Susceptibility of koi x crucian carp and koi x goldfish hybrids to koi herpesvirus (KHV) and the development of KHV disease (KHVD).

Hybrids of koi, Cyprinus carpio x crucian carp, Carassius carassius and koi x goldfish, Carassius auratus, proved to be susceptible to koi herpesvirus (KHV, syn. CyHV-3) and developed KHV disease (KHVD). While hybrids of koi x goldfish were partly resistant to mortality following infection by immersion, most koi x crucian carp hybrids died after bath infection. KHV DNA was detected in dead fish but also in all surviving animals by different polymerase chain reactions (PCRs). According to these results, hybrid crossbreeding does not seem to prevent severe losses associated with KHV in terms of inducing KHVD. The present study showed severe losses after a waterborne KHV infection of between 35% and 100% in koi x goldfish and koi x crucian carp hybrids as well as in SPF carp.

Highly sensitive fetal goat tongue cell line for detection and isolation of foot-and-mouth disease virus.

A fetal goat cell line (ZZ-R 127) supplied by the Collection of Cell Lines in Veterinary Medicine of the Friedrich Loeffler Institute was examined for susceptibility to infection by foot-and-mouth disease (FMD) virus (FMDV) and by two other viruses causing clinically indistinguishable vesicular conditions, namely, the viruses of swine vesicular disease and vesicular stomatitis. Primary bovine thyroid (BTY) cells are generally the most sensitive cell culture system for FMDV detection but are problematic to produce, particularly for laboratories that infrequently perform FMD diagnostic tests and for those in countries where FMD is endemic that face problems in sourcing thyroid glands from FMD-negative calves. Strains representing all seven serotypes of FMDV could be isolated in ZZ-R 127 cells with a sensitivity that was considerably higher than that of established cell lines and within 0.5 log of that for BTY cells. The ZZ-R 127 cell line was found to be a sensitive, rapid, and convenient tool for the isolation of FMDV and a useful alternative to BTY cells for FMD diagnosis.

Protection of gruntlings against classical swine fever virus-infection after oral vaccination of sows with C-strain vaccine.

The objective of this study was to investigate the maternal protection of gruntlings derived from wild sows vaccinated orally against classical swine fever (CSF) using C-strain vaccine. Three vaccinated sows and one unvaccinated control sow were included. Challenge infection of the progeny was carried out either intranasally or by contact at the beginning of the third month of life (61-65 days post-natum). Whereas, two of three litters had maternal antibodies, the progeny of one vaccinated sow was seronegative at challenge. The progeny of the control sow, which was challenged by contact infection, developed moderate clinical signs except for one animal which became ill and died. Two gruntlings derived from the vaccinated sows also died of CSF, although one of them had a relatively high maternal antibody titre (128 ND(50)). The transient infection and partial virus shedding observed in a small number of gruntlings with maternal antibodies and the fact that one animal with maternal antibodies became ill and died confirm the incomplete maternal protection at this age. The reason for this incomplete protection is discussed. As none of the surviving gruntlings could be shown to carry CSFV or viral RNA at the end of the experiment (36 or 70 d.p.i.), it may be concluded that these animals do not represent a potential CSFV reservoir.

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.

Replication of classical swine fever virus strains and isolates in different porcine cell lines.

Classical swine fever virus (CSFV) is an economically important pathogen of domestic pigs and wild boar. Due to the highly variable clinical picture of CSF, laboratory methods are essential for an unambiguous diagnosis. Virus isolation using cell culture is still considered the gold standard. It is based on the incubation of permissive cells with organ or leukocyte preparations followed by antigen detection. In the "EU Diagnostic Manual for CSF Diagnosis", the permanent cell line PK(15) (porcine kidney) is recommended. In the European Reference Laboratory (EURL) a clone of this cell line, PK(15)A, and the STE (swine testicular epitheloid) cell line are in use for propagation of CSFV. The aim of this work was to assess the relative ability of eleven permanent cell lines derived from various organs of wild boar and domestic pig, respectively, to support the replication of different strains and isolates in comparison to these cell lines. An avirulent and a highly virulent laboratory CSFV strain, and several recent field isolates from domestic pigs and wild boars were used. Titers were determined after one, two and three virus passages, and after 48 and 120 h of incubation. Of the eleven cell lines analyzed, two were found that replicated all the tested CSFV strains and field isolates. Those may be useful for improving diagnosis of CSFV and for preparing low-passaged virus stocks of new isolates.

