Morphometrical and molecular identification of Echinococcus granulosus genotypes in wild canids in north of Iran.

BACKGROUND/OBJECTIVE: The cestode Echinococcus granulosus causes cystic echinococcosis, a zoonotic parasitic infection that constitutes a significant public health risk. This parasite has been documented to have potential reservoirs and carriers among wild canids, namely wolves, foxes and jackals. This study aimed to determine the prevalence and molecular characteristics of E. granulosus sensu lato species/genotypes among wild canids in three northern, northeastern and north-western Iran regions. METHODS: From 2019 to 2022, 93 wild canid carcasses (69 jackals), (22 foxes) and (2 wolves) were collected that were killed in car accidents or illnesses. Analyses of morphology and morphometry were performed to verify the presence of E. granulosus. To determine E. granulosus s.l. species/genotypes, polymerase chain reaction (PCR)-RFLP (ITS1) was performed utilizing the Bsh1236I (BstUI) restriction enzyme. COX1, NADH1 and ITS1 gene sequencing were also performed to confirm the PCR-RFLP results. RESULTS: During this study, 93 wild canids were examined, and 3.2% (95% CI: 0%-7%) of the 93 were infected with Echinococcus. The north-western region of Iran showed two out of 30 jackals (6.6%) infected with adult Echinococcus compared to one out of 35 jackals (2.8%) in the northern region. DNA from Echinococcus was detected in these individuals by PCR. Based on PCR-RFLP analysis of the ITS1 gene and sequencing of COX1, NADH1 and ITS1 gene, E. granulosus sensu stricto genotype was confirmed in the jackals that had been infected. CONCLUSION: Evidence shows that E. granulosus occurs in jackals in Iran, with the E. granulosus s.s. genotype being the most common. This parasite has been identified as a zoonotic parasite with a genotype that can be transmitted to livestock and humans. Establishing effective control measures to prevent the spread of echinococcosis and ensure public health is crucial.

Trichinella spp. in wolves (Canis lupus) and red foxes (Vulpes vulpes) of southern Italy.

Trichinella spp. are cosmopolitan parasites that infect a wide range of hosts, with wildlife being the main reservoir of these zoonotic nematodes, especially red foxes (Vulpes vulpes) and wolves (Canis lupus) due to their apex position in the food chain in most European countries. The aim of this study is to investigate the prevalence of Trichinella spp. in these wild canids and their epidemiological role in the Campania region (southern Italy). From 2017 to 2023, the carcasses of red foxes (n = 352) and wolves (n = 41) were collected as part of a health surveillance plan. Muscle samples were analysed individually by artificial digestion and four (1.1%) red foxes and nine (21.9%) wolves tested positive for Trichinella britovi. All Trichinella isolates were identified as T. britovi by multiplex PCR. Statistically significant differences in prevalence were found by province (p-value = 0.05) for red foxes and sampling years (p-value = 0.01) for wolves. The prevalence was lower in red foxes than in wolves, probably due to the longer life expectancy of wolves compared to red foxes and the role of wolves as apex predators compared to red foxes as meso-carnivores. The results obtained confirm the important role that these wild canids play in the circulation of the parasite.

First report of occurrence of Babesia gibsoni in captive Indian wolves.

Blood samples from fifteen captive Indian wolves (Canis lupus pallipes) maintained at Arignar Anna Zoological Park, Vandalur, Chennai were screened for the presence of Babesia spp., Ehrlichia canis and Trypnosoma evansi DNA by PCR. Out of 15 wolf samples, 3 samples were found positive for Babesia spp. The amplified 18S rRNA gene fragments from 3 wolves were sequenced and confirmed as Babesia gibsoni. A maximum likelihood tree was constructed using the three sequences along with other Babesia spp. sequences derived from GenBank adopting HKY nucleotide substitution model based on the Bayesian Information Criterion. The phylogenetic analysis confirmed that the three sequences were of Babesia gibsoni and highly divergent from Babesia canis, B. vogeli and B. vulpes. This might be a possible spill over event of B. gibsoni from community dogs through blood feeding dog ticks. This is the first report and molecular confirmation of B. gibsoni infection in captive Indian wolves.

