Corrigendum: Comparing copromicroscopy to intestinal scraping to monitor red fox intestinal helminths with zoonotic and veterinary importance.

[This corrects the article DOI: 10.3389/fvets.2022.1085996.].

Description of a Sarcoptic Mange Outbreak in Alpine Chamois Using an Enhanced Surveillance Approach.

Since 1995, the Alpine chamois (Rupicapra r. rupicapra) population of the Dolomites has been affected by sarcoptic mange with considerable management concerns. In this study, 15 years (2006-2020) of passive surveillance and demographic data were analyzed in order to describe a mange outbreak. Furthermore, an enhanced passive surveillance protocol was implemented in order to evaluate the efficiency of ordinary vs. enhanced surveillance protocol in identifying dead chamois in the field and in reaching a correct diagnosis. Our results confirm the role of mange as a determining factor for chamois mortality, while stressing the importance of a wider view on the factors affecting population dynamics. The enhanced passive surveillance protocol increased the probability of carcass retrieval and identification of the cause of death; however, its adoption may be too costly if applied for long periods on a wide scale. Passive surveillance, in both ordinary and enhanced surveillance protocol, should encompass the use of other strategies in the future to study the eco-epidemiology of the disease in wild Caprinae.

Clinical Tick-Borne Encephalitis in a Roe Deer (Capreolus capreolus L.).

Tick-borne encephalitis virus (TBEV) is the causative agent of tick-borne encephalitis (TBE), a severe zoonosis occurring in the Palearctic region mainly transmitted through Ixodes ticks. In Italy, TBEV is restricted to the north-eastern part of the country. This report describes for the first time a case of clinical TBE in a roe deer (Capreolus capreolus L.). The case occurred in the Belluno province, Veneto region, an area endemic for TBEV. The affected roe deer showed ataxia, staggering movements, muscle tremors, wide-base stance of the front limbs, repetitive movements of the head, persistent teeth grinding, hypersalivation and prolonged recumbency. An autopsy revealed no significant lesions to explain the neurological signs. TBEV RNA was detected in the brain by real-time RT-PCR, and the nearly complete viral genome (10,897 nucleotides) was sequenced. Phylogenetic analysis of the gene encoding the envelope protein revealed a close relationship to TBEV of the European subtype, and 100% similarity with a partial sequence (520 nucleotides) of a TBEV found in ticks in the bordering Trento province. The histological examination of the midbrain revealed lymphohistiocytic encephalitis, satellitosis and microgliosis, consistent with a viral etiology. Other viral etiologies were ruled out by metagenomic analysis of the brain. This report underlines, for the first time, the occurrence of clinical encephalitic manifestations due to TBEV in a roe deer, suggesting that this pathogen should be included in the frame of differential diagnoses in roe deer with neurologic disease.

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.

Comparing copromicroscopy to intestinal scraping to monitor red fox intestinal helminths with zoonotic and veterinary importance.

The red fox acts as reservoir for several helminthic infections which are of interest for both public and animal health. Huge efforts have been made for the assessment of the sensitivity of coprological tests for the detection of Echinococcus multilocularis, while less attention has been paid to other helminthic species. This study aimed at assessing the performance of two copromicroscopic techniques in the detection and prevalence estimation of gastrointestinal helminths in the red fox. Helminths were isolated from the small intestines of 150 red foxes from Bolzano province, Italy, with a scraping, filtration and counting technique (SFCT) and morphologically identified. Rectal contents were collected and submitted to simple flotation (FT) and, only for Taenids, a method based on the concentration of eggs and identification with multiplex PCR (CMPCR). Using SFCT as a reference standard, we estimated the sensitivity of the copromicroscopic tests. Three species of nematodes (namely, Toxocara canis, Uncinaria stenocephala and Pterygodermatites sp.) and five species of cestodes (E. multilocularis, Taenia crassiceps, T. polycantha, Hydatigera taeniaeformis, Mesocestoides sp.) were identified with SFCT, whereas eggs referable to the same taxa were detected with fecal diagnostics, except for Pterygodermatites sp. and Mesocestoides sp. The sensitivity of FT was low for all taxa, ranging from 9.8 to 36.3%, with lower values for Taeniidae. CMPCR was confirmed to perform better for the detection of Taeniidae eggs (23.5%) and the multiplex PCR on retrieved eggs was effective in the identification of the species. A meta-analysis of literature also suggested that our results are consistent with existing data, indicating that copromicroscopy tends to underestimate the prevalence of helminthic infections. The extent of such underestimation varies with taxon, being higher at high prevalence levels, in particular for cestodes. Irregular dynamics of egg shedding, and routine deep freezing of red fox feces may explain the frequency of false negatives with copromicroscopy. Low sensitivity of copromicroscopic tests should be accounted for when estimating prevalence and when defining the correct sample size for the detection of the parasites.

