Prevalence of Pentatrichomonas hominis in foxes and raccoon dogs and changes in the gut microbiota of infected female foxes in the Hebei and Henan Provinces in China.

Pentatrichomonas hominis (P. hominis) is a zoonotic parasite that affects a wide range of hosts, causing gastrointestinal diseases. The present study aimed to evaluate the prevalence of P. hominis among caged foxes and raccoon dogs and the effect of P. hominis on the gut microbiota in female foxes. A total of 893 fresh fecal samples were collected from the Hebei and Henan Provinces in China. P. hominis was screened based on 18S rRNA gene expression via nested PCR. The difference in the gut microbiota between nine P. hominis-positive and nine P. hominis-negative samples was investigated by 16S rRNA gene sequencing. The total prevalence of P. hominis infection in foxes and raccoon dogs was 31.7% (283/893). The prevalence rates of P. hominis infection were 28.2% (88/312) and 33.6% (195/581) in foxes and raccoon dogs, respectively. Phylogenetic analysis revealed that all P. hominis strains detected in foxes and raccoon dogs in the present study were the zoonotic genotype CC1. Moreover, compared with those in the P. hominis-negative group, the diversity of the gut microbiota in the P. hominis-positive group was lower, and the abundance of Firmicutes and the ratio of Firmicutes/Bacteroidetes (F/B) in the P. hominis-positive group were lower than those in the P. hominis-negative group. We speculate that these differences may be due to indigestion and diarrhea in infected female foxes. Overall, the present study evaluated the prevalence of P. hominis in foxes and raccoon dogs in the Henan and Hebei Provinces and revealed that P. hominis infection interrupted the diversity of the gut microbiota in female foxes.

Molecular characterization of oriental eyeworm (Thelazia callipaeda) detected from raccoon (Procyon lotor) and Japanese raccoon dog (Nyctereutes viverrinus) in Kanto region, Japan.

BACKGROUND: The oriental eyeworm Thelazia callipaeda (Spirurida: Thelaziidae) is an emerging parasitic ocular nematode of carnivores and humans. In domestic animals and humans, the infection causes varying degrees of inflammation and lacrimation, and wild carnivores represent an important reservoir. In this study we examined the infection status and molecular characterization of T. callipaeda in two urban carnivores, raccoons Procyon lotor and wild Japanese raccoon dogs Nyctereutes viverrinus, in the Kanto region of Japan. METHODS: From January 2020 to December 2021, 193 carcasses including 178 raccoons and 15 raccoon dogs were examined for the presence of worms in the eye. The worms from infected animals (one worm per host) were morphologically identified as T. callipaeda. Worms (1-5 worms per host) were subjected to genetic analysis using mitochondrial cytochrome c oxidase subunit I gene sequences. RESULTS: The prevalence of T. callipaeda in raccoons and Japanese raccoon dogs was 20.2% (36/178) and 13.3% (2/15), respectively. The cox1 sequences from 56 worms from 38 animals revealed three haplotypes (h9, h10, and h12). Analysis of multiple worms for five raccoons showed co-infection of two different haplotypes (h9 and h10) in a single host. Comparing our data with published sequences, three sequences obtained from raccoons and raccoon dogs shared the same haplotypes as those reported in humans, dogs, and cats in Japan. CONCLUSIONS: Our findings show a high prevalence of T. callipaeda in raccoons, suggesting that this invasive carnivore species serves as an important natural reservoir of T. callipaeda in the Kanto region of Japan, an area with the highest human population of the country.

First detection of Trichinella spiralis in raccoon (Procyon lotor) in Germany.

