Genotyping of European Toxoplasma gondii strains by a new high-resolution next-generation sequencing-based method.

PURPOSE: A new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains. METHODS: T. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples. RESULTS: Among type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing. CONCLUSION: The new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.

A ring trial to harmonize Toxoplasma gondii microsatellite typing: comparative analysis of results and recommendations for optimization.

A ring trial among five European laboratories was organized to reach consistency in microsatellite (MS) typing of the zoonotic parasite Toxoplasma gondii. Three sample sets were circulated and analyzed by each laboratory following a previously published method that is based on fragment length polymorphism of 15 MS markers. The first sample set compared typing results in general and focused on effects of DNA concentration; the second sample set focused on the polymorphic fingerprinting markers that can differentiate T. gondii strains within the same archetypal lineage; and the third set focused on non-archetypal genotypes. Methodological variations between laboratories, including the software programs used to determine MS fragment length, were collated using a questionnaire. Overall, lineage-level typing results reached a high level of agreement, especially in samples with the highest DNA concentrations. However, laboratory-specific differences were observed for particular markers. Major median differences in fragment length, of up to 6 base pairs, were related to the fluorophore used to label fragment-specific primers. In addition, primer pairs with identical sequences obtained from different suppliers resulted in fragments of differing length. Furthermore, differences in the way the sequencing profiles were assessed and interpreted may have led to deviating results in fragment length determination. Harmonization of MS typing, for example, by using the same fluorophores or by numerical adjustments applied to the fragment-lengths determined, could improve the uniformity of the results across laboratories. This is the first interlaboratory comparison, providing guidelines (added as a supplement) for the optimization of this technique.

Chicken line-dependent mortality after experimental infection with three type IIxIII recombinant Toxoplasma gondii clones.

Three genetically different clones of Toxoplasma gondii, also different in mouse virulence, were studied by experimental infection in chickens. For the experiments, four chicken lines were used, which differed in phylogenetic origin and performance level: two white egg layer lines, one with high laying performance (WLA), one with low (R11) and two brown layer lines, also displaying high (BLA) and low (L68) egg number. Chickens were intraperitoneally infected with three different T. gondii isolates representing type IIxIII recombinant clones, i.e. showing both, type II- and type III-specific alleles. These clones (K119/2 2C10, B136/1 B6H6, K119/2 A7) had exhibited virulence differences in a mouse model. In chickens, a significantly higher mortality was observed in white layer lines, but not in brown layer lines, suggesting that differences in the phylogenetic background may influence the susceptibility of chickens for toxoplasmosis. In addition, antibody (IgY) levels varied in surviving chickens at 31 days post infection. While low to intermediate antibody levels were observed in white layers, intermediate to high levels were measured in brown layers. Infection with a T. gondii clone showing low chicken virulence resulted in higher antibody levels in all chicken lines compared to infection with T. gondii clones of intermediate or high chicken virulence. This was in agreement with the parasite load as determined by real-time PCR. Overall, results show that progeny resulting from natural sexual recombination of T. gondii clonal lineages, may differ in their virulence for mice and chickens.

Sarcocystis sinensis is the most prevalent thick-walled Sarcocystis species in beef on sale for consumers in Germany.

