Tularemia - possible increase and new risk factors

Purpose: Tularemia is a zoonotic disease caused by the bacterium Francisella tularensis. In Europe each year approximately 1200 human cases are reported. Four subspecies are currently known: tularensis (the most virulent form), holarctica (the most widespread form), mediasiatic, and novicida. In Austria Francisella tularensis supsp. holarctica is endemic in the eastern part of the country (Lower Austria and Burgenland), and is known to have a 5-year cycle. Zoonotic transmission from pet species in Europe has only been described in Norway due to a cat bite, as well as after an accidental exposure to the disease while spaying a cat. In 2014 first reports of clinically ill dogs were reported from Norway. Methods & Materials: As hunting with dogs has a long tradition in Austria, and as there are endemic areas for the disease a first serological screening of 80 hunting dogs used in the hunt for European brown hares (Lepus europaeus) was conducted. Results: Of these 80 dogs 5 tested positive for tularemia (6.25%, CI 2.1% - 14%). One positive dog had shown some clinical symptoms, however this female dog also tested positive for Brucella canis. Conclusion: This result shows that dogs not only have contact to the pathogen, but also seroconvert. The occurrence of the disease is thought to increase in the next years due to our changing climate, and this year there is a new hotspot of the disease in Austria (i.e. Salzburg). These changes, as well as the result of this study highlight the need to raise the awareness level of the disease, its possible increase and new risk factors

Immunoblotting for the serodiagnosis of alveolar echinococcosis in alive and dead Eurasian beavers (Castor fiber)

A novel species-specific anti-beaver-IgG-alkaline-phosphatase conjugate was synthesized for the development of a new serological test for echinococcosis in beavers. Two different ELISAs conventionally used for human Echinococcus multilocularis serology (Em18-ELISA and Em2-ELISA) yielded diagnostic sensitivities of 0% and 46%, respectively. In contrast, the subsequently developed immunoblotting assay gave an 85% diagnostic sensitivity (11 out of 13 beavers with alveolar echinococcosis were immunoblotting-positive, i.e. showed reactivity with a specific 21 Mr band), and maximal specificity. In conclusion, this immunoblotting assay should be the method of choice for use in serological studies on E. multilocularis in Eurasian beavers, and the test proved suitable to investigate both animals alive and post-mortem

Widespread occurrence of the non-pathogenic hare calicivirus (HaCV Lagovirus GII.2) in captive-reared and free-living wild hares in Europe

The Lagovirus genus comprises both pathogenic viruses as European brown hare syndrome virus (EBHSV- GII.1) and rabbit hemorrhagic disease viruses (RHDV-GI.1 and RHDV2-GI.2), that principally infect European brown hares (Lepus europeaus) and European rabbits (Oryctolagus cuniculus), respectively, causing severe necrotic hepatitis, spleen enlargement and disseminated haemorrhage. This genus includes also non-pathogenic agents, such as rabbit calicivirus (RCV-E1 – GI.3) and the non-pathogenic hare Lagovirus, provisionally named hare calicivirus (HaCV – GII.2). The latter had been identified for the first time in 2012 in the gut contents and faeces of healthy young hares raised in a breeding farm. In this study, we further investigated the presence of HaCV by testing the intestinal tract of 621 wild hares collected between 2010 and 2018 in Northern and Central Italy, and in 2011 in Austria, Germany and Spain. These wild hares were found dead for causes other than EBHS or were healthy hares shot during the hunting season. Forty-three out of 322 hare samples from Italy and 14 out of 299 samples from Austria and Germany were positive for HaCV–GII.2 by RT-PCR using universal primers for lagoviruses and primers specific for HaCV. Sequence analysis of the full capsid protein gene conducted on 12 strains representative of different years and locations indicated that these viruses belong to the same, single cluster as the prototype strain initially identified at the hares’ farm (HaCV_Bs12_1). The relatively high level of genetic variation (88% nt identity) within this cluster suggests HaCVs may have been circulating widely in Europe for some time

Development and validation of a portable, point-of-care canine distemper virus qPCR test.

