Echinococcus P29 antigen: molecular characterization and implication on post-surgery follow-up of CE patients infected with different species of the Echinococcus granulosus complex.

The protein P29 is a potential serological marker for post-treatment monitoring of cystic echinococcosis (CE) especially in young patients. We now have demonstrated that P29 is encoded in the Echinococcus genus by a single gene consisting of 7 exons spanning 1.2 kb of DNA. Variability of the p29 gene at inter- and intra-species level was assessed with 50 cDNA and 280 genomic DNA clones isolated from different E. granulosus s.l. isolates (E. granulosus sensu stricto (G1), E. equinus (G4), E. ortleppi (G5), E. canadensis (G6), E. canadensis (G7) and E. canadensis (G10)) as well as four E. multilocularis isolates. Scarce interspecies polymorphism at the p29 locus was observed and affected predominantly E. granulosus s.s. (G1), where we identified two alleles (A1 and A2) coding for identical P29 proteins and yielding in three genotypes (A1/A1, A2/A2 and A1/A2). Genotypic frequencies expected under Hardy-Weinberg equilibrium revealed a high rate of heterozygosity (47%) that strongly supports the hypothesis that E. granulosus s.s. (G1) is predominantly outbreeding. Comparative sequence analyses of the complete p29 gene showed that phylogenetic relationships within the genus Echinococcus were in agreement with those of previous nuclear gene studies. At the protein level, the deduced P29 amino acid (AA) sequences exhibited a high level of conservation, ranging from 97.9% AA sequence identity among the whole E. granulosus s.l. group to 99.58% identity among E. multilocularis isolates. We showed that P29 proteins of these two species differ by three AA substitutions without implication for antigenicity. In Western-blot analyses, serum antibodies from a human CE patient infected with E. canadensis (G6) strongly reacted with recombinant P29 from E. granulosus s.s. (G1) (recEg(G1)P29). In the same line, human anti-Eg(G1)P29 antibodies bound to recEcnd(G6)P29. Thus, minor AA sequence variations appear not to impair the prognostic serological use of P29.

First cases of besnoitiosis in cattle in Switzerland

Bovine besnoitiosis has been diagnosed in neighboring countries but not in Switzerland so far. This disease occurs endemically in France and focal outbreaks have been reported in Germany and Italy. To determine if Besnoitia besnoiti is introduced into Switzerland through the import of breeding cattle from France, a systematic serological survey was performed. A total of 412 breeding cattle (from 114 farms) imported from France into Switzerland between 2005 and 2011, were serologically examined for antibodies against B. besnoiti using a commercial ELISA kit (PrioCHECK© Besnoitia Ab 2.0, Prionics AG, Zurich, Switzerland). Sixty-four (15.5 %) animals reacted positive in ELISA. The serologic diagnosis was confirmed by an indirect immunfluorescence test (IFAT) and a Western blot (WB) in only 2 Limousin cows imported from France on a farm in Eastern Switzerland. Subsequently, this whole herd (n = 16) was examined clinically and serologically and 2 additional Limousin cows imported from Germany also reacted positive in the three serological tests. One of these cows presented B. besnoiti tissue cysts in the scleral conjunctiva and typical skin lesions in the head region. The infection was further confirmed cytologically, histopathologically and by PCR. It can be concluded that the parasite is most likely being introduced into Switzerland through the import of infected animals.

Off-pump compared to minimal extracorporeal circulation surgery in coronary artery bypass grafting.

