Enikő Wehmann

Newcastle disease outbreaks in recent years in Western Europe were caused by an old (VI) and a novel genotype (VII)

SummaryNewcastle disease virus (NDV) strains, isolated from outbreaks during epizootics between 1992 and 1996 in Western European countries, were compared by restriction enzyme cleavage site mapping of the fusion (F) protein gene between nucleotides 334 and 1682 and by sequence analysis between nucleotides 47 and 435. Both methods revealed that NDV strains responsible for these epizootics belong to two distinct genotypes. Strains derived from sporadic cases in Denmark, Sweden, Switzerland and Austria were classified into genotype VI [6], the same group which caused outbreaks in the Middle East and Greece in the late 1960’s and in Hungary in the early 1980’s. In contrast, viruses that caused epizootics in Germany, Belgium, The Netherlands, Spain and Italy could be classified into a novel genotype (provisionally termed VII), hitherto undetected in Europe. It is possible that the genotype VII viruses originated in the Far East because they showed a high genetic similarity (97%) to NDV strains isolated from Indonesia in the late 1980’s.

Identification and grouping of Newcastle disease virus strains by restriction site analysis of a region from the F gene

SummaryA 75% region of the F gene (between nucleotides 334 and 1682) of Newcastle disease virus (NDV) RNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR). PCR products were cleaved by three restriction endonucleases and the positions of thirty cleavage sites were mapped in more than 200 NDV strains. Restriction site analysis established six major groups of NDV isolates and unique fingerprints of vaccine strains. Group I comprised lentogenic strains isolated mainly from waterfowl with some from chickens. “Old” (prior to 1960s) North American isolates of varying virulence including lentogenic and mesogenic vaccine strains belonged to group II. Group III included two early isolates from the Far East. Early European strains (Herts 33 and Italien) of the first panzootic (starting in the late 1920s) and their descendants with some modifications were placed into group IV. NDV strains isolated during the second panzootic of chickens (starting in the early 1960s) were classified into two groups. Group V included strains originating in imported psittacines and in epizootics of chickens at the early 1970s. Group VI comprised strains from the Middle East in the late 1960s and later isolates from Asia and Europe. Pigeon paramyxovirus-1 strains that were responsible for the third panzootic formed a distinct subgroup in group VI. Our grouping of NDV strains has confirmed group differences established by monoclonal antibodies. It is concluded that restriction site analysis of F gene PCR amplicons is a relatively fast, simple and reliable method for the differentiation and identification of NDV strains.

Two novel genetic groups (VIIb and VIII) responsible for recent Newcastle disease outbreaks in Southern Africa, one (VIIb) of which reached Southern Europe

Summary 34 strains of Newcastle disease virus (NDV) isolated during epizootics in the Republic of South Africa and in Mozambique between 1990 and 1995, and in Bulgaria and Turkey in 1995–1997 were identified by restriction enzyme and partial sequence analysis of the fusion (F) protein gene. The majority of isolates in southern Africa and those from Bulgaria and Turkey were placed into a novel group which has been termed VIIb. Group VIIb is part of a larger genetic cluster (VII) that also includes NDV strains from the Far East and some western European countries (VIIa). The genetic distance of 7–8, 5% between genotype VIIa and VIIb viruses excludes the existence of a direct epidemiological link between recent southern African epizootics and outbreaks in either western Europe in the 1990’s or those of the Far East. Another hitherto unrecorded genotype (VIII) was also found in South Africa with descendants of putative ancestral members isolated in the 1960’s. The genetic distance of recent group VIII strains from the major epizootic genotype (VIIb) is over 11%, therefore outbreaks caused by them were epidemiologically unrelated. Genotype VIII viruses must have been maintained in South Africa by endemic infections during the past decades while group VIIb appears to be introduced more recently.

The occurrence of five major Newcastle disease virus genotypes (II, IV, V, VI and VIIb) in Bulgaria between 1959 and 1996.

Partial sequence and restriction enzyme cleavage site analyses of the fusion protein gene were used to genotype 47 Newcastle disease virus strains isolated between 1959 and 1996 in Bulgaria. Viruses belonged to five major genotypes that appeared to be associated with epizootics characterized by temporal and/or geographical restrictions. Genotype IV viruses (responsible for the European branch of the first panzootic) dominated the scene up to the early 1980s, interspersed with sporadic outbreaks caused by genotype II (US strains causing pneumoencephalitis) viruses. Genotype V viruses (transmitted by psittacines from South America) were first shown in 1973 and persisted until the late 1980s. Genotype VI (earliest members from the Middle-East 1968/70 outbreaks) was represented by scattered isolations between 1974 and 1996. A genotype VIIb (recent Middle East epizootic) virus was isolated as early as in 1984. Newcastle disease epizootics in Bulgaria were highlighted by multiple infection with more than one genotype at any one time.

Genetic analysis of Newcastle disease virus strains isolated in Bosnia-Herzegovina, Croatia, Slovenia and Yugoslavia, reveals the presence of only a single genotype, V, between 1979 and 2002.