Classical swine fever virus Strain 'C'. How long is it detectable after oral vaccination?

To determine the persistence period of C-strain vaccine virus in immunized animals, domestic pigs and wild boars were vaccinated orally and killed on different days post vaccinationem (dpv). Tissue samples were taken at necropsy from both species for detection of C-strain virus. From domestic pigs nasal swabs and faeces were also collected. During the investigation period (2-12 dpv) vaccine virus could never be detected in nasal secretions and in faeces of vaccinated domestic pigs. In contrast, C-strain virus was found in organs until day 8 pv in domestic pigs and until day 9 pv in wild boars. Whereas in domestic pigs virus was detected in tonsils, Ln. mandibularis or in spleen, in wild boar it only was found in tonsils. We conclude that C-strain vaccine virus is not detectable in wild boars longer than 10-12 days after intake of the vaccine baits.

Establishment of a new bovine leukosis virus producing cell line.

Due to the prevalence of different bovine leukosis virus (BLV) species in the cattle population in Europe, problems may arise in the serological diagnosis of BLV infections. In addition, earlier investigations demonstrated that contamination of the BLV antigen-producing cell culture systems by bovine viral diarrhea virus (BVDV) may give rise to misinterpretation of serological test results after BVDV vaccination of cattle. By co-cultivation of peripheral leukocytes of a BLV-infected cow with a permanent sheep kidney cell line, a new BLV-producing cell line named PO714 was established. This line carries a BLV provirus of the Belgian species and has been tested to be free of a variety of possibly contaminating viruses and mycoplasms. Investigations of a panel of well-characterised sera by agar gel immunodiffusion (AGID) and capture ELISA (cELISA) tests using antigen prepared from this new cell line in comparison with antigen of the well-known cell line FLK/BLV yielded comparable results. False positive results caused by BVDV cross-reactions could be eliminated when tests were carried out with antigen derived from the new cell line.

A Hammondia-like parasite from the European fox (Vulpes vulpes) forms biologically viable tissue cysts in cell culture.

Tissue cysts of parasites of the genus Hammondia are rarely described in naturally or experimentally infected intermediate hosts. However, ultrastructural examinations on tissue cyst stages of Hammondia sp. are needed, e.g. to compare these stages with those of Neospora caninum and other related parasites. We describe a cell culture system employed to examine the in vitro development of tissue cysts of a Hammondia sp.-like parasite (isolate FOX 2000/1) which uses the European fox as a definitive host. Cells of a diploid finite cell line from embryonal bovine heart (KH-R; CCLV, RIE 090) were infected by inoculation of sporozoites und cultivated for up to 3 months. Transmission electron microscopic examination of 17 day old cell culture material revealed the presence of cyst walls. Infected cell cultures cultivated for 2 months were used to feed a fox. Six to 13 days post infection the fox shed large numbers (n=1.2 x 10(7)) of Hammondia-sp. like oocysts which could not be distinguished from those used to infect the cell culture as determined by DNA sequencing of the internal transcribed spacer 1 and the D2/D3 domain of the large subunit ribosomal DNA. To find out the proportion of parasitophorous vacuoles that had developed into tissue cysts, the expression of bradyzoite markers was examined by probing infected cell cultures with mouse polyclonal antibodies against Toxoplasma gondii bradyzoite antigen 1 (anti-BAG1) and rat monoclonal antibodies against a cyst wall protein (mAbCC2). Nineteen and 90 days post infection all parasitophorous vacuoles in the cell cultures were positive with anti-BAG1 and mAbCC2. This shows that biologically viable (i.e. infectious) tissue cysts of a fox-derived Hammondia sp. isolate (FOX 2000/1) can be efficiently produced in this cell culture system. Since in vitro cystogenesis of dog-derived Hammondia heydorni has not been observed yet, in vitro cyst formation might be one trait to separate fox-derived Hammondia sp. from H. heydorni on a species level.