OBJECTIVE Coronary artery bypass grafting (CABG) using extracorporeal circulation (ECC) is still the gold standard. However, alternative techniques have been developed to avoid ECC and its potential adverse effects. These encompass minimal extracorporeal circulation (MECC) or off-pump coronary artery bypass grafting (OPCAB). However, the prevailing potential benefits when comparing MECC and OPCABG are not yet clearly established. METHODS In this retrospective study we investigated the potential benefits of MECC and OPCABG in 697 patients undergoing CABG. Of these, 555 patients had been operated with MECC and 142 off-pump. The primary endpoint was Troponin T level as an indicator for myocardial damage. RESULTS Study groups were not significantly different in general. However, patients undergoing OPCABG were significantly older (65.01 years ± 9.5 vs. 69.39 years ± 9.5; p value <0.001) with a higher Logistic EuroSCORE I (4.92% ± 6.5 vs. 5.88% ± 6.8; p value = 0.017). Operating off pump significantly reduced the need for intra-operative blood products (0.7% vs. 8.6%; p-value <0.001) and the length of stay in the intensive care unit (ICU) (2.04 days ± 2.63 vs. 2.76 days ± 2.79; p value <0.001). Regarding other blood values a significant difference could not be found in the adjusted calculations. The combined secondary endpoint, major cardiac or cerebrovascular events (MACCE), was equal in both groups as well. CONCLUSIONS Coronary artery bypass grafting using MECC or OPCABG are two comparable techniques with advantages for OPCABG regarding the reduced need for intra-operative blood products and shorter length of stay in the ICU. However serological values and combined endpoint MACCE did not differ significantly in both groups.

Q fever outbreak in the terraced vineyards of Lavaux, Switzerland

Coxiella burnetii infection (Q fever) is a widespread zoonosis with low endemicity in Switzerland, therefore no mandatory public report was required. A cluster of initially ten human cases of acute Q fever infections characterized by prolonged fever, asthenia and mild hepatitis occurred in 2012 in the terraced vineyard of Lavaux. Epidemiological investigations based on patients' interviews and veterinary investigations included environmental sampling as well as Coxiella-specific serological assay and molecular examinations (real-time PCR in vaginal secretions) of suspected sheep. These investigations demonstrated that 43% of sheep carried the bacteria whereas 30% exhibited anti-Coxiella antibodies. Mitigation measures, including limiting human contacts with the flock, hygiene measures, flock vaccination and a public official alert, have permitted the detection of four additional human cases and the avoidance of a much larger outbreak. Since November 2012, mandatory reporting of Q fever to Swiss public health authorities has been reintroduced. A close follow up of human cases will be necessary to identify chronic Q fever.

Characterization of small ruminant lentivirus A4 subtype isolates and assessment of their pathogenic potential in naturally infected goats.

BACKGROUND Small ruminant lentiviruses escaping efficient serological detection are still circulating in Swiss goats in spite of a long eradication campaign that essentially eliminated clinical cases of caprine arthritis encephalitis in the country. This strongly suggests that the circulating viruses are avirulent for goats.To test this hypothesis, we isolated circulating viruses from naturally infected animals and tested the in vitro and in vivo characteristics of these field isolates. METHODS Viruses were isolated from primary macrophage cultures. The presence of lentiviruses in the culture supernatants was monitored by reverse transcriptase assay. Isolates were passaged in different cells and their cytopathogenic effects monitored by microscopy. Proviral load was quantified by real-time PCR using customized primer and probes. Statistical analysis comprised Analysis of Variance and Bonferroni Multiple Comparison Test. RESULTS The isolated viruses belonged to the small ruminant lentiviruses A4 subtype that appears to be prominent in Switzerland. The 4 isolates replicated very efficiently in macrophages, displaying heterogeneous phenotypes, with two isolates showing a pronounced cytopathogenicity for these cells. By contrast, all 4 isolates had a poor replication capacity in goat and sheep fibroblasts. The proviral loads in the peripheral blood and, in particular, in the mammary gland were surprisingly high compared to previous observations. Nevertheless, these viruses appear to be of low virulence for goats except for the mammary gland were histopathological changes were observed. CONCLUSIONS Small ruminant lentiviruses continue to circulate in Switzerland despite a long and expensive caprine arthritis encephalitis virus eradication campaign. We isolated 4 of these lentiviruses and confirmed their phylogenetic association with the prominent A4 subtype. The pathological and histopathological analysis of the infected animals supported the hypothesis that these A4 viruses are of low pathogenicity for goats, with, however, a caveat about the potentially detrimental effects on the mammary gland. Moreover, the high proviral load detected indicates that the immune system of the animals cannot control the infection and this, combined with the phenotypic plasticity observed in vitro, strongly argues in favour of a continuous and precise monitoring of these SRLV to avoid the risk of jeopardizing a long eradication campaign.

