Tularaemia seroprevalence of captured and wild animals in Germany: the fox (Vulpes vulpes) as a biological indicator.

A total of 2475 animals from Germany, both captive and wild, were tested for antibodies against Francisella tularensis to obtain more knowledge about the presence of this pathogen in Germany. An indirect and a competitive ELISA served as screening methods, positive and inconclusive samples were confirmed by Western blot. Of the zoo animals sampled between 1992 and 2007 (n = 1122), three (0·3%) were seropositive. The seroconversion of a hippopotamus in Berlin Zoo was documented. From 1353 serum samples of wild foxes (Vulpes vulpes), raccoon dogs (Nyctereutes procyonoides) and wild boars (Sus scrofa), collected between 2005 and 2009 in the federal state of Brandenburg (surrounding Berlin), a total of 101 (7·5%) tested positive for antibodies to F. tularensis lipopolysaccharide. Our results indicate a higher seroprevalence of F. tularensis in wildlife in eastern Germany than commonly assumed. Furthermore, we found foxes and raccoon dogs to be biological indicators for tularaemia.

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.

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).

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.

Monitoring of laying capacity, immunoglobulin Y concentration, and antibody titer development in chickens immunized with ricin and botulinum toxins over a two-year period.

One of the key benefits in using chickens for immunization is the high yield of antibodies obtainable. It is known that egg production decreases over time, while animal maintenance costs remain stable. It would, however, be desirable to keep hens as long as possible to obtain maximal amounts of antibodies. To identify a suitable length of time that animals can be kept and to optimize the cost:yield ratio, we monitored the number of eggs laid, the total amount of chicken IgY, and the specific antibody titer from individually prepared eggs over a 2-yr period. The plant toxin ricin and the Clostridium botulinum neurotoxins type A and B were used to immunize 4 chickens. The number of eggs laid in 2 yr was approximately 600 per hen (about 80% of the maximum egg number), yielding about 20 to 40 g of total IgY per hen. A stable antibody titer of 1:100,000 to 1:1,000,000, as measured by ELISA, was obtained following up to 11 injections of 10 to 20 microg of immobilized native toxin. Laying capacities were found to decrease, on average, from 7 eggs/wk at the point of first immunization to 2 eggs/wk after more than 2 yr. In parallel, the yield of total and specific IgY increased over time, so that the antibody recovery remained high, even after prolonged immunization times. Using purified IgY preparations, classical immunological assays such as ELISA and Western blotting were performed. Furthermore, the IgY showed neutralizing capacity when used to block the functional activity of the toxins both in vitro and in vivo. Analysis of the total IgY content over time demonstrated a complex biological oscillation (and the antigen-specific titer), with a shorter time period of around 7 d (circaseptan rhythm). In summary, we successfully immunized chickens with ricin and botulinum neurotoxins and monitored laying capacity, IgY concentration, and specific antibody titer over an extended period of 2 yr.

Retrospective serological survey on selected viral pathogens in wild boar populations in Germany.

The objective of this study was to retrospectively evaluate the occurrence of porcine parvovirus (PPV), Aujeszky's disease virus (ADV), transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV), porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza virus (SIV) in selected wild boar populations in Germany (n = 1,221). Commercial enzyme linked immunosorbent assay and hemagglutination inhibition tests were used for serological monitoring. The serosurvey revealed seroprevalence rates of 64.28%, 11.26%, 7.87%, 7.84%, 3.82% and 1.59% for PPV, ADV, PRCV, SIV, PRRSV and TGEV, respectively. The seroprevalence rates differed between populations and age classes with the highest number of antibody-positive wild boars in older animals (>1 year old). No antibodies to TGEV were found in Baden-Wuerttemberg and in Mecklenburg-Western Pomerania (investigation period 1997/1998). In addition, sera collected in Mecklenburg-Western Pomerania in 1997/1998 were negative for SIV. Even though the seroprevalence rates established for these viruses, except for PPV, were relatively low, wild boars may act as a reservoir for pathogens and a source of infection for domestic pigs and humans. Based on the epidemiological situation, no risk of a spread of these viruses should emanate from wild boars, neither for wildlife nor for livestock. However, effective and science-based disease monitoring programmes should continuously be carried out in wild boar populations.

Diagnostic procedures after completion of oral immunisation against classical swine fever in wild boar.

The purpose of this paper is to define diagnostic procedures for wild boar after the completion of oral immunisation against classical swine fever (CSF). Epidemiological analysis of CSF in wild boar in Germany demonstrated that it is vital to carry out virological investigations on all animals found dead, sick or involved in traffic accidents. In principle, this should ensure an effective and prompt diagnosis of CSF. In addition, a defined number of wild boar, especially young animals < or = 6 months old, should also be tested for CSF virus to guarantee a high confidence level in the virological monitoring. Which animals should be examined serologically depends on the age class investigated, the season in which vaccination was stopped and the period of time since completion of vaccination. Therefore, different serological procedures have been defined for different situations during the first three years after completion of oral immunisation.

