Effects of monensin and essential oils on immunological, haematological and biochemical parameters of cows during the transition period.

Using a model to generate experimental groups with different manifestations of post-partum (p.p.) fat mobilization and ketogenesis, the effects of a dietary and a medical intervention on biochemical and haematological parameters, antibody titre, leucocytes subsets and function of transition cows were examined. In total, 60 German Holstein cows were allocated 6 weeks antepartum (a.p.) to 3 high-body condition score (BCS) groups (BCS 3.95) and 1 low-BCS group (LC, BCS 2.77). High-BCS cows received a monensin controlled-release capsule (HC/MO) or a blend of essential oils (HC/EO) or formed a control group (HC). Parameters were evaluated in 3 periods (day (d) -42 until calving, 1 until 14 days in milk (DIM), 15 until 56 DIM). Over the course of trial, various parameters were influenced by period with greatest variability next to calving. White blood cell count was higher in the HC (8.42 × 103 /µl) and HC/EO (8.38 × 103 /µl) groups than in the HC/MO group (6.81 × 103 /µl) considering the whole trial. Supplementation of monensin decreased aspartate aminotransferase in comparison with the HC group similar to LC treatment. Bilirubin concentration was nearly doubled in all high-BCS cows in period 2. In period 3, essential oils increased γ-glutamyltransferase (80.4 Units/l) in comparison with all other groups and glutamine dehydrogenase (61 Units/l) in comparison with the LC (19 Units/l) and the HC/MO group (18 Units/l). Results suggest that parameters were generally characterized by a high variability around calving. Based on biochemical characteristics, it appeared that the HC cows seemed to have compromised hepatocyte integrity when compared to the LC cows. From the immune parameters investigated, the BVDV antibody response was more pronounced in HC/MO compared to HC/EO.

Genetic and antigenic characterization of Bungowannah virus, a novel pestivirus.

Bungowannah virus, a possible new species within the genus Pestivirus, has been associated with a disease syndrome in pigs characterized by myocarditis with a high incidence of stillbirths. The current analysis of the whole-genome and antigenic properties of this virus confirms its unique identity, and further suggests that this virus is both genetically and antigenically remote from previously recognized pestiviruses. There was no evidence of reactivity with monoclonal antibodies (mAbs) that are generally considered to be pan-reactive with other viruses in the genus, and there was little cross reactivity with polyclonal sera. Subsequently, a set of novel mAbs has been generated which allow detection of Bungowannah virus. The combined data provide convincing evidence that Bungowannah virus is a member of the genus Pestivirus and should be officially recognized as a novel virus species.

Limited interlaboratory comparison of Schmallenberg virus antibody detection in serum samples.

Eight veterinary institutes in seven different countries in Europe participated in a limited interlaboratory comparison trial to evaluate laboratory performances of Schmallenberg virus (SBV) antibody detection in serum. Seven different sheep sera and three different cattle sera were circulated, and all participating institutes were asked to test these sera using SBV antibody detection assay(s) in place in their laboratories. All laboratories within the trial performed a virus neutralisation test (VNT) as well as one or two ELISAs on all samples, and swiftly detected SBV antibodies using these assays. VNT was more sensitive in detecting SBV antibodies than several of the used ELISA assays. Based on the test results, one cattle and one sheep SBV antibody-positive serum were selected to serve as reference sera, which now can be supplied to other laboratories on request.

Atypical 'HoBi'-like pestiviruses--recent findings and implications thereof.

In 2004, an atypical pestivirus named D32/00_'HoBi', isolated from foetal calf serum (FCS) originating from Brazil, was described (Schirrmeier et al., 2004). A few years later, a closely related virus (Th/04_KhonKaen) was detected in serum from a calf in Thailand, indicating that this group of atypical pestiviruses already is spread in cattle populations in various regions of the world. At the Friedrich-Loeffler-Institute, Insel Riems, Germany, FCS batches are regularly tested for pestivirus contamination, in general with positive PCR results, and in some cases the contaminants have been typed as 'HoBi'-like. At the National Veterinary Institute (SVA) in Uppsala, Sweden, a recent event with contaminated FCS ruined much of the ongoing cell culture work. From the FCS and the contaminated cells we were able to amplify and sequence nucleic acid from three different pestivirus strains, including BVDV-1, -2 and 'HoBi'-like; this in a commercial FCS that had been tested free from pestivirus by the manufacturer. In this short communication we review the current status of atypical 'HoBi'-like pestiviruses, describe recent findings and discuss the implications thereof.

Recombination in rabbit haemorrhagic disease virus: possible impact on evolution and epidemiology.

