Antimicrobial resistance in Escherichia coli isolates from Japanese raccoon dogs (Nyctereutes viverrinus) in Kanagawa Prefecture, Japan: Emergence of extended-spectrum cephalosporin-resistant human-related clones.

Introduction. Wild animals are one of the putative reservoirs of antimicrobial-resistant bacteria, but the significance of raccoon dogs remains to be investigated.Hypothesis. Raccoon dogs can be a reservoir of antimicrobial-resistant bacteria.Aim. This study aimed to explore the prevalence of antimicrobial resistance, mainly extended-spectrum cephalosporins resistance, in Escherichia coli isolates from faeces of 80 Japanese raccoon dogs in Kanagawa Prefecture, Japan.Methodology. All of the 80 faecal samples were streaked onto deoxycholate-hydrogen sulfate-lactose (DHL) and cefotaxime (CTX)-supplemented DHL (DHL-CTX) agars. Susceptibilities to ten antimicrobials were determined using the agar dilution method. Additionally, extended-spectrum ß-lactamases (ESBLs) and AmpC-type ß-lactamases (ABLs) were identified in addition to sequence types (STs), in ESC-resistant isolates by a polymerase chain reaction and sequencing.Results. Out of all the samples, 75 (93.8 %) and 20 (25.0 %) E. coli isolates were isolated by DHL and DHL-CTX agars, respectively. Significantly higher resistance rates to most of the drugs were found in DHL-CTX-derived isolates than DHL-derived isolates (P<0.01). Genetic analysis identified CTX-M-14 (n=6), CTX-M-2 (n=2), CTX-M-1 (n=1) and CTX-M-55 (n=1) as ESBLs, and CMY-2 (n=8) and DHA-1 (n=1) as ABLs in 20 DHL-CTX-derived isolates. Most of the detected STs were related to Japanese humans (i.e. ST10, ST58, ST69, ST131, ST357, ST648 and ST4038). Notably, this is the first report on ST69, ST131, ST155 and ST648, which are well-known international high-risk clones in Japanese raccoon dogs.Conclusion. Our findings underscore the need to understand the significance of raccoon dogs as an antimicrobial-resistant bacteria reservoir using one health approach.

Wildlife Carnivorous Mammals As a Specific Mirror of Environmental Contamination with Multidrug-Resistant Escherichia coli Strains in Poland.

In recent decades, the number of studies on the occurrence of resistant strains in wildlife animals has increased significantly, but data are still fragmentary. The aim of this study was to evaluate drug resistance of Escherichia coli strains isolated from wild carnivorous mammals, common in Poland. Selective media with antimicrobials (tetracycline, kanamycin, chloramphenicol, and cefotaxime) were used for isolation. Of 53 isolates shown to be distinct by the amplification of DNA fragments surrounding rare restriction site-fingerprinting method, 77.8% were multidrug-resistant (multidrug-resistant). All strains were resistant to ampicillin and many of them also exhibited resistance to tetracycline (76.2%), sulfamethoxazole (57.1%), streptomycin and kanamycin (49.2%), chloramphenicol (30.1%), and nalidixic acid (46%). In most cases, the phenotypic resistance profile was confirmed by detection of relevant genes mostly occurring in strains isolated from livestock animals and humans. Extended-spectrum ß-lactamase-producing strains were detected in one mink and three martens. The strains were carriers of blaTEM-1, blaTEM-135, and blaCTX-M-15 genes. Our research confirmed a high carrier rate of MDR E. coli, even more than one MDR strain in a single individual; therefore, wider monitoring in this group of animals should be considered.

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.

Comparative analysis of the gut microbiota of the blue fox (Alopex lagopus) and raccoon dog (Nyctereutes procyonoides).

The goal of this study is to compare the gut microbiota of domestic blue fox (Alopex lagopus) and raccoon dog (Nyctereutes procyonoides) to provide better understanding of their intestinal gut microbiota. We analyzed the structure of fecal microbes in 40 blue foxes and 40 raccoon dogs that were raised under same conditions, using high-throughput Illumina sequencing targeting the V3-V4 region of the 16S rRNA gene. In total, 295,146 sequence reads were obtained. The average number of operational taxonomical units in the two group samples was 194 to 286. Firmicutes (blue fox 73.40%, raccoon dog 46.90%) and Bacteroidetes (blue fox 21.92%, raccoon dog 44.25%) were the most abundant phyla in the gut of blue fox and raccoon dog. At the genus level, Prevotella (blue fox 16.89%, raccoon dog 36.22%), Blautia (blue fox 9.02%, raccoon dog 13.72%), and Peptostreptococcaeae_incertae_sedi (blue fox 22.41%, raccoon dog 2.84%) were commonly presented in the gut of two kinds of animal. Principal coordinates analysis showed that the microbial communities were different between blue fox and raccoon dog. The Firmicutes-to-Bacteroidetes ratio was higher in blue foxes (3:1) than in raccoon dogs (1:1). Moreover, Peptostreptococcaeae_incertae_sedi and Prevotella, were more abundant in the gut of blue fox, whereas the abundance of Prevotella and Blautia were higher in the gut of raccoon dog. In conclusion, the present study revealed the difference of the gut microbial composition between blue fox and raccoon dog under the same diet conditions.