Establishment of human Ig producing heterohybridomas by fusion of mouse myeloma cells with human lymphocytes derived from peripheral blood, bone marrow, spleen, lymph node, and synovial fluid. Effect of polyclonal prestimulation and cryopreservation.

50 fusion experiments were carried out to analyse heterohybridization efficiencies on mouse myeloma cells of the P3 X63 Ag8/653 line with human lymphocytes derived from peripheral blood, bone marrow, lymph node, spleen or synovial fluid. We found higher yields of growing and human Ig-producing hybridoma lines when lymphocytes from spleen or lymph node were fused. Although primary hybridomas could be established from fusions with bone marrow-derived cells, only in nine out of 1616 initially seeded wells was Ig production registered. Four fusions using immune cells from synovial fluid were made without success. Independently of the source of lymphocytes pokeweed mitogen (PWM) prestimulation had no enhancing effect on the percentage of wells with cell growth and this did not alter the IgM:IgG ratio in primary hybridomas (9:1), although cells from all compartments used here (with the exception of bone marrow cells) could be stimulated with PWM to produce both IgG and IgM in cultures. Cryopreserved lymphocytes from different sources could be used for fusions with comparable results registered for the fresh material.

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.

Feeder cells from different sources and conditioned media for recloning of human--mouse and mouse--mouse hybridomas.

The use of different feeder cell layers (peritoneal macrophages from mice and rats, spleen cells and thymocytes from mice) for recloning of human--mouse and mouse--mouse hybridomas has been described. Optimal numbers of feeder cells from different sources required for high cloning efficiencies were determined. It was possible to use cryopreserved rat and mouse macrophages as feeders for cell cloning. However, the resulting cloning efficiency was much lower in comparison to the fresh material. Culture supernatants from human endothelial cells (added in a final concentration of 40% v/v) and from chicken embryo fibroblasts (25%) could replace the feeder cell layer in recloning experiments with both human--mouse and mouse--mouse hybridomas. Therefore, conditioned media (prepared in large quantities) may be used for generating standardized conditions for high-efficient cloning and recloning of hybridoma cell lines.

Cell biology of human IgM-producing hybridomas derived from a fusion of human spleen lymphocytes with mouse myeloma cells.

Hybrids were derived from the fusion of mouse myeloma cells with human spleen cells from a patient with active idiopathic thrombocytopenia. Of 288 initially seeded cultures, 186 were found to produce human Ig. The growth and Ig production rates, cloning efficiencies using different feeder layers and the karyotype were determined for 9 clones that stably produced human monoclonal IgM (2-100 micrograms/ml) for at least 9 months. All cells of the Ig-producing hybridoma clones were positive for cytoplasmic-Ig, whereas only 20-65% of cells expressed surface Ig (mu and chains). Human monoclonal antibodies in mass cultures were derived in serum-free PRMI 1640 medium. Two clones produced human IgM (nearly 2 mg/ml) in the ascitic fluid of nude mice. Feeder cells of peritoneal macrophages from Balb/c mice enabled more efficient recloning of human x mouse hybrids than did thymocytes. Nearly all subclones derived from 2 clones were found to produce the same monoclonal antibodies as the parental lines. Information on the individual parameters of a hybridoma cell line may be helpful in the large-scale production of human monoclonal antibodies.

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.

Replacement of fetal calf serum in cell cultures by an egg yolk factor with cholecystokinin/gastrin-like immunoreactivity.

The in vitro culture of various cell types is an important scientific tool and is becoming increasingly acceptable as a viable alternative to animal experiments. Fetal calf serum (FCS) is a supplement used in many cell culture media, and provides cells with growth factors and cytokines necessary for successful culture. In view of the animal welfare issues surrounding the production of FCS, an alternative agent allowing the replacement or reduction in the use of FCS is desirable. A yolk extract factor (EYF-X) obtained from chicken eggs is described, which facilitates the in vitro culture of a variety of cell types. When the extract was added to a culture medium used for in vitro fertilisation, the number of successful fertilisations was significantly increased. In a further in vitro model (permanent neuronal cell line N2A), the yolk extract significantly stimulated cell proliferation as well as the growth of cell processes. A set of specific antibodies against different parts of the prepro-cholecystokinin reacted with the extract. The intensity of the reaction depends on the age of the egg (time after the laying date). Analysis by gel chromatography recorded a main protein fraction with an apparent molecular mass of 20-30kDa. This fraction was labelled by Western blot with an antibody with specificity against CCK-octapeptide. These findings suggest that the yolk factor may be a CCK/gastrin-like molecule. Since CCK/gastrin-like molecules have also been detected in the spermatozoa of mammals, the influence on in vitro fertilisation could be explained by the yolk factor replacing the endogenous CCK/gastrin-like molecule destroyed in sperm freezing. The results of this study suggest that it might be possible to replace FCS with EYF-X. The application of the yolk factor to a broad spectrum of cell types remains to be investigated.

