István Kiss

Prevalence and characterization of Salmonella infantis isolates originating from different points of the broiler chicken-human food chain in Hungary

During the 10-month study period Salmonella contamination of broiler houses and the flocks reared in three farms (A, B and C), the slaughter houses where the flocks were slaughtered, as well as the carcass and retail raw meat products originating from them was investigated. In the broiler farm A five consecutive flocks, in the B and C farms one flock was sampled. Environmental samples were taken prior to the introductions. Environmental, drinking water, feed and faecal samples were collected regularly using standard methods. Before and during processing of the flocks, environmental and carcass samples were taken at the abattoirs. Salmonella contamination of the carcass, retail meat, as well as stool samples of farm and abattoir workers and from human illnesses registered in the same period and region were also examined. Isolation, sero-, phage- and antibiotic resistance typing, class 1 integron and plasmid profiling of the strains were performed; their genetic relationship was assessed by PFGE. Although the broiler house and the faecal samples of the 5 flocks of the farm A were negative for Salmonella, S. infantis was isolated from 20-100% of the abattoir carcass samples. The retail raw meat samples were 0-100% S. infantis positive. The environmental samples of farm B were Salmonella negative, but the examined flock was contaminated: S. infantis was identified from 43% of the faecal samples. This serotype was identified in 100% of the carcass and retail raw meat samples. From environmental samples taken before the arrival of the 1-day-old chicks in the broiler house C, S. infantis was cultured. S. infantis prevalence in the faecal samples was 35% and all the carcass and retail raw meat samples were S. infantis contaminated. Altogether 164 S. infantis strains were isolated out of which 145 were further characterized. The vast majority (142/145) of the strains belonged to phage types 217 and 213. All but one were characterized by the nalidixic acid-streptomycin-sulphonamide-tetracycline resistances, had an 885 bp class 1 integron and a large plasmid of > 168 kb in size. The strains showed > or = 88.7% genetic similarity. The results obtained shows that the same multi-drug resistant S. infantis clone was spread from the examined broiler farms contaminating the slaughter and the retail meat and appeared in the human illnesses of the examined region that was earlier detected as the dominant clone characteristic of the broiler and human population of the whole country.

Incidence, Diversity, and Molecular Epidemiology of Sapoviruses in Swine across Europe

ABSTRACT Porcine sapovirus is an enteric calicivirus in domestic pigs that belongs to the family Caliciviridae. Some porcine sapoviruses are genetically related to human caliciviruses, which has raised public health concerns over animal reservoirs and the potential cross-species transmission of sapoviruses. We report on the incidence, genetic diversity, and molecular epidemiology of sapoviruses detected in domestic pigs in a comprehensive study conducted in six European countries (Denmark, Finland, Hungary, Italy, Slovenia, and Spain) between 2004 and 2007. A total of 1,050 swine fecal samples from 88 pig farms were collected and tested by reverse transcription-PCR for sapoviruses, and positive findings were confirmed by sequencing. Sapoviruses were detected in 80 (7.6%) samples collected on 39 (44.3%) farms and in every country. The highest prevalence was seen among piglets aged 2 to 8 weeks, and there was no significant difference in the proportion of sapovirus-positive findings for healthy animals and animals with diarrhea in Spain and Denmark (the only countries where both healthy animals and animals with diarrhea were tested). On the basis of the sequence of the RNA polymerase region, highly heterogeneous populations of viruses representing six different genogroups (genogroups III, VI, VII, and VIII, including potential new genogroups IX and X) were identified, with a predominance of genogroup GIII (50.6%). Genogroup VIII, found in five of the six countries, had the highest degree of homology (up to 66% at the amino acid level) to human sapovirus strains. Sapoviruses are commonly circulating and endemic agents in swine herds throughout Europe. Highly heterogeneous and potential new genogroups of sapoviruses were found in pigs; however, no “human-like” sapoviruses were detected.

Molecular characterization of highly pathogenic H5N1 avian influenza viruses isolated in Sweden in 2006

BackgroundThe analysis of the nonstructural (NS) gene of the highly pathogenic (HP) H5N1 avian influenza viruses (AIV) isolated in Sweden early 2006 indicated the co-circulation of two sub-lineages of these viruses at that time. In order to complete the information on their genetic features and relation to other HP H5N1 AIVs the seven additional genes of twelve Swedish isolates were amplified in full length, sequenced, and characterized.ResultsThe presence of two sub-lineages of HP H5N1 AIVs in Sweden in 2006 was further confirmed by the phylogenetic analysis of approximately the 95% of the genome of twelve isolates that were selected on the base of differences in geographic location, timing and animal species of origin. Ten of the analyzed viruses belonged to sub-clade 2.2.2. and grouped together with German and Danish isolates, while two 2.2.1. sub-clade viruses formed a cluster with isolates of Egyptian, Italian, Slovenian, and Nigerian origin. The revealed amino acid differences between the two sub-groups of Swedish viruses affected the predicted antigenicity of the surface glycoproteins, haemagglutinin and neuraminidase, rather than the nucleoprotein, polymerase basic protein 2, and polymerase acidic protein, the main targets of the cellular immune responses. The distinctive characteristics between members of the two subgroups were identified and described.ConclusionThe comprehensive genetic characterization of HP H5N1 AIVs isolated in Sweden during the spring of 2006 is reported. Our data support previous findings on the coincidental spread of multiple sub-lineage H5N1 HPAIVs via migrating aquatic birds to large distance from their origin. The detection of 2.2.1. sub-clade viruses in Sweden adds further data regarding their spread in the North of Europe in 2006. The close genetic relationship of Swedish isolates sub-clade 2.2.2. to the contemporary German and Danish isolates supports the proposition of the introduction and spread of a single variant of 2.2.2. sub-clade H5N1 avian influenza viruses in the Baltic region. The presented findings underline the importance of whole genome analysis.

