[Effect of experimental classical swine fever (CSF) infection on libido and ejaculate parameters in boars]. / Einfluss einer experimentellen Infektion mit dem Virus der klassischen Schweinepest auf Libido und Ejakulat- parameter beim Eber.

The objective of this study was to characterize the effect of an experimental infection with the classical swine fever (CSF) virus on libido and ejaculate parameters of adult boars. Four boars 10 month old were infected with a CSF field isolate (Visbek/Han95). Semen was collected every second day after infection and daily during the pyrexic phase. The only clinical signs in the boars were an increase in body temperature, but never above 39.9 degrees C and a temporally reduction of food intake. The libido was always good, so semen collection was performed in three boars without difficulty and the semen quality was always in the range of the minimum requirements for sperm that is used for artificial insemination. Although one boar had a good libido only a sperm free ejaculate could be collected on one day. The results show that a CSF virus infection of adult boars hardly causes any clinical symptoms and that sperm can be obtained despite fever. Insemination boars may thus be of special epidemiological relevance for the dissemination of the CSF virus.

[Analysis of bulk milk samples using polymerase chain reaction: an additional tool for bovine viral diarrhea monitoring]. / Untersuchung von Sammelmilchproben mittels Polymerasekettenreaktion: Eine ergänzende Methode für die BVD-Uberwachung.

Programmes for the eradication and control of infections with bovine viral diarrhea virus (BVDV) concentrate on the identification and elimination of persistently infected (PI) animals. The identification of these animals is mainly based on the detection of viral antigen using ELISA techniques. Protocols detecting viral nucleic acid using RT-PCR have been described recently. Due to high costs the German model recommends screening of animals of 9 up to 36 months of age. Screening of bulk milk samples using RT-PCR technology would allow a system independent of age. The aim of the present study was to test whether bulk milk samples (1433 including max. 50 animals each) collected in four counties of Lower Saxony are suitable for a complementary identification of PI animals via RT-PCR. Thirty-one bulk milk samples derived from 27 dairy herds were BVDV positive, corresponding to 2.3 % of the herds analysed in this study. Two samples first scored doubtful. Follow up tests revealed lactating PI animals in most cases (18). In other cases the epidemiological status of the herd, i.e. high sero-prevalence and/or presence of PI animals among non-lactating cattle, suggested a transient infection detected in the first bulk milk sample. These results demonstrate that monitoring of lactating cattle of any age using RT-PCR is a very sensitive, economically effective additional method for the identification of PI animals.

Epidemiology of classical swine fever in Germany in the 1990s.

In Germany, 424 outbreaks of CSF in domestic pigs and a great number of cases in wild boar were recorded between 1990 and 1998. Most of the federal states ('Bundesländer') were affected. Epidemiological data from field investigations combined with genetic typing allowed to distinguish seven unrelated epidemics and a number of sporadic outbreaks in domestic pigs. Detailed epidemiological data was available for 327 outbreaks. It was found that 28% of these were primary outbreaks. Most of them were due to indirect or direct contact to wild boar infected with CSF virus or swill feeding. Infected wild boar remain the main risk for domestic pigs. The most frequent sources of infection in secondary or follow up outbreaks were the trade with infected pigs, neighbourhood contacts to infected farms and other contacts via contaminated persons and vehicles, respectively. An increased risk of virus transmission from infected herds to neighbourhood farms was observed up to a radius of approximately 500m. More than two thirds of the infected herds were discovered due to clinical signs. About 20% were identified by epidemiological tracing on and back. These were scrutinised because contacts to infected herds were evident. In conclusion, tracing of contact herds and clinical examination combined with carefully targeted virological testing of suspicious animals is likely to be the most important measure to immediately uncover secondary outbreaks. Obligatory serological screening in the surveillance and the restriction zones do not seem to be efficient measures to detect follow-up outbreaks.

Molecular epidemiology of a large classical swine fever epidemic in the European Union in 1997-1998.

