Meat juice as diagnostic sample for virological and serological diagnosis of classical swine fever.

The objective of this paper was to assess if meat juice is a suitable substrate for virological and serological diagnosis of classical swine fever (CSF). Fifty-six domestic pigs and 21 wild boars experimentally vaccinated and/or infected as well as 129 field samples from wild boars were involved in this study. Meat juice from diaphragm, forequarter and hindquarter was used for investigations. CSFV and viral RNA were detected in meat juice between days 5 and 21 post infection (pi). Animals which had survived the infection were diagnosed virologically negative and antibody-positive in muscle fluid. After vaccination or vaccination and subsequent infection of animals (n = 42), meat juice samples scored serologically positive. The antibody titres of these samples were significantly lower than in serum. Serological investigations of field samples derived from wild boars (n = 75) shot in Mecklenburg-Western Pomerania showed a clear correlation between the antibody-positive samples in serum and in meat juice, whereas the serological results of meat juice samples (n = 54) from wild boars collected in Lower Saxony were slightly different. The reasons for these differences are discussed. Nevertheless, meat juice seems to be a suitable substrate for CSF diagnosis, especially for wild boars.

Epidemiological survey of swine influenza A virus in selected wild boar populations in Germany.

The aim of this study was to evaluate the epidemiological situation of swine influenza virus (SIV) infections in different wild boar populations in Germany based on a serological surveillance in some Bundeslaender (federal states) in connection with virological investigations in wild boar shot in Northern Germany (Mecklenburg-Western Pomerania, district of Nordvorpommern). Altogether, 1245 sera from wild boar were tested using the hemagglutination inhibition test. The established seroprevalence rate was low (on average 5.2%). Antibodies were only detected against the subtypes H1N1 and H3N2 showing differences between wild boar populations and age classes. The virological investigation of samples derived from lungs of wild boar shot in Mecklenburg-Western Pomerania, district of Nordvorpommern (n=242), revealed that the virus prevalence (two virologically positive animals, 0.8%) was very low. Based on serological typing, the isolated SIV was identified as subtype H3N2. Molecular biological investigations of the hemagglutinin (HA) and neuraminidase (NA) genes confirmed this result. This study suggests that SIV infections in wild boar seem to be no serious threat for domestic pigs.

Retrospective analysis of the oral immunisation of wild boar populations against classical swine fever virus (CSFV) in region Eifel of Rhineland-Palatinate.

In the present study the effect of control measures implemented during the classical swine fever (CSF) epidemic in wild boar in the Eifel region of the German federal state of Rhineland-Palatinate from 1999 to 2005 was assessed. During the first 3 years after official confirmation of virus detection these measures comprised intensive hunting, especially of young animals and hygiene measures. Subsequently oral immunisation (o.i.) using a modified live virus vaccine was introduced as an additional control tool. All shot wild boar from the restricted area were tested virologically and serologically for CSF. The laboratory results from over 110,000 animals accompanied by information about age, gender and geographical origin of the animals were collected in a relational database. In total about 82% of all virologically positive wild boars were piglets, thus confirming the importance of this age group in the perpetuation of the epidemic. An analysis of the hunting bag showed that piglets were underrepresented compared to older animals throughout the eradication programme. This finding indicated that hunters did not comply with the control strategy of intense targeting of young animals. Before as well as after the implementation of o.i. a significantly higher virological prevalence and a significantly lower serological prevalence were observed in piglets compared to yearlings and adults. Shortly after the beginning of the vaccination campaign in February 2002 CSFV prevalence decreased significantly whereas the serological prevalence increased markedly in all age classes. In order to test the influence of age and vaccination on the serological prevalence a logistic regression model was used. Our results strongly suggest that under the field conditions in the Eifel region vaccination against CSFV had a crucial influence on the increase of seroprevalence rate and the elimination of CSFV. The last virus-positive pig was found 13 months after start of o.i.

Susceptibility of sheep to European bat lyssavirus type-1 and -2 infection: a clinical pathogenesis study.