Anaplasma phagocytophilum DNA amplified from lesional skin of seropositive dogs.

Canine granulocytic anaplasmosis (CGA) is caused by the rickettsial microorganism Anaplasma phagocytophilum. CGA is typically characterized by fever, thrombocytopenia, lethargy, anorexia, arthropy, and other nonspecific clinical signs. Skin lesions have been described in naturally infected lambs and humans. The pathophysiology of CGA is not entirely clear, and the persistence of the organism after the resolution of clinical signs has been described. The aim of the study was to investigate if A. phagocytophilum can be detected in canine lesional skin biopsies from A. phagocytophilum-seropositive dogs with etiologically unclear skin lesions that improved after the treatment with doxycycline. Paraffin-embedded lesional skin biopsies were allocated into separate groups: biopsies from A. phagocytophilum-seropositive dogs responsive to treatment with doxycycline (n=12), biopsies from A. phagocytophilum-seronegative dogs (n=2), and biopsies in which skin lesions histopathologically resembled a tick bite (n=10). The serological status of the latter group was unknown. Histology of the seropositive and seronegative dog skin lesions did not indicate an etiology. DNA was extracted, and a conventional PCR for partial 16S rRNA gene was performed. Anaplasma phagocytophilum DNA was amplified from 4/12 seropositive dogs' skin biopsies. All sequences were 100% identical to the prototype A. phagocytophilum human strain (GenBank accession number U02521). Anaplasma phagocytophilum was not amplified from the 2 seronegative and 10 suspected tick bite dogs. Serum antibody titers of the PCR-positive dogs ranged from 1:200 to 1:2048. Histopathologically, a mild-to-moderate perivascular to interstitial dermatitis composed of a mixed cellular infiltrate and mild-to-moderate edema was seen in all seropositive dogs. In 8/12 seropositive dogs, vascular changes as vasculopathy, fibrinoid necrosis of the vessel walls, and leukocytoclastic changes were observed. In summary, our results support the hypothesis that the persistence of A. phagocytophilum in the skin may be causative for otherwise unexplained skin lesions in seropositive dogs.

Vesicular stomatitis virus replicon expressing the VP2 outer capsid protein of bluetongue virus serotype 8 induces complete protection of sheep against challenge infection.

Bluetongue virus (BTV) is an arthropod-borne pathogen that causes an often fatal, hemorrhagic disease in ruminants. Different BTV serotypes occur throughout many temperate and tropical regions of the world. In 2006, BTV serotype 8 (BTV-8) emerged in Central and Northern Europe for the first time. Although this outbreak was eventually controlled using inactivated virus vaccines, the epidemic caused significant economic losses not only from the disease in livestock but also from trade restrictions. To date, BTV vaccines that allow simple serological discrimination of infected and vaccinated animals (DIVA) have not been approved for use in livestock. In this study, we generated recombinant RNA replicon particles based on single-cycle vesicular stomatitis virus (VSV) vectors. Immunization of sheep with infectious VSV replicon particles expressing the outer capsid VP2 protein of BTV-8 resulted in induction of BTV-8 serotype-specific neutralizing antibodies. After challenge with a virulent BTV-8 strain, the vaccinated animals neither developed signs of disease nor showed viremia. In contrast, immunization of sheep with recombinant VP5 - the second outer capsid protein of BTV - did not confer protection. Discrimination of infected from vaccinated animals was readily achieved using an ELISA for detection of antibodies against the VP7 antigen. These data indicate that VSV replicon particles potentially represent a safe and efficacious vaccine platform with which to control future outbreaks by BTV-8 or other serotypes, especially in previously non-endemic regions where discrimination between vaccinated and infected animals is crucial.

