Diagnostic response to a cross-border challenge for the Swiss caprine arthritis encephalitis virus eradication program.

INTRODUCTION: Animal trading between countries with different small ruminant lentivirus infectious status is a potential danger for the reintroduction of eradicated genotypes. This was the case in 2017 with the importation of a large flock of seropositive goats into Switzerland. The handling of this case permitted us to test the preventive measures in place. The coordination between the local veterinarian and the cantonal and federal veterinary authorities worked efficiently and rapidly involved the national reference center in the investigations. This case posed a challenge for the reference center and enabled scrutiny of the applied diagnostic tests. ELISA and western blot provided consistent results and pointed to an unusually high infection rate in the flock. This was confirmed by the isolation of several viruses from different organs and cells, demonstrating that the spleen is particularly well suited for isolation of small ruminant lentiviruses. The SU5-ELISA, designed to predict the subtype of the infecting virus, correctly pointed to a B1 subtype as the infectious agent. We confirmed that with this test it is necessary to analyze a representative number of samples from a flock and not just individual sera to obtain reliable results. This analysis permitted us to identify particular amino acid residues in the SU5 peptides that may be crucial in determining the subtype specificity of antibody binding. Different gag-pol and env regions were amplified by PCR using primers designed for this purpose. The phylogenetic analysis revealed a surprisingly high heterogeneity of the sequences, pointing to multiple infections within single animals and the entire flock. In conclusion, this case showed that the defense of the CAEV negative status of the Swiss goat population with respect to the virulent, prototypic B1 subtype of small ruminant lentiviruses, requires, among other measures, a diagnostic facility capable of performing a thorough analysis of the collected samples.

INTRODUCTION: Le commerce d'animaux entre pays où le statut infectieux des lentivirus des petits ruminants est différent constitue un danger potentiel pour la réintroduction de génotypes éradiqués. Ce fut le cas en 2017 avec l'importation d'un grand troupeau de chèvres séropositives en Suisse. Le traitement de cette affaire nous a permis de tester les mesures préventives mises en place. La coordination entre le vétérinaire local et les autorités vétérinaires cantonales et fédérales a été efficace et a impliqué rapidement le centre de référence national dans les enquêtes. Ce cas a constitué un défi pour le centre de référence et a permis d'examiner de près les tests de diagnostic appliqués. Les tests ELISA et Western blot ont fourni des résultats cohérents et ont mis en évidence un taux d'infection anormalement élevé dans le troupeau. Cela a été confirmé par l'isolement de plusieurs virus provenant d'organes et de cellules différents, démontrant que la rate est particulièrement bien adaptée à l'isolement des lentivirus des petits ruminants. Le SU5-ELISA, conçu pour prédire le sous-type du virus infectant, désignait correctement un sous-type B1 en tant qu'agent infectieux. Nous avons confirmé qu'avec ce test, il était nécessaire d'analyser un nombre représentatif d'échantillons d'un troupeau et pas seulement des sérums individuels pour obtenir des résultats fiables. Cette analyse nous a permis d'identifier des résidus d'acides aminés particuliers dans les peptides SU5 qui pourraient jouer un rôle crucial dans la détermination de la spécificité de sous-type de la liaison à l'anticorps. Différentes régions gag-pol et env ont été amplifiées par PCR en utilisant des amorces conçues à cet effet. L'analyse phylogénétique a révélé une hétérogénéité étonnamment élevée des séquences, indiquant de multiples infections chez les animaux isolés et dans l'ensemble du troupeau. En conclusion, cette affaire a montré que la défense du statut négatif CAEV de la population de chèvres suisses vis-à-vis du virus virulent, sous-type B1 des lentivirus des petits ruminants, nécessite, entre autres mesures, un système de diagnostic capable d'effectuer une analyse approfondie des échantillons collectés.

Influence of border disease virus (BDV) on serological surveillance within the bovine virus diarrhea (BVD) eradication program in Switzerland.