European bat lyssaviruses (EBLVs) have been known to cross the species barrier from their native bat host to other terrestrial mammals. In this study, we have confirmed EBLV-1 and EBLV-2 susceptibility in sheep (Ovis ammon) following intracranial and peripheral (intramuscular) inoculation. Notably, mild clinical disease was observed in those exposed to virus via the intramuscular route. Following the intramuscular challenge, 75% of the animals infected with EBLV-1 and 100% of those that were challenged with EBLV-2 developed clinical signs of rabies and then recovered during the 94-day observation period. Disease pathogenesis also varied substantially between the two viruses. Infection with EBLV-1 resulted in peracute clinical signs, which are suggestive of motor neuron involvement. Antibody induction was observed and substantial inflammatrory infiltrate in the brain. In contrast, more antigen was detected in the EBLV-2-infected sheep brains but less inflammatory infiltrate and no virus neutralising antibody was evident. The latter involved a more protracted disease that was behaviour orientated. A high infectious dose was required to establish EBLV infection under experimental conditions (> or =5.0 logs/ml) but the infectious dose in field cases remains unknown. These data confirm that sheep are susceptible to infection with EBLV but that there is variability in pathogenesis including neuroinvasiveness that varies with the route of infection. This study suggests that inter-species animal-to-animal transmission of a bat variant of rabies virus to a terrestrial mammal host may be limited, and may not always result in fatal encephalitis.

Value of skin punch biopsies for the diagnosis of acute classical swine fever.

The objective of this study was to determine if skin punch biopsies are appropriate for the diagnosis of classical swine fever. For this purpose, 6 wild boars and 2 domestic pigs were experimentally infected with the highly virulent classical swine fever virus (CSFV) Koslov and 5 domestic pigs with a CSFV field isolate (genotype 2.3 Uelzen) derived from wild boar. Skin biopsy specimens were tested using virus isolation, real-time reverse transcription polymerase chain reaction (rtRT-PCR), and fluorescent antibody test (FAT) on cryosections. Whereas CSFV Koslov was first detected at 4 days postinfection (DPI) by rtRT-PCR and virus isolation, FAT failed to detect CSFV antigen until 9 DPI. In domestic pigs infected with CSFV 2.3 Uelzen, viral RNA and CSFV were detected at 7 DPI. FAT was negative until 11 DPI. CSFV antigen was detected in endothelial cells of the vascular plexus in the upper dermis as shown by confocal laser-scanning microscopy and double labeling with von Willebrand factor. At 18 DPI, CSFV antigen was present diffusely in capillaries and spindle shaped cells of the dermis, multifocally within keratinocytes of the epidermis and in numerous cells of the inner and outer root sheath epithelium, hair bulb, and intravascular leukocytes. The rtRT-PCR proved to be the test with the highest sensitivity followed by virus isolation and FAT. Taken together, this study demonstrates that skin is easy to sample antemortem and is also suitable as postmortem tissue, and suggests that rtRT-PCR of skin should be included for CSF diagnosis in the acute period of disease.

Nictitating membrane as a potentially useful postmortem diagnostic specimen for classical swine fever.

The gold standard for diagnosis of classical swine fever (CSF) is cell culture virus isolation combined with reverse transcriptase-polymerase chain reaction (RT-PCR) and fluorescent antibody test (FAT) in cryosections of tonsils, spleen, various lymph nodes, ileum, and kidney. Autolytic and heterolytic samples render correct FAT evaluation difficult and can even yield false-negative or ambiguously positive results. To extend the spectrum of CSF diagnostic specimens, the authors tested whether the nictitating membrane (NM) might be a useful adjunct diagnostic specimen in wild boars and domestic pigs. To accomplish this, results of virus isolation, FAT, and RT-PCR were compared on NM samples and lymphoid tissues, which are the routine specimens of choice for CSF diagnosis. Wild boars (n = 30) and domestic pigs (n = 8) were experimentally challenged with various CSF virus (CSFV) strains or isolates of different virulence. The FAT revealed CSFV antigen in surface and tubular adenoid epithelium as well as in lymphatic follicles of the NM. In wild boars and domestic pigs with CSF, a strong agreement was found between results of FAT, virus isolation, and RT-PCR on NM and lymphoid tissues. These results suggest that NM is a useful additional specimen that can provide valuable data for postmortem diagnosis of CSF. The NM is relatively easy to sample at necropsy, and postmortem autolysis and heterolysis of this tissue is minimal compared with internal organs.

Hepatitis E virus genotype 3 diversity: phylogenetic analysis and presence of subtype 3b in wild boar in Europe.

An increasing number of indigenous cases of hepatitis E caused by genotype 3 viruses (HEV-3) have been diagnosed all around the word, particularly in industrialized countries. Hepatitis E is a zoonotic disease and accumulating evidence indicates that domestic pigs and wild boars are the main reservoirs of HEV-3. A detailed analysis of HEV-3 subtypes could help to determine the interplay of human activity, the role of animals as reservoirs and cross species transmission. Although complete genome sequences are most appropriate for HEV subtype determination, in most cases only partial genomic sequences are available. We therefore carried out a subtype classification analysis, which uses regions from all three open reading frames of the genome. Using this approach, more than 1000 published HEV-3 isolates were subtyped. Newly recovered HEV partial sequences from hunted German wild boars were also included in this study. These sequences were assigned to genotype 3 and clustered within subtype 3a, 3i and, unexpectedly, one of them within the subtype 3b, a first non-human report of this subtype in Europe.