Trichinella spp. are foodborne parasites that can cause severe and potentially fatal disease in humans. Infections occur through consumption of meat containing the infectious stage (L1). In Germany the domestic cycle has been eradicated. In wild animals sporadic occurrence is observed in species such as wild boar, red foxes and raccoon dogs. The omnivore raccoon which is an invasive species in Europe is known as a potential host but has not been studied intensely regarding this parasite in Germany until now, thus resulting in a lack of knowledge about its role in the sylvatic cycle. Raccoons from the urban area of Leipzig were investigated for several pathogens including Trichinella spp. in a cooperative project. Muscle samples of 88 individuals were examined using the artificial digestion method (ADM). One animal was found positive, which is the first detection of this parasite in a raccoon in Germany.

Divergent Cryptosporidium species and host-adapted Cryptosporidium canis subtypes in farmed minks, raccoon dogs and foxes in Shandong, China.

Cryptosporidium spp. are common parasitic pathogens causing diarrhea in humans and various animals. Fur animals are widely farmed in Shandong Province, China, but the prevalence and genetic identity of Cryptosporidium spp. in them are unclear. In this study, 1,211 fecal samples were collected from 602 minks, 310 raccoon dogs and 299 foxes on two farms in Shandong and analyzed for Cryptosporidium spp. by nested PCR and sequence analyses of the small subunit rRNA gene. The overall infection rate of Cryptosporidium spp. was 31.5% (381/1,211), with a higher infection rate in raccoon dogs (37.7%, 117/310) than in foxes (32.4%, 97/299) and minks (27.7%, 167/602). By age, the highest infection rates of Cryptosporidium spp. were observed in raccoon dogs of 1-2 months, minks of 5-6 months, and foxes of > 12 months. Three Cryptosporidium species and genotypes were detected, including C. canis (n = 279), C. meleagridis (n = 65) and Cryptosporidium mink genotype (n = 37). Among the three major host species, raccoon dogs were infected with C. canis only (n = 117), while foxes were infected with both C. canis (n = 32) and C. meleagridis (n = 65), and minks with C. canis (n = 130) and Cryptosporidium mink genotype (n = 37). Subtyping of C. canis by sequence analysis of the 60 kDa glycoprotein gene identified eight subtypes. They belonged to two known subtype families, XXa and XXd, and two novel subtype families XXf and XXg, with host adaptation at the subtype family level. Notably, C. canis from foxes was genetically distant from those in other hosts. Further subtyping analysis identified three subtypes (IIIeA21G2R1, IIIeA19G2R1 and IIIeA17G2R1) of C. meleagridis and two novel subtype families Xf and Xg of the Cryptosporidium mink genotype. The presence of zoonotic C. canis subtypes in raccoon dogs and C. meleagridis subtypes in foxes suggests that these fur animals might be potential reservoirs for human-pathogenic Cryptosporidium spp.

Development of a rapid scabies immunodiagnostic assay based on transcriptomic analysis of Sarcoptes scabiei var. nyctereutis.

Scabies is a highly contagious skin disease caused by the mite Sarcoptes scabiei that affects many mammals. However, the sensitivity of traditional tests for scabies diagnosis in humans is less than 50%. To simplify the diagnosis of scabies, methods that are simple, sensitive, specific, and cost-effective are required. We developed an immunodiagnostic test based on S. scabiei var. nyctereutis RNA-seq data collected from Japanese raccoon dogs with sarcoptic mange. Three candidate antigens-a highly expressed hypothetical protein "QR98_0091190," another mite allergen known as "SMIPP-Cc," and an abundant "vitellogenin-like protein"-were evaluated by western-blot analysis. A lateral flow immunoassay, using specific antibodies against the vitellogenin-like protein, successfully detected scabies in the skin flakes of S. scabiei-infected raccoon dogs. This assay can potentially diagnose scabies more accurately in wildlife, as well as in humans.

Molecular characterization of Cryptosporidium spp. in minks (Neovison vison), blue foxes (Vulpes lagopus), and raccoon dogs (Nyctereutes procyonoides) in farms from Xinjiang, Northwest China.