Real-Time PCR-Based Pathotyping of Newcastle Disease Virus by Use of TaqMan Minor Groove Binder Probes

ABSTRACT A real-time reverse-transcription PCR was developed to detect and pathotype Newcastle disease viruses (NDV) in clinical samples. Degenerate oligonucleotide primers and TaqMan probes with nonfluorescent minor groove binder (MGB) quencher amplified and hybridized to a region in the fusion protein (F) gene that corresponds to the cleavage site of the F0 precursor, which is a key determinant of NDV pathogenicity. The application of degenerate primers and TaqMan MGB probes provided high specificity to the assay, as was shown by the successful and rapid pathotype determination of 39 NDV strains representing all the known genotypes (I to VIII) and pathotypes (lentogens/mesogens/velogens). The PCR assays specific for lentogenic and velogenic/mesogenic strains had high analytical sensitivity, detecting approximately 10 and 20 copies of the target molecule per reaction, respectively. The detection limit was also determined in terms of 50% egg infective dose (EID50) by using dilution series of virus stock solutions to be approximately 101.0 and 10−1.3 EID50/ml for lentogens and velogens/mesogens, respectively. Organ, swab, and stool specimens from experimentally infected animals were tested to prove the clinical suitability of the method. The results of this study suggest that the described real-time PCR assay has the potential to be used for the rapid detection/pathotyping of NDV isolates and qualitative/quantitative measurement of the virus load.

Real-time reverse transcription-polymerase chain reaction detection of Newcastle disease virus using light upon extension fluorogenic primers.

A real-time reverse transcriptase (RT)-PCR assay, applying light upon extension (LUX) fluorogenic primers, was developed for rapid and efficient detection of Newcastle disease virus (NDV). The method, which targets the fusion (F) protein gene of the viral genome, gave positive signal with all NDV isolates tested (32/32), while negative results were obtained with heterologous pathogens (35/35), including 13 avian influenza virus isolates. The detection limit of the assay was approximately 10+1.2 egg infectious dose (EID)50/0.2 ml and 10+2.2 EID50/0.2 ml for virus suspensions and spiked chicken fecal samples, respectively. As expressed in plasmid copy number, the procedure has a sensitivity of approximately 20 copies of the plasmid harboring the target gene. Due to its high specificity, sensitivity, and relative simplicity, the LUX RT-PCR assay provides a novel, rapid, and practical tool for the detection of NDV.

From farm to fork follow-up of thermotolerant campylobacters throughout the broiler production chain and in human cases in a Hungarian county during a ten-months period

A study tracking thermotolerant campylobacters from the setting of the broilers throughout the whole rearing period, slaughter and sale of chicken products in five consecutive broiler rotations of the same henhouse as well as in two different other farms was conducted in a well-defined geographic area (Hajdú-Bihar county, Hungary) between March 2006 and Feb 2007. All notified cases of human campylobacteriosis in this area during the study period were also included. One hundred and one, 44, 23 and 282 Campylobacter jejuni and 13, 15, 20 and 60C. coli were isolated from broiler houses, slaughterhouses, retail shops and human samples, respectively. Sixty-two isolates collected from broilers or their environment selected from different flocks (57C. jejuni, 5C. coli), 92 isolates collected from abattoirs and retail shops (72C. jejuni, 20C. coli), as well as 85 randomly selected human isolates (74C. jejuni, 11C. coli) were subjected to PFGE analysis using restriction enzymes KpnI and SmaI. Sixty-six of the isolates produced unique Sma-Kpn profiles; the majority (46) of these were of human origin. The remaining isolates formed PFGE clusters of between 2-25 isolates with 14 (12C. jejuni and 2C. coli) main clusters comprised of five or more isolates with identical KpnI-SmaI patterns. Two genetic clones of C. jejuni (clone A, n=25; clone B, n=20) included 18% of isolates from different sources. Generally, isolates from one cluster were found in 1-3 different flocks, notably, clone B was present in three rotations including those from the two independent farms. Six of the seven investigated flocks had one or two characteristic prevalent clones. Transmission of clones between consecutive flocks was frequently seen. Spread of both C. jejuni and C. coli was traced multiple times along the food chain; eight C. jejuni, but no C. coli clones were detected both in broilers and humans. These data suggest that broilers were the major source for C. jejuni but not for C. coli in the studied area and period. For C. jejuni the carryover of strains between consecutive flocks may be a common event, but the strain is eventually replaced by another and consecutive carryover events seem to be infrequent. The majority of the human disease was due to nonepidemic strains; some clones were transmitted from more than one broiler flocks (including epidemiologically unrelated flocks) to humans multiple times.

