A total of 272 staphylococcal isolates from cases of bovine mastitis (159 Staphylococcus aureus) belonging to 12 different species were identified with ID32 STAPH galleries, and 51 of them were confirmed by 16S rRNA gene (rrs) sequencing. The same isolates were examined for their hemolytic activity on sheep blood agar, DNase activity, and coagulase activity and with two rapid identification kits (Slidex Staph Plus kit and RAPIDEC Staph from Bio-Merieux). The results of this study confirm those obtained by other groups with hemolysis, DNase, and coagulase. Only 50% of S. aureus isolates from mastitis cases show coagulase activity after 4 h of incubation, and a 24-h incubation is necessary for the full sensitivity of this test. In contrast to results from other studies with human isolates, the Slidex Staph Plus kit was not sensitive enough for the identification of S. aureus from bovine mastitis samples. The aurease test of the RAPIDEC Staph kit showed 100% sensitivity and 100% specificity. Used in conjunction with hemolysis patterns, the RAPIDEC Staph kit is therefore very well adapted to rapid, efficient, and cost-effective identification of S. aureus in cultures from bovine mastitis samples. Sequencing of rrs genes also proved very efficient in identifying the Staphylococcus species encountered in these samples and confirming phenotypical identification results with unsatisfactory scores. With continuously improving technologies and decreasing costs, genetic identification methods like rrs gene sequencing will soon find a place in routine veterinary diagnostics.

A fluorogenic PCR specific for ovine herpesvirus 2 (OvHV-2) DNA was developed and compared to a previously established conventional seminested PCR. Testing of a total of 152 blood samples from both positive and negative animals revealed that the results of both assays corresponded to each other in 100% of the cases. A second fluorogenic PCR for genomic sheep DNA was required to normalize the quantity of viral DNA in the sample. Separate standard curves had to be constructed for each PCR. The analytical sensitivity of the new PCRs ranged between at least 10 copies and sometimes even 1 copy of target DNA per reaction mixture. In dilution series of the target DNAs, linear decreases of the signals were observed over 7 orders of magnitude. Thus, it was possible to calculate the amounts of viral DNA in relation to the amounts of cellular DNA by normalizing the absolute quantity of OvHV-2 DNA with the amount of genomic sheep DNA. By this technique, it was possible for the first time to quantitatively characterize the course of OvHV-2 replication in naturally infected sheep.Malignant catarrhal fever (MCF) is a sporadic but usually fatal infectious disease of cattle and other ruminant species (8,9,10,12). Ovine herpesvirus 2 (OvHV-2) is believed to be the etiologic agent of the sheep-associated form of MCF (SA-MCF) that occurs almost worldwide. The agent of the wildebeest-associated form of MCF, alcelaphine herpesvirus 1 (AlcHV-1), was readily isolated and propagated in cell culture (4, 13). In contrast, the isolation of OvHV-2 in cell cultures has not been reported (5), while our own attempts remained unsuccessful (unpublished data). The establishment of a conventional PCR for the detection of OvHV-2 DNA was therefore important (2, 3). This conventional PCR was applied for detection of OvHV-2 in vivo. It turned out that the natural reservoir of this virus is sheep. Among cattle, only samples from animals suffering from MCF and some rare survivors of MCF (9, 10) react positively in this PCR (U. U. Müller-Doblies, J. Egli, H. Li, U. Braun, and M. Ackermann, in press). On the basis of these observations, PCR was established as the new "gold standard" for the diagnosis of MCF in cattle (11). Thus, without being able to grow the virus in cell culture, the technical requirements for tracing of this agent in a qualitative manner had been developed.However, to get a better insight into the pathogenesis and replication of OvHV-2, it is essential to trace the agent in a quantitative manner along the time axis of a natural infection.Conventional quantitative PCRs are time-consuming and carry a high risk of false-positive results due to contamination, especially when a high sample throughput is required. Therefore, a fluorogenic PCR was developed for quantitative determination of OvHV-2 DNA in samples obtained from sheep. The assay makes use of a dually labeled fluorogenic probe, which is designed to hybridize to the sequence between the primers (User's Manual, ABI PRISM 7700 Sequence Detection System, Perkin-Elmer Applied Biosyste...