The objective of this study was to determine the infection rate and genetic diversity of Cryptosporidium spp. in minks, foxes, and raccoon dogs, farmed in the Xinjiang Uygur Autonomous Region, Northwest China. Fresh fecal specimens were collected from individual cages of farmed minks (n = 214), blue foxes (n = 35), and raccoon dogs (n = 39) and examined using nested PCR based on the Cryptosporidium spp. small subunit rRNA gene. Cryptosporidium spp. was detected in 35 cages (12.2%, 35/288), with a higher infection rate detected in raccoon dogs (20.5%) compared with minks (12.1%) and blue foxes (2.9%). Sequence analysis showed that Cryptosporidium canis was the only species identified in blue foxes and raccoon dogs, while in the 26 Cryptosporidium-positive mink specimens, Cryptosporidium mink genotype (n = 17), C. canis (n = 7), and Cryptosporidium parvum (n = 2) were identified. Further analysis based on the 60-kDa glycoprotein (gp60) gene determined that both C. parvum isolates belonged to the subtype IIdA15G1, while eight of the 17 Cryptosporidium mink genotype isolates were a novel subtype that we have named XeA5G1. To the best of our knowledge, this is the first report of C. parvum subtype IIdA15G1 infection in minks. Since all the Cryptosporidium species/genotypes identified in minks, foxes, and raccoon dogs from Xinjiang have been previously found in humans, our results suggest that these fur animals may play a role in the transmission of zoonotic Cryptosporidium.

Molecular identification of Sarcocystis lutrae (Apicomplexa: Sarcocystidae) from the raccoon dog, Nyctereutes procyonoides, and the common raccoon, Procyon lotor, in the Czech Republic.

BACKGROUND: Apicomplexan parasites of the genus Sarcocystis have an obligate two-host life-cycle and comprise about 200 species, which infect different cold- and warm-blooded hosts, including humans. Recently, morphological and molecular studies of sarcocysts in broadly spread carnivore hosts have been on the rise. The description of muscular tissues infection by Sarcocystis in the raccoon dog and the common raccoon from the Czech Republic is herein presented. METHODS: During January-August 2019, 15 raccoon dogs and 1 common raccoon were examined from 5 districts (Ceská Lípa, Liberec, Mladá Boleslav, Trutnov and Ústí nad Labem) of the Czech Republic. Muscle parts (diaphragm, forearm, hind-limb, tongue and heart) were examined in wet preparations under compression by light microscopy. After finding Sarcocystis sp., morphological characteristics and molecular analyses of 18S rRNA, 28S rRNA, ITS1 and cox1 loci were used to identify the species. RESULTS: Sarcocysts were detected and identified in 1 out of 15 raccoon dogs and in the single common raccoon. Preferential infection sites were diaphragm and tongue, followed by forearm and hind limb. To our knowledge, this is the first identification of microscopic sarcocysts by multi-locus genetic analysis from both host species. Molecular analyses revealed 100% similarity at 18S rRNA, 28S rRNA, and cox1 genes with S. lutrae for both hosts and 98-100% identity at the ITS1 region of the isolate from the common raccoon. CONCLUSIONS: Both widely distributed non-indigenous wild carnivores represent new intermediate host records for S. lutrae and the first report of this parasite in a member of the family Procyonidae, but still with an unknown natural definitive host. Molecular data revealed that this parasite species appears more closely related to the Sarcocystis spp. using raptorial birds as definitive hosts. Therefore, further studies aimed at its identification, including the complete life-cycle, remain necessary.

Prevalence and genotype distribution of Enterocytozoon bieneusi in farmed raccoon dogs (Nyctereutes procyonoides) in Shandong Province, eastern China.