Dietary probiotics have different effects on the composition of fecal microbiota in farmed raccoon dog (Nyctereutes procyonoides) and silver fox (Vulpes vulpes fulva).

BACKGROUND: The abuse of antibiotics in animal husbandry imposes a serious threat to both animal health and the environment. As a replacement for antibiotics, probiotic products have been widely used in livestock farming to promote growth of animals. However, no products specifically developed for farmed raccoon dogs and foxes are commercially available at the moment. This study was conducted to investigate the effects of mixed probiotics on farmed raccoon dogs and foxes. RESULTS: Two feeding trials on farmed raccoon dogs and foxes were performed. A mixed probiotic preparation composed of Bifidobacterium bifidum, Clostridium butyricum, Bacillus subtilis and Bacillus licheniformis was fed to these two canine species in order to assess whether such a mixed probiotics can be an alternative to antibiotics (control group). The body weight of raccoon dogs exhibited an increasing tendency with mixed probiotics administration, while that of foxes did not. The serum antioxidant activity was evaluated, and a significantly increase of total antioxidative capacity (T-AOC) was observed in both species. Illumina MiSeq was used for the sequencing of 16S rRNA genes to compare the composition of fecal microbiota between the control and mixed probiotics groups. Although α-diversity did not change, ß-diversity of the fecal microbiota showed a distinct dissimilarity between the control and probiotics groups of both raccoon dogs and foxes. Dietary mixed probiotics increased the abundance of the genus Bifidobacterium in the fecal samples of raccoon dogs, and the genus Bacillus in the fecal samples of foxes. The different responses of raccoon dogs and foxes to probiotics might be the result of differences in the composition of the native gut microbiota of the two species. CONCLUSIONS: The mixed probiotics preparation composed of Bifidobacterium bifidum, Clostridium butyricum, Bacillus subtilis and Bacillus licheniformis could be an effective feed additive for the improvement of the health of farmed raccoon dogs, but it may not be suitable for foxes.

The occurrence of Anaplasmataceae in European populations of invasive carnivores.

The raccoon (Procyon lotor) and the raccoon dog (Nyctereutes procyonoides) belong to a group of the invasive species. The introduced species as potential reservoirs for vector-borne pathogens have been the subject of recent research, though there are still no data with reference to the European population of the raccoon, and few studies concern only the raccoon dog. This study shows the occurrence of Anaplasmataceae representatives in raccoons and a sympatric population of the raccoon dogs obtained from the area of Poland and Germany. During the study, the occurrence of Anaplasma phagocytophilum ecotype I in the introduced raccoon in northwestern Poland was revealed. Additionally, Candidatus Neoehrlichia sp. (FU98) was identified for the first time in the raccoon dog in Central Europe and thereby the raccoon dog is a new host for this pathogen.

Molecular Detection of Anaplasma, Bartonella, and Borrelia theileri in Raccoon Dogs (Nyctereutes procyonoides) in Korea.

Anaplasmosis, cat-scratch disease, and Lyme disease are emerging vector-borne infectious diseases in Korea. Although the prevalence of vector-borne pathogens (VBPs) in domestic animals and vector arthropods has been documented, there is limited information on the presence of VBPs in wild animals. The raccoon dog (Nyctereutes procyonoides), a wild canid found in East Asia and Europe, represents a potential wildlife reservoir for zoonotic diseases. To investigate the prevalence of VBPs in raccoon dogs, 142 carcasses and 51 blood samples from captured raccoon dogs were collected from 2003 to 2010 and from 2008 to 2009, respectively, in Korea. In addition, 105 Haemaphysalis flava (14 larvae, 43 nymphs, 32 males, and 16 females) and nine Haemaphysalis longicornis (all female) were collected from three raccoon dogs. Samples of the spleen and blood were tested for the presence of VBPs by using nested polymerase chain reaction. Among the samples collected from 193 raccoon dogs and 114 ticks, two samples were positive for Anaplasma phagocytophilum, four for Anaplasma bovis, two for Borrelia theileri, and two for Bartonella henselae. To the best of our knowledge, this study is the largest survey of raccoon dogs aimed at the analysis of VBPs in this species. Moreover, the present study represents the first identification of A. phagocytophilum, B. henselae, and B. theileri in raccoon dogs in their native habitat (East Asia).