BACKGROUND: In 2008, a program to eradicate bovine virus diarrhea (BVD) in cattle in Switzerland was initiated. After targeted elimination of persistently infected animals that represent the main virus reservoir, the absence of BVD is surveilled serologically since 2012. In view of steadily decreasing pestivirus seroprevalence in the cattle population, the susceptibility for (re-) infection by border disease (BD) virus mainly from small ruminants increases. Due to serological cross-reactivity of pestiviruses, serological surveillance of BVD by ELISA does not distinguish between BVD and BD virus as source of infection. RESULTS: In this work the cross-serum neutralisation test (SNT) procedure was adapted to the epidemiological situation in Switzerland by the use of three pestiviruses, i.e., strains representing the subgenotype BVDV-1a, BVDV-1h and BDSwiss-a, for adequate differentiation between BVDV and BDV. Thereby the BDV-seroprevalence in seropositive cattle in Switzerland was determined for the first time. Out of 1,555 seropositive blood samples taken from cattle in the frame of the surveillance program, a total of 104 samples (6.7%) reacted with significantly higher titers against BDV than BVDV. These samples originated from 65 farms and encompassed 15 different cantons with the highest BDV-seroprevalence found in Central Switzerland. On the base of epidemiological information collected by questionnaire in case- and control farms, common housing of cattle and sheep was identified as the most significant risk factor for BDV infection in cattle by logistic regression. CONCLUSION: This indicates that pestiviruses from sheep should be considered as a source of infection of domestic cattle and might well impede serological BVD surveillance.

Epidemiological survey on Cryptosporidium in an Equine Perinatology Unit.

The present study aims to evaluate the prevalence, pattern of spread and risk factors for the transmission of cryptosporidiosis in foals and mares hospitalized in a University Equine Perinatology Unit, where a new subtype family of Cryptosporidium horse genotype was described by Caffara et al. (2013). Mares (36) and foals (37) hospitalized during the 2012 foaling season were included. Multiple sampling from each animal was performed (a total of 305 stool samples were collected). One hundred and eleven environmental samples (gauze swabs) were also collected before and after the breeding season. Fourteen foals were found positive for Cryptosporidium spp. by PCR in at least one sample; a total of 35 foal stool specimens were confirmed for the presence of the protozoa. Instead none of the stool specimens from mares were found positive. PCR-RFLP analysis shows Cryptosporidium parvum in 5 stool samples and Cryptosporidium horse genotype in 21. In 9 specimens, from 4 different foals, the profile was suggestive for a mixed infection. The subtyping at gp60 locus showed 2 strains as members of the subtype family IId and six of the subfamily IIa of C. parvum. Twenty isolates were identified as Cryptosporidium horse genotype subtype VIaA15G4. Five gauze swabs collected from the walls of the boxes where the animals were hosted out of 111 environmental samples examined were PCR positive for Cryptosporidium spp. Cryptosporidium parvum was detected in one sample collected before the foaling season, while Cryptosporidium horse genotype profile was observed in 4 wall samples collected at the end of the 2012 foaling season. The prevalence observed in foals (37.8%) was higher than that reported in other studies. These features and the diffusion of the same genotype point out as the EPU, where critically ill foals are hospitalized, can support the spread of cryptosporidiosis. Therefore, the manual tasks and the activities carried out in these facilities are of great importance, as they might favor the diffusion of the infection.

Short communication: Transmission of border disease virus to seronegative cows inseminated with infected semen.

The goal of this study was to investigate the transmissibility of border disease (BD) virus to seronegative cows via artificial insemination with cryopreserved semen from a bull persistently infected with BD virus. Five pestivirus naive cows were inseminated with BD virus-infected semen. Blood was collected for detection of pestivirus antibody by means of an ELISA on day 0 (day of insemination) and then every 7 days until day 56, at which time a serum neutralisation test (SNT) for differentiation of BD and BVD virus was carried out. Seroconversion was first noticed in two cows on day 14, in two cows on day 21 and in one cow on day 28. In the SNT, all cows had distinctly positive titres against BD virus. Therefore, BD virus is readily transmitted by infected semen, but none of the cows conceived, most likely because of poor semen quality.

[Development of a real-time RT-PCR for detection of equine influenza virus]. / Entwicklung einer real-time RT-PCR zum Nachweis von equinem Influenzavirus.