Echinococcus granulosus Sensu Stricto and Echinococcus multilocularis in a Gray Wolf (Canis lupus) in Turkey: Further Evidence for Increased Risk of Alveolar Echinococcosis in Urban Areas.

OBJECTIVE: The aim of this study was to identify Echinococcus species by morphological and molecular means. METHODS: A dead gray wolf (Canis lupus) was found near Erzurum province and brought to the parasitology laboratory. Sedimentation and counting technique (SCT) and polymerase chain reaction (PCR) analysis were conducted. RESULTS: The SCT implications indicated that the wolf had a substantial worm burden (62,720 and 49,280 parasites) due to a co-infection of E. granulosus s.l. and E. multilocularis. Genus/species-specific PCR was used to analyze DNA extracted from adult worms and confirmed as E. granulosus s.s. and E. multilocularis, utilizing COI and 12S rRNA gene sequence analysis, respectively. CONCLUSION: This report presents the first co-detection of E. granulosus s.s. and E. multilocularis in a gray wolf found in an urban area in a highly endemic area for human echinococcosis in northeastern Turkey. The results emphasize that AE is not only a problem of rural areas, but also occurs in urban areas, which may pose a threat to public health. Therefore, surveillance in urban areas is crucial. The need to develop new control strategies for domestic and wildlife in the study area is also highlighted.

High genetic diversity of Echinococcus canadensis G10 in northeastern Asia: is it the region of origin?

Echinococcus canadensis consists of 4 genotypes: G6, G7, G8 and G10. While the first 2 predominantly infect domestic animals, the latter are sylvatic in nature involving mainly wolves and cervids as hosts and can be found in the northern temperate to Arctic latitudes. This circumstance makes the acquisition of sample material difficult, and little information is known about their genetic structure. The majority of specimens analysed to date have been from the European region, comparatively few from northeast Asia and Alaska. In the current study, Echinococcus spp. from wolves and intermediate hosts from the Republic of Sakha in eastern Russia were examined. Echinococcus canadensis G10 was identified in 15 wolves and 4 cervid intermediate hosts. Complete mitochondrial cytochrome c oxidase subunit 1 (cox1) sequences were obtained from 42 worm and cyst specimens from Sakha and, for comparison, from an additional 13 G10 cysts from Finland. For comparative analyses of the genetic diversity of G10 of European and Asian origin, all available cox1 sequences from GenBank were included, increasing the number of sequences to 99. The diversity found in northeast Asia was by far higher than in Europe, suggesting that the geographic origin of E. canadensis (at least of G10) might be northeast Asia.

Endoparasites of the Iberian wolf (Canis lupus signatus) and mesocarnivores in Central Portugal.

At the end of the nineteenth century, massive population declines were observed in carnivores due to the emergence of infectious diseases. This study aims to investigate, by means of coprological analysis, the prevalence and intensity of the parasites that infect the endangered Iberian wolf Canis lupus signatus and two mesocarnivores (the red fox Vulpes vulpes and the stone marten Martes foina) in Central Portugal. In total, 67.2% of the samples screened were infected; Toxascaris leonina (40.6%) was the parasite with the highest prevalence, followed by Ancylostomatidae and Eimeria spp. (28.1%). Eimeria spp. was found in stone marten with the highest infection rate (37,800 OPG), followed by T. leonina (10,100 EPG) in a red fox sample. Moderate to high levels of parasitic infections were identified in 73.3% of red foxes from the western area. Our results highlight the possibility of cross-infection among these carnivore species and cross-contamination in the wildlife-livestock-human interface.

Evaluating noninvasive methods for estimating cestode prevalence in a wild carnivore population.