Development of maternal antibodies after oral vaccination of young female wild boar against classical swine fever.

An experimental study was performed to investigate the development of maternal antibodies after oral immunisation of young female wild boar against classical swine fever (CSF) using C-strain vaccine. Our results demonstrated that maternal antibodies do not persist in the offspring for more than 3 months. Based on the neutralising serum antibody titres, we assume that piglets of wild sows vaccinated orally twice or immunised once a long time before conception have protective antibodies for approximately 2 months. Furthermore, it seems that the level and the duration of maternal antibodies in the offspring are depend on the age of the female animals at the moment of vaccination as demonstrated in our experiment. The recent vaccination procedure consists of three double vaccinations in spring, summer and autumn. Especially vaccinations in summer and autumn could be crucial for transfer of high maternal antibody titres to the offspring.

An update on safety studies on the attenuated "RIEMSER Schweinepestoralvakzine" for vaccination of wild boar against classical swine fever.

The RIEMSER Schweinepestoralvakzine is an attenuated vaccine for oral vaccination of wild boar against classical swine fever (CSF). The safety of this licensed bait vaccine which is based on the CSF virus (CSFV) strain "C" was investigated in eight animal species, e.g. weaner pigs (n=111), wild boar (n=11), ruminants (cattle, goats and sheep, n=11), foxes (n=5), rabbits (n=12), and mice (n=10). Animals were vaccinated either with a single vaccine dose containing at least 10(4.5) TCID(50), or with overdoses, i.e. the 10-fold dose, or they were subjected to repeated application schemes. During the entire observation period none of the animals which were given the vaccine virus showed clinical signs, with the exception of rabbits. These reacted to the vaccination with fever. Orally vaccinated pigs did not transmit vaccine virus to susceptible contact animals (sentinels). In none of the species examined neither vaccine virus nor viral RNA could be detected in blood after vaccination. In one wild boar viral RNA could be established in the tonsil 21 days post-vaccination (dpv); all other organ samples tested virologically negative. Up to 77.5% of the pigs and wild boar developed virus neutralising antibodies (VNA) already 14 dpv. The mean VNA titres observed in the vaccination groups seemed to depend rather on individual factors than on the administered virus dose (virus titre per dose) or the vaccination scheme. These results are comparable with findings obtained during oral vaccination campaigns in wild boar and after parenteral vaccination with this C-strain virus. From the results presented here it can be concluded that RIEMSER Schweinepestoralvakzine is safe for target and non-target species.

Chimeric pestiviruses: candidates for live-attenuated classical swine fever marker vaccines.

The use of attenuated classical swine fever virus (CSFV) strains as live vaccines is no longer allowed for the control of classical swine fever in Europe, due to the inability to differentiate between infected and vaccinated animals (Differentiating Infected from Vaccinated Animals; DIVA), except as emergency vaccines or as bait vaccines for wild boars. Thus, the establishment of a DIVA vaccine(s) is of pivotal importance for the control of this infectious disease. In this study, recombinant versions of the live-attenuated vaccine strain CSFV Riems were generated by replacing parts of the E2 gene with the corresponding sequence of border disease virus strain Gifhorn. Three cDNA clones were constructed: pRiems-ABC-Gif, pRiems-A-Gif and pRiems-BC-Gif. Infectious particles were obtained from clones pRiems-ABC-Gif and pRiems-BC-Gif only, whereas transfected RNA from clone pRiems-A-Gif behaved like a replicon. Based on its ability to be differentiated in vitro from wild-type CSFV by mAbs, vRiems-ABC-Gif was assessed for immunogenicity and protection against challenge infection in pigs. Before challenge, no CSFV-specific anti-E2 antibodies could be detected with commercial E2-blocking ELISAs in vRiems-ABC-Gif-vaccinated animals, whereas vRiems-vaccinated pigs developed high titres of anti-E2 antibodies, confirming the marker properties of this vaccine candidate. After oral vaccination, only partial protection against challenge infection was observed in the vRiems-ABC-Gif vaccinees, whereas all intramuscularly vaccinated animals and all vRiems-vaccinated animals were fully protected. These experiments suggest that the strategy of exchanging specific antigenic epitopes among pestiviruses is a promising tool for the development of new CSFV marker vaccines.