Enterocytozoon bieneusi is a common microsporidian species, which can infect humans and various species of animals. However, little is known about E. bieneusi prevalence and genotypes in farmed raccoon dogs (Nyctereutes procyonoides) in Shandong Province, China. In this study, a total of 356 fecal samples were collected from farmed raccoon dogs in Weihai, Weifang, and Yantai cities in Shandong Province, China. A total of 23 (6.5%) samples were E. bieneusi-positive by nested PCR amplification of the internal transcribed spacer (ITS) region of ribosomal DNA. Statistical analysis showed that E. bieneusi prevalence in male raccoon dogs was higher than that in female raccoon dogs, and the highest E. bieneusi prevalence was detected in adult raccoon dogs. Sequence analysis revealed four known E. bieneusi genotypes (D, type IV, CHG1, and Peru8), and type IV (11/23) was the predominant genotype. The genotypes type IV, Peru8, and CHG1 were reported in raccoon dogs for the first time in China. Phylogenetic analysis showed that three human-pathogenic genotypes (D, type IV, and Peru8) were clustered into group 1, and the CHG1 belonged to group 2. These findings expand the current understanding of E. bieneusi prevalence and genotype distribution in raccoon dogs in China. Our study also shows that raccoon dogs are hosts for E. bieneusi belonging to several genotypes, including zoonotic ones, highlighting the possibility of transmission of this pathogen between raccoon dogs and humans.

MOLECULAR SURVEY OF BARTONELLA ROCHALIMAE IN JAPANESE RACCOON DOGS (NYCTEREUTES PROCYONOIDES VIVERRINUS).

Wild carnivores serve as reservoirs of several zoonotic Bartonella species such as Bartonella henselae, Bartonella vinsonii subsp. berkhoffii, and Bartonella rochalimae. The raccoon dog (Nyctereutes procyonoides viverrinus) is the most common native carnivore in Japan, but epidemiologic studies of Bartonella infections have not been performed in this animal species yet. Here, we report a molecular survey of B. rochalimae prevalence in 619 wild raccoon dogs captured from 2009 to 2017 in western Japan. Bartonella rochalimae DNA was detected in 7.1% (44/619) of the raccoon dogs examined by PCR targeting the rpoB and ssrA genes. All of the sequences obtained were identical in each of the genes. The prevalence of B. rochalimae by sex of the animals was 6.1% (21/344) in male and 8.4% (23/275) in female. The prevalence by year varied from 2% (1/45) in 2011 to 14% (4/28) in 2016. The prevalence (7.9%) of B. rochalimae in the raccoon dogs with sarcoptic mange tended to be higher than the prevalence (4.0%) in the animals without the infestation of mites, although the differences were not significant. Sequence analysis indicated that Japanese raccoon dogs in the area examined were infected with B. rochalimae carrying identical sequences in the rpoB and ssrA genes. In addition, the raccoon dog strain had few sequence variations in both genes compared to other known B. rochalimae strains detected in other parts of the world.

Prevalence and Genotyping of Toxoplasma gondii Infection in Raccoon Dogs (Nyctereutes procyonoides) in Northern China.

Toxoplasma gondii is one of protozoan parasites resulting in zoonosis, which can infect nearly all of warm-blooded hosts, including humans and raccoon dogs (Nyctereutes procyonoides). However, related reports on prevalence and genetic characterization of T. gondii strains in raccoon dogs were few in China. The aim of this study was to survey the prevalence and genetic characterization of T. gondii strains in domestic raccoon dogs from Jilin, Liaoning, and Hebei provinces, northern China. During April 2016 to November 2017, a total of 337 tissue samples collected from domestic raccoon dogs were detected with B1 gene using a nested PCR. And the positive samples were genotyped at 11 genetic markers (SAG1, 5'-and 3'-SAG2, alternative SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2, and Apico) using multilocus PCR-restriction fragment length polymorphism technology. Sixteen out of 337 sika deer (4.75%) were positive with B1 gene by nest PCR. Furthermore, four positive DNA samples were completely typed through further being genotyped, in which three samples were identified as ToxoDB Genotype #9, and one sample was confirmed as ToxoDB Genotype #10. The results of molecular detection not only revealed the existence of T. gondii in domestic raccoon dogs in Jilin, Liaoning, and Hebei for the first time, but also provided the information of genetic diversity. This study also indicated that ToxoDB Genotype #9 as a kind of potential reservoir for T. gondii transmission, may be main genotype in domestic raccoon dogs in China, posing a risk of infection in human health.