Bovines are intermediate hosts of Sarcocystis cruzi, Sarcocystis hirsuta, and Sarcocystis hominis, which use canids, felids, or primates as definitive hosts, respectively. Cattle represent also intermediate hosts of Sarcocystis sinensis, but the definitive hosts of this parasite are not yet known. Sarcocystosis in cattle is frequently asymptomatic. The infection is characterized by the presence of thin-walled (S. cruzi) or thick-walled muscle cysts or sarcocysts (S. hominis, S. sinensis, and S. hirsuta). Recent reports suggest high prevalence of the zoonotic S. hominis in beef in Europe. We therefore aimed at differentiating Sarcocystis spp. in beef offered to consumers in Germany using molecular and microscopical methods, focusing on those species producing thick-walled sarcocysts. A total of 257 beef samples were obtained from different butcheries and supermarkets in Germany and processed by conventional and multiplex real-time PCR. In addition, 130 of these samples were processed by light microscopy and in 24.6% thick-walled cysts were detected. Transmission electron microscopical analysis of six of these samples revealed an ultrastructural cyst wall pattern compatible with S. sinensis in five samples and with S. hominis in one sample. PCR-amplified 18S ribosomal DNA (rDNA) fragments of 28 individual thick-walled cysts were sequenced, and sequence identities of ≥98% with S. sinensis (n = 22), S. hominis (n = 5) and S. hirsuta (n = 1) were observed. Moreover, nine Sarcocystis sp. 18S rDNA full length gene sequences were obtained, five of S. sinensis, three of S. hominis, and one of S. hirsuta. Out of all samples (n = 257), 174 (67.7%) tested positive by conventional PCR and 179 (69.6%) by multiplex real-time PCR for Sarcocystis spp. Regarding individual species, 134 (52%), 95 (37%), 17 (6.6%), and 16 (6.2%) were positive for S. cruzi, S. sinensis, S. hirsuta, and S. hominis, respectively. In conclusion, S. sinensis is the most prevalent thick-walled Sarcocystis species in beef offered for consumption in Germany. Further studies are needed to identify the final host of S. sinensis as well as the potential role of this protozoan as a differential diagnosis to the zoonotic species S. hominis.

'Schmallenberg virus'--a novel orthobunyavirus emerging in Europe.

In 2011, a novel orthobunyavirus of the Simbu serogroup, the Schmallenberg virus (SBV), was discovered using a metagenomic approach. SBV caused a large epidemic in Europe in ruminants. As with related viruses such as Akabane virus, it appears to be transmitted by biting midges. Transplacental infection often results in the birth of malformed calves, lambs and goat kids. In more than 5000 farms in Germany, The Netherlands, Belgium, France, UK, Italy, Spain, Luxembourg, Denmark and Switzerland acute infections of adult ruminants or malformed SBV-positive offspring were detected, and high seroprevalences were seen in adult ruminants in the core regions in The Netherlands, Germany and Belgium. The discovery of SBV, the spread of the epidemic, the role of vectors, the impact on livestock, public health issues, SBV diagnosis and measures taken are described in this review. Lessons to be learned from the Schmallenberg virus epidemic and the consequences for future outbreaks are discussed.

Towards a new, ecologically targeted approach to monitoring wild bird populations for avian influenza viruses.

Prevalence monitoring of avian influenza in wild bird populations is important to estimate risks for the occurrence of potentially zoonotic and economically disastrous outbreaks of highly pathogenic avian influenza virus (AIV) in poultry worldwide. A targeted, cost-effective monitoring method for AIV in wild birds was developed, which is based on monitoring results for AIV in Germany and information on the distribution and abundance of wild bird species in selected habitat types. Spatial data were combined with virological and outbreak data for the period of 1 January 2006 to 31 December 2010. Using Germany as an example, we identified 11 indicator species. By concentrating monitoring efforts on these species in spatially confined locations, we propose a targeted and more cost-effective risk-based AIV monitoring approach that can be adapted universally for the identification of wild bird indicator species worldwide with the perspective of reducing sample sizes (and costs) without impairing the validity of the results.

A long-term serological survey on Aujeszky's disease virus infections in wild boar in East Germany.