Rabbit haemorrhagic disease (RHD) was first recognised in 1984 following the introduction of apparently healthy rabbits into China from Germany. The aetiological agent Rabbit haemorrhagic disease virus (RHDV) has subsequently killed hundreds of millions of domestic and wild rabbits particularly in Europe, China and Australia. Previously, using phylogenetic analysis we have attempted to understand the underlying factors that determine why this virus emerged, and why it has such an unpredictable epidemiology. Here we report the use of tree congruency supported by bootscanning analysis to detect recombination amongst both closely related, and widely divergent strains of RHDV. We show that recombination occurs commonly and in several different regions of the RHDV genome. Moreover, the first identified strain of RHDV, i.e. from China in 1984, showed evidence of recombination in the capsid gene, with a virus or viruses containing lineages in German strains. These observations imply that recombination may play a significant role in the evolution, epidemiology and diversity of RHDV.

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.

Persistence of viral RNA in rabbits which overcome an experimental RHDV infection detected by a highly sensitive multiplex real-time RT-PCR.

An internally controlled multiplex real-time RT-PCR using TaqMan probes and external standards for absolute RNA quantification was developed as a new diagnostic tool for the detection of rabbit haemorrhagic disease virus (RHDV). The test revealed a specificity of 100%, an analytical sensitivity of 10 copies/well and a linearity over a range from 10(1) to 10(10) copies. The viral loads in organs, leukocytes, sera and excretions of seropositive, convalescent rabbits which were overcoming an experimental infection with RHDV were determined using the validated assay. As a result, viral RNA was demonstrated and quantified for at least 15 weeks. Thus, a persistence of viral RNA after experimental infection of rabbits could be shown for the first time. In contrast, neither antigen nor infectious virus could be detected by antigen-ELISA, immunohistochemistry or experimental transmission. Therefore, further experiments are necessary to prove that the persistence of RNA is linked with the persistence of infectious virus particles.

Persistence of rabbit haemorrhagic disease virus genome in vaccinated rabbits after experimental infection.

Rabbit haemorrhagic disease (RHD) is usually a fatal disease in rabbits which has spread rapidly across the continents. While previous studies suggested persistence in rabbits to be an important factor in the epidemiology, the relevance of field virus infection of immune rabbits has not been investigated in experimentally infected animals before. This report describes for the first time the persistence of rabbit haemorrhagic disease virus (RHDV) genome for at least 15 weeks in rabbits immunized with an inactivated vaccine as well as a subunit vaccine and subsequently challenged with virulent RHDV. The viral RNA loads were determined by real-time reverse transcription-polymerase chain reaction. No conspicuous association of the detectable amount of RHDV RNA with the type of vaccine, the time after infection and--with one exception--the level of RHDV-specific antibodies in the immunized animals was observed. The results presented in this study are an urgent evidence for the existence of carrier animals as an important factor in the epidemiology of RHD.

Establishment of a competitive ELISA (cELISA) system for the detection of influenza A virus nucleoprotein antibodies and its application to field sera from different species.

A recombinant baculovirus (RBV) encoding the nucleoprotein (NP) of avian influenza virus (AIV) was generated and the appropriate protein was expressed in Sf9 cells. Purified recombinant NP and the NP-specific monoclonal antibody HB65 were used to establish a competitive ELISA (cELISA) system for the detection of NP-specific antibodies in sera of ducks, geese and wild birds. Tests to evaluate this method were carried out using sera of ducks experimentally infected with AIV, pre-immune duck and chicken sera, and poultry field sera, which tested negative in the haemagglutination inhibition (HI) assay, and field sera of several poultry species experimentally infected with other viruses. The evaluation of the test demonstrated a high sensitivity and specificity of this method. Tests carried out using field sera of duck and goose flocks revealed widely corresponding results obtained by HI assay and cELISA indicating that this test is applicable for flock diagnosis. Differing results were obtained for individual samples. It can be assumed that for the most part this was because of a better recognition of the conserved NP antigen by serum antibodies, although some results remained unclear.

A universal heterologous internal control system for duplex real-time RT-PCR assays used in a detection system for pestiviruses.

A heterologous in vitro transcript based on a specific primer-probe HEX system was generated as a universal internal control (IC) to improve virus-specific real-time reverse-transcriptase PCR (RT-PCR) assays. By using a set of different primers, several PCR fragments of desired sizes of an in vitro transcript of the enhanced green fluorescent protein (EGFP) gene were generated, and the fragments were detected using a HEX-labelled probe. For long-term storage of the in vitro transcript a special RNA-safe buffer (RSB) was developed. Freezing and thawing of the IC diluted in RSB did not result in any substantial loss of detectable IC copy numbers. The new IC system was used for the first time in a duplex real-time RT-PCR assay for the detection of pestivirus-derived RNA, in particular from bovine viral diarrhea virus (BVDV). Primers and TaqMan probes for the 'panpesti' assay were selected by analysing the consensus sequence of the 5' non-translated region (5' NTR) of more than 600 different pestiviruses. Finally, the optimised primer probe combination showed an analytical sensitivity of less than 10 copies/reaction. In the duplex set-up, the analytical sensitivity of the validated real-time RT-PCR was identical to the sensitivity of the single assay without IC, and the diagnostic sensitivity of the duplex assay was equal or higher if compared to virus isolation.