Toxoplasma gondii infection in selected species of free-living animals in Poland.

INTRODUCTION AND OBJECTIVE: Free-living animals can play an important role as a reservoir of Toxoplasma gondi;, however, data concerning this issue in Poland are still limited.The aim of study was to assess the occurrence of T. gondii infection by using molecular methods in free-living animals in selected regions of Poland. MATERIAL AND METHODS: Tissues samples of 396 animals (foxes, muskrats, birds, martens, badgers, polecats, raccoons, minks, raccoon dogs, otters, small rodents and insectivores, and grass snakes were collected from various regions of Poland. After samples digestion, DNA was isolated using QIAmp DNA Mini Kit (Qiagen). DNA extraction from small rodents and insectivores samples was performed without digestion. Next, nested PCR (B1 gene) and, for a part of nested PCR positive amplicons, RFLP PCR, were performed according to the method by Grigg and Boothroyd (2001). The other part of nested PCR positive DNA isolates were genotyped using 5 genetic markers: SAG1, SAG2 (5'- and 3'), SAG3, BTUB and GRA6, based on the method by Dubey et al. (2006). These PCR products were sequenced and compared with the NCBI database using Blast. RESULTS: In total, in 50 of the 396 examined animals DNA of T. gondii was detected (12.6%). The highest percentages of positive results in PCR was obtained in martens (40.9%) and badgers (38.5%), lower in birds (27.3%) and the lowest in foxes (7.4%). The RFLP and multilocus PCR analysis showed the dominance of T. gondii clonal type II (or II/III). CONCLUSIONS: The results of this study indicate the frequent T. gondii infection among free-living animals in Poland, especially martens and badgers, which may indirectly indicate that these animals contribute to the spread of the parasite in the sylvatic environment in Poland. The genotyping analysis showed the dominance of T. gondii clonal type II (or II/III).

Clinical efficacy of orally administered fluralaner for treatment of scabies in six free-ranging raccoon dogs (Nyctereutes procyonoides).

BACKGROUND: Sarcoptic mange in free-ranging raccoon dogs (Nyctereutes procyonoides) caused by Sarcoptes scabiei is a widespread zoonotic disease that causes severe skin lesions with significant morbidity and mortality. Fluralaner is a member of the isoxazoline chemical class and is an acaricide and insecticide widely used in flea, tick and mite infections of dogs (Canis lupus familiaris). OBJECTIVE: To evaluate the clinical efficacy of orally administered fluralaner in free-ranging raccoon dogs naturally infected with sarcoptic mange. ANIMALS: Six raccoon dogs rescued at the Seoul Wildlife Center between November 2017 and April 2018. METHODS AND MATERIALS: Raccoon dogs were treated with a single dose of a chewable fluralaner tablet. Clinical lesion scoring and superficial skin scraping were performed weekly for three weeks to assess treatment efficacy; the general health was assessed daily to monitor response and observe any adverse drug reactions. RESULTS: Within seven days of treatment, a marked reduction in skin lesions was observed and mites were no longer present in skin scrapings. There was no evidence of re-infestation and no additional drug administration was required. CONCLUSIONS AND CLINICAL IMPORTANCE: Although this was a nonrandomized, uncontrolled study of a small number of animals, it demonstrated that fluralaner may be suitable for treating sarcoptic mange in raccoon dogs.

Seroprevalence and risk factors of Toxoplasma gondii infection in domestic raccoon dogs in four provinces in northern China.