Between 1985 and 2008, a total of 102,387 wild boar sera originating from Eastern Germany covering an area of 108 589 km2 were tested for the presence of Aujeszky's disease virus (ADV)-specific antibodies. From 1985 until 1991 and from 1992 until 2008, wild boar sera were exclusively investigated using either conventional seroneutralization assays (n=39 621) or commercial gB and full antigen ELISAs (n=62,766), respectively. Spatial-temporal analysis revealed an increasing ADV seroprevalence from 0·4% to 15·9%, on average, during the 24-year observation period that went along with a continuous spread of the infection in a western direction. During 2006 and 2008, 18% of the 66 affected districts had ADV seroprevalences >30%. There was a significant correlation between ADV seroprevalence and the hunting index of population density (HIPD) of wild boar in the entire study area, although this did not hold true for some regions. Seroprevalences did not differ between sexes but were age-dependent. East Germany has been officially free of Aujeszky's disease (pseudorabies) in domestic pigs since 1985. Although a risk for domestic pigs cannot be completely ruled out, experience has shown that ADV in domestic pigs could be eliminated although the virus was present in the wild boar population. Despite increasing ADV seroprevalence in the East German wild boar population no spillover infections from wild boar to domestic pigs have been reported. To further trace ADV infections in the wild boar population in Germany, a nationwide serological monitoring programme should be implemented.

The trade network in the dairy industry and its implication for the spread of contamination.

In case of an outbreak of a foodborne disease, administrative decisions in the context of crisis management are only efficient if they follow standard practices and are specifically adapted to the outbreak situation in a timely manner. These goals are hard to achieve. The complexity of national and global trade structures obscures a clear view of trade flows and, consequently, it is often impossible to unravel complex trade links quickly. Furthermore, increasing public concerns about possible health hazards caused by global trade put additional pressure on decision makers. The aim of this paper was to unveil the specific trade structures of the German milk supply chain, to highlight how these structures could affect the spatial spread of a hypothetical contaminant, and to quantify the risk of the contaminant reaching the consumer. To achieve this goal, the vertical and horizontal trade links between milk producers, dairies, and consumers were taken into account. The horizontal flow of milk between dairies (inter-dairy trade), which is intended to compensate a temporary over- or undersupply of milk, is of special importance in this respect. We hypothesized that the extent of inter-dairy trade would significantly influence the spatial spread of contaminated milk and the contamination risk. This hypothesis was tested using a computer simulation model that predicts the hypothetical spread of a contaminant via trade of milk. The model parameters were estimated using trade data collected in 2004 and 2010. The results of our study indicate that inter-dairy trade significantly influenced the contamination risk. Compared with a scenario with no inter-dairy trade, the risk that contaminated milk will reach the consumer was up to 4 times higher, even with moderate inter-dairy trade. The contamination risk depended on the extent of inter-dairy trade in a nonlinear way and reached its maximum asymptotically when inter-dairy trade increased. The contamination risk exhibited considerable spatial variation, which could be utilized to implement more accurate food control interventions in times of crisis caused by a foodborne disease.

Bovine tuberculosis: making a case for effective surveillance.

In 2008, a cow with marked gross lesions suspicious for bovine tuberculosis (bTB) was identified by meat inspection at home slaughtering in north-western Germany. Epidemiological investigations led to the identification of another 11 affected farms with a total of 135 animals which reacted positive to the skin test. Eight affected farms had been in trade contact with the putative index farm. While the source for the initial introduction remained unknown, it was shown that all isolates tested shared the same molecular characteristics suggesting a common source of infection. The findings demonstrate that bTB can easily be transmitted via animal trade and may remain undetected for years in herds in the absence of tuberculin testing. Hence, we believe that bTB surveillance should not rely only on meat inspection, but on a combination of both meat inspection and intradermal tuberculin testing.

Characterization of pseudorabies virus of wild boar origin from Europe.