Helminth infections are cryptic and can be difficult to study in wildlife species. Helminth research in wildlife hosts has historically required invasive animal handling and necropsy, while results from noninvasive parasite research, like scat analysis, may not be possible at the helminth species or individual host levels. To increase the utility of noninvasive sampling, individual hosts can be identified by applying molecular methods. This allows for longitudinal sampling of known hosts and can be paired with individual-level covariates. Here we evaluate a combination of methods and existing long-term monitoring data to identify patterns of cestode infections in gray wolves in Yellowstone National Park. Our goals were: (1) Identify the species and apparent prevalence of cestodes infecting Yellowstone wolves; (2) Assess the relationships between wolf biological and social characteristics and cestode infections; (3) Examine how wolf samples were affected by environmental conditions with respect to the success of individual genotyping. We collected over 200 wolf scats from 2018-2020 and conducted laboratory analyses including individual wolf genotyping, sex identification, cestode identification, and fecal glucocorticoid measurements. Wolf genotyping success rate was 45%, which was higher in the winter but decreased with higher precipitation and as more time elapsed between scat deposit and collection. One cestode species was detected in 28% of all fecal samples, and 38% of known individuals. The most common infection was Echinococcus granulosus sensu lato (primarily E. canadensis). Adult wolves had 4x greater odds of having a cestode infection than pups, as well as wolves sampled in the winter. Our methods provide an alternative approach to estimate cestode prevalence and to linking parasites to known individuals in a wild host system, but may be most useful when employed in existing study systems and when field collections are designed to minimize the time between fecal deposition and collection.

Dirofilaria immitis antigenemia and microfilaremia in Iberian wolves and red foxes from Portugal.

BACKGROUND: Dirofilaria immitis is a parasitic nematode endemic in the Mediterranean countries, which causes cardiopulmonary dirofilariosis in wild and domestic animals. Despite being recognized hosts of D. immitis, wild carnivores such as wolves and foxes are frequently disregarded when considering a potential role in the transmission of these zoonotic nematodes. In Portugal, studies available regarding D. immitis circulation are scarce, likely underestimating its relevance. To add knowledge on this, we sought to assess Iberian wolves (Canis lupus signatus) and red foxes (Vulpes vulpes) from northern Portugal for D. immitis antigenemia and microfilaremia. METHODS: Blood samples from 42 Iberian wolves and 19 red foxes were collected, during 2010-2012, in Peneda-Gerês National Park. Antigenemia was searched for by rapid antigen detection test kits (Uranotest Dirofilaria ®). Microfilaremia was assessed by polymerase chain reaction (PCR). Nucleic acids were extracted from blood using QIAamp® DNA Mini Kit (Qiagen), and DNA was screened for the presence of microfilaria using a conventional PCR targeting the 5.8S-internal transcribed spacer 2-28S regions, followed by bidirectional sequencing, Basic Local Alignment Search Tool analysis and phylogenetic analysis. RESULTS: Three red foxes had antigenemia, with an occurrence of 15.8% (95% confidence interval [CI] 3.4-39.6), while showing no evidence for the presence of microfilaremia. No wolf samples presented evidence for D. immitis antigenemia. Nevertheless, two wolves were positive for D. immitis microfilaremia (4.8%; 95% CI 0.6-16.2%) as revealed by PCR and confirmed by bidirectional sequencing. CONCLUSIONS: Although Dirofilaria microfilaremia in wolves does not necessarily correlate to an endangerment of the infected animal's health, positive individuals can act as a reservoir for further infection if the intermediate mosquito hosts are present. To the best of our knowledge, one single study had reported that wolves were suitable Dirofilaria hosts, but microfilaremia have never been reported.

Zoonotic Thelazia callipaeda eyeworm in brown bears (Ursus arctos): A new host record in Europe.

Thelazia callipaeda (Spirurida, Thelaziidae) eyeworm, a zoonotic parasite transmitted by lachryphagus drosophilids, causes subclinical to clinical ocular disease in a wide range of vertebrates, including humans. In the past 20 years, the infection spread primarily in areas where the vector thrives, and now is endemic in many European countries. Recently, this nematode has been reported also in the United States. Here, we describe the first record of the brown bear (Ursus arctos) as a host of T. callipaeda. Eight (26.7%) out of 30 bears were found to be infected with T. callipaeda adult worms. In addition, the parasite was detected in 13 wolves (Canis lupus) sharing the same environment with the brown bears. At the molecular characterization, all nematodes belonged to the haplotype 1, which is the only one recorded in Europe, as yet. This finding demonstrates that brown bears may act as hosts for this parasite, suggesting its potential role in the maintenance of the cycle not only in the wild but also in captivity. Under the above circumstances, the brown bear may represent a potential zoonotic risk for humans, both in rural and urban areas where other reservoirs may be infected.

Occurrence and genetic diversity of Cryptosporidium spp. in wild foxes, wolves, jackals, and bears in central Europe.