[Case report: Porcine circovirus type 2 infection in an European wild boar (Sus scrofa) in the state of Brandenburg, Germany]. / Porzine Circovirus Typ 2-Infektion bei einem Wildschwein (Sus scrofa) im Land Brandenburg.

This case represents the first case of Porcine Circovirus Type 2 (PCV-2)--infection in a free living European wild boar associated with morphological lesions, which are regarded as characteristic for Postweaning Multisystemic Wasting Syndrome (PMWS) in domestic pigs. The animal, an approximately 10 month old male, was found dead in a rural area within the state of Brandenburg, Germany. The closest commercial pig farm is located in 3 km distance from the spot where the carcass was found. At necropsy, the animal was found to be in a runted condition. Morphological investigation revealed two lesion complexes. Firstly, lymphatic depletion was present in different organs. Mainly the white pulp of the spleen was affected, where lymph follicles and periarteriolar lymphatic sheaths were nearly completely depleted of lymphoid cells. The former lymphatic areas could only be identified by the presence of histiocytic cells. Secondly, there were widely distributed lesions indicative of a bacterial septicemia i.e. purulent-necrotizing lymphadenitis, pulpous hyperplasia of the spleen, miliary lytic liver necroses and foci of fibrinous pneumonia. Within the lesions, bacterial colonies were found (short Gram-negative rods). Bacteriology revealed a septicemic Salmonella choleraesuis var. Kunzendorf--infection. Virologically, the animal was tested with negative results for Classical Swine Fever Virus and PRRSV. The unusual depletion of the lymphatic tissue mainly in the spleen led to the suspicion of a PCV-2 infection. Typical circoviral particles were found by negative-contrast electron microscopy in samples from spleen and lymph nodes. Using a commercial antiserum against Porcine Circovirus, positive staining was found by fluorescence microscopy in tonsils, spleen and lymph nodes. Finally, the virus was identified to be PCV-2 by species-specific PCR. The presented case rises the questions if PCV-2 is endemic in the European wild boar population at least in certain areas, if it is of pathogenetic importance for wild boars and if the virus present in wild boars is identical to that present in domestic pigs with PMWS.

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.

Generalized fatal Cowpox virus infection in a cat with transmission to a human contact case.

A 4-month-old female domestic shorthair cat was infected by a virus of the Poxvirus family. The animal developed a severe pneumonia and generalized ulcerating lesions of the skin. Histologically, typical eosinophilic intracytoplasmic inclusion bodies indicative of an Orthopoxvirus (OPV) infection were present. The lung showed grey-white to haemorrhagic nodular lesions with a central zone of complete necrosis of alveolar and bronchial tissue. Electron microscopy from skin and lung nodules revealed typical square-shaped OPV particles. Cultivation of the virus on chorio-allantoic membranes of embryonated chicken eggs resulted in haemorrhagic plaques. Restriction enzyme analysis, PCR and sequencing of the D8L gene identified the OPV isolate as a typical Cowpox virus. It was transmitted by the cat to a human contact person who developed a local nodular dermatitis at the inoculation site in association with signs of general infection and had an increase of OPV-specific neutralizing antibodies in paired serum samples.

A virological survey in migrating waders and other waterfowl in one of the most important resting sites of Germany.

Wild birds are considered a potential reservoir or a carrier of viral diseases and may therefore play a role in the epidemiology of economically important or zoonotic diseases. In 2001 and 2002, a survey with special emphasis on virus isolation in migrating waders and some other birds were conducted. In one of the most important inland resting sites for migratory waterfowl, tracheal and cloacal swabs were collected from 465 waders representing 19 different species, and 165 other birds that were not captured on purpose. A total of 42 avian viruses were isolated, 34 of these were identified as paramyxoviruses (PMVs). The majority of isolates came from waders and wild ducks, and were characterized as PMV-1. In contrast, PMV-4 was found in wild ducks only, PMV-6 was mainly detected in wader species. Four avian influenza viruses (AIVs), belonging to H4 and H3 haemagglutinin subtype, were isolated from wild duck species. Furthermore, four reo-like viruses were isolated from one particular wader species for the first time. The majority of virus positive birds were <1 year old and did not show any clinical symptoms. There was no evidence for the presence of West Nile virus in these birds. These results confirm that the restricted resting sites in Western Europe must be considered as important locations for the intra- and interspecies transmission of avian viruses.

[The occurrence of virus enteritis (duck plague) in wild birds]. / Untersuchungen zum Vorkommen der Virusenteritis (Entenpest) bei Wildvögeln.

296 samples from wild birds of 15 species were incorporated into long term exploration of duck plague on ducks in farms. By using virological and serological standard methods 9 virus carriers and 20 serum samples showing positive antibody titers could be detected. The epidemiology as well as the relation of the incidence of duck plague in wild birds and farm poultry is discussed.