Pseudorabies virus (PrV) infections appear to be more widely distributed in the European wild boar (Sus scrofa) population than assumed. In Europe, attempts to isolate and characterize the causative agents have been limited so far. We therefore collected and examined a total of 35 PrV isolates obtained from wild boar or hunting dogs in Germany, France, Spain, Italy, Slovakia and Hungary between 1993 and 2008. Restriction enzyme analysis of genomic DNA using BamHI showed that all isolates, except one, belonged to genogroup I but different subtypes were evident. For further investigations of the phylogenetic relationships, a 732-bp fragment of the glycoprotein C (gC) gene was amplified by PCR. Sequence analysis revealed about 40 variant positions within this fragment. Comparison of the nucleotide sequences supported the separation into a clade containing isolates from North-Rhine Westphalia, Rhineland-Palatinate (Germany), France and Spain (clade B) and an apparently more variable clade comprising isolates from Brandenburg, Baden-Wurttemberg, Saxony, Saxony-Anhalt (Germany), Slovakia, Hungary, Italy and France (clade A).

First isolation of Neospora caninum from the faeces of a dog from Portugal.

We report the in vitro isolation of Neospora caninum from the faeces of a naturally infected 8-year-old male stray boxer from Portugal. Vero cell cultures were infected using parasite stages obtained after oral inoculation of gamma-interferon knockout mice with 10(2) sporulated oocysts. The isolate was identified by microscopical examination, as well as histological, immunological and molecular methods including a DNA-microsatellite-based typing technique, and was subsequently named NC-P1. The DNA-microsatellite pattern observed in the NC-P1 isolate was not previously reported for any N. caninum isolate. To our knowledge, this is the first isolation of N. caninum from the faeces of a naturally infected dog from Portugal.

Molecular comparison of Neospora caninum oocyst isolates from naturally infected dogs with cell culture-derived tachyzoites of the same isolates using nested polymerase chain reaction to amplify microsatellite markers.

Neospora caninum infection is an important cause of bovine abortion. The infection can be transmitted transplacentally or by ingestion of oocysts shed by definitive hosts. There are few reports of dogs naturally shedding N. caninum oocysts and only some oocyst isolates were transferred into cell culture. The aim of the present study was to analyse N. caninum oocysts from the faeces of naturally infected dogs using a microsatellite-based typing technique and to compare them with cell culture-derived tachyzoites of the same isolates. To this end, N. caninum oocysts from six naturally infected dogs were inoculated into gamma-interferon knockout mice. After these mice had developed disease, tissue samples or peritoneal washings from necropsied mice were transferred into cell culture. Nested-PCR techniques were developed for the sensitive and specific amplification of N. caninum microsatellite-containing regions (MS1B, MS2, MS3, MS4, MS5 and MS10). DNA was extracted from oocysts and cell culture tachyzoites of each isolate, followed by amplification and sequence analysis of microsatellite-containing regions. Each parasite isolate examined yielded a unique microsatellite genotype, while no differences were revealed when data for N. caninum oocysts were compared with cultured tachyzoites of the same isolate. Our technique may allow the typing of clinical samples and different strains of N. caninum at the molecular level. This method may prove useful for the identification of infection sources in molecular epidemiological studies.

Virological monitoring of white storks (Ciconia ciconia) for avian influenza.

Between 2003 and 2008, more than 600 white stork (Ciconia ciconia) nestlings in the German federal state of Brandenburg were ringed and examined for influenza A viruses. With the spread of highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 among wild birds in Germany in spring 2006, dead wild birds, including 88 white storks, were tested for infection with HPAIV. Furthermore, fresh fecal samples were examined by RT-PCR to monitor the occurrence of HPAIV in adult storks. While the monitoring of nestlings and adult white storks failed to yield evidence of influenza A virus infections in these birds, two storks found dead in April 2006 in the same location tested positive for HPAIV H5N1. Sequence analysis revealed that the virus isolated from one of the storks belonged to clade 2.2, which was commonly found in wild birds in the north of Germany and other European countries during the epidemic in 2006. Despite these two cases, white storks seemed to serve as neither a vector nor as a reservoir for HPAIV in Germany. The risk of white storks transmitting HPAIV to domestic poultry and humans is low.

Validation of PCR-based protocols for the detection of Echinococcus multilocularis DNA in the final host using the Intestinal Scraping Technique as a reference.

