Isolates of bovine viral diarrhoea virus (BVDV) collected in Germany were examined for their genomic heterogeneity in sequences from the 5'untranslated region (UTR) of the viral genome. Polymerase chain reaction (PCR) tests based on the 5'UTR and the region coding for the NS2-3/4A polypeptide were used to differentiate between BVDV I and BVDV II genotypes. Eleven out of 96 BVDV-isolates were identified as BVDV II. Virus neutralization tests with BVDV I- or II-specific antisera raised in cattle were done. The mean titers were reduced by 7.2-fold (BVDV I-antiserum versus type II-isolates) or 35-fold (BVDV II-antiserum versus type I-isolates) when using the respective heterologous virus.

Bovine vaginal cytobrush specimens were analyzed for the presence of Chlamydia spp. by a high-sensitivity, high-specificity quantitative PCR. The 53% prevalence of low-level Chlamydia psittaci and C. pecorum genital infection detected in virgin heifers suggests predominantely extragenital transmission of Chlamydia in cattle and conforms to the high seroprevalence of anti-Chlamydia antibodies.Over the last 40 years, evidence has accumulated to suggest the ubiquitous presence of infections with intracellular bacteria of the genus Chlamydia in cattle and other livestock species. Despite some improvement in diagnostic techniques, our understanding about the prevalence and pathogenetic significance of these infections, succinctly reviewed by Shewen (11) in 1980, has not substantially changed since that time. In cattle, enzyme-linked immunosorbent assay examinations of sera for the antibody against Chlamydia psittaci suggest a high level of exposure to C. psittaci (6,8,10). The application of nested PCR to bovine clinical specimens substantiated such widespread, but mostly clinically inapparent, presumably low-level infections (3, 7), similar to the findings for human C. pneumoniae infections (1). However, due to high technical demands, these PCR methods were rarely transferred from research settings to systematic epidemiological investigations and diagnostic use. A simple, highly specific, fluorescentprobe-based single-tube LightCycler quantitative PCR (qPCR) platform was recently developed for the detection of Chlamydia DNA. This platform optimizes sample nucleic acid preservation, extraction, and recovery, as well as qPCR methodology, for maximum sensitivity in the detection of Chlamydia (2). This has opened the possibility for the sensitive routine diagnosis of chlamydial infection as well as for systematic epidemiological studies. Such investigations would benefit from both the high sensitivity and the ability to ascertain quantitative differences in chlamydial burdens between animals, which may be required to understand disease mechanisms. In this initial epidemiological application of the Chlamydia qPCR platform, we addressed the question of chlamydial infection of the bovine genital tract in animals that had not previously been exposed to the possibility of sexual transmission of chlamydiae. We report here a high prevalence of genital tract infection with C. psittaci and C. pecorum in clinically normal virgin cattle.A herd of 51 virgin Holstein heifers, 14 to 16 months old, was sampled four times at weekly intervals. These animals were clinically normal, but low-grade vaginitis was common.Vaginal cytobrush specimens (Histobrush; Fisher Scientific, Suwanee, Ga.) were obtained by 10-s rotation in the vaginal vestibulum, brushes were immediately transferred to 400 l of RNA-DNA stabilization reagent (Roche Applied Science, Indianapolis, Ind.) in a 1.5-ml screw-cap microcentrifuge tube, samples were stirred, and the brushes were cut off. Brushes were removed after a 5-min centrifugation at 3,000 ϫ g at room tempera...

Highly pathogenic avian influenza (HPAI) is a striking disease in susceptible poultry, which leads to severe economic losses. Inactivated vaccines are the most widely used vaccines in avian influenza virus (AIV) vaccination programs. However, these vaccines interfere with the serological detection of wild-type AIV infections in immunized populations. The use of vaccines that allow differentiation between infected and vaccinated animals (DIVA strategy) would stop current stamping-out policies. Therefore, novel vaccination strategies are needed to allow improved protection of animals and humans against HPAI virus (HPAIV) infection. The presented study analyzed for the first time the immunogenic capacity of plant-expressed full-length hemagglutinin (rHA0) of HPAIV H5N1 in several vaccine formulations within the highly relevant host species chicken. We were able to express plant-expressed rHA0 at high levels and could show that, when administered with potent adjuvants, it is highly immunogenic and can fully protect chicken against lethal challenge infection. Real-time reverse transcription (RT)-PCR and serological tests demonstrated only marginally increased virus replication in animals vaccinated with plant-derived rHA0 compared to animals immunized with an inactivated reference vaccine. In addition, the use of plant-expressed rHA0 also allowed an easy serological differentiation of vaccinated from AIV-infected animals based on antibodies against the influenza virus NP protein.Highly pathogenic avian influenza (AI) (HPAI) is a striking disease in susceptible poultry, which leads to severe economic losses (21). Since 2003, the H5N1 HPAI epidemic has claimed over 220 million poultry and other birds either through direct mortality from infection or from preemptive culling (22). The implementation of vaccination of poultry as a tool for the reduction of the viral load in the environment and, thus, for decreasing the risk of transmission within poultry-and, as a consequence, to humans-is still a discussed topic. Inactivated vaccines are the most widely used vaccines in AI vaccination programs. They are particularly addressed to protect adult chickens, turkeys, and other birds in emergency situations, e.g., when ring vaccination is used in an area of an HPAI virus (HPAIV) outbreak or when prophylactic vaccination is used in a region where H5 or H7 AI virus (AIV) infections are endemic. However, these vaccines limited the serological detection of wild-type AIV infections in immunized populations, as wild-type infection could be detectable only through higher antibody titers to nonstructural proteins or if the neuraminidase subtype of the vaccine differed from the subtype of the introduced wild-type virus (28).The use of vaccines that fit in any case to the strategy of differentiating infected from vaccinated animals (DIVA) would make a strong case for turning away from current stamping-out policies in many countries. Vaccines that consist of only one major antigenic protein related to influenza A virus (e.g., hemagglutini...

Classical methods for detection of Chlamydophila species, and of antibodies against these agents, have indicated that these bacteria are highly prevalent in cattle and associated with numerous disease conditions. These methods demonstrated acute Chlamydophila-induced diseases such as epizootic bovine abortion, as well as worldwide variable, but generally high, Chlamydophila seroprevalence. However, it was impossible to consistently detect the low levels of these organisms which were suspected to be present in endemic infections. Application of highly sensitive real-time PCR and ELISA methods for detection of Chlamydophila spp. DNA and of antibodies against Chlamydophila spp., respectively, in a series of prospective cohort studies revealed a high prevalence of Chlamydophila spp. genital infections in female calves (61%) and adult heifers (53%). These infections were acquired by extragenital transmission in the first weeks of life, and infection frequency was increased by crowding of the animals. A challenge study demonstrated that infection with C. abortus resulted in decreased fertility of heifers. The experimental use of a C. abortus vaccine provided evidence for immunoprotection against C. abortus-induced suppression of bovine fertility. The results of these investigations suggest that bovine Chlamydophila infection should be viewed more as pervasive, low-level infection of cattle than as rare, severe disease. Such infections proceed without apparent disease or with only subtle expressions of disease, but potentially have a large impact on bovine herd health and fertility.

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