Newcastle disease (ND) epizootics in some European countries after the World War II were caused by ND virus (NDV) of multiple genotypes (IV-VIIa) occurring sequentially and/or simultaneously. This study was carried out to characterise the genetic composition of NDV strains during the outbreaks in the territory of the former Yugoslavia in order to enhance our understanding of the relationships of past epizootics in Europe. Sixty-eight NDV strains isolated between 1979 and 2002 were analysed by restriction enzyme digestion and partial sequencing of the fusion protein gene. All isolates were placed in genotype V, an exotic type, that was introduced to western Europe in 1970. Residue substitution analysis has allowed the recognition of four genetic variants, Vb1-Vb4, and the tracing of their movements. Vb1, a dominant variant in Bulgaria from the late 1970s, was also wide spread in the former Yugoslavia throughout the period under investigation. Vb2, a variant occurring in the neighbouring countries in the early 1970s could be the founder of the epidemic in Yugoslavia and it was present up to the late 1980s. Variants Vb3 and Vb4 could be found only after 1987. In conclusion, the ND outbreaks in Yugoslavia were part of the epizootic wave due to genotype V viruses that started in western Europe in 1970 and became endemic in the region. Inter-country transmission occurred for all variants, and Vb3 and Vb4 might have evolved during the endemic period.

Genetic relatedness of Brucella suis biovar 2 isolates from hares, wild boars and domestic pigs.

Porcine brucellosis generally manifests as disorders in reproductive organs potentially leading to serious losses in the swine industry. Brucella suis biovar 2 is endemic in European wild boar (Sus scrofa) and hare (Lepus europeus, Lepus capensis) populations, thus these species may play a significant role in disease spread and serve as potential sources of infection for domestic pigs. The aim of this study was an epidemiologic analysis of porcine brucellosis in Hungary and a comparative analysis of B. suis bv. 2 strains from Europe using multiple-locus variable-number tandem repeat analysis (MLVA). MLVA-16 and its MLVA-11 subset were used to determine the genotypes of 68 B. suis bv. 2 isolates from Hungary and results were then compared to European MLVA genotypes. The analyses indicated relatively high genetic diversity of B. suis bv. 2 in Hungary. Strains isolated from hares and wild boars from Hungary showed substantial genetic divergence, suggesting separate lineages in each host and no instances of cross species infections. The closest relatives of strains from Hungarian wild boars and domestic pigs were mainly in the isolates from German and Croatian boars and pigs. The assessment of the European MLVA genotypes of wild boar isolates generally showed clustering based on geographic origin. The hare strains were relatively closely related to one another and did not cluster based on geographic origin. The limited relationships between geographic origin and genotype in isolates from hares might be the result of cross-border live animal translocation. The results could also suggest that certain B. suis strains are more adapted to hares. Across Europe, isolates from domestic pigs were closely related to isolates originating from both hares and wild boars, supporting the idea that wild animals are a source of brucellosis in domestic pigs.

Rapid identification of Newcastle disease virus vaccine strains LaSota and B-1 by restriction site analysis of their matrix gene

A region constituting 88% of the matrix gene of Newcastle disease virus vaccine strains LaSota and B-1 was amplified by reverse transcription-polymerase chain reaction. Amplified products of LaSota and B-1 strains derived from vaccine serials of different companies were digested with restriction enzymes MboI and HinfI. Strain characteristic cleavage site maps were obtained that allowed for a reliable and rapid differentiation between strains LaSota and B-1.

Lentogenic field isolates of Newcastle disease virus isolated in Canada and Hungary are identical with the vaccine type used in the region

Lentogenic field isolates of Newcastle disease virus were examined by restriction enzyme analysis of RT-PCR products generated from the matrix protein gene that discriminates between strains LaSota and B-1, the two most widely used lentogenic vaccine viruses. Isolates were derived from regions where, exclusively or predominantly, only one type of vaccine was employed. Viruses collected in Hungary for two decades were exclusively of LaSota-type while the Canadian collection predominantly included B-1, which corresponded to the vaccine types used in the regions. Isolation of vaccine type lentogenic viruses from unvaccinated flocks supports the occurrence of area spread of these lentogenic viruses.

Mutations Associated with Decreased Susceptibility to Seven Antimicrobial Families in Field and Laboratory-Derived Mycoplasma bovis Strains

ABSTRACT The molecular mechanisms of resistance to fluoroquinolones, tetracyclines, an aminocyclitol, macrolides, a lincosamide, a phenicol, and pleuromutilins were investigated in Mycoplasma bovis. For the identification of mutations responsible for the high MICs of certain antibiotics, whole-genome sequencing of 35 M. bovis field isolates and 36 laboratory-derived antibiotic-resistant mutants was performed. In vitro resistant mutants were selected by serial passages of M. bovis in broth medium containing subinhibitory concentrations of the antibiotics. Mutations associated with high fluoroquinolones MICs were found at positions 244 to 260 and at positions 232 to 250 (according to Escherichia coli numbering) of the quinolone resistance-determining regions of the gyrA and parC genes, respectively. Alterations related to elevated tetracycline MICs were described at positions 962 to 967, 1058, 1195, 1196, and 1199 of genes encoding the 16S rRNA and forming the primary tetracycline binding site. Single transversion at position 1192 of the rrs1 gene resulted in a spectinomycin MIC of 256 μg/ml. Mutations responsible for high macrolide, lincomycin, florfenicol, and pleuromutilin antibiotic MICs were identified in genes encoding 23S rRNA. Understanding antibiotic resistance mechanisms is an important tool for future developments of genetic-based diagnostic assays for the rapid detection of resistant M. bovis strains.

Characterisation of Pasteurella dagmatis-like isolates recovered from the feline oral cavity

Three Pasteurella dagmatis-like strains recovered from the feline oral cavity were analysed by traditional biochemical tests, the Biolog Microstation™ ID System, and 16S rRNA and sodA gene sequence analysis. The molecular biological methods revealed that these strains differ from P. dagmatis, forming a new genomospecies in the genus Pasteurella sensu stricto. Furthermore, sequence analysis and multiple alignments of 16S rRNA and the sodA gene established that the P. pneumotropica NCTC10827 and the P. dagmatis-like strains described here possess high genetic similarity.