Oral uptake of infectious Echinococcus multilocularis eggs shed by canids with their faeces may lead to development of alveolar echinococcosis in humans, which is clinically similar to a malignant infiltrative tumor and may be fatal if left untreated. E. multilocularis is therefore regarded as one of the most important and neglected metazoan parasites in the Northern hemisphere. The diagnosis of this tapeworm in the final host plays a key role in the epidemiology of E. multilocularis. The diagnostic performance of a magnetic-capture (MC) DNA extraction protocol in combination with a minor groove-binder real time PCR (MC-MGBqPCR) for the detection of E. multilocularis eggs was determined relative to a highly sensitive variant of the Intestinal Scraping Technique (IST) using faecal samples of foxes. In addition, we compared results obtained by MC-MGBqPCR with those of a previously validated protocol (QIAamp Fast DNA Stool Mini Kit (QT) combined with a TaqMan qPCR). Furthermore, a workflow using the NucleoMagVet DNA extraction kit (NM) in combination with MGBqPCR and TaqMan-qPCR was also included in the comparisons. To estimate the analytical sensitivity, phosphate-buffered saline and fox faecal samples were spiked with different numbers of eggs and tested in defined combinations of DNA extraction and PCR protocols. To assess the diagnostic sensitivity of the different workflows, samples were used that had been collected from the ampulla recti or the rectum of 120 foxes hunted in Brandenburg, Germany. The samples represented five IST categories formed according to the E. multilocularis worm burden of the foxes. For DNA extraction by MC or using two other commercial extraction kits, the supernatants obtained from 3 g of bead-beaten faecal samples were used. The extracted DNAs were then processed in the respective PCR protocols. The MC-MGBqPCR showed the highest diagnostic sensitivity (93%; 95% Confidence Interval (CI): 86-97%) relative to IST. The QT extraction protocol in combination with TaqMan-qPCR had the second highest sensitivity (89%; 95% CI: 80-94%), followed by NM with MGBqPCR (86%; 95% CI: 77-93%) in comparison to IST. The lowest diagnostic sensitivity was found for the NM combined with the TaqMan-qPCR protocol (72%; 95% CI: 62-82%). In conclusion, the MC-MGBqPCR seems to represent a suitable alternative to IST. However, applied to 3 g faecal samples, the less costly QT-TaqMan-qPCR workflow yielded a similar diagnostic sensitivity relative to IST. However, differences between these two workflows were not statistically significant.

Toxoplasma gondii infection and toxoplasmosis in farm animals: Risk factors and economic impact.

The protozoan parasite Toxoplasma gondii is a zoonotic parasite that can be transmitted from animals to humans. Felids, including domestic cats, are definitive hosts that can shed oocysts with their feces. In addition to infections that occur by accidental oral uptake of food or water contaminated with oocysts, it is assumed that a large proportion of affected humans may have become infected by consuming meat or other animal products that contained infective parasitic stages of T. gondii. Since farm animals represent a direct source of infection for humans, but also a possible reservoir for the parasite, it is important to control T. gondii infections in livestock. Moreover, T. gondii may also be pathogenic to livestock where it could be responsible for considerable economic losses in some regions and particular farming systems, e.g. in areas where the small ruminant industry is relevant. This review aims to summarize actual knowledge on the prevalence and effects of infections with T. gondii in the most important livestock species and on the effects of toxoplasmosis on livestock. It also provides an overview on potential risk factors favoring infections of livestock with T. gondii. Knowledge on potential risk factors is prerequisite to implement effective biosecurity measures on farms to prevent T. gondii infections. Risk factors identified by many studies are cat-related, but also those associated with a potential contamination of fodder or water, and with access to a potentially contaminated environment. Published information on the costs T. gondii infections cause in livestock production, is scarce. The most recent peer reviewed reports from Great Britain and Uruguay suggest annual cost of about 5-15 million US $ per country. Since these estimates are outdated, future studies are needed to estimate the present costs due to toxoplasmosis in livestock. Further, the fact that T. gondii infections in livestock may affect human health needs to be considered and the respective costs should also be estimated, but this is beyond the scope of this article.