Angiostrongylosis in northeastern Italy: First report of two autochthonous fatal cases in dogs and first detection in a wild red fox.

Canine angiostrongylosis is an emergent cardio-pulmonary gastropod-borne helminthic infection caused by the metastrongyloid nematode Angiostrongylus vasorum. Clinically, it is characterized by a wide spectrum of non-specific signs and the red fox serves as the most important reservoir for dog infections. In Italy, this disease has been well documented both in northwestern and central-southern regions, whereas it is apparently poorly recognized in the northeastern area of the country. This report describes the diagnostic findings of two autochthonous cases of fatal canine angiostrongylosis and of one case in a wild red fox detected in northeastern Italy. Reporting cases is relevant to clinicians in order to increase their awareness for the prompt diagnosis of a potentially life-threatening disease that may go unnoticed or misdiagnosed.

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.

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.

Ticks are more suitable than red foxes for monitoring zoonotic tick-borne pathogens in northeastern Italy.

BACKGROUND: Northeastern Italy is a hotspot for several tick-borne pathogens, transmitted to animals and humans mainly by Ixodes ricinus. Here we compare the results of molecular monitoring of ticks and zoonotic TBPs over a six-year period, with the monitoring of red foxes (Vulpes vulpes) in an endemic area. RESULTS: In the period 2011-2016, 2,578 ticks were collected in 38 sites of 20 municipalities of Belluno Province. Individual adults (264), pooled larvae (n = 330) and nymphs (n = 1984) were screened for tick-borne encephalitis virus, Borrelia burgdorferi (s.l.), Rickettsia spp., Babesia spp., Anaplasma phagocytophilum and "Candidatus Neoehrlichia mikurensis" by specific SYBR green real-time PCR assays and sequencing. The spleens of 97 foxes, culled in the period 2015-2017 during sport hunting or population control programs, were also screened. Overall, nine different pathogens were found in I. ricinus nymph and adult ticks: Rickettsia helvetica (3.69%); R. monacensis (0.49%); four species of the B. burgdorferi (s.l.) complex [B. afzelii (1.51%); B. burgdorferi (s.s.) (1.25%); B. garinii (0.18%); and B. valaisiana (0.18%)]; A. phagocytophilum (3.29%); "Candidatus N. mikurensis" (1.73%); and Babesia venatorum (0.04%). Larvae were collected and screened in the first year only and two pools (0.6%) were positive for R. helvetica. Tick-borne encephalitis virus was not found in ticks although human cases do occur in the area. The rate of infection in ticks varied widely according to tick developmental stage, site and year of collection. As expected, adults were the most infected, with 27.6% harboring at least one pathogen compared to 7.3% of nymphs. Pathogens with a minimum infection rate above 1% were recorded every year. None of the pathogens found in ticks were detectable in the foxes, 52 (54%) of which were instead positive for Babesia cf. microti (also referred to as Babesia microti-like, "Theileria annae", "Babesia annae" and "Babesia vulpes"). CONCLUSIONS: The results show that foxes cannot be used as sentinel animals to monitor tick-borne pathogens in the specific epidemiological context of northeastern Italy. The high prevalence of Babesia cf. microti in foxes and its absence in ticks strongly suggests that I. ricinus is not the vector of this pathogen.

Phenotypic features and phylogenetic background of extraintestinal hemolytic Escherichia coli responsible of mortality in puppies.

A 10-day-old litter of five puppies of Bracco Italiano dog breed showed weakness and diarrhea and, 2 days later, four of them died. At the same time, the bitch showed high hyperthermia (40 °C) and endometritis. The necropsy of a puppy revealed a severe lobar pneumonia accompanied with a bilateral nephrosis. No gross lesions were detected in other organs. Histopathology of the lung revealed severe multifocal fibrino-suppurative necrotizing bronchiolar-alveolitis associated with rod-shaped bacterial aggregates and diffuse interstitial lymphocytic infiltration. The kidney showed severe multifocal necrosis of the tubular epithelium and diffuse severe congestion of the parenchyma. A pure culture of hemolytic Escherichia coli carrying the Cnf-1 gene was identified, from both the puppy organs and bitch's milk. Moreover, phylo-typing assigned them to the phylogroup B2. Two weeks later, fecal samples from the bitch and the survived puppy were collected for a second microbiological analysis, identifying two hemolytic E. coli strains, Cnf positive and Cdt negative and Cnf and Cdt negative, respectively. Some E. coli pathogenic strains may cause enteric or extraintestinal disease. In dogs and cats, strains of extraintestinal pathogenic E. coli (ExPEC) produce specific virulent factors such as hemolysis and cytotoxin necrotizing factors (Cnf). In this episode, we hypothesize that the bitch's milk could be the main source of ExPEC infection causing high puppies mortality. The role of the bitch as a carrier could not be excluded: stressful conditions, such as pregnancy and delivery, would change the host-pathogen dynamics possibly increasing the release of the infectious burden.