New findings on echinococcosis

The following new aspects of cystic and alveolar echinococcosis (= infections with the metacestode stages of Echinococcus granulosus and E. multilocularis respectively) are reported: identification of a Swiss E. granulosus isolate as "cattle strain" which differs from the "sheep strain"; new observations on proliferation and metastasis formation of larval E. multilocularis; information on chemotherapy of human echinococcosis; recent developments in immunoserology. The latter includes a new technique for serological differential diagnosis of cystic and alveolar echinococcosis, the determination of parasite-specific immunoglobulin classes (IgG, IgM, IgA and IgE) and circulating antigens in ELISA, and the introduction of arc-5 detection in routine serodiagnosis. A highly purified, species-specific antigen from E. multilocularis is now available for seroepidemiological studies.

Molecular Epidemiology Issues of BVDV Infection in the Eastern Ukraine

Aim. This study was focused on (i) detection of specific BVDV-antibodies within selected cattle farms, (ii) identification of persistently infected (PI) animals and (iii) genetic typing of selected BVDV isolates. Methods. RNA extraction, real-time polymerase chain reaction, ELISA technique, sequencing. Results. Specific BVDV-antibodies were detected in 713 of 1,059 analyzed samples (67.3 per cent). This number is in agreement with findings in many cattle herds around the world. However, the number of positive samples differed in the herds. While 57 samples out of 283 (20.1 per cent) were identified in the first herd, 400 out of 475 (84.2 per cent) and 256 out of 301 (85 per cent) animals were positive in the second and third herd. High number of animals with BVDV RNA was detected in all herds. The real-time PCR assay detected BVDV RNA in 5 of 1068 samples analyzed (0.5 per cent). 4 positive samples out of 490 (0.8 per cent) and 1 out of 301 (0.33 per cent) were found in the second and third herd. The genetic materials of BVDV were not found in the first herd. Data on the number of PI animals were in accord with serological findings in the cattle herds involved in our study. The genetic typing of viral isolates revealed that only BVDV, Type 1 viruses were present. The hylogenetic analysis confirmed two BVDV-1 subtypes, namely b and f and revealed that all 4 viruses from the second farm were typed as BVDV-1b and all of them were absolutely identical in 5’-UTR, but virus from the third farm was typed as BVDV-1f. Conclusion. Our results indicated that the BVDV infection is widespread in cattle herds in the eastern Ukraine, that requires further research and development of new approaches to improve the current situation.

Detection of one VH antibody sequence in both healthy donors and urticaria patients.

We have previously isolated anti-FcepsilonRIalpha autoantibodies from phage libraries of healthy donors and urticaria patients. Strikingly, the same antibody, LTMalpha15, was isolated from both libraries. Sequence analysis revealed a germline configuration of the LTMalpha15 variable heavy (V(H)) chain with a slightly mutated variable light (V(L)) chain supporting its classification as a natural autoantibody. Distribution analysis of anti-FcepsilonRIalpha autoantibodies by functional or serological tests delivered conflicting data. For this reason we have developed a new real-time PCR to analyse the distribution of LTMalpha15V(H) in healthy donors and urticaria patients. Our new bioinformatic program permitted the design of a minor groove binder (MGB) TaqMan probe that specifically detected the LTMalpha15V(H). We were able to demonstrate a broad range of rearranged V(H) gene copy number without any correlation to the state of health. Monitoring LTMalpha15V(H) gene copy number in a single donor over a period of 70 days revealed a time-related fluctuation of circulating B cells carrying LTMalpha15V(H). We propose that our real-time PCR may serve as a model for the quantification of natural antibody sequences at a monoclonal level.