Equine influenza is a highly contagious respiratory disease in horses caused by influenza A viruses. In this work a real-time RT-PCR for fast and sensitive diagnosis of equine influenza viruses (EIV) targeting a highly conserved region of the matrix gene was developed. In addition two RT-PCR methods for the amplification of large parts of the matrix- and HA gene were adapted for molecular-epidemiological characterization of viruses. The primers of the real-time RT-PCR had homologies of 99.4% to EIV- and 97.7% to all influenza A viral sequences, whereas the minor groove binder (MGB) probe showed homologies of 99.3% and 99.6%, respectively. These high values allow application of the assay for influenza viruses in other species. Using 20 equine, 11 porcine and 2 avian samples, diagnostic suitability of the assay was confirmed. High specificity for influenza viruses was shown both experimentally and by software simulation. The assay analytical sensitivity was at 10(2)-10(3) copies of RNA and 10(0)-10(1) copies of DNA, respectively. This allows virus detection also in circumstances of minor viral shedding. All amplified EIV sequences were classified phylogenetically within the known lineages.

Graphitic carbon in a nanostructured titanium oxycarbide thin film to improve implant osseointegration.

A nanostructured coating layer on titanium implants, able to improve their integration into bones and to protect against the harsh conditions of body fluids, was obtained by Ion Plating Plasma Assisted, a method suitable for industrial applications. A titanium carbide target was attached under vacuum to a magnetron sputtering source powered with a direct current in the 500-1100 W range, and a 100 W radio frequency was applied to the sample holder. The samples produced at 900 W gave the best biological response in terms of overexpression of some genes of proteins involved in bone turnover. We report the characterization of a reference and of an implant sample, both obtained at 900 W. Different micro/nanoscopic techniques evidenced the morphology of the substrates, and X-ray Photoelectron Spectroscopy was used to disclose the surface composition. The layer is a 500 nm thick hard nanostructure, composed of 60% graphitic carbon clustered with 15% TiC and 25% Ti oxides.

Real-Time RT-PCR for the Detection of Lyssavirus Species.

The causative agents of rabies are single-stranded, negative-sense RNA viruses in the genus Lyssavirus of Rhabdoviridae, consisting of twelve classified and three as yet unclassified species including classical rabies virus (RABV). Highly neurotropic RABV causes rapidly progressive encephalomyelitis with nearly invariable fatal outcome. Rapid and reliable diagnosis of rabies is highly relevant for public and veterinary health. Due to growing variety of the genus Lyssavirus observed, the development of suitable molecular assays for diagnosis and differentiation is challenging. This work focused on the establishment of a suitable real-time RT-PCR technique for rabies diagnosis as a complement to fluorescent antibody test and rabies tissue culture infection test as gold standard for diagnosis and confirmation. The real-time RT-PCR was adapted with the goal to detect the whole spectrum of lyssavirus species, for nine of which synthesized DNA fragments were used. For the detection of species, seven probes were developed. Serial dilutions of the rabies virus strain CVS-11 showed a 100-fold higher sensitivity of real-time PCR compared to heminested RT-PCR. Using a panel of thirty-one lyssaviruses representing four species, the suitability of the protocol could be shown. Phylogenetic analysis of the sequences obtained by heminested PCR allowed correct classification of all viruses used.

Presence of Campylobacter and Arcobacter species in in-line milk filters of farms authorized to produce and sell raw milk and of a water buffalo dairy farm in Italy.

