incidence of Flavobacterium columnare in wild and cultured freshwater fish species in Hungary. This bacterium usually causes disease in waters of more than 25 °C temperature. However, with the introduction of intensive fish farming systems, infected fish exposed to stress develop disease signs also at lower temperatures; in addition, the temperature of natural waters rises to the critical level due to global warming. Twenty-five isolates from wild and cultured freshwater fishes were identified as F. columnare by specific PCR, although both the fragment lengths and the results of PCRRFLP genotyping with BsuRI (HaeIII) and RsaI restriction enzymes raised doubts regarding this species classification. Sequencing of the 16S ribosomal RNA gene revealed that 23 isolates belonged to the species F. johnsoniae and two represented Chryseobacterium spp. The isolates were found to have high-level multidrug resistance: all were resistant to ampicillin and polymyxin B, the 23 F. johnsoniae strains to cotrimoxazole, 88% of them to gentamicin, and 72% to chloramphenicol. The majority of the 25 isolates were sensitive to erythromycin (88%), furazolidone (76%), and florfenicol (68%).

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