Parasites of the genus Cryptosporidium Tyzzer, 1910 are one of the most common protistan parasites of vertebrates. Faecal samples from 179 red foxes (Vulpes vulpes [Linnaeus]), 100 grey wolves (Canis lupus Linnaeus), 11 golden jackals (Canis aureus Linnaeus), and 63 brown bears (Ursus arctos Linnaeus) were collected in the Czech Republic, Poland and Slovakia. Samples were examined for the presence of Cryptosporidium spp. using microscopy and PCR/sequence analysis. Phylogenetic analysis based on the small subunit ribosomal RNA (SSU), actin and 60-kDa glycoprotein (gp60) genes using the maximum likelihood method revealed the presence of Cryptosporidium tyzzeri Ren, Zhao, Zhang, Ning, Jian et al., 2012 (n = 1) and C. andersoni Lindsay, Upton, Owens, Morgan, Mead et Blackburn, 2000 (n = 2) in red foxes, C. canis Fayer, Trout, Xiao, Morgan, Lai et Dubey, 2001 (n = 2) and C. ubiquitum Fayer, Santín et Macarisin, 2010 (n = 2) in grey wolves, and C. galli Pavlásek, 1999 in brown bears (n = 1) and red foxes (n = 1). Subtyping of isolates of C. ubiquitum and C. tyzzeri based on sequence analysis of gp60 showed that they belong to the XIId and IXa families, respectively. The presence of specific DNA of C. tyzzeri, C. andersoni and C. galli, which primarily infect the prey of carnivores, is probably the result of their passage through the gastrointestinal tract of the carnivores. Finding C. ubiquitum XIId in wolves may mean broadening the host spectrum of this subtype, but it remains possible this is the result of infected prey passing through the wolf - in this case deer, which is a common host of this parasite. The dog genotype of C. canis was reported for the first time in wolves.

Taeniid cestodes in a wolf pack living in a highly anthropic hilly agro-ecosystem.

The Italian wolf population in human-modified landscapes has increased greatly in the last few decades. Anthropisation increases the risk of transmission of many zoonotic infections and in this context, control of taeniid cestode species needs to be addressed from a One Health perspective. Predator-prey interactions are at the root of taeniid cestode transmission, and the wolf plays a key role in the maintenance and transmission of taeniids. To date, all available data on the taeniids of wolves in Italy refer to populations living in a wild habitat. Between 2018 and 2019, we investigated taeniids in a wolf pack living in a highly anthropic hilly agro-ecosystem. Thirty-eight faecal samples were collected and analysed, 4 of which were also genetically characterised for individual wolves and belonged to three different animals. Samples collected were analysed microscopically and by molecular analysis in order to identify the taeniid species. Taeniid eggs were detected in 34.2% (13/38) of samples. Within samples positive to taeniid eggs only Echinococcus granulosus s.s. and Taenia hydatigena were identified in 26.3% and 10.5% of the samples, respectively. On microscopic examination, Capillaria spp., Ancylostomatidae and Toxocara canis eggs, Crenosoma vulpis larvae, and coccidian oocysts were also found. The combination of low biodiversity of taeniid species with a high occurrence of E. granulosus s.s. recorded in this study could be the consequence of a deeper link occurring between wolves and livestock in human-modified landscapes than in wild settings.