[Production of maedi-visna virus antigen for the immunodiffusion test and its use for field studies]. / Herstellung von Maedi-Visna-Virusantigen für den Immundiffusionstest und sein Einsatz für Felduntersuchungen.

Using the Maedi-Visna virus strain WLC1 (Weybridge) and different cell lines of sheep plexus chorioideus we were able to establish and improve a production procedure of MV-antigen for use in immunodiffusion assay. Our attention was focused mainly on the efficient virus multiplication in cell cultures and on standardisation of the procedure to find a method keeping the antigen loss as low as possible. Investigations with our antigen in 39 farms of 5 of the former districts in East Germany revealed a seropositive reagent rate between 0 and more than 60%, underlining the need for a complex diagnostic and eradication programme.

[Is feeding of green silage in areas with hog cholera in wild boar a danger for domestic swine herds? Experimental study]. / Ist die Verfütterung von Grünfuttersilage in Gebieten mit Schweinepest beim Schwarzwild eine Gefahr für die Hausschweinebestände? Experimentelle Studie.

In an experimental study we tested the survival of hog cholera virus (HCV) contained in pieces of muscular tissue and organs from experimentally infected swine after incubation in silage. In big (diameter greater than 20 cm) muscular pieces HCV survived even in excellent mineral acid silage (pH 3.8-4.0) after a storage of 5 months. On the other hand in smaller parts (musculature tissue, organs less than 20 cm diameter) we never found virulent HCV after 3 months of incubation. Independent of the size of the tested organs we did not find any virulent HCV in silage with pH 5.2 after 3 months. The results of our investigations show, that the feeding of green silage in areas with hog cholera among wild boar is a potential risk for the domestic swine population. In conclusion we propose to feed green silage to unvaccinated pigs in such areas only after a storage of 9 month.

[Retroviruses. Problems in electron microscopic diagnosis]. / Retroviren. Probleme bei der elektronenmikroskopischen Diagnostik.

A department of electron microscopy with specialisation in virus diagnosis evaluates the results concerning retrovirus findings. In the course of research work already classified retroviruses were observed, numerous unexpected retrovirus diagnoses being of special interest. This concerned exclusively to ultra-thin sections of permanent and immune cells. Expounded in this paper are principles by which examiners should abide in any case. Reference is also made to the importance of individual findings.

[A hitherto unrecognized pattern of reactions in vertebrate cells (RiV). 3. Experimental therapy with the help of RiV particle preparations in mice with mammary carcinomas]. / Ein bisher unbekanntes Reaktionsmuster in Vertebratenzellen (RiV). 3. Mitteilung: Therapieversuche mit Hilfe von RiV-Partikelpräparaten bei Mäusen mit Mammakarzinom.

The growth of spontaneous mammary carcinomas in mice was significantly suppressed by administration of RiV-particle-preparations of different species origin (duck, cattle), the survival rate being essentially improved. Despite long-time application of RiV-preparations no side effects could be observed.

[A hitherto unknown reaction pattern in vertebrate cells (RiV). 2. The protective effect of RiV particle preparations against foot-and-mouth disease in guinea pigs]. / Ein bisher unbekanntes Reaktionsmuster in Vertebratenzellen (RiV). 2. Mitteilung: Die Schutzwirkung von RiV-Partikel-Präparaten gegen Maul- und Klauenseuche bei Meerschweinchen.

Further observations concerning the previously described RiV-particles are reported. They were isolated from a diploid cell line of bovine origin, embryonal duck fibroblasts and BHK-21 cells. A protective effect against foot-and-mouth-disease virus in guinea pigs could be observed following inoculation with the RiV-preparation of bovine origin. All 3 preparations isolated from the 3 cell lines showed immunologic cross reactions.