A highly endemic area of Echinococcus multilocularis identified through a comparative re-assessment of prevalence in the red fox (Vulpes vulpes), Alto Adige (Italy: 2019-2020).

Surveillance of Echinococcus multilocularis at the edge of its range is hindered by fragmented distributional patterns and low prevalence in definitive hosts. Thus, tests with adequate levels of sensitivity are especially important for discriminating between infected and non-infected areas. In this study we reassessed the prevalence of E. multilocularis at the southern border of its distribution in Province of Bolzano (Alto Adige, northeastern Alps, Italy), to improve surveillance in wildlife and provide more accurate estimates of exposure risk. We compared the diagnostic test currently implemented for surveillance based on coproscopy and multiplex PCR (CMPCR) to a real-time quantitative PCR (qPCR) in 235 fox faeces collected in 2019 and 2020. The performances of the two tests were estimated using a scraping technique (SFCT) applied to the small intestines of a subsample (n = 123) of the same foxes as the reference standard. True prevalence was calculated and the sample size required by each faecal test for the detection of the parasite was then estimated. True prevalence of E. multilocularis in foxes (14.3%) was markedly higher than reported in the last decade, which was never more than 5% from 2012 to 2018 in the same area. In addition, qPCR showed a much higher sensitivity (83%) compared to CMPCR (21%) and agreement with the reference standard was far higher for qPCR (0.816) than CMPCR (0.298) meaning that for the latter protocol, a smaller sample size would be required to detect the disease. Alto Adige should be considered a highly endemic area. Routine surveillance on definitive hosts at the edges of the E. multilocularis distribution should be applied to smaller geographic areas, and rapid, sensitive diagnostic tools using directly host faeces, such as qPCR, should be adopted.

Assessing Red Fox (Vulpes vulpes) Demographics to Monitor Wildlife Diseases: A Spotlight on Echinococcus multilocularis.

The assessment of red fox population density is considered relevant to the surveillance of zoonotic agents vectored by this species. However, density is difficult to estimate reliably, since the ecological plasticity and elusive behavior of this carnivore hinder classic methods of inference. In this study, red fox population density was estimated using a non-invasive molecular spatial capture-recapture (SCR) approach in two study areas: one in a known hotspot of the zoonotic cestode Echinococcus multilocularis, and another naïve to the parasite. Parasitological investigations on collected samples confirmed the presence of the parasite exclusively in the former area; the SCR results indicated a higher fox population density in the control area than in the hotspot, suggesting either that the relationship between fox density and parasite prevalence is not linear and/or the existence of other latent factors supporting the parasitic cycle in the known focus. In addition, fox spotlight count data for the two study areas were used to estimate the index of kilometric abundance (IKA). Although this method is cheaper and less time-consuming than SCR, IKA values were the highest in the areas with the lower molecular SCR density estimates, confirming that IKA should be regarded as a relative index only.

Business intelligence tools to optimize the appropriateness of the diagnostic process for clinical and epidemiologic purposes in a multicenter veterinary pathology service.

Laboratory tests provide essential support to the veterinary practitioner, and their use has grown exponentially. This growth is the result of several factors, such as the eradication of historical diseases, the occurrence of multifactorial diseases, and the obligation to control endemic and epidemic diseases. However, the introduction of novel techniques is counterbalanced by economic constraints, and the establishment of evidence- and consensus-based guidelines is essential to support the pathologist. Therefore, we developed standardized protocols, categorized by species, type of production, age, and syndrome at the Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe), a multicenter institution for animal health and food safety. We have 72 protocols in use for livestock, poultry, and pets, categorized as, for example, "bovine enteric calf", "rabbit respiratory", "broiler articular". Each protocol consists of a panel of tests, divided into 'mandatory' and 'ancillary', to be selected by the pathologist in order to reach the final diagnosis. After autopsy, the case is categorized into a specific syndrome, subsequently referred to as a syndrome-specific panel of analyses. The activity of the laboratories is monitored through a web-based dynamic reporting system developed using a business intelligence product (QlikView) connected to the laboratory information management system (IZILAB). On a daily basis, reports become available at general, laboratory, and case levels, and are updated as needed. The reporting system highlights epidemiologic variations in the field and allows verification of compliance with the protocols within the organization. The diagnostic protocols are revised annually to increase system efficiency and to address stakeholder requests.

Echinococcus multilocularis and other cestodes in red foxes (Vulpes vulpes) of northeast Italy, 2012-2018.