Pathological findings in the red fox (Vulpes vulpes), stone marten (Martes foina) and raccoon dog (Nyctereutes procyonoides), with special emphasis on infectious and zoonotic agents in Northern Germany.

Anthropogenic landscape changes contributed to the reduction of availability of habitats to wild animals. Hence, the presence of wild terrestrial carnivores in urban and peri-urban sites has increased considerably over the years implying an increased risk of interspecies spillover of infectious diseases and the transmission of zoonoses. The present study provides a detailed characterisation of the health status of the red fox (Vulpes vulpes), stone marten (Martes foina) and raccoon dog (Nyctereutes procyonoides) in their natural rural and peri-urban habitats in Schleswig-Holstein, Germany between November 2013 and January 2016 with focus on zoonoses and infectious diseases that are potentially threatening to other wildlife or domestic animal species. 79 red foxes, 17 stone martens and 10 raccoon dogs were collected from traps or hunts. In order to detect morphological changes and potential infectious diseases, necropsy and pathohistological work-up was performed. Additionally, in selected animals immunohistochemistry (influenza A virus, parvovirus, feline leukemia virus, Borna disease virus, tick-borne encephalitis, canine adenovirus, Neospora caninum, Toxoplasma gondii and Listeria monocytogenes), next-generation sequencing, polymerase chain reaction (fox circovirus) and serum-neutralisation analysis (canine distemper virus) were performed. Furthermore, all animals were screened for fox rabies virus (immunofluorescence), canine distemper virus (immunohistochemistry) and Aujeszky's disease (virus cultivation). The most important findings included encephalitis (n = 16) and pneumonia (n = 20). None of the investigations revealed a specific cause for the observed morphological alterations except for one animal with an elevated serum titer of 1:160 for canine distemper. Animals displayed macroscopically and/or histopathologically detectable infections with parasites, including Taenia sp., Toxocara sp. and Alaria alata. In summary, wildlife predators carry zoonotic parasitic disease and suffer from inflammatory diseases of yet unknown etiology, possibly bearing infectious potential for other animal species and humans. This study highlights the value of monitoring terrestrial wildlife following the "One Health" notion, to estimate the incidence and the possible spread of zoonotic pathogens and to avoid animal to animal spillover as well as transmission to humans.

Comparison of fecal microbiota of three captive carnivore species inhabiting Korea.

This study aimed at characterizing fecal microbiota of three captive carnivore species of leopard cats Prionailurus bengalensis, Eurasian otters Lutra lutra and raccoon dogs Nyctereutes procyonoides. We used DNA barcoding sequencing to analyze 16S rRNA genes of uncultured bacteria in the feces collected in the Seoul Zoo. The sequencing analyses revealed that: 1) Firmicutes was the most dominant phylum for all three animals; 2) bacterial genus-rank compositions were distinct across species of the animals; and 3) bacterial community memberships were different across species of the studied animals. We expect such baseline information is useful for better understanding of these endangered species and future management of their health in zoos.

Differential associations of Borrelia species with European badgers (Meles meles) and raccoon dogs (Nyctereutes procyonoides) in western Poland.