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that can infect almost all homoiothermal animals, including domestic raccoon dogs (Nyctereutes procyonoides). However, related reports on T. gondii infection in domestic raccoon dogs were limited in China. Therefore, a serological investigation was undertaken to investigate the seroprevalence and risk factors for T. gondii infection in domestic raccoon dogs. A total of 962 serum samples were collected from Jilin, Liaoning, Heilongjiang and Hebei provinces, northern China between April 2016 and November 2017, and were detected by the indirect hemagglutination test (IHA). The seroprevalence of T. gondii infection was 7.28% in the overall surveyed raccoon dogs by IHA, which was different among the four provinces ranging from 6.54% to 7.57%. The seroprevalence of T. gondii infection in male and female raccoon dogs was 6.62% and 7.79%, respectively. Based on statistical analysis, age was regarded as an important risk factor for T. gondii infection in raccoon dogs in this study (P < 0.05). This study reported the seroprevalence and risk factors of T. gondii infection in domestic raccoon dogs in northern China, which provided essential data for prevention and control of T. gondii infection in raccoon dogs in Jilin province, Liaoning province, Heilongjiang province and Hebei province.

The complete mitochondrial genome of Sarcoptes scabiei var. nyctereutis from the Japanese raccoon dog: Prediction and detection of two transfer RNAs (tRNA-A and tRNA-Y).

Sarcoptes scabiei (Acari: Sarcoptidae) causes a common contagious skin disease that affects many mammals. Here, the complete mitochondrial genome of a mite, S. scabiei var. nyctereutis, from Japanese wild raccoon dogs was analyzed. The 13,837bp circular genome contained 13 protein-coding genes, two rRNA genes, and 22 tRNA genes. For the first time, two tRNAs (alanine and tyrosine), that were thought to be absent in scabies mites from other animals, were predicted to have short, non-cloverleaf structures by in silico annotation and detected by RT-PCR, sequencing, and northern analysis. The mitochondrial genome structure of S. scabiei is similar to that of Psoroptes cuniculi and Dermatophagoides farinae. While small and unusual tRNA genes seem to be common among acariform mites, further experimental evidence for their presence is needed. Furthermore, through an analysis of the cox1 gene, we have provided new evidence to confirm the transmission of this mite between different animal hosts.

First report of Neospora caninum seroprevalence in farmed raccoon dogs in China.

Neospora caninum is an intracellular protozoan parasite with a worldwide distribution, which can cause abortion and stillbirth in ruminants. However, little is known of N. caninum infection in raccoon dogs in China. Thus, a total of 1181 serum samples of farmed raccoon dogs were collected from the major production areas of Jilin, Changchun in Jilin province, and Rizhao, Weihai and Yantai in Shandong province in China. The antibodies to N. caninum were examined by a competitive-inhibition enzyme-linked immunoassay (cELISA). While the N. caninum seroprevalence in farmed raccoon dogs from different regions (cities) were not statistically significant, farmed raccoon dogs in Shandong province had a significantly higher N. caninum seroprevalence (8.99%, 95% CI 7.13-10.85) than those in Jilin province (4.46%, 95% CI 1.99-6.93) (P < 0.05). N. caninum seroprevalence in male and female raccoon dogs were 7.91% (95% CI 5.99-9.83) and 8.06% (95% CI 5.46-10.66), respectively. In addition, the seroprevalence of N. caninum infection in infancy, youth and adult raccoon dogs was 7.32% (95% CI 4.31-10.33), 7.93% (95% CI 5.44-10.42) and 8.41% (95% CI 5.82-11.00), respectively. This is the first report of N. caninum seroprevalence in farmed raccoon dogs in China, which provided baseline data for the prevention of N. caninum infection in raccoon dogs in China.

The host age related occurrence of Alaria alata in wild canids in Latvia.