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.

Validation of a real-time RT-PCR assay for sensitive and specific detection of classical swine fever.

A fully validated, ready-to-use, real-time reverse transcription-polymerase chain reaction (RT-PCR) assay, multiplexed for simultaneous detection of an internal control, for the simple and rapid diagnosis of classical swine fever (CSF) was developed. Primers and FAM-labeled TaqMan-probes specific for classical swine fever virus (CSFV) were selected from the consensus sequence of the 5' non-translated region (5' NTR) of 78 different CSFV strains. For determining analytical sensitivity, an in vitro transcript (T7-PC3alf) of the 5' NTR was constructed and tested. In addition, the T7-PC3alf transcript was further used as a positive control and a standard for quantitation of CSFV genome copies. A second heterologous in vitro transcript based on a specific primer-probe HEX-system was designed as an internal positive control for the RNA isolation step and RT-PCR. By using limited primer concentrations for the internal control, no adverse effects on the sensitivity of the CSF-system could be observed, and the newly designed duplex real-time RT-PCR proved to have a sensitivity of approximately eight copies. The primer-probe combination selected was strictly CSFV-specific and no amplification was observed in all non-CSFV pestiviruses tested.

Subacute liver necrosis after experimental infection with rabbit haemorrhagic disease virus (RHDV).

Rabbit haemorrhagic disease (RHD) is usually peracute to acute, while subacute to chronic disease is rare. This paper describes gross and histopathological findings in four out of 20 rabbits aged 14 weeks, experimentally infected with one of two German field isolates of RHD virus. Eight rabbits survived the infection for 10 days and were killed after four of them, infected with 100 to 10 000 haemagglutination units, had started to develop progressive jaundice. Histopathologically, icteric livers showed severe subacute centrilobular bridging necrosis with calcification, and proliferation of periportal hepatocytes and bile ducts. Positive-strand RHDV RNA was detected by in-situ hybridization, mainly in periportal macrophages. Loss of the normal hepatic architecture, reparation (fibrosis) and hepatocellular regeneration, together with moderate inflammatory reaction, are signs of liver cirrhosis. These signs, observed in young rabbits given small doses of RHD virus, are interpreted as an unusual outcome of experimental inoculation.

[Detection of border disease virus in a sheep flock in Saxony]. / Nachweis von Border Disease Virus in einem Schafbestand in Sachsen.

A pestivirus has been isolated from brain samples of two lambs suffering from clinical signs of border disease. The two identical isolates were allocated to the "true" border disease virus group concerning their reaction pattern with monoclonal antibodies and the 5'UTR sequence data. Nevertheless, alterations of phenotype and genotype in comparison with references of both BDV-subgroups have been shown. The existence of an infection with border disease virus in the flock has been confirmed by serological studies.

Porcine reproductive and respiratory syndrome virus (PRRSV): kinetics of infection in lymphatic organs and lung.

Pigs were infected by the oronasal route with European isolates of the porcine reproductive and respiratory syndrome virus (PRRSV; I10 and Cobbelsdorf). The kinetics of infection in lymphatic organs and the lung were analysed by immunofluorescence detection of virus antigen, re-isolation of the virus and reverse transcription--polymerase chain reaction (RT-PCR) for PRRSV-specific RNA. The kinetics of PRRSV infection proceeded in three phases, irrespective of the varying infestation of lymphatic organs within the first days post-infection (p.i.). First, an early acute infection of lymphatic organs developed within the first week and was characterized by a high number of antigen-positive macrophages. Second, a delayed acute infection of the lung was observed, which was most pronounced during the second and third week p.i. when a high number of infected alveolar macrophages was observed. The acute infection of lymphatic organs had resolved at this time. Infected cells in the lung were predominantly located in pneumonic lesions. Third, a persistent infection was demonstrated by RT-PCR and immunohistology when the experiments were terminated at day 49 p.i. The virus persisted in lymphatic organs, especially in the tonsils, and in the lung. At this stage, indications for a re-occurrence of acute infection were observed in restricted areas of the lung.

Pathogenic, antigenic and molecular properties of rabbit haemorrhagic disease virus (RHDV) isolated from vaccinated rabbits: detection and characterization of antigenic variants.