TITLE: Cestodes Taeniidae dans une meute de loups vivant dans un agroécosystème vallonné hautement anthropique. ABSTRACT: La population de loups italiens dans les paysages modifiés par l'homme a considérablement augmenté au cours des dernières décennies. L'anthropisation augmente le risque de transmission de nombreuses infections zoonotiques et, dans ce contexte, le contrôle des espèces de cestodes Taeniidae doit être abordé dans une perspective One Health. Les interactions prédateurs-proies sont à la base de la transmission des Cestodes Taeniidae, et le loup joue un rôle clé dans le maintien et la transmission des Taeniidae. À ce jour, toutes les données disponibles sur les Taeniidae du loup en Italie se réfèrent aux populations vivant dans un habitat sauvage. Entre 2018 et 2019, les Taeniidae d'une meute de loups vivant dans un agro-écosystème vallonné hautement anthropique ont été étudiés. Trente-huit échantillons fécaux ont été collectés et analysés, dont quatre ont également été génétiquement caractérisés pour des loups individuels et appartenaient à trois animaux différents. Les échantillons prélevés ont été analysés au microscope et par analyse moléculaire afin d'identifier les espèces de Taeniidae. Des œufs de Taeniidae ont été détectés dans 34,2 % (13/38) des échantillons. Dans les échantillons positifs aux œufs de Taeniidae, seul Echinococcus granulosus s.s. et Taenia hydatigena ont été identifiés dans 26,3 % et 10,5 % des échantillons, respectivement. Lors de l'examen microscopique, des œufs de Capillaria spp., d'Ancylostomatidae et de Toxocara canis, des larves de Crenosoma vulpis et des oocystes de coccidie ont également été trouvés. La combinaison d'une faible biodiversité d'espèces de Taeniidae avec une forte occurrence d'E. granulosus s.s. rapportées dans cette étude pourrait être la conséquence d'un lien plus profond entre le loup et le bétail dans les paysages humains que dans les paysages sauvages.

Survey of coyotes, red foxes and wolves from Wyoming, USA, for Echinococcus granulosus s. l.

The paraphyletic group Echinococcus granulosus sensu lato is comprised of parasitic tapeworms of wild and domestic canids such as wolves (Canis lupus) and coyotes (Canis latrans), which serve as definitive hosts, and ungulates, which are the intermediate hosts. Members of this tapeworm group are characterized by both cosmopolitan distribution and zoonotic disease potential. This survey (conducted from 2012 through 2017) was designed to provide insight into the prevalence and distribution of this parasite in wild canids in Wyoming. Echinococcus sp. infections were documented in 14 of 22 gray wolves (63.6%), 1 of 182 coyotes (0.55%) and 0 of 5 red foxes (Vulpes fulva). Echinococcus granulosus s. l. was confirmed in 4 of these 14 specimens obtained from wolves with two parasite specimens corresponding morphologically with E. canadensis (G8/G10). These results suggest that wolves serve as the major definitive host of E. granulosus s. l. in Wyoming, while coyotes do not play an equivalent role. Limited sample size precludes evaluation of the importance of the red fox as a favorable definitive host. Whereas this study documents the occurrence of E. granulosus s. l. in Wyoming, the zoonotic disease risk does not appear to be high. Education remains the key to disease prevention, coupled with good hygienic practices by humans and anthelmintic treatment of domestic dogs exhibiting elevated risk of exposure.

Patterns and processes of pathogen exposure in gray wolves across North America.

The presence of many pathogens varies in a predictable manner with latitude, with infections decreasing from the equator towards the poles. We investigated the geographic trends of pathogens infecting a widely distributed carnivore: the gray wolf (Canis lupus). Specifically, we investigated which variables best explain and predict geographic trends in seroprevalence across North American wolf populations and the implications of the underlying mechanisms. We compiled a large serological dataset of nearly 2000 wolves from 17 study areas, spanning 80° longitude and 50° latitude. Generalized linear mixed models were constructed to predict the probability of seropositivity of four important pathogens: canine adenovirus, herpesvirus, parvovirus, and distemper virus-and two parasites: Neospora caninum and Toxoplasma gondii. Canine adenovirus and herpesvirus were the most widely distributed pathogens, whereas N. caninum was relatively uncommon. Canine parvovirus and distemper had high annual variation, with western populations experiencing more frequent outbreaks than eastern populations. Seroprevalence of all infections increased as wolves aged, and denser wolf populations had a greater risk of exposure. Probability of exposure was positively correlated with human density, suggesting that dogs and synanthropic animals may be important pathogen reservoirs. Pathogen exposure did not appear to follow a latitudinal gradient, with the exception of N. caninum. Instead, clustered study areas were more similar: wolves from the Great Lakes region had lower odds of exposure to the viruses, but higher odds of exposure to N. caninum and T. gondii; the opposite was true for wolves from the central Rocky Mountains. Overall, mechanistic predictors were more informative of seroprevalence trends than latitude and longitude. Individual host characteristics as well as inherent features of ecosystems determined pathogen exposure risk on a large scale. This work emphasizes the importance of biogeographic wildlife surveillance, and we expound upon avenues of future research of cross-species transmission, spillover, and spatial variation in pathogen infection.