The trematode Alaria alata has a complex life cycle in which carnivore mammals may become infected by feeding on mesocercariae-infected, second intermediate hosts or paratenic hosts. Afterwards, young flukes migrate through various organs of the definitive host, including the diaphragm and the lungs, before reaching the small intestine. The aim of the present study was to establish the prevalence and infection intensity of A. alata in carnivore mammals, the host age and related occurrence of different A. alata development stages in particular animals in Latvia. Overall, samples from 992 animals (539 red foxes, 411 raccoon dogs and 42 grey wolves) were examined using sedimentation and counting techniques to observe the presence of A. alata metacercariae and adult trematodes in each particular host. The present study, based on data collected over five hunting seasons (2010/2011-2014/2015), is a comprehensive study on the circulation of A. alata in wild canids in Latvia. A. alata was present in all species examined and the overall prevalence reached 86.2%. A significant positive correlation was observed between the burden of A. alata metacercariae and adult trematodes in raccoon dogs (rs = 0.27, p = 0.001) and red foxes (rs = 0.17, p = 0.0002). A significantly higher (p = 0.003) A. alata metacercariae mean intensity was observed in juvenile raccoon dogs compared to the one observed in adult raccoon dogs and red foxes from both age groups. The presence of both A. alata development stages in a particular animal concurrently and at different intensity levels may indicate primary infection and re-cured infection of the host.

Echinococcus multilocularis in Denmark 2012-2015: high local prevalence in red foxes.

In Western Europe, the Echinococcus multilocularis lifecycle is predominantly sylvatic, typically involving red foxes (Vulpes vulpes) as the main definitive hosts with Microtus spp. and Arvicola spp. as intermediate hosts. During a 4-year surveillance study (2012-2015), Danish red foxes and raccoon dogs (n = 1345) were examined for E. multilocularis. Moreover, 134 insectivores and rodents collected in South Jutland during spring and summer 2016 were examined for the presence of metacestodes. The sedimentation and counting technique and molecular typing were used to identify E. multilocularis infections in the carnivores, while the rodent livers were examined macro- and microscopically for parasite lesions. Following morphological identification of E. multilocularis adult worms, the identity was verified by sequence analysis of the 12S rRNA gene in most cases (n = 13). Echinococcus multilocularis infection was demonstrated in 19 red foxes (Vulpes vulpes) originating from only two specific areas of South Jutland, namely Højer and Grindsted, and in two raccoon dogs (Nyctereutes procyonoides), originating from Højer. In Højer, 28.5% (CI 95% 11.7-45.3) of the examined red foxes were E. multilocularis positive per year. Moreover, positive red foxes were identified each year from 2012 to 2015, while E. multilocularis positive red foxes were only identified in Grindsted in 2013 (4.0%) and 2014 (6.4%). In contrast, all collected rodents were negative for E. multilocularis. We conclude that E. multilocularis is locally endemic in South Jutland with a high local prevalence in Højer.

Identification and genetic characterization of Sarcocystis arctica and Sarcocystis lutrae in red foxes (Vulpes vulpes) from Baltic States and Spain.

BACKGROUND: Typically, carnivores serve as definitive hosts for Sarcocystis spp. parasites; currently, their role as intermediate hosts is being elucidated. The present study aimed to identify and molecularly characterize Sarcocystis cysts detected in striated muscle of red foxes from different populations in Latvia, Lithuania and Spain. METHODS: Muscle samples from 411 red foxes (Vulpes vulpes) and 269 racoon dogs (Nyctereutes procyonoides) from Latvia, 41 red foxes from Lithuania and 22 red foxes from Spain were examined for the presence of Sarcocystis sarcocysts by light microscopy (LM). Sarcocystis spp. were identified by transmission electron microscopy (TEM) and molecular biology techniques. RESULTS: Sarcocystis cysts were detected in 11/411 (2.7%) Latvian, 3/41 (7.3%) Lithuanian, and 6/22 (27.3%) Spanish red foxes, however, cysts were not observed in the muscles of racoon dogs. Based on LM, TEM, 18S rDNA, 28S rDNA, ITS1, cox1 and rpoB sequences, Sarcocystis arctica and Sarcocystis lutrae cysts were identified in red fox muscles from Latvia and Lithuania, whereas only S. arctica was detected in Spain. The 18S rDNA, 28S rDNA and ITS1 sequences from the 21 isolates of S. arctica from Latvia, Lithuania and Spain were identical. By contrast, two and four haplotypes were determined based on mtDNA cox1 and apicoplast rpoB sequences, respectively. Polymorphisms were not detected between the two isolates of S. lutrae from Latvia and Lithuania. Based on phylogenetic results, S. arctica and S. lutrae were most closely related to Sarcocystis spp. using predatory mammals as intermediate hosts and to Sarcocystis species with a bird-bird life-cycle. CONCLUSIONS: Based on current knowledge, the red fox and Arctic fox (Vulpes lagopus) could act as intermediate host for the same two Sarcocystis species. Molecular results suggest the existence of two genetic lineages of S. arctica, and such divergence relies on its geographical distribution but not on their intermediate host species.