The objectives of this study were to investigate the presence of Campylobacter spp. and Arcobacter spp. in dairy herds authorized for the production and sale of raw milk and in a water buffalo dairy farm, and to test the antimicrobial susceptibility of the isolates. A total of 196 in-line milk filters were collected from 14 dairy farms (13 bovine and 1 water buffalo) for detection of Campylobacter spp. and Arcobacter spp. by microbiological culture. For each farm investigated, 1 isolate for each Campylobacter and Arcobacter species isolated was tested using the Etest method (AB Biodisk, Solna, Sweden) to evaluate the susceptibility to ciprofloxacin, tetracycline, chloramphenicol, ampicillin, erythromycin, and gentamicin. A total of 52 isolates were detected in 49 milk filters in 12 farms (85.7%) out of 14 and the isolates were identified as Campylobacter jejuni (6), Campylobacter hyointestinalis ssp. hyointestinalis (8), Campylobacter concisus (1), Campylobacter fetus ssp. fetus (1), Arcobacter butzleri (22), and Arcobacter cryaerophilus (14). The small number of isolates tested for antimicrobial susceptibility precludes any epidemiological consideration but highlights that all Campylobacter isolates were susceptible to macrolides, which are the first-choice drugs for the treatment of campylobacteriosis, and that resistance to fluoroquinolones and tetracycline was detected; for Arcobacter isolates, resistance to ampicillin and chloramphenicol was detected. The sale of raw milk for human consumption by self-service automatic vending machines has been allowed in Italy since 2004 and the presence of C. jejuni in in-line milk filters confirms that raw milk consumption is a significant risk factor for human infection. The high occurrence of emerging Campylobacter spp. and Arcobacter spp. discovered in dairy farms authorized for production and sale of raw milk represents an emerging hazard for human health.

Imported human rabies in Switzerland, 2012: a diagnostic conundrum.

Human rabies is rare in Western Europe. It is not easily recognized in the absence of a history of exposure. We describe the clinical course, diagnosis and follow-up of an imported human rabies case in Switzerland. The patient, a U.S. citizen, presented at an outpatient clinic in Iraq with pain in his right shoulder on July 5, 2012. On July 8 he was transferred to a hospital in the United Arab Emirates, where he exhibited progressive encephalitis with coma. On July 29, he was transferred to a hospital in Switzerland, where he died on July 31, 2012. The autopsy showed severe encephalitis. Rabies was diagnosed by the rapid fluorescent focus inhibition test (RFFIT) and confirmed by fluorescence antibody testing (FAT) in brain smears and immunohistochemistry on paraffin-embedded brain sections. The viral strain was characterized by RT-PCR followed by sequencing and phylogenetic analysis as an American bat rabies strain associated with Tadarida brasiliensis. Close contacts and exposed health care workers received postexposure prophylaxis (PEP).

Occurrence of ε-proteobacterial species in rabbits (Oryctolagus cuniculus) reared in intensive and rural farms.

In order to investigate the occurrence of Campylobacter, Helicobacter and Arcobacter species in caecal contents of rabbits reared in intensive and rural farms, a total of 87 samples from animals belonging to 29 farms were analysed by both cultural and PCR analyses. PCR analysis directly from faecal samples detected 100% positive samples for Campylobacter genus, 3.4% for Helicobacter genus and none for Arcobacter genus. 83 out of 87 animals (95.4%) and all the 29 farms were positive for Campylobacter cuniculorum as also determined by cultural examination. Campylobacter coli and Campylobacter jejuni were isolated only from three animals reared in two rural farms. No Helicobacter and Arcobacter species were isolated. To evaluate a possible genetic variability, one strain of C. cuniculorum from each farm was analysed by Pulsed Field Gel Electrophoresis (PFGE) and Amplified Fragment Length Polymorphism (AFLP). Genotyping revealed that C. cuniculorum population is heterogeneous among the different sources and no dominant clone has spread in the investigated farms. This survey highlighted a high presence of C. cuniculorum with a high rate of intestinal colonization, low presence of C. jejuni-coli, Helicobacter spp. and any Arcobacter spp. in farmed rabbits.

Quantitative risk assessment of verocytotoxin-producing Escherichia coli O157 and Campylobacter jejuni related to consumption of raw milk in a province in Northern Italy.