European badgers and raccoon dogs and their associated ticks and lice were assayed for the presence of Lyme borreliosis and relapsing fever-group spirochete DNA in western Poland. Analyses of blood, ear-biopsy and liver samples revealed that 25% of 28 raccoon dogs and 12% of 34 badgers were PCR positive for borreliae. Borrelia garinii was the dominant species in raccoon dogs (62.5%), followed by B. afzelii (25%) and B. valaisiana (12.5%). PCR-positive badgers were infected only with B. afzelii. A total of 351 attached ticks was recovered from 23 (82%) of the raccoon dogs and 13 (38%) of the badgers. Using a nested PCR targeting the ITS2 fragments of Ixodes DNA, four Ixodes species were identified: I. ricinus, I. canisuga, I. hexagonus, and one provisionally named I. cf. kaiseri. Ixodes canisuga and I. ricinus prevailed on both host species. The highest infection prevalence was detected in I. ricinus, followed by I. canisuga and I. cf. kaiseri. Borrelia garinii and B. afzelii accounted for 61.6% and 30.1% of the infections detected in all PCR-positive ticks, respectively. Four other Borrelia species (B. burgdorferi sensu stricto, B. valaisiana, B. lusitaniae and B. miyamotoi) were detected only in I. ricinus from raccoon dogs. Moreover, Borrelia DNA, mostly B. garinii, was detected in 57 (81.4%) of 70 Trichodectes melis lice derived from 12 badgers. The detection of B. afzelii in one-half of PCR-positive biopsies reconfirms previous associations of this species with mammalian hosts, whereas the high prevalence of B. garinii in feeding lice and I. ricinus ticks (including larvae) demonstrates that both carnivores serve as hosts for B. garinii. The lack of B. garinii DNA in the tissues of badgers versus its prevalence in raccoon-dog biopsies, however, incriminates only the latter carnivore as a potential reservoir host.

Prevalence, risk factors and molecular characterization of Enterocytozoon bieneusi in raccoon dogs (Nyctereutes procyonoides) in five provinces of Northern China.

The disease microsporidiosis is found worldwide and is mainly caused by Enterocytozoon bieneusi. E. bieneusi can infect a wide range of hosts; however, information regarding the prevalence and genotyping of E. bieneusi infection in raccoon dogs (Nyctereutes procyonoides) is limited. Therefore, in 2015, we examined 305 faecel samples from 80 farmed raccoon dogs in Jilin Province, from 54 in Hebei Province, from 72 in Liaoning Province, from 29 in Shandong Province, and from 40 in Heilongjiang Province. The overall prevalence of E. bieneusi infection in farmed raccoon dogs was 22.30%. Logistic regression analysis suggests that age, gender and region of raccoon dogs were highly related to the prevalence of E. bieneusi infection. Moreover, six E. bieneusi internal transcribed spacer (ITS) region sequences, including four known genotypes, namely D, CHN-DC1, NCF2, and CHN-F1, and two novel genotypes (NCR1 and NCR2), were identified in the present study. The present study firstly indicated the existence of E. bieneusi genotypes NCF2, NCR1, NCR2and CHN-F1 in infected raccoon dogs in Northern China. Integrated control strategies should be implemented to limit E. bieneusi infection in farmed raccoon dogs, and to prevent transmission of this disease to other animals and humans.

Genetic variation of mini- and microsatellites and a clonal structure in Enterocytozoon bieneusi population in foxes and raccoon dogs and population differentiation of the parasite between fur animals and humans.

Enterocytozoon bieneusi is an obligate intracellular protozoan parasite that infects a wide range of mammal hosts and birds. Previous genotypic surveys were limited to measure the polymorphisms at the ribosomal internal transcribed spacer (ITS) that evolved slowly. Data on population structure are available only on E. bieneusi isolates from primates. This study explored the genotypic and phylogenetic characteristics of four mini- and microsatellites and performed a population genetic analysis in 39 E. bieneusi isolates of potentially zoonotic ITS genotype D from farmed foxes and raccoon dogs in China. Sequence polymorphisms facilitated determination of six, two, four, and five genotypes at markers MS1, MS3, MS4, and MS7, respectively. Patterns of phylogeny revealed different levels of diversity within and among the genetic markers. Clear genotypic and phylogenetic divergences between E. bieneusi isolates of ITS genotype D from fur animals and humans were observed at individual markers. Complete linkage disequilibrium and very limited recombination in subsequent population genetic analysis supported a clonal structure for E. bieneusi population from fur animals (FID). Phylogenetic analysis, genetic network, and measures of F ST and gene flow demonstrated population differentiation of FID from two known human E. bieneusi populations HID (with a clonal structure) and HIA (with an epidemic structure). The data indicated an ideal resolving power of MLST compared to the previously widely used ITS genotyping and confirmed the clonal nature and population differentiation of E. bieneusi in various hosts.

Genotyping of Enterocytozoon bieneusi in Farmed Blue Foxes (Alopex lagopus) and Raccoon Dogs (Nyctereutes procyonoides) in China.