Phylogenetic analyses of highly pathogenic avian influenza virus isolates from Germany in 2006 and 2007 suggest at least three separate introductions of H5N1 virus.

In spring 2006, highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 was detected in Germany in 343 dead wild birds, as well as in a black swan (Cygnus atratus) kept in a zoo, three stray cats, one stone marten (Martes foina), and in a single turkey farm. In 2007 (June-July) the virus reoccurred in 96 wild birds at six geographically separate locations in the Southeast of Germany. In addition, a backyard mixed duck and goose holding was affected. Real-time RT-PCR [Hoffmann, B., Harder, T., Starick, E., Depner, K., Werner, O., Beer, M., 2007. Rapid and highly sensitive pathotyping of avian influenza A H5N1 virus by using real-time reverse transcription-PCR. J. Clin. Microbiol. 45, 600-603] and nucleotide sequencing confirmed that these H5-viruses belonged to the Qinghai lineage of HPAIV H5N1 (clade 2.2). For a more detailed analysis, the hemagglutinin and neuraminidase genes of 27 selected German H5N1 viruses isolated 2006 or 2007 and originating from different regions and animal species were sequenced and analysed phylogenetically. As a result, three closely related but distinguishable H5N1 subclades could be defined: In 2006 a 'Northern type' (subclade 2.2.2), representing virus isolates from the German federal states Mecklenburg-Western Pomerania, Schleswig-Holstein, Brandenburg, and Lower Saxony, and a 'Southern type' (subclade 2.2.1) from Baden-Württemberg and Bavaria were detected. Interestingly, representatives of both types were present in Central Germany and caused the outbreak in turkeys (subclade 2.2.2) and in a case in a tufted duck (Aythya fuligula) (subclade 2.2.1) in Saxony. Furthermore, one isolate from the South of Germany was identified as 2.2.2 and vice versa a 2.2.1-like isolate was found in Northern Germany. H5N1 viruses isolated in 2007 belonged to a third type (subclade 2.2.3) which was not detected in 2006. Our data suggest the introduction of three distinct H5N1 variants into the wild bird population of Germany. The source of these viruses and the exact time of introduction remain obscure. Based on the identification of closely related H5N1 viruses from Southern and Central Russia, a recent introduction via wild birds on winter escape from these regions, early in 2006 constitutes the most likely scenario for the 2006 outbreaks. The viruses detected in 2007 most likely represent another new incursion from an as yet unknown source.

Neospora caninum infection in Greek dairy cattle herds detected by two antibody assays in individual milk samples.

A survey to demonstrate the presence and the extent of Neospora caninum infection in dairy cattle was carried out in Greece. Seven hundred and seventy-seven (777) individual milk samples from all milking cows in 10 dairy herds were tested using an ELISA (p38-milk-ELISA) and immunoblot analysis. The herd prevalence was 80.0% and 100.0%, while the overall prevalence was 15.2% (ranging between 0.0% and 38.9%) and 27.9% (ranging between 3.4% and 61.6%) based on the results of the ELISA and the immunoblot test, respectively. In conclusion, N. caninum infection is present among dairy cattle in Greece, deserving more studies to determine its contribution to abortion problems in Greek dairy herds.

Occurrence of Toxoplasma gondii and Hammondia hammondi oocysts in the faeces of cats from Germany and other European countries.