[Laboratory diagnosis of avian influenza by reverse transcription (RT)-PCR]. / Labordiagnose der Aviären Influenza mittels Reverser Transkription (RT)-PCR.

RT-PCR assays which amplify conserved regions of the influenza A virus gene are useful tools for the rapid and specific detection of infections of poultry with avian influenza virus (AIV) and for the investigation of large numbers of samples, e.g. within the framework of surveillance programs. Here, we present findings on the efficiency and on the limits of an RT-PCR assay which amplifies a part of the matrix protein gene. Sensitivity and specificity of the method were increased by the additional use of nested PCR. Parameters which may have an essential influence on the detection limit are outlined and discussed. A major focus of the study is the detection of AIV RNA from organ samples and swabs.

The "Friedrich-Loeffler-Institut": past, present and future of research in infectious diseases of animals.

The Friedrich-Loeffler-Institut, founded in 1910 by Friedrich Loeffler, the discoverer of the first animal virus, foot-and-mouth disease virus, is the oldest virological research facility in the world. Beyond viruses, its area of competence has significantly expanded since its foundation and now also covers bacterial, parasitic and prion diseases of livestock, poultry and aquatic animals. Presently located at four sites within Germany (Insel Riems, Jena,Tübingen,Wusterhausen) the tasks of the institute as delineated in the Animal Disease Act encompass research on infectious animal diseases including zoonoses, import/export examinations, epidemiological studies in case of outbreaks of notifiable animal diseases, acting as reference laboratory for notifiable animal diseases and nationwide quality management of diagnosis of notifiable animal diseases. It is obliged to publish and maintain up-to-date diagnostic regimes for notifiable animal diseases, and it publishes a yearly report on animal health in Germany. With the increasing importance of infectious diseases of animals, in particular those potentially harmful to man (zoonoses), the Friedrich-Loeffler-Institut will be moving into new facilities including laboratories and animal facilities up to the highest biosafety level at its main site Insel Riems on the occasion of its 100th anniversary.

[Vaccination of weaner pigs against classical swine fever with the subunit vaccine "Porcilis Pesti": influence of different immunization plans on excretion and transmission of challenge virus]. / Vakzination von Absatzferkeln gegen Klassische Schweinepest mit der Subunit-Vakzine "Porcilis Pesti": Einfluss des Vakzinationsschemas auf Challengevirusausscheidung und -übertragung.

Excretion and transmission of CSFV after vaccination with the CSF subunit marker vaccine "Porcilis Pesti" have been studied using the following different vaccination schedules: Group A--two vaccinations with an interval of 28 d, challenge 14 d after second vaccination (p.v2.); group B--two vaccinations with an interval of 14 d, challenge 14 d later; group C--two vaccinations with an interval of 28 d, challenge at time of booster vaccination; group D--two vaccinations with an interval of 14 d, challenge 7 d p.v2.; group E--single vaccination and infection 14 d later. After infection one sentinel pig was added to the vaccinated and infected pigs of each group. A single vaccination did not induce protective immunity against a CSFV challenge. Double vaccination at a four-week interval protected piglets from clinical infection, and neither viraemia and leukopenia nor virus excretion were detected if infected 14 d p.v2. Two vaccinations at a two-week interval followed by a challenge 7 d p.v2. led to a short viraemia on day 5 p.i. but without excretion of CSFV. Though all other vaccination schedules induced a reduction in virus shedding and a decrease in CSFV replication, in all these cases in-contact controls became infected. The results of transmission of CSFV are discussed in relation to a potential use of subunit marker vaccines in CSF control.

Evaluation of the oral immunisation of wild boar against classical swine fever in Baden-Württemberg.

The oral immunisation of wild boar against classical swine fever (CSF) in Baden-Württemberg is described and evaluated. The bait vaccine based on the CSF virus (CSFV) strain "C" proved to be safe in wild boar of all age classes. The modified immunisation procedure consisting of three double vaccinations per year was very effective. CSFV was not detected beyond the second immunisation campaign. The average rate of seropositive wild boar diagnosed over all immunisation periods was 49.2%. The seroprevalence rate increased significantly during the first year of immunisation and reached its maximum after the third vaccination period with 72% antibody positive animals. The higher percentage of seropositive young boars in this field trial compared to the seroprevalence rates in this age class in other field trials in Germany may be attributed to the new vaccination scheme. Factors that may be responsible for the decreased herd immunity after the fourth or sixth immunisation period are discussed.