Molecular detection and genetic characterizations of Cryptosporidium spp. in farmed foxes, minks, and raccoon dogs in northeastern China.

Cryptosporidium spp. are common intestinal protozoa causing diarrhea in humans and a variety of animal species. With the recent development of fur industry, a large number of fur animals are farmed worldwide, especially in China. The existence of identical Cryptosporidium species/genotypes in humans and fur animals suggests zoonotic potential. In order to assess the presence of zoonotic Cryptosporidium species and/or genotypes in farmed fur animals, 367 fecal specimens were collected from 213 foxes, 114 minks and 40 raccoon dogs farmed in Heilongjiang, Jilin, and Liaoning provinces, northeastern China, during the period from June 2014 to October 2016. By PCR and sequencing of the partial small subunit (SSU) rRNA gene of Cryptosporidium, 20 of 367 (5.4%) animal samples were found to be infected, corresponding to 12 of 213 fox samples (5.6%) and 8 of 114 mink samples (7.0%) screened. Three Cryptosporidium species/genotypes were identified: C. canis (n = 17), C. meleagridis (n = 1) and Cryptosporidium mink genotype (n = 2). Two host-adapted C. canis types (C. canis dog genotype and C. canis fox genotype) were found. By PCR and sequencing of the partial 60 kDa glycoprotein (gp60) encoding gene, one mink genotype isolate was successfully subtyped as XcA5G1R1. The three Cryptosporidium species/genotypes identified in this study have been previously reported in humans suggesting that fur animals infected with Cryptosporidium spp. may pose a risk of zoonotic transmission of cryptosporidiosis, especially for the people working in fur animal farming and processing industry.

Trichinella spp. biomass has increased in raccoon dogs (Nyctereutes procyonoides) and red foxes (Vulpes vulpes) in Estonia.

BACKGROUND: Raccoon dogs and red foxes are well-adapted hosts for Trichinella spp. The aims of this study were to estimate Trichinella infection prevalence and biomass and to investigate which Trichinella species circulated in these indicator hosts in Estonia. METHODS: From material collected for evaluating the effectiveness of oral vaccination program for rabies eradication in wildlife, samples from 113 raccoon dogs and 87 red foxes were included in this study. From each animal, 20 g of masseter muscle tissue was tested for the presence of Trichinella larvae using an artificial digestion method. The Trichinella larvae were identified to species level by multiplex polymerase chain reaction method. RESULTS: The majority of tested animals were infected with Trichinella spp. The parasite species identified were T. nativa and T. britovi. The apparent infection prevalence was 57.5% in raccoon dogs and 69.0% in red foxes, which were higher than previous estimates. In addition, the larval burden had also increased in both hosts. We estimated that in 2011-2012, the Trichinella spp. biomass was more than 15 times higher in raccoon dogs and almost two times higher in red foxes than in 1992-2000 (based on mean larval burden), and almost 20 times higher in raccoon dogs and almost five times higher in red foxes than in 2000-2002 (based on median larval burden). CONCLUSIONS: Raccoon dogs and red foxes are relevant reservoirs for Trichinella spp. in Estonia. The biomass of Trichinella circulating in sylvatic cycles was substantial and had increased: there is substantial infection pressure in the sylvatic cycle.