A quantitative risk assessment was developed to describe the risk of campylobacteriosis and hemolytic uremic syndrome (HUS) linked to consumption of raw milk sold in vending machines in Northern Italy. Exposure assessment considered the microbiological status of dairy farms, expected milk contamination, storage conditions from bulk tank to home storage, microbial growth during storage, destruction experiments, consumption frequency of raw milk, age of consumers, serving size, and consumption preference. The differential risk between milk handled under regulation conditions (4°C throughout all phases) and the worst field handling conditions was considered. The probability of Campylobacter jejuni infection was modeled with a single-hit dose-response beta-Poisson model, whereas for HUS an exponential dose-response model was chosen and two probabilities were used to model the higher susceptibility of children younger than 5 years old. For every 10,000 to 20,000 consumers each year, the models predicted for the best and worst storage conditions, respectively, 2.12 and 1.14 campylobacteriosis cases and 0.02 and 0.09 HUS cases in the 0- to 5-year age group and 0.1 and 0.5 HUS cases in the >5-year age group. The expected pediatric HUS cases do not differ considerably from those reported in Italy by the Minister of Health. The model developed may be a useful tool for extending the assessment of the risk of campylobacteriosis and HUS due to raw milk consumption at the national level in Italy. Considering the epidemiological implications of this study, the risk of illness linked to raw milk consumption should not be ignored and could be reduced by the use of simple measures. Boiling milk before consumption and strict control of temperatures by farmers during raw milk distribution have significant effects on campylobacteriosis and HUS and are essential measures for risk management.

C-C bond unsaturation degree in monosubstituted ferrocenes for molecular electronics investigated by a combined near-edge x-ray absorption fine structure, x-ray photoemission spectroscopy, and density functional theory approach.

We present the results of an experimental and theoretical investigation of monosubstituted ethyl-, vinyl-, and ethynyl-ferrocene (EtFC, VFC, and EFC) free molecules, obtained by means of synchrotron-radiation based C 1s photoabsorption (NEXAFS) and photoemission (C 1s XPS) spectroscopies, and density functional theory (DFT) calculations. Such a combined study is aimed at elucidating the role played by the C-C bond unsaturation degree of the substituent on the electronic structure of the ferrocene derivatives. Such substituents are required for molecular chemical anchoring onto relevant surfaces when ferrocenes are used for molecular electronics hybrid devices. The high resolution C 1s NEXAFS spectra exhibit distinctive features that depend on the degree of unsaturation of the hydrocarbon substituent. The theoretical approach to consider the NEXAFS spectrum made of three parts allowed to disentangle the specific contribution of the substituent group to the experimental spectrum as a function of its unsaturation degree. C 1s IEs were derived from the experimental data analysis based on the DFT calculated IE values for the different carbon atoms of the substituent and cyclopentadienyl (Cp) rings. Distinctive trends of chemical shifts were observed for the substituent carbon atoms and the substituted atom of the Cp ring along the series of ferrocenes. The calculated IE pattern was rationalized in terms of initial and final state effects influencing the IE value, with special regard to the different mechanism of electron conjugation between the Cp ring and the substituent, namely the σ/π hyperconjugation in EtFC and the π-conjugation in VFC and EFC.

Isolation of glucosidase and phospholipase positive Bacillus circulans on ALOA medium.

AIM: To report the growth of glucosidase and phospholipase positive bacteria on agar Listeria according to Ottaviani and Agosti (ALOA) different from Listeria monocytogenes, Listeria ivanovii and Bacillus cereus. METHODS AND RESULTS: Raw water-buffalo milk was analysed according to EN ISO 11290. Streaking of Fraser broth on ALOA resulted in green colonies with an opaque halo after 48 h at 30°C. Colonies were transferred onto Tryptone soya yeast extract agar and showed cultural characteristics atypical for L. monocytogenes. Results of confirmation tests according to EN ISO 11290 method: negative haemolysis test, weak positive camp test in correspondence with Staphylococcus aureus, no fermentation of rhamnose, fermentation of xylose. Gram staining showed tapered, curved, Gram-positive rods with subterminal to terminal ellipsoidal spores, 0.5-0.7 µm diameter 4-5 µm. API 50CH CHB kit (99.9% percentage of identification) and the sequence of the 833 bp PCR product (portion of 16S rRNA, generic primers 1492-r; p27-f) showed 97% identity with Bacillus circulans ATCC 4513 (GenBank AY724690). CONCLUSIONS: Some B. circulans strains can grow on ALOA, according to ISO 11290, confirmation tests readily differentiate B. circulans from L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The different morphology of the colonies must be kept in mind to select true L. monocytogenes for confirmation test and to avoid overestimation of L. monocytogenes count.

Walking the dog and moving the cat: rabies serology in the context of international pet travel schemes.

