A. Hlinak

Organ distribution of Schmallenberg virus RNA in malformed newborns

A novel orthobunyavirus was first detected in German dairy cows in autumn 2011 and was subsequently found in the brains of malformed lambs, kids and calves in the Netherlands, Germany, Belgium, France, Italy, Great Britain, Luxembourg and Spain. For rapid detection of this novel virus, named Schmallenberg virus, a real time quantitative reverse transcription PCR (RT-qPCR) was developed at the Friedrich-Loeffler-Institut and provided to the federal veterinary state laboratories in Germany. For diagnostic purposes, the organ distribution of this new virus was analyzed in several organs and body fluids of 15 lambs and two calves showing typical malformations. Spleen, cerebrum, meconium, spinal cord, rib cartilage, umbilical cord, placental fluid out of the stomach as well as external placental fluid scraped from the coat of the foetuses were collected during necropsy. All animals were tested RT-qPCR positive in the external placental fluid, and all but one were also RT-qPCR positive in the cerebrum, the umbilical and the spinal cord. Our results suggest that both the external placental fluid and the umbilical cord could be suitable sample materials for the confirmation of an infection with Schmallenberg virus in malformed newborns, at least in lambs. This is of special interest since those samples can be collected very easily on the farm without the need of a necropsy.

Highly pathogenic avian influenza virus (H5N1) in frozen duck carcasses, Germany, 2007.

Article summary line: Phylogenetic and epidemiologic evidence shows incursion of HPAIV into the food chain.

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).

SEROLOGICAL SURVEY OF VIRAL PATHOGENS IN BEAN AND WHITE-FRONTED GEESE FROM GERMANY

Sera from wild geese were tested for antibodies to selected viral pathogens at a resting site for wild waterfowl in Germany. Serum samples from both bean geese (Anser fabalis) and white-fronted geese (Anser albifrons) collected in October 1991 were examined using serological methods licensed for routine diagnosis in domestic poultry. Of 130 sera tested, antibodies to several infectious agents were found including Newcastle disease virus (45%), goose parvovirus (48%), avian reovirus (29%) and avian adenovirus or egg drop syndrome 76 virus (6%). Antibodies against duck hepatitis virus were not detected. Differences in seroprevalences were not detected between the two geese species. While role and significance of wild geese in the epidemiology of avian diseases remains to be determined, it is possible that they could be of some importance as reservoirs and carriers of certain viral diseases of domestic poultry.

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 Aujeszkys 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 Aujeszkys 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.

A descriptive analysis of the potential association between migration patterns of bean and white-fronted geese and the occurrence of Newcastle disease outbreaks in domestic birds.

The sightings and migration patterns of 65 bean (Anser fabalis) and 65 white-fronted geese (Anser albifrons) are reported. In the past, these geese were serologically screened for the occurrence of Newcastle disease virus (NDV) and other avian viral diseases by Hlinak et al. (3). Of the 130 birds originally tagged and serologically screened in 1991, 53 birds were resighted between 1991 and 1996. Most of the sightings were reported from main wintering and resting sites in Germany and The Netherlands. It is noteworthy that 19 of the 53 birds sighted had serologic evidence that they had been exposed to NDV before the time of marking in 1991. Although the origin of these infections in bean geese and white-fronted geese is still unknown, the sightings reported in this study indicate that, once infected, wild geese may be involved in the dissemination and spread of avian viral diseases, specifically Newcastle disease. The migration patterns of the wild geese provided further evidence that the main resting and wintering areas of migratory waterfowl are likely to be important for the inter- and intraspecies transmission of avian diseases, thereby representing risk areas for the poultry industry.

Virological Monitoring of White Storks (Ciconia ciconia) for Avian Influenza

Abstract 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.

Spontaneous Aujeszky's disease (pseudorabies) in European wild boars (Sus scrofa) in the federal state of Brandenburg, Germany.

In this report two cases of spontaneous Aujeszkys disease (AD, syn. pseudorabies) in European wild boars (Sus scrofa), in the federal state of Brandenburg, Germany, are described. Both animals displayed severe central nervous disturbances, including loss of fear of humans, disorientation, and tremors of head and limbs, and were shot by hunters for laboratory exclusion of rabies. The main finding in the well nourished, juvenile (approx. 7-8 months) animals was a non-suppurative panencephalitis characterized by neuronal necroses, intranucelar eosinophilic inclusion bodies in necrotic neurons, spongiosis of the neuropil, gliosis and perivascular cuffing of lymphocytes, plasma cells and eosinophilic granulocytes. Pseudorabies virus (PrV)-antigen was detected by immunohistochemistry in typically affected brain areas and was isolated from pooled tissues (brain and tonsil) in both cases. The molecular characterization of the virus isolates revealed that they belonged to the wild boar-associated PrV subtype Iw. These cases indicate that spontaneous AD can sporadically occur in free living wild boars under natural conditions. However, factors triggering the disease, e. g. social stress, age related change from passive to active immunity, individual susceptibility to PrV infection and environmental conditions, have to be clarified by future experimental studies.

A novel alphaherpesvirus associated with fatal diseases in banded penguins

A novel avian alphaherpesvirus, preliminarily designated sphenicid alphaherpesvirus 1 (SpAHV-1), has been independently isolated from juvenile Humboldt and African penguins (Spheniscus humboldti and Spheniscus demersus) kept in German zoos suffering from diphtheroid oropharyngitis/laryngotracheitis and necrotizing enteritis (collectively designated as penguin-diphtheria-like disease). High-throughput sequencing was used to determine the complete genome sequences of the first two SpAHV-1 isolates. SpAHV-1 comprises a class D genome with a length of about 164 kbp, a G+C content of 45.6 mol% and encodes 86 predicted ORFs. Taxonomic association of SpAHV-1 to the genus Mardivirus was supported by gene content clustering and phylogenetic analysis of herpesvirus core genes. The presented results imply that SpAHV-1 could be the primary causative agent of penguin-diphtheria-like fatal diseases in banded penguins. These results may serve as a basis for the development of diagnostic tools in order to investigate similar cases of penguin diphtheria in wild and captive penguins.

Serological and virological survey and resighting of marked wild geese in Germany

In order to investigate the potential role of arctic geese in the epidemiology, the spatial and temporal spread of selected avian diseases, in autumn 2002, a virological and serological survey designed as capture-mark-resighting study was conducted in one of the most important coastal resting sites for migratory waterfowl in Germany. Oropharyngeal, cloacal swabs and blood samples were collected from a total of 147 birds comprising of three different arctic geese species including White-fronted Goose (Anser albifrons), Tundra Bean Goose (Anser fabalis rossicus), Pink-footed Goose (Anser brachyrhynchus) as well as from 29 non-migratory Canada Geese (Branta canadensis). Altogether, six adeno-like viruses (ALV; 95% CI, 1.74–9.92%) and two avian paramyxoviruses (APMV-4; 95% CI, 0.19–5.53%) were isolated mainly from juvenile White-fronted Geese. In addition, four Canada Geese were infected with lentogenic APMV-1 (95% CI, 3.89–31.66%) at the date of sampling. No avian influenza viruses, reo-like viruses could be isolated despite serological evidence. Likewise, no evidence of current or previous infection by West Nile virus was found. Of the 147 birds tagged in the following years, 137 birds were re-sighted between 2002 and 2008 accumulating to 1925 sightings. About 90% of all sightings were reported from the main wintering and resting sites in Germany and The Netherlands. Eight of the resighted geese were virus positive (ALV and APMV-4) at the time point of sampling in 2002.