Canine distemper virus (CDV) is a multi-host pathogen that can cause significant mortality in domestic, wild terrestrial and marine mammals. It is a major conservation threat in some endangered species. Infection can result in severe respiratory disease and fatal encephalitis. Diagnosis and disease monitoring in wildlife, and differentiation of CDV from rabies (a life-threatening zoonotic disease that can produce similar neurologic signs), would benefit from the availability of a portable, point-of-care (POC) diagnostic test. We therefore developed a quantitative RT-PCR assay for CDV using shelf-stable, lyophilized reagents and target-specific primers and probes for use with the handheld Biomeme two3™ qPCR thermocycler. Biomeme's extraction methodology, lyophilized reagents, and thermocycler were compared to our standard laboratory-based methods to assess sensitivity, efficiency and overall test performance. Results using a positive control plasmid for CDV showed comparable sensitivity (detection of 50 copies) and PCR efficiency between the two platforms, and CDV detection was similar between platforms when tested using a modified live CDV vaccine. Significantly higher Ct values (average Ct = 5.1 cycles) were observed using the Biomeme platform on known CDV positive animal samples. CDV detection using the Biomeme platform was similar in 25 of 26 samples from suspect CDV cases when compared to standard virology laboratory testing. One false positive was observed that was negative upon retest. The Biomeme methodology can be adapted for detection of specific targets, and this portable technology saves time by eliminating the need for local or international sample transport for laboratory-based diagnostics. However, results of our testing suggest that decreased diagnostic sensitivity (higher Ct values) relative to laboratory-based methods was observed using animal samples, so careful validation and optimization are essential. Portable qPCR platforms can empower biologists and wildlife health professionals in remote and low-resource settings, which will greatly improve our understanding of CDV disease ecology and associated conservation threats in wildlife

Positive selection and precipitation effects on the mitochondrial NADH dehydrogenase subunit 6 gene in brown hares (Lepus europaeus) under a phylogeographic perspective

Previous studies in hares and jackrabbits have indicated that positive selection has shaped the genetic diversity of mitochondrial genes involved in oxidative phosphorylation, which may affect cellular energy production and cause regional adaptation to different environmental (climatic) pressures. In the present study, we sequenced the NADH dehydrogenase subunit 6 (MT-ND6) gene of 267 brown hares (L. europaeus) from Europe and Asia Minor and tested for positive selection and adaptations acting on amino acid sequences (protein variants). Molecular diversity indices and spatial clustering were assessed by DnaSP, Network, and Geneland, while the presence of selection signals was tested by codeml in PAML, and by using the Datamonkey Adaptive Evolution web server. The SPSS software was used to run multinomial regression models to test for possible effects of climate parameters on the currently obtained protein variants. Fifty-eight haplotypes were revealed with a haplotype diversity of 0.817, coding for 17 different protein variants. The MT-ND6 phylogeographic pattern as determined by the nucleotide sequences followed the earlier found model based on the neutrally evolving D-loop sequences, and reflected the earlier found phylogeographic Late Pleistocene scenario. Based on several selection tests, only one codon position consistently proved to be under positive selection. It did occur exclusively in the evolutionarily younger hares from Europe and it gave rise to several protein variants from the southeastern and south-central Balkans. The occurrence of several of those variants was significantly favored under certain precipitation conditions, as proved by our multinomial regression models. Possibly, the great altitudinal variation in the Balkans may have lead to bigger changes in precipitation across that region and this may have imposed an evolutionarily novel selective pressure on the protein variants and could have led to regional adaptation. © 2019 Stefanović et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Characterization of mecC gene-carrying coagulase-negative Staphylococcus spp. isolated from various animals.

The presence of the methicillin resistance gene mecC in coagulase-negative Staphylococcus spp. (CoNS) is scarce. The aim of this study was to characterize mecC-positive CoNS isolated from various wild and domestic animals. The presence of the mecC gene was screened in 4299 samples from wild animals and domestic animals. Fifteen coagulase-negative staphylococci, that displayed a cefoxitin-resistant phenotype, were tested mecC-positive by PCR. Antimicrobial susceptibility testing was performed for all isolates. The 15 isolates were genotyped by sequencing of the entire class E mec gene complex (blaZ-mecC-mecR1-mecI), the ccrA and ccrB recombinase genes and other determinants within the type XI SCCmec element. DNA microarray analysis was performed and five selected isolates were additionally whole genome sequenced and analyzed. S. stepanovicii (n = 3), S. caprae (n = 1), S. warneri (n = 1), S. xylosus (n = 1) and S. sciuri (n = 9) were detected. All but the S. sciuri isolates were found to be susceptible to all non-beta lactams. The entire class E mec gene complex was detected in all isolates but ccrA and ccrB genes were not identified in S. stepanovicii and S. xylosus. The genes erm(B) and fexA (n = 4, each) were the most predominant non-beta lactam resistance genes detected in the S. sciuri isolates. Even though the presence of the mecC gene among CoNS is a rare observation, this study further expands our knowledge by showing that the mecC gene, including its allotypes, are present in more staphylococcal species from different animal species than has been previously described