Faecal samples of 24,106 cats from Germany and other European countries were examined microscopically in a veterinary laboratory in Germany between October 2004 and November 2006 to estimate the prevalence of animals shedding Toxoplasma gondii or Hammondia hammondi oocysts. Oocysts of 9-15 microm size with a morphology similar to that of H. hammondi and T. gondii were found in 74 samples (0.31%). A total of 54 samples were further characterised to achieve a species diagnosis and to determine the genotype of T. gondii isolates by PCR and PCR-RFLP. From these samples, 48 isolates were obtained: 26 (0.11%) were finally identified as T. gondii and 22 (0.09%) as H. hammondi. T. gondii-positive samples came from Germany, Austria, France and Switzerland while H. hammondi was detected in samples from Germany, Austria and Italy. In two samples (one T. gondii and one H. hammondi), PCR indicated the presence of Hammondia heydorni DNA. No Neospora caninum DNA was detected in any of the feline faecal samples. Twenty-two of the 26 T. gondii isolates could be genotyped. A PCR-RFLP analysis for the SAG2, SAG3, GRA6 and BTUB genes revealed T. gondii genotype II in all cases. Morphologically, H. hammondi oocysts exhibited a statistically significantly smaller Length-Width-Ratio than T. gondii oocysts.

Molecular and serological surveillance of Hepatitis E virus in wild and domestic carnivores in Brandenburg, Germany.

Hepatitis E virus (HEV) is a zoonotic virus which circulates in pigs and wild boars as main reservoir species. To reveal the infection rate in carnivores, we have carried out a monitoring study of raccoons, raccoon dogs, dogs and cats sampled in Brandenburg, Germany. In summary, 53.8% (43 of 80) of the raccoons, 34.3% (25 of 73) of the raccoon dogs, 56.6% (47 of 83) of dogs and 32.3% (21 of 65) of cats were tested positive for HEV-specific antibodies. No viral RNA could be detected. This first description of anti-HEV antibodies in raccoons and raccoon dogs worldwide and in dogs and cats in Germany highlights the natural host range expansion of HEV.

Analysis of Toxoplasma gondii clonal type-specific antibody reactions in experimentally infected turkeys and chickens.

Due to their ground-feeding behaviour, free-ranging chickens and turkeys are exposed to oocysts and are good indicators of the presence of Toxoplasma gondii in the environment. In addition, poultry may become infected by ingestion of tissues of infected intermediate hosts such as small rodents. Free-ranging poultry are considered an important source of T. gondii infection in humans, especially in developing countries. Knowledge on T. gondii genotypes in infected animals and humans is important for understanding the epidemiology of T. gondii infections. The aim of the present study was to analyse the ability of experimentally infected turkeys and chickens to develop a T. gondii clonal type-specific antibody response (IgY) after i.v. inoculation with tachyzoites of three T. gondii clonal lineages, types I, II and III. A peptide microarray displaying a panel of 101 different synthetic peptides was used for serotyping. Peptide sequences were derived from polymorphic regions of 16 T. gondii proteins (GRA1, GRA3-7, SAG1, SAG2A, SAG3, SAG4, SRS1, SRS2, ROP1, NTPase I and NTPase III and BSR4). The array was probed with 120 sera from experimentally infected chickens and turkeys inoculated with different doses of T. gondii tachyzoites (104, 103 and 102) collected from isolates representative for T. gondii clonal types I (RH), II (ME49) or III (NED) and uninfected controls. After screening of the peptides with reference sera from chickens and turkeys, and evaluation of data by Receiver Operating Characteristics analysis, 41 and 40 peptides were identified that appeared suitable to detect type-specific reactions with sera collected at 2, 5, 7 and 9 weeks p.i. Selected peptides allowed the identification of T. gondii clonal types, until 9 week p.i., which the chickens or turkeys had been inoculated with. At 9 weeks p.i., a high proportion of the experimentally infected chickens (67% (12/18)) and turkeys (61% (11/18)) no longer reacted with the selected peptides. Serotyping of the infection in individual chickens or turkeys was only possible when the whole peptide panel was applied. Clonal type-specific antibody responses were dynamic in both poultry species and depended on the individual animal and the time after infection.