Enterocytozoon bieneusi is the most common species of microsporidia found both in humans and animals. Farmed animals, particularly closely associated to humans, may play an important role of zoonotic reservoir in transmitting this disease to humans. The fur industry is a major economic component in some parts of China. To understand the prevalence, genotype variety and zoonotic risk of E. bieneusi in farmed foxes and raccoon dogs, two species of fur animals, fecal specimens of 110 blue foxes and 49 raccoon dogs from Heilongjiang and Jilin provinces in China were examined by internal transcribed spacer (ITS)-based PCR. E. bieneusi was detected in 16.4% (18/110) blue foxes and 4.1% (2/49) raccoon dogs. Altogether, four genotypes of E. bieneusi were identified, including two known genotypes D (n = 13) and EbpC (n = 5), and two novel genotypes named as CHN-F1 (n = 1) in a fox and CHN-R1 (n = 1) in a raccoon dog. Phylogenetic analysis revealed that all the four genotypes were the members of zoonotic group 1. Genotypes D and EbpC were found in humans previously. The findings of zoonotic genotypes of E. bieneusi in the foxes and raccoon dogs suggest these animals infected with E. bieneusi may pose a threat to human health.

Widespread presence of human-pathogenic Enterocytozoon bieneusi genotype D in farmed foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) in China: first identification and zoonotic concern.

Enterocytozoon bieneusi is a well-known causative agent of microsporidial infections in a variety of mammal hosts including humans in China, whereas there were no epidemiological data on wild animals bred in captivity, and the role of the neglected hosts in transmission of zoonotic microsporidiasis remains unknown. Herein, we investigated feces from 191 farmed foxes (Vulpes vulpes) and 162 farmed raccoon dogs (Nyctereutes procyonoides) for the prevalence and genotypic characteristics of E. bieneusi in Harbin City, northeast China. Polymerase chain reaction (PCR) targeting the internal transcribed spacer (ITS) of the rRNA gene enabled the identification of 53 (27.7%) and 17 (10.5%) positives from fox and raccoon dog specimens, respectively. There was only minor difference in prevalence between juvenile and adult foxes. Adult raccoon dogs have an infection rate significantly higher than juveniles. The most common human-pathogenic E. bieneusi, genotype D, is widespread among foxes and raccoon dogs of various ages by sequence analysis of the ITS locus. Genotypes CHN-DC1 and mixed CHN-DC1/WildBoar3 were detected in one adult raccoon dog each. Here is the first report describing the presence of zoonotic E. bieneusi genotypes in farmed foxes and raccoon dogs. The widespread existence of genotype D in surveyed animals is of great concern for public health.

Detection of Anaplasma phagocytophilum in red foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) from Brandenburg, Germany.

Anaplasma phagocytophilum is an obligate intracellular and tick-transmitted bacterium, which causes granulocytic anaplasmosis in animals and humans. Although infection with A. phagocytophilum in domestic animals and vector ticks is documented, there is sparse information on the occurrence of A. phagocytophilum in wild animals. Red foxes (Vulpes vulpes) as well as raccoon dogs (Nyctereutes procyonoides) are wildlife species highly abundant in certain areas of Germany and represent a potential wildlife reservoir for zoonotic diseases. To obtain data about the occurrence of A. phagocytophilum in these animals, red fox and raccoon dog carcasses (hunted or found dead) were collected from January to September 2009 in the Federal State of Brandenburg, Germany. Lung tissue samples were subjected to DNA extraction and were examined for the presence of A. phagocytophilum DNA by means of real-time PCR. Anaplasma phagocytophilum was detected in 10 out of 122 (8.2%) lungs of red foxes and in 3 out of 13 (23%) lungs of raccoon dogs. To the best of our knowledge, A. phagocytophilum was detected for the first time in red foxes and raccoon dogs in Germany.

Detection of spotted fever and typhus group rickettsial infection in wild raccoon dogs (Nyctereutes procyonoides koreensis) in Chonbuk Province, Korea.

Both the spotted fever and the typhus group rickettsioses are potential emerging arthropod-borne infectious disease complexes in Korea. Wild raccoon dogs (Nyctereutes procyonoides koreensis) sheltered at the Korean Society for the Protection of Wild Animals, Chonbuk Branch, Korea, were sampled for the presence of spotted fever and typhus group rickettsial pathogens. Thirty-six serum samples of wild raccoon dogs were examined by an indirect fluorescent antibody test using type-specific YH strain Rickettsia japonica (spotted fever group rickettsia) and Wilmington strain Rickettsia typhi (typhus group rickettsia). The prevalence of sera that were seropositive was 30.5% (11/36) for the spotted fever group and 41.6% (15/36) for the typhus group. Five samples were seropositive for both antigens. This study is first to demonstrate rickettsial antibodies in wild raccoon dogs, and it provides seroepidemiologic evidence of the endemic presence of rickettsial agents in Korea.