Preliminary studies on feline coronavirus distribution in naturally and experimentally infected cats.

Abstract The shedding, tissue distribution and quasispecies composition of feline coronaviruses were studied in naturally and experimentally infected cats. The infection remained subclinical, but the majority of the animals shed the virus via faeces throughout the experiment. Sequences corresponding to the viral nucleocapsid region were amplified by reverse-transcription polymerase chain reaction from the cortex, dura mater, pancreas, lungs, third eyelid, and the heart muscle in four cases. Interestingly, the ORF7b viral region – a supposed virulence factor – was detected in fewer organs, raising the possibility that this region can be affected by deletions during virus replication in vivo. It is demonstrated that the composition of the viral quasispecies differs between organs, and that genomic regions with different functions undergo distinct processes of selection, which should be considered during the evolution of feline coronaviruses.

Novel means of viral antigen identification: Improved detection of avian influenza viruses by proximity ligation

Recent outbreaks of avian influenza in different parts of the world have caused major economic losses for the poultry industry, affected wildlife seriously and present a significant threat even to human public health, due to the risk for zoonotic transmission. The ability to recognize avian influenza viruses (AIVs) early is of paramount importance to ensure that appropriate measures can be taken quickly to contain the outbreak. In this study, the performance of a proximity ligation assay (PLA) for the detection of AIV antigens in biological specimens was evaluated. It is shown that PLA: (i) as a novel principle of highly sensitive antigen detection is extending the arsenal of tools for the diagnosis of AIV; (ii) is very specific, nearly as sensitive as a commonly used reference real-time PCR assay, and four orders of magnitude more sensitive than a sandwich ELISA, utilizing the same antibody; (iii) avoids the necessity of nucleic acids extraction, which greatly facilitates high-throughput implementations; (iv) allows the use of inactivated samples, which safely can be transported from the field to diagnostic laboratories for further analysis. In summary, the results demonstrate that PLA is suited for rapid, accurate and early detection of AIV.

Four different sublineages of highly pathogenic avian influenza H5N1 introduced in Hungary in 2006-2007

Highly pathogenic avian influenza (HPAI) H5N1 viruses were introduced to Hungary during 2006-2007 in three separate waves. This study aimed at determining the full-length genomic coding regions of the index strains from these epizootics in order to: (i) understand the phylogenetic relationship to other European H5N1 isolates, (ii) elucidate the possible connection between the different outbreaks and (iii) determine the putative origin and way of introduction of the different virus variants. Molecular analysis of the HA gene of Hungarian HPAI isolates obtained from wild birds during the first introduction revealed two groups designated Hungarian1 (HUN1) and Hungarian2 (HUN2) within sublineage 2.2B and clade 2.2.1, respectively. Sequencing the whole coding region of the two index viruses A/mute swan/Hungary/3472/2006 and A/mute swan/4571/Hungary/2006 suggests the role of wild birds in the introduction of HUN1 and HUN2 viruses: the most similar isolates to HUN1 and HUN2 group were found in wild avian species in Croatia and Slovakia, respectively. The second introduction of HPAI H5N1 led to the largest epizootic in domestic waterfowl in Europe. The index strain of the epizootic A/goose/Hungary/14756/2006 clustered to sublineage 2.2.A1 forming the Hungarian3 (HUN3) group. A common ancestry of HUN3 isolates with Bavarian strains is suggested as the most likely scenario of origin. Hungarian4 (HUN4) viruses isolated from the third introduction clustered with isolate A/turkey/United Kingdom/750/2007 forming a sublineage 2.2.A2. The origin and way of introduction of HUN4 viruses is still obscure, thus further genetic, phylogenetic, ecological and epidemiological data are required in order to elucidate it.

New Pig Disease in Hungary: Postweaning Multisystemic Wasting Syndrome Caused by Circovirus (Short Communication)

Postweaning multisystemic wasting syndrome (PMWS), a new disease in Hungary, was recognized in a swine herd located in Southeast Hungary, during the early winter of 1999. The first clinical signs of paleness, anaemia, and leanness appeared immediately after weaning, at the age of 40-50 days. Pustules were frequently observed on the skin of the trunk, and signs of necrotic dermatitis were also visible. A syndrome of poor growth and wasting was characteristic of the affected pigs. A porcine circovirus (PCV), the suspected causative agent, was detected by polymerase chain reaction (PCR). Sequencing data and restriction endonuclease (RE) analysis of the PCR products suggested that the virus belonged to the PCV-II group where all the causative agents of PMWS are also grouped.