The risk of transmission of pathogens from free-ranging wild boars (Sus scrofa scrofa) to outdoor domestic pigs (S. scrofa domesticus) is of increasing concern in many European countries. We assess this risk, using Switzerland as an example. We estimated 1) the prevalence of important pathogens in wild boars and 2) the risk of interactions between wild boars and outdoor pigs. First, we tested 252 wild boars from selected areas between 2008 and 2010 for infection with Brucella spp. Bacterial prevalence was estimated to 28.8% (confidence interval [CI] 23.0-34.0) when using bacterial culture (B. suis Biovar 2) and real-time polymerase chain reaction. Antibody prevalence was 35.8% (CI 30.0-42.0), which was significantly higher than in previous studies in Switzerland. We also tested 233 wild boars for porcine reproductive and respiratory syndrome virus (PRRSV). Antibody prevalence was 0.43% (CI 0.01-2.4) for EU-PRRSV and real-time reverse transcription polymerase chain reaction results were negative. These findings suggest that B. suis is increasingly widespread in wild boars and PRRSV is currently not of concern. Second, we documented the spatial overlap between free-ranging wild boars and outdoor piggeries by mapping data on their respective occurrence. Wild boars are most widespread in the mountain range along the western and northern Swiss borders, while most piggeries are located in central lowlands. A risk of interaction is mainly expected at the junction between these two bioregions. This risk may increase if wild boars expand eastward and southward beyond anthropogenic barriers believed to limit their range. Therefore, we evaluated the potential of expansion of the wild boar population. Population trends suggest a continuous increase of wild boars for the past 15 yr. Surveillance of selected wildlife passages using cameras on highways and main roads indicates that these barriers are permeable (average of up to 13 wild boar crossings per 100 days). Thus an increase of wild boar range should be considered. There may be a risk of B. suis spillover from wild boars in Switzerland, which could increase in the future. Data on the occurrence of interactions between pigs and wild boars are needed to assess this risk.

No abstract

Ovine herpesvirus 2 (OvHV-2), a member of the viral subfamily Gammaherpesvirinae, shares numerous similarities with human herpesvirus 8 (HHV-8). Both viruses are apathogenic in their healthy original host, may cause lymphoprolipherative diseases, cannot routinely be propagated in cell culture, and may be sexually transmitted. However, the pathways of sexual transmission of these viruses, as well as the underlying pathogenetic dynamics, are not well understood. Organs from naturally OvHV-2-infected, as well as OvHV-2-free, sheep were quantitatively analyzed for OvHV-2 by the DNA amplification techniques. The dynamics of OvHV-2 multiplication and excretion were monitored after experimental infections and, most importantly, subsequent to vasectomy. The OvHV-2 DNA load in various tissues and internal organs was not merely reflecting the viral DNA load in the bloodstream, which suggested compartmentalization of OvHV-2. Moreover, OvHV-2 DNA was detected at several portals for virus shedding, i.e., the respiratory, alimentary, and urogenital tracts. Transient OvHV-2 excretion was detected in ejaculates of experimentally infected rams. Upon vasectomy, OvHV-2 DNA reappeared in the ejaculatory plasma, but the titers did not decline after reaching a peak. Spiking and fractionation experiments revealed an inhibitory activity, associated with the spermatozoa, which was able to suppress detection of viral DNA but which was no longer present in samples from vasectomized animals. Therefore, epidemiological studies on viruses that may be transmitted by the ejaculatory pathway and for whose tracing nucleic acid amplification methods are used, i.e., OvHV-2, HHV-8, and the human immunodeficiency virus, should include vasectomized males.The hallmarks of gamma herpesviruses, i.e., ovine herpesvirus 2 (OvHV-2; the causative agent of malignant catarrhal fever) and the tumorigenic human herpesvirus 8 (HHV-8), include restricted host range and causing host-specific, proliferative, immunopathological diseases (13,15,19). HHV-8 and OvHV-2 share at least three important features:(i) Normally, they are not associated with disease in the healthy original host. However, in the immunologically ill adapted host, they are causative agents of lymphoproliferative diseases. (ii) Natural isolates cannot routinely be propagated in cell culture (7,18). Therefore, efficient tracing of these viruses relies on DNA amplification techniques. (iii) Several lines of evidence suggest that they may be sexually transmitted (2, 3, 14, 16). However, the reported prevalences of HHV-8 in prostate and semen range from zero to Ͼ90% (reviewed in reference 9). To date, hardly any information concerning the pathogenesis and shedding of OvHV-2 has been presented.Good animal models for studying HHV-8 infections are not available (4). However, specific-OvHV-2-free sheep may be employed to gain significant information on infection, pathogenesis, and transmission of gammaherpesviruses.To gain insight into the pathogenetic basis for gammaherpesvirus excretion, organs from natu...