BACKGROUND: Echinococcus multilocularis is a small tapeworm affecting wild and domestic carnivores and voles in a typical prey-predator life cycle. In Italy, there has been a focus of E. multilocularis since 1997 in the northern Italian Alps, later confirmed in red foxes collected from 2001 to 2005. In this study, we report the results of seven years of monitoring on E. multilocularis and other cestodes in foxes and describe the changes that occurred over time and among areas (eco-regions) showing different environmental and ecological features on a large scale. METHODS: Eggs of cestodes were isolated from feces of 2872 foxes with a sedimentation/filtration technique. The cestode species was determined through multiplex PCR, targeting and sequencing ND1 and 12S genes. Analyses were aimed to highlight variations among different eco-regions and trends in prevalence across the study years. RESULTS: Out of 2872 foxes, 217 (7.55%) samples resulted positive for cestode eggs at coproscopy, with differences of prevalence according to year, sampling area and age class. Eight species of cestodes were identified, with Taenia crassiceps (2.65%), Taenia polyacantha (1.98%) and E. multilocularis (1.04%) as the most represented. The other species, Mesocestoides litteratus, Taenia krabbei, T. serialis, T. taeniaeformis and Dipylidium caninum, accounted for < 1% altogether. Echinococcus multilocularis was identified in foxes from two out of six eco-regions, in 30 fecal samples, accounting for 1.04% within the cestode positives at coproscopy. All E. multilocularis isolates came from Bolzano province. Prevalence of cestodes, both collectively and for each of the three most represented species (T. crassiceps, T. polyacantha and E. multilocularis), varied based on the sampling year, and for E. multilocularis an apparent increasing trend across the last few years was evidenced. CONCLUSIONS: Our study confirms the presence of a focus of E. multilocularis in red foxes of northeast Italy. Although this focus seems still spatially limited, given its persistence and apparent increasing prevalence through the years, we recommend research to be conducted in the future on the ecological factors that, on a smaller scale, allow this zoonotic species to persist. On the same scale, we recommend a health education campaign to inform on the measures to prevent this zoonosis, targeted at people living in the area, especially hunters, dog owners, forestry workers and other potentially exposed categories.

Canine leishmaniosis in the Italian northeastern Alps: A survey to assess serological prevalence in dogs and distribution of phlebotomine sand flies in the Autonomous Province of Bolzano - South Tyrol, Italy.

The Autonomous Province of Bolzano-South Tyrol (APB), located in the northernmost territory of the Italian eastern Alps, is still considered non-endemic for canine leishmaniosis (CanL) despite clinical cases being observed and a competent Leishmania infantum vector (Phlebotomus perniciosus) having been recorded since 2008. A serological survey of leishmaniosis among a randomly-selected subpopulation of registered owned dogs was carried in 2018, followed by entomological investigations performed in 2019 and driven by canine survey results. A total of 457 resident dogs from all over the APB territory were examined through IFAT for antibodies against L.infantum, of which 63 (13.8%) tested positive. Thirty-five seropositive cases (7.7%) were considered autochthonous to APB, i.e. dogs born and lived in the province, or imported dogs with no travel history in the past 5 years. Most of these animals showed an antibody titre at the threshold level of 1:40, suggesting a low degree of parasite transmission/contacts. In 2 autochthonous cases with moderately high IFAT titre, the infection was confirmed by nested-PCR in peripheral blood. Thirty-one georeferenced sites were monitored for sand flies by means of interception (sticky papers) and attraction (CDC miniature light traps) collection devices. Traps were set during summer approximately on monthly basis, and extended up to October for positive sites. Only 2 sites were found positive for a total of 317 phlebotomine specimens collected by sticky traps, which included a previously known P. perniciosus-endemic site near Bolzano town. Sergentomyia minuta was by far the most prevalent (98.1%) and the only recorded sand fly species in the most northerly Italian site ever investigated (Coldrano municipality in Venosta valley). For the first time, Leishmania serology and n-PCR positive dogs autochthonous to APB were identified, however the spread of sand flies competent for L. infantum transmission could not be demonstrated in several places where endemic seropositive cases were recorded. APB can be considered a territory of low CanL endemicity, however awareness and continuous monitoring are needed to detect changes in the epidemiological status of the zoonosis.

Two waves of canine distemper virus showing different spatio-temporal dynamics in Alpine wildlife (2006-2018).

Canine distemper virus (CDV) represents an important threat for both wild and domestic carnivores. Since 2006, the North-Eastern regions in Italy have been experiencing severe and widespread recurring outbreaks of CDV affecting the wild carnivore population. In this study we performed an extensive phylogeographic analysis of CDV strains belonging to the Wildlife-Europe genetic group identified between 2006 and 2018 in Veneto, Trentino Alto Adige and Friuli Venezia Giulia regions. Our analysis revealed that viruses from the first (2006-2009) and the second (2011-2018) epidemic wave cluster separately, suggesting the introduction of two distinct genetic variants. These two events were characterized by different diffusion rates and spatial distribution, thus suggesting the existence of a connection between infection spread and host population dynamics. We also report the first spillover event of this strain to a non-vaccinated dog in a rural area of Friuli Venezia Giulia. The increasing prevalence of the infection in wildlife population, the broad host range of CDV circulating in the Alpine wildlife and the first reported transmission of a wild-adapted strain to a domestic dog in this region raise concerns over the vulnerability of wildlife species and the exposure of our pets to new threatening strains. Understanding the dynamic of CDV epidemics will also improve preparedness for re-emerging diseases affecting carnivore species.