A big epidemic of classical swine fever (CSF) occurred in the European Community in 1997. The first case was reported at the beginning of January 1997 from Germany. The disease presumably spread to the Netherlands, and from there to Italy, Spain and eventually to Belgium. About 30 isolates from these outbreaks were analysed by comparison of the nucleotide sequence data generated from fragments of both the E2 glycoprotein gene (190 nucleotides) and from the 5'-nontranslated region (5'-NTR; 150 nucleotides). By combining epidemiological data with genetic typing, it was found that the outbreaks were related and caused by a virus belonging to the genetic subgroup 2.1. As this type of virus had been reported infrequently in Europe and not at all since 1993, we postulate that it was newly introduced into the European Union (EU).

Detection of classical swine fever virus in semen of infected boars.

During the Classical Swine Fever (CSF) epidemic in 1997 in the EU member states Germany, Italy, Spain and The Netherlands, boars in an artificial insemination (AI) centre were found to be infected with CSF virus. This raised a question of epidemiological importance which could not be answered immediately. Can CSF virus be shed by semen of infected boars and what conclusions concerning the risk of spreading CSF infection by semen can be drawn. Experimental studies were conducted to answer this question. Four young boars were infected with a CSF field virus isolate from Germany, which had been characterised in a previous animal experiment. Semen was collected at least every other day after infection. The semen was subjected to the standard diagnostic procedure for the detection of CSF virus and to semen quality assessment. The boars were euthanized at day 8, 12, 16 and 21 post infection, respectively. A post mortem examination was done and organ samples were taken from the CSF reference organs and genital organs for the detection of virus and antigen. The course of CSF infection of the boars was mild but detectable during the second week of infection. CSF virus could be isolated from semen of two animals during the pyrexic phase and from the epididymis but not from the testes. Since CSF virus shedding via semen could be proven, it was concluded that the disease may also be transmitted by artificial insemination. However analysis of semen in cell culture for the presence of CSF virus is not suitable as a routine method for CSF diagnosis.

Structure and presentation of a World Wide Web database of CSF virus isolates held at the EU reference laboratory.

A computerized database was generated with the epidemiological data of more than 600 CSF virus strains and isolates kept in the EU Reference Laboratory for Classical Swine Fever in Hanover. In addition, as sequence data from defined regions of the genome are increasingly being used for genetic typing of new isolates and are thus being published, it was decided to integrate them into the database. In order to make the epidemiological and the sequence data available to other laboratories through the World Wide Web, a searchable web interface was programmed, which can be accessed using an Internet browser like Netscape or Internet Explorer. The possibility to exchange data via the web has the potential to increase our knowledge concerning genetic and epidemiological links between outbreaks worldwide.

Application of a computer program for genetic typing of classical swine fever virus isolates from Germany.

The commercial software program HLA SequiTyper (Amersham Pharmacia Biotech), designed originally for human leukocyte antigen typing, was adapted for rapid typing of classical swine fever (CSF) virus isolates. The program compares new sequence data with those stored in a database file and calculates the most probable assignment. For generating the CSF virus sequence database, 150 bp of the 5' nontranslated genomic region (5'-NTR) from 88 German classical swine fever virus isolates from outbreaks between 1984 and 1997 were solid-phase sequenced directly after RT-PCR amplification. Sequence alignments showed that they all belonged to the previously defined genetic group 2. Within this group, six different subgroups could be distinguished, and were designated according to the geographic location where they are either still endemic or where they appeared most commonly. The advantage of using the HLA SequiTyper program is that it reads directly the sequence files as generated by the ALF sequencer (Amersham Pharmacia Biotech), making any manipulations unnecessary. In addition, a constant quality control of the raw sequence data can be achieved, as more than one sequence from the same isolate can be evaluated at once. Using this approach, new CSF isolates can be typed within 2 days.