Multiple infections of rodents with zoonotic pathogens in Austria

Rodents are important reservoirs for a large number of zoonotic pathogens. We examined the occurrence of 11 viral, bacterial, and parasitic agents in rodent populations in Austria, including three different hantaviruses, lymphocytic choriomeningitis virus, orthopox virus, Leptospira spp., Borrelia spp., Rickettsia spp., Bartonella spp., Coxiella burnetii, and Toxoplasma gondii. In 2008, 110 rodents of four species (40 Clethrionomys glareolus, 29 Apodemus flavicollis, 26 Apodemus sylvaticus, and 15 Microtus arvalis) were trapped at two rural sites in Lower Austria. Chest cavity fluid and samples of lung, spleen, kidney, liver, brain, and ear pinna skin were collected. We screened selected tissue samples for hantaviruses, lymphocytic choriomeningitis virus, orthopox viruses, Leptospira, Borrelia, Rickettsia, Bartonella spp., C. burnetii, and T. gondii by RT-PCR/PCR and detected nucleic acids of Tula hantavirus, Leptospira spp., Borrelia afzelii, Rickettsia spp., and different Bartonella species. Serological investigations were performed for hantaviruses, lymphocytic choriomeningitis virus, orthopox viruses, and Rickettsia spp. Here, Dobrava-Belgrade hantavirus-, Tula hantavirus-, lymphocytic choriomeningitis virus-, orthopox virus-, and rickettsia-specific antibodies were demonstrated. Puumala hantavirus, C. burnetii, and T. gondii were neither detected by RT-PCR/PCR nor by serological methods. In addition, multiple infections with up to three pathogens were shown in nine animals of three rodent species from different trapping sites. In conclusion, these results show that rodents in Austria may host multiple zoonotic pathogens. Our observation raises important questions regarding the interactions of different pathogens in the host, the countermeasures of the host's immune system, the impact of the host–pathogen interaction on the fitness of the host, and the spread of infectious agents among wild rodents and from those to other animals or humans.

Molecular epidemiology of hepatitis B virus infection in Switzerland: a retrospective cohort study.

BACKGROUND Chronic hepatitis B virus (HBV) infection affects up to 7 % of the European population. Specific HBV genotypes are associated with rapid progression to end-stage liver disease and sub-optimal interferon treatment responses. Although the geographic distribution of HBV genotypes differs between regions, it has not been studied in Switzerland, which lies at the crossroads of Europe. METHODS In a retrospective analysis of 465 HBV samples collected between 2002 and 2013, we evaluated the HBV genotype distribution and phylogenetic determinants, as well as the prevalence of serological evidence of hepatitis delta, hepatitis C and HIV infections in Switzerland. Baseline characteristics of patients were compared across their region of origin using Fisher's exact test and ANOVA, and risk factors for HBeAg positivity were assessed using logistic regression. RESULTS The Swiss native population represented 15.7 % of HBV-infected patients living in Switzerland. In the overall population, genotype D was most prevalent (58.3 %), whereas genotype A (58.9 %) was the predominant genotype among the Swiss native population. The prevalence of patients with anti-HDV antibodies was 4.4 %. Patients of Swiss origin were most likely to be HBeAg-positive (38.1 %). HBV genotypes of patients living in Switzerland but sharing the same original region of origin were consistent with their place of birth. CONCLUSIONS The molecular epidemiology of HBV infection in Switzerland is driven by migration patterns and not by the genotype distribution of the native population. The prevalence of positive anti-HDV antibodies in our cohort was very low.

First isolation of a rhabdovirus from perch Perca fluviatilis in Switzerland

Perca fluviatilis is a fish species of increasing interest to the Swiss fish farming industry. In recent years, recirculation systems have been specifically set up to increase production. In one of these farms, abnormal spiral swimming associated with elevated mortalities occurred in repeated batches of imported perch shortly after stocking on several occasions. No bacterial or parasitic etiology was detected, but a virus grown in bluegill fry (BF-2) cells was identified as perch rhabdovirus. Subsequent investigations of other samples suggested a viral tropism for the central nervous system (CNS). Phylogenetic analysis of the partial N and entire G gene sequences positioned this isolate in genogroup C of the species Perch rhabdovirus, with high nucleotide and amino acid (aa) sequence identities with the DK5533 strain isolated in Denmark in 1989. Comparative studies using other closely related isolates allowed the distinction of 2 serological Patterns among perch rhabdoviruses and the identification of a proline substitution by a serine in Position 147 of the glycoprotein potentially involved in antigenic differentiation. Even if perch imported onto the farm tested negative by virus isolation prior to transport, they may have been the origin of this outbreak since CNS tissue was not included in the samples that were analyzed. Another possibility might be a sub-clinical infection with a viral load in resident fish too low to be detected. This study reports the first isolation of a perch rhabdovirus in Switzerland, and emphasizes the necessity of optimizing diagnostic tools that facilitate better control of the risks associated with fish translocation.