Rabbit haemorrhagic disease virus (RHDV) isolates were obtained from several animals previously vaccinated with an inactivated vaccine. Seven isolates were analyzed by immunological and molecular biological methods and compared to reference strains. Antigenic characterization with monoclonal antibodies as well as haemagglutination assays demonstrated considerable differences between individual isolates. However, sequencing of the capsid protein genes revealed a high degree of homology between five of these isolates and the reference strain FRG. In contrast, two isolates specified remarkably different capsid proteins with a degree of variation not observed so far in RHDV. Amino acid alterations were found clustered between residues 301 and 328 (region C), 344 and 434 (region E) and also in the 3' region of the capsid protein gene. Interestingly, experimental vaccination of rabbits followed by challenge with the heterologous variant strains showed restricted cross-protection against one of the strains. In summary, we found a level of antigenic variation not detected in RHDV so far, and describe two distinct new antigenic variants.

Arterivirus PRRSV. Experimental studies on the pathogenesis of respiratory disease.

Pigs were infected with the porcine respiratory and reproductive syndrome virus (PRRSV) by the oronasal route. We studied the development of histological lesions, sites of virus infection and of inflammatory infiltrates by quantitative evaluation of reactive cells. The animals developed a multifocal interstitial pneumonia. Clinical signs of pneumonia were observed from day 7 to 21. In the first stage, an acute alveolitis was found, which was characterised by a hyperplasia of type II pneumocytes within the septa and an accumulation of macrophages in the alveolar spaces. Within 2-4 days p.i., virus infected cells were prominent in lymphatic organs, but their number declined rapidly during the following days. In the following period, the number of virus antigen positive cells increased in the lung. An interesting discrepancy existed between the relatively small number of virus specific cells and the degree of intensive pneumonia. As a first step to analyse mechanisms leading to the induction of pneumonia, we studied transcriptional expression of cytokines and other immunomodulatory molecules by semiquantitative RT-PCR.

Rabbit hemorrhagic disease virus (RHDV): ultrastructure and biochemical studies of typical and core-like particles present in liver homogenates.

Calicivirus particles isolated from rabbits suffering from acute RHD were compared with virions found in rabbits with chronic disease. Liver homogenates of rabbits with the protracted disease display no hemagglutinating activity and contain viral particles with diameters of 25-27 nm. These virions contain only one structural protein of 30 kDa and are distinctly smaller than intact rabbit hemorrhagic disease virus (RHDV) (32-40 nm). To prove the RHDV identity of the smaller virions, their reactivity with RHDV specific antibodies was investigated by immunoblots of the virion protein and by immunoelectron microscopy. Proteolytic digestion of RHDV particles with alpha-chymotrypsin did not transform RHDV into the smaller form. We assume that these core-like particles (CLPs) are not a result of proteolytic digestion but arise from a truncated RHDV genome or defective expression.

[Experimental reproduction of the respiratory form of infection with the agent of epidemic late abortion of swine]. / Experimentelle Reproduktion der respiratorischen Verlaufsform der Infektion mit dem Erreger des Seuchenhaften Spätabortes der Schweine.

Porcine reproductive and respiratory syndrome has been observed in a large swine-farm. Diagnostic investigations were undertaken. Piglets infected with homogenate from the lung of a sick gilt developed biphasic fever up to 42 degrees C, inappetence and apathy. A few piglets showed red-blue discolored parts of the ears and of the skin in the abdominal region. A cytopathogenic agent has been isolated on cultivated alveolar-macrophages. Infection of another group of piglets with macrophages-propagated agent resulted in similar clinical signs too. Organs of euthanatized piglets have been examined anatomical, histological and with the fluorescent antibody technique. Specific fluorescence could be seen in tonsils, bronchial lymph nodes, spleen and in pathological-changed parts of the lungs. Antibodies against PRRS-virus were detected in blood samples of experimentally infected pigs.

Some physicochemical properties of the virus of rabbit haemorrhagic disease.

Purified and concentrated preparations of virus from liver extracts of infected rabbits contain virus specific components with sedimentation coefficients of about 175, 110 and sometimes 133S and more slow units. Full and empty virus particles with a diameter of about 34 nm were shown electron microscopically in the corresponding 175 and 110S fractions of the sucrose density gradient. The average of buoyant density of the 175, 133, 110S and more slow units are 1.36, 1.32 and 1.31 g/ml respectively. The extinction coefficient E260 nm is 4.3 +/- 0.7 cm2/mg. The RNA content is 17 +/- 4%. SDS-PAGE shows a "65" kD protein as a single or major component. Beside smaller polypeptides with lower intensities, the 67 kD polypeptide reacts positively in the Western blot with polyclonal antibodies of rabbits. The molecular weight of the virus is 15 +/- 4 x 10(6)D. The pH stability of the 175S unit was also tested.