No abstract

A die-off of passerine birds, mostly Eurasian siskins (Carduelis spinus), occurred in multiple areas of Switzerland between February and March 2010. Several of the dead birds were submitted for full necropsy. Bacteriological examination was carried out on multiple tissues of each bird. At gross examination, common findings were light-tan nodules, 1 to 4 mm in diameter, scattered through the esophagus/crop. Histologically, a necroulcerative transmural esophagitis/ingluvitis was observed. Bacterial cultures yielded Salmonella enterica subsp. enterica serovar Typhimurium. At the same time, 2 pet clinics reported an unusual increase of domestic cats presented with fever, anorexia, occasionally dolent abdomen, and history of presumed consumption of passerine birds. Analysis of rectal swabs revealed the presence of S. Typhimurium in all tested cats. PFGE (pulsed field electrophoresis) analysis was performed to characterize and compare the bacterial isolates, and it revealed an indistinguishable pattern between all the avian and all but 1 of the feline isolates. Cloacal swabs collected from clinically healthy migrating Eurasian siskins (during autumn 2010) did not yield S. Typhimurium. The histological and bacteriological findings were consistent with a systemic infection caused by S. Typhimurium. Isolation of the same serovar from the dead birds and ill cats, along with the overlapping results of the PFGE analysis for all the animal species, confirmed a spillover from birds to cats through predation. The sudden increase of the number of siskins over the Swiss territory and their persistency during the whole winter of 2009-2010 is considered the most likely predisposing factor for the onset of the epidemic.

Canine herpesvirus (CHV-1) causes neonatal deaths as well as infertility due to embryonal death, abortion and stillbirths in breeding kennels. The aim of this study was to determine the prevalence of antibodies against canine herpesvirus in the serum of dogs older than 1 year in breeding kennels in the Gauteng Province of South Africa. A serum neutralization test (SNT) and a newly developed enzyme linked immunosorbent assay (ELISA) were used to test the serum samples of 328 dogs in 38 breeding kennels. With SNT as well as ELISA, 22% of sera were positive (P > 0.9). Seventeen kennels (45% of total kennels) each had at least one positive dog on SNT compared with twenty kennels (53% of total kennels) that each had at least one positive dog on ELISA (P = 0.6). The prevalence of positive dogs in positive kennels was 42 ± 26% (n = 17 kennels) for SNT and 39 ± 26% (n = 20 kennels) for ELISA. Pairwise comparison of kennels showed that the prevalence of SNT positive dogs was similar to the prevalence of ELISA positive dogs (P = 0.3, n = 38 kennels). Seroprevalence was independent of age, gender or colony size. This study suggests that canine herpesvirus is sufficiently common in breeding dogs in the Gauteng Province of South Africa to pose a threat to neonatal survival and fertility.