Postnatal persistent infection with classical Swine Fever virus and its immunological implications.

It is well established that trans-placental transmission of classical swine fever virus (CSFV) during mid-gestation can lead to persistently infected offspring. The aim of the present study was to evaluate the ability of CSFV to induce viral persistence upon early postnatal infection. Two litters of 10 piglets each were infected intranasally on the day of birth with low and moderate virulence CSFV isolates, respectively. During six weeks after postnatal infection, most of the piglets remained clinically healthy, despite persistent high virus titres in the serum. Importantly, these animals were unable to mount any detectable humoral and cellular immune response. At necropsy, the most prominent gross pathological lesion was a severe thymus atrophy. Four weeks after infection, PBMCs from the persistently infected seronegative piglets were unresponsive to both, specific CSFV and non-specific PHA stimulation in terms of IFN-γ-producing cells. These results suggested the development of a state of immunosuppression in these postnatally persistently infected pigs. However, IL-10 was undetectable in the sera of the persistently infected animals. Interestingly, CSFV-stimulated PBMCs from the persistently infected piglets produced IL-10. Nevertheless, despite the addition of the anti-IL-10 antibody in the PBMC culture from persistently infected piglets, the response of the IFN-γ producing cells was not restored. Therefore, other factors than IL-10 may be involved in the general suppression of the T-cell responses upon CSFV and mitogen activation. Interestingly, bone marrow immature granulocytes were increased and targeted by the virus in persistently infected piglets. Taken together, we provided the first data demonstrating the feasibility of CSFV in generating a postnatal persistent disease, which has not been shown for other members of the Pestivirus genus yet. Since serological methods are routinely used in CSFV surveillance, persistently infected pigs might go unnoticed. In addition to the epidemiological and economic significance of persistent CSFV infection, this model could be useful for understanding the mechanisms of viral persistence.

Field-Applicable Recombinase Polymerase Amplification Assay for Rapid Detection of Mycoplasma capricolum subsp. capripneumoniae.

Contagious caprine pleuropneumonia (CCPP) is a highly contagious disease caused by Mycoplasma capricolum subsp. capripneumoniae that affects goats in Africa and Asia. Current available methods for the diagnosis of Mycoplasma infection, including cultivation, serological assays, and PCR, are time-consuming and require fully equipped stationary laboratories, which make them incompatible with testing in the resource-poor settings that are most relevant to this disease. We report a rapid, specific, and sensitive assay employing isothermal DNA amplification using recombinase polymerase amplification (RPA) for the detection of M. capricolum subsp. capripneumoniae. We developed the assay using a specific target sequence in M. capricolum subsp. capripneumoniae, as found in the genome sequence of the field strain ILRI181 and the type strain F38 and that was further evidenced in 10 field strains from different geographical regions. Detection limits corresponding to 5 × 10(3) and 5 × 10(4) cells/ml were obtained using genomic DNA and bacterial culture from M. capricolum subsp. capripneumoniae strain ILRI181, while no amplification was obtained from 71 related Mycoplasma isolates or from the Acholeplasma or the Pasteurella isolates, demonstrating a high degree of specificity. The assay produces a fluorescent signal within 15 to 20 min and worked well using pleural fluid obtained directly from CCPP-positive animals without prior DNA extraction. We demonstrate that the diagnosis of CCPP can be achieved, with a short sample preparation time and a simple read-out device that can be powered by a car battery, in <45 min in a simulated field setting.