Data of 13'469 blood samples from 10'999 dogs and 2'470 cats tested for rabies neutralizing antibodies within the framework of pet travel schemes were analysed for single and combined factors influencing antibody titres and failures. The time span between vaccination and drawing the blood sample was confirmed as a major source of failure in dogs with a proportion of 23 % at 4 months after primary vaccination (single dose). Failures in dogs and cats (titre < 0.5 IU) were significantly reduced after double primary vaccination (2 doses within 7 - 10 days), although failures reached comparable levels in dogs as early as 6 months after vaccination. In contrast, failure after vaccination was generally below 5 % in dogs and absent in cats after a booster applied at earliest 12 months after single primary vaccination. Statistically significant differences between the failures of the vaccine brands «Rabisin¼ (1.5 %), «Defensor¼ (6.7 %), «Nobivac Rabies¼ (11.0 %) and «Rabdomun¼ (18.2 %) were found in dogs but also between the titres induced in cats. Significant differences were found between different dog breeds with some small breeds showing a significantly higher responsiveness. Taken together, a new regimen for rabies vaccination consisting of double primary vaccination with a short interval of 7 - 10 days and a one-year booster appears to be highly recommended for dogs and cats.

Surveillance for European bat lyssavirus in Swiss bats.

Most countries in Western Europe are currently free of rabies in terrestrial mammals. Nevertheless, rabies remains a residual risk to public health due to the natural circulation of bat-specific viruses, such as European bat lyssaviruses (EBLVs). European bat lyssavirus types 1 and 2 (EBLV-1 and EBLV-2) are widely distributed throughout Europe, but little is known of their true prevalence and epidemiology. We report that only three out of 837 brains taken from bats submitted to the Swiss Rabies Centre between 1976 and 2009 were found by immunofluorescence (FAT) to be positive for EBLVs. All three positive cases were in Myotis daubentoni, from 1992, 1993 and 2002. In addition to this passive surveillance, we undertook a targeted survey in 2009, aimed at detecting lyssaviruses in live bats in Switzerland. A total of 237 bats of the species M. daubentoni, Myotis myotis, Eptesicus serotinus and Nyctalus noctula were captured at different sites in western Switzerland. Oropharyngeal swabs and blood from each individual were analysed by RT-PCR and rapid fluorescent focus inhibition test (RFFIT), respectively. RNA corresponding to EBLV-2 was detected from oropharyngeal swabs of a single M. daubentoni bat, but no infectious virus was found. Molecular phylogenetic analysis revealed that the corresponding sequence was closely related to the other EBLV-2 sequences identified in previous rabies isolates from Swiss bats (particularly to that found at Geneva in 2002). Three M. daubentoni bats were found to be seropositive by RFFIT. In conclusion, even though the prevalence is low in Switzerland, continuous management and surveillance are required to assess the potential risk to public health.

Epidemiological study of pestiviruses in South American camelids in Switzerland.

BACKGROUND: In the context of the ongoing eradication campaign for bovine viral diarrhea virus (BVDV) in cattle in Switzerland, the role of South American camelids (SAC) as a possible virus reservoir needed to be evaluated. OBJECTIVE: To assess and characterize the prevalence of pestivirus infections in SAC in Switzerland. ANIMALS: Serum samples collected from 348 animals (40 herds) in 2008 and from 248 animals (39 herds) in 2000 were examined for antibodies against pestiviruses and for the presence of BVDV viral RNA. METHODS: Cross-sectional study using stratified, representative herd sampling. An indirect BVDV-ELISA was used to analyze serum samples for pestivirus antibodies, and positive samples underwent a serum neutralization test (SNT). Real-time RT-PCR to detect pestiviral RNA was carried out in all animals from herds with at least 1 seropositive animal. RESULTS: In 2008, the overall prevalence of animals positive for antibodies (ELISA) and pestiviral RNA or was 5.75 and 0%, respectively. In 2000, the corresponding prevalences were 3.63 and 0%, respectively. The seroprevalences (SNT) for BVDV, border disease virus or undetermined pestiviruses were estimated to be 0, 1.73, and 4.02% in 2008, and 0.40, 1.21, and 2.02% in 2000, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: At the present time, SAC appear to represent a negligible risk of re-infection for the BVDV eradication program in cattle in Switzerland.