Mycobacterium aviumsubsp. paratuberculosis isolated from wild red deer (Cervus elaphus) in Northern Italy.

Paratuberculosis (or Johne's disease) is an infectious disease which affects mainly ruminants and it is caused by Mycobacterium avium subsp. paratuberculosis (MAP). During a culling program (years 2011-2015) aimed at controlling the red deer (Cervus elaphus) population in Stelvio National Park (Italian Alps), where paratuberculosis was already described in this species, 382 tissue samples from the Lombardy Region and 102 fecal specimens from the Autonomous Province of Bolzano were analyzed by PCR. Of these, 77 samples (20.16%) from the Lombardy area and 19 specimens (18.63%) from the Bolzano area resulted PCR positive. The cultural test was carried out on PCR positive samples (n = 96), enabling the isolation of 19 MAP field strains which were genotyped using MIRU-VNTR typing and Short Sequence repeats (SSRs). Our results suggest that all isolates share an identical VNTR profile corresponding to the INMV1 genotype. The only variation was on the locus SSR2, but the utility of this last locus has already been questioned because of its instability. Overall, these data suggest a common clonal origin and host adaptation during the diffusion of paratuberculosis in this population. Finally, this profile is the same as that which has already been described in the cattle population in Northern Italy, suggesting a possible inter-species disease transmission pattern from wildlife to domestic ruminants and vice versa.

Phenotypic characterization and ERIC-PCR based genotyping of Paenibacillus larvae isolates recovered from American foulbrood outbreaks in honey bees from Italy.

BACKGROUND: Paenibacillus larvae is the etiological agent of American foulbrood (AFB), a widespread and severe bacterial brood disease of honey bees. The genomic characterization of P. larvae strains by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) is able to differentiate four genotypes (ERIC I, ERIC II, ERIC III, ERIC IV). The information on the presence of P. larvae ERIC genotypes worldwide is few. OBJECTIVES: We have characterized P. larvae strains isolated in Italy from AFB outbreaks to obtain information on ERIC genotypes and phenotypes of the strains circulating in the country. METHODS: A total of 117 P. larvae isolates from 115 AFB outbreaks occurring in 2008-2012 were subjected to phenotypic and genetic characterization. RESULTS: The genomic characterization allowed the identification of ERIC I and ERIC II genotypes. Examining the data of Northern and Central Italy separately it was noted that in Northern Italy most outbreaks were caused by the ERIC I genotype (78.6%), followed by the ERIC II genotype (18.6%) and by co-infections (ERIC I + ERIC II) (2.6%). In Central Italy, only outbreaks caused by the ERIC I genotype were observed. With regard to phenotypic characteristics all examined strains of ERIC II genotype fermented fructose while no strains of ERIC I genotype possessed this ability. CONCLUSION: Both P. larvae ERIC I and ERIC II genotypes were isolated from the AFB outbreaks, but ERIC II genotype was isolated only in Northern Italy. The fermentation of fructose seems to be a genotype-specific biochemical marker.

Serological evidence for Parapoxvirus infection in chamois from the Tyrol regions of Austria and Italy.

Orf-virus (ORFV) is a parapoxvirus that infects small ruminants worldwide causing sporadic zoonotic infections, mainly transmitted by direct contact with sheep and goats. Following an ORFV case in a hunter of Alpine chamois (Rupicapra rupicapra), who did not report previous contact to domestic animals, a serological survey in Western Austria was conducted to assess the seroprevalence of ORFV in this species. In addition, this study also tested blood/tissue samples of chamois from different areas of the adjacent province of Bolzano/Northern Italy for antibodies against ORFV using immunofluorescence and ELISA. The observed seropositivity rates in the chamois tested on the Austrian and Italian side of the Alps were 23.5% and 9.5%, respectively, with a combined 95% confidence interval ranging from 0.0678 to 0.238. Although the prevalence was significantly lower than the one observed in Austrian sheep flocks, this study provided the first evidence that parapoxviruses have spilled over into chamois populations to a significant degree in the Tyrol regions of Austria and Italy.

A distinct CDV genotype causing a major epidemic in Alpine wildlife.

Canine distemper virus (CDV) infection represents an important conservation threat to many carnivore species and has contributed to the population decline of several wild terrestrial and aquatic mammalian species. Since 2006, the Alpine region of North-Eastern (NE) Italy has been experiencing a severe and widespread outbreak of CDV affecting the wild carnivore population. In this study we performed an extensive phylogenetic and molecular evolutionary analysis of CDV identified during the recent wildlife epidemic in the Alpine region. Our analysis yielded data on the evolutionary dynamics of the Alpine wildlife CDV epidemic and revealed the emergence and spread of a single genetic cluster of CDV. The wide distribution of the novel cluster combined with the identification of a specific amino acid mutation, which is believed to increase the ability of the virus to replicate in a wider host range, raises concerns over the possible implications of the spread of this virus on the conservation of endangered wildlife species.