The first records of Spirometra erinaceieuropaei (Cestoda: Diphyllobothriidae), a causative agent of human sparganosis, in Latvian wildlife.

Diphyllobothriid tapeworms of the genus Spirometra are causative agents of sparganosis, food-borne zoonotic parasitic disease. They have been recorded in broad spectrum of hosts, including humans, in all continents except Antarctica. Spirometra tapeworms have been intensively studied in several Asian countries; however, they have been rather neglected in Europe. The aim of this study was to provide a pilot screening of Spirometra spp. in Latvia, where data on sparganosis are not available. Tapeworms morphologically identified as diphyllobothriid species were isolated from grey wolves Canis lupus and Eurasian lynxes Lynx lynx from Latvia during the hunting periods 2013-2019. The parasites were subjected to molecular genotyping using sequences of the partial large (LSU rDNA; 615 bp) and small (SSU rDNA; 720 bp) subunits of the nuclear ribosomal RNA gene and complete (1566 bp) cytochrome c oxidase subunit I gene of the mitochondrial DNA (cox1 mtDNA). Analyses of both ribosomal subunits of 13 tapeworms revealed no intraspecific variation within the respective rDNA subunits. On the other hand, sequence analysis of mitochondrial cox1 revealed intraspecific polymorphism displayed by 12 cox1 haplotypes. Comparison of the current data with sequences of the corresponding DNA regions deposited in the GenBank revealed 99.3-99.5% (LSU rDNA), 99.2% (SSU rDNA) and 99.6-100% (cox1 mtDNA) identity of studied tapeworms with Spirometra erinaceieuropaei, which provided the first confirmation of this diphyllobothriid tapeworm in Latvia. Since S. erinaceieuropaei is probably prevalent in Latvian wildlife and may also occur in other potential host species, further studies are needed in order to acquire complex data on its geographic distribution and transmission in the natural environment of Latvia, as well as on the spectrum of its intermediate, paratenic, and definitive hosts.

Dirofilaria immitis in wolves recolonizing northern Italy: are wolves competent hosts?

BACKGROUND: Wild carnivores such as the grey wolf (Canis lupus), red fox (Vulpes vulpes) and golden jackal (Canis aureus) are recognized hosts of Dirofilaria immitis. However, few studies have focused on their actual role in the epidemiology of heartworm infection. This study describes the prevalence and distribution of D. immitis in wolves in a heartworm-endemic area in northern Italy where wolves have recently returned after long-time eradication, and investigates the fertility status of the collected adult nematodes. METHODS: In the frame of a long-term wolf monitoring programme in northwestern Italy, 210 wolf carcasses from four provinces were inspected for the presence of filarioid nematodes in the right heart and pulmonary arteries. Female heartworms were measured, and their uterine content analyzed according to a previously described "embryogram" technique. RESULTS: Three wolves, all originating from a single province (Alessandria), were positive for D. immitis (1.42%, 95% CI: 0.48-4.11%, in the whole study area; 13.6%, 95% CI: 4.7-33.3%, limited to the single province from which infected wolves originated). Mean intensity was 5 worms (range: 3-7) and the female worms measured 21-28 cm in length. Six out of 9 female worms harbored uterine microfilariae: 5 were classified as gravid; 1 showed a "discontinuous gradient"; and 3 were non-gravid. CONCLUSIONS: The present data show that heartworm infection is already prevalent in wolves that have recolonized the known heartworm-endemic area. Based on "embryogram" results, wolves were shown suitable heartworm hosts. Interestingly, investigated wolves appeared similarly exposed to heartworm infection as sympatric unprotected dogs (owned dogs that have never received any heartworm prevention treatment) sampled at the beginning of the wolf return process.

The fox tapeworm, Echinococcus multilocularis, in grey wolves and dogs in Slovakia: epidemiology and genetic analysis.