[Transmissible spongiform encephalopathies]. / Ubertragbare spongiforme Enzephalopathien.

Transmissible spongiform encephalopathies are a unique group of brain diseases of different animal species and man that can be transmitted between individuals by inoculation or ingestion of diseased nervous system tissues. The causative agents induce non inflammatory spongiform degeneration of the central nervous system which leads to a progressive loss of brain function including ataxia and paralysis resulting always in death. The agent might consist only of a conformationally altered cellular protein of the host species devoid of nucleic acid. The natural occurrence of transmissible spongiform encephalopathies is restricted to a few species. Thus, under certain conditions, i. e. the feeding of infectious bone meal or scientific experiments, the agents can be transmitted to other species causing lethal disease.

The development of early vs. late onset mucosal disease is a consequence of two different pathogenic mechanisms.

Bovine viral diarrhea (BVD) virus is the causative agent of fatal mucosal disease (MD) of cattle. Experimental induction of MD can be achieved by superinfection of calves persistently viremic with a noncytopathic (ncp) BVD virus using an antigenically similar cytopathic (cp) BVD virus. Here we describe the characterisation of BVD viruses isolated from three cases of experimentally induced MD. One animal developed clinical symptoms two weeks after superinfection (early onset MD), while the onset of disease in the other two cases occurred with a delay of months (late onset MD). Antigenic characterisation of the viruses was performed using a panel of monoclonal antibodies against the E2 glycoprotein. For genetic analysis, RT-PCR was applied to amplify specific insertions and duplications in the NS2-3 genomic region of the cp BVD viruses. In addition, these amplicons and fragments of the viral E2 genes were sequenced. The results showed that in the case of early onset MD the cp BVD virus isolated after begin of disease was identical to the one used for superinfection. In contrast, the cp BVD viruses isolated from the two animals with late onset MD were obviously the result of genetic recombinations between the persistent ncp and the superinfecting cp BVD viruses. We conclude that early and late onset MD are the consequence of different pathogenic mechanisms.

Experimentally induced "late-onset" mucosal disease--characterization of the cytopathogenic viruses isolated.

Antigenic and genetic analyses were performed in order to establish relationships between the noncytopathogenic (ncp) and the cytopathogenic (cp) bovine viral diarrhoea viruses (BVDV) involved in the induction of a case of experimentally induced "late-onset" mucosal disease (MD) symptoms. The persistent ncpBVDV, the cpBVDV used for superinfection (strain TGAC) and the virus isolates from faeces (cpX) were examined using an immunoplaque test (IPT) to distinguish between cp and ncp virus populations. The cp populations were cloned by plaque purification and found to be free of ncpBVDV when using the IPT. The cpBVDV clones and the persistent ncpBVDV were analysed in an enzyme immunoassay on heat-fixed infected cells (IM-EIA) and in a neutralization test using a panel of 27 monoclonal antibodies against the E0 (gp48) and E2 (gp53) viral glycoproteins. It was found that strain TGAC contained two antigenically distinct subpopulations of cpBVDV (TGAC-B1 and TGAC-B2). The endogenous ncpBVDV and the cpX clones had the same reactivity pattern in both tests. In addition, p80 gene duplications in the genomes of the cpBVDV clones were analysed using the polymerase chain reaction and subsequent restriction enzyme analysis of the amplicons. The clones analysed from TGAC-B1 and those from cpX had gene duplications of identical sizes showing the same restriction enzyme patterns. Our results suggest that the cpBVDV which finally lead to "late-onset" MD arose by recombination and/or by mutations of the cpBVDV used for superinfection.

Effect of sodium acetate on expression of K99 pili by Escherichia coli.

Sodium acetate suppressed K99 production in Escherichia coli strains cultured on a minimal medium, as determined by seroagglutination and enzyme-linked immunosorbent assay. The greatest suppression occurred when the medium contained both sodium acetate and glucose. Glucose alone did not suppress K99 production.