Echinococcus multilocularis, the causative agent of human alveolar echinococcosis, is an important emerging parasite in the northern hemisphere. In epidemiological studies, the highest attention is being paid to foxes as the main reservoir hosts responsible for geographic expansion from multiple focal populations and the invasion of urban habitats, but little information is available on the parasite distribution in other carnivores. Hence, the study was designed to obtain updated information about the occurrence and genetic diversity of E. multilocularis in grey wolves and dogs in Slovakia. Faecal samples of wolves were collected from three locations under a certain level of environmental protection in the central and eastern parts of the country, and the presence of the parasite DNA was detected in 35.7% of 112 samples, with the highest rate (51.2%) recorded in the Poloniny National Park in north-eastern Slovakia. Among 110 faecal dog samples, E. multilocularis was detected in three faeces from segregated Roma settlements in the eastern part of the country, which accounted for an overall positivity of 2.7%. Sequence analysis of two mitochondrial genes, 12S rRNA and NADH dehydrogenase subunit 1, revealed four haplotypes in 13 isolates from wolves and dogs originating from four sites in eastern and central Slovakia, with all samples bearing a European-type pattern of E. multilocularis. The more than one-third positivity rate of E. multilocularis in wolf faecal samples dispersed over a large part of the country has corroborated the extensive circulation of the parasite in wildlife and confirmed the need to improve intervention control strategies.

First report of Thelazia callipaeda in a free-ranging Iberian wolf (Canis lupus signatus) from Spain.

Thelazia callipaeda (Spirurida, Thelaziidae) is a vector-borne zoonotic eyeworm able to infect a broad spectrum of carnivores. Here, we describe the first case of bilateral infection by T. callipaeda in the eyes of an adult female Iberian wolf (Canis lupus signatus) in central Spain. Nematodes collected were morphologically identified (n = 42), and two specimens were molecularly characterized. At the sequence analysis of the partial mitochondrial cytochrome c oxidase subunit 1 gene, T. callipaeda haplotype 1 (the only haplotype circulating in Europe) was detected. The role of the Iberian wolf as a natural reservoir for T. callipaeda in the life cycle of this emerging zoonosis and the implications in conservation are discussed.

Detection of DNA from the zoonotic raccoon roundworm Baylisascaris procyonis in a French wolf.

Baylisascaris procyonis is a zoonotic nematode whose main definitive host is the raccoon, an invasive carnivore in Europe introduced from the United States. B. procyonis causes larva migrans with poor prognosis in humans. This parasite was unexpectedly detected in France for the first time upon molecular screening of wolf faecal samples. Because no patent infection was found, the wolf cannot be considered as a definitive host. This discovery of B. procyonis in France nonetheless raises questions about the parasite status of the expanding raccoon populations in the country, which will be investigated in the future.

Echinococcus multilocularis and Echinococcus granulosus in canines in North-Khorasan Province, northeastern Iran, identified using morphology and genetic characterization of mitochondrial DNA.

BACKGROUND: Canids are definitive hosts of Echinococcus multilocularis and Echinococcus granulosus. This study aimed to survey these two Echinococcus species in canids of North-Khorasan Province, northeastern Iran, using morphological criteria and genetic characterization of mitochondrial DNA. METHODS: The carcasses of 106 canids, namely 61 jackals (Canis aureus), 23 foxes (Vulpes vulpes), 19 dogs (Canis familiaris) and three wolves (Canis lupus) were collected from the study area in 2013-2014 and examined for Echinococcus species. Morphological features were assessed by microscopy of adult worms. For molecular characterization, DNA was extracted, mostly from the adult worms but also from eggs. DNA fragments of the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) mitochondrial genes were amplified and sequenced. Sequences were aligned and compared with reference sequences. Intraspecific and interspecific diversity were calculated and phylogenetic analysis was performed. RESULTS: Overall, 9.4% of the canids (eight jackals and two foxes) were found infected with E. multilocularis by molecular methods, of which seven cases were also confirmed using morphological description of the adult worms. Echinococcus granulosus was found in 6.6% of the canines (four dogs, two jackals and one wolf) as determined by both molecular methods and adult cestode morphology. All E. granulosus isolates were identified as the G1 genotype. Comparative sequence analysis indicated 0-0.7% and 0% intraspecific divergence within E. granulosus isolates and 0% and 0-0.2% within E. multilocularis isolates for cox1 and nad1, respectively. CONCLUSIONS: This study revealed the presence of E. multilocularis and E. granulosus in canids of North-Khorasan Province of Iran. Jackals were found infected with both E. multilocularis and E. granulosus, but infection with the former species was higher.