Isolation and genetic characterization of Staphylococcus aureus from wild animal feces and game meats.

The populations of Japanese deer and boar have increased dramatically and have a serious impact on farming and mountain villages. Although the Japanese government promotes the use of captured wild animals, game meat is not subject to sanitary control considering that it is not subject to meat inspection or quality control. Here, we have attempted to isolate Staphylococcus aureus, a typical foodborne pathogen, as a part of an investigation of contamination in the meats of wild animals and their processing stages. We examined 390 samples of deer feces, 117 samples of wild boar feces, and 75 samples of disemboweled deer meat for isolation of S. aureus; ultimately, 30 (positive rate: 7.7%), 2 (1.7%), and 21 (28.0%) strains were isolated, respectively, from the samples. The genome sequences of these isolates were analyzed and were subjected to multilocus sequence typing. We identified 12 new sequence types (STs) and a dominant population of S. aureus with a characteristic genetic background in wild animals, namely, the ST groups derived from CC121 (number of strains = 39). These strains did not harbor the enterotoxin gene or only harbored egc-related enterotoxin, which is of low involvement in Staphylococcal food poisoning. However, one ST2449 strain, which produces causative enterotoxins, was isolated from a deer's feces. Since there are several common STs isolated from feces and dismembered meat and because fecal contamination during dismemberment is suspected, continuous monitoring and guidance for improving sanitary management conditions during processing and handling of the meat are highly warranted with immediate effect.

Mitogenomic exploration supports the historical hypothesis of anthropogenic diffusion of a zoonotic parasite Echinococcus multilocularis.

Animal movement across regions owing to human activity can lead to the introduction of pathogens, resulting in disease epidemics with medical and socioeconomic significance. Here, we validated the hypothesis that human activity, such as the transportation of infected animals, has played a significant role in introducing the zoonotic parasite Echinococcus multilocularis into Hokkaido, Japan, by synthesizing and evaluating parasite genetic data in light of historical records. Our analysis indicates that a major genetic group in Hokkaido originated from St. Lawrence Island, USA, which is in accordance with the route suggested by historical descriptions. Moreover, we identified a minor genetic group closely related to parasites found in Sichuan, China. This fact implies that parasite invasion in Japan may result from complex and inadvertent animal translocations. These findings emphasize the anthropogenic impacts on zoonotic parasite spread and provide a crucial perspective for preventing future potential epidemics.

Seroprevalence of severe fever with thrombocytopenia syndrome virus in medium-sized wild mammals in Miyazaki, Japan.

Severe fever with thrombocytopenia syndrome (SFTS) is a fatal emerging tick-borne zoonotic disease caused by the SFTS virus (SFTSV). SFTSV infection in humans and companion animals is a matter of concern in endemic areas. Various wild animals are involved in the transmission cycle of SFTSV with vector ticks. Because the home range of medium-sized wild mammals commonly overlaps with humans' living spheres, this study aimed to reveal the endemicity of SFTSV in such mammals. This study investigated the prevalence of antibodies against SFTSV and viral RNA in medium-sized wild mammals in Miyazaki Prefecture, Japan where human cases have been most frequently reported in Japan and performed a phylogenetic analysis to compare the detected SFTSV with those previously reported. Forty-three of 63 (68%) Japanese badgers (Meles anakuma) and 12 of 53 (23%) Japanese raccoon dogs (Nyctereutes procyonoides viverrinus) had antibodies against SFTSV. Japanese marten (n = 1), weasels (n = 4), and Japanese red fox (n = 1) were negative. Two of 63 (3%) badgers tested positive for SFTSV RNA, whereas the other species were negative. Phylogenetic analysis of the partial nucleotide sequence of SFTSV revealed that viral RNA detected from badgers exhibited 99.8% to 100% similarity to SFTSV, as previously reported in humans, cat, and ticks in the study area. This study demonstrated high seropositivity of antibodies in medium-sized wild mammals and suggested that SFTSV could be shared among these mammals, humans, and companion animals in endemic areas.

Tongue worm (subclass: Pentastomida) infection and treatment in two domesticated reptiles - A case report.

Tongue worms (Subclass: Pentastomida) are endoparasites found in carnivorous reptiles, fish, amphibians, birds, and mammals. Several pentastomids cause pentastomiasis, a zoonotic disease. We encountered tongue worm infection in two reptiles imported into Japan: a Wahlberg's velvet gecko (Homopholis wahlbergii) bred in Germany and a wild-caught green tree python (Morelia viridis) from the Republic of Indonesia. The infecting tongue worms were identified as Raillietiella sp. and Armillifer sp. in the gecko and python, respectively, based on morphological and molecular analyses. Gecko is a newly recorded definitive host harboring Raillietiella tongue worm. Live tongue worms were first detected in domestic reptiles in Japan, and this indicates a risk that these tongue worms could be spread to other animals, as well as humans. Therefore, the diagnosis and treatment of infected pets are of importance. In the case of the two infected reptiles, the availability of fecal egg examination for diagnosis was shown. In addition, the effectiveness of treatment with 200 µg/kg/day ivermectin orally once weekly was demonstrated, without no side-effects and no re-detection of eggs after the cessation of treatment.

Anthelmintic Baiting of Foxes against Echinococcus multilocularis in Small Public Area, Japan.

We distributed anthelmintic baits on a university campus in Japan inhabited by foxes infected with Echinococcus multilocularis to design an effective baiting protocol for small public areas. High-density baiting can reduce the risk for human exposure to the parasite to near zero. However, monthly baiting is recommended to maintain this effect.

Diagnostic value of recombinant nanoluciferase fused Toxoplasma gondii antigens in Luciferase-linked Antibody Capture Assay (LACA) for Toxoplasma infection in pigs.

Toxoplasmosis is a widespread protozoan zoonosis. Since ingesting undercooked meat harboring Toxoplasma gondii cyst is considered one of the major transmission routes to humans, the screening of T. gondii in meat-producing animals can reduce the risk of food-borne toxoplasmosis in humans. Among serological diagnostic methods, Luciferase-linked Antibody Capture Assay (LACA) has been found to be a promising platform with high sensitivity and specificity. In this study, we aimed to evaluate recombinant nanoluciferase fused-T. gondii antigens (rNluc-GRA6, rNluc-GRA7, rNluc-GRA8 and rNluc-BAG1) for their potential use in LACA for pigs. As a result, the sensitivity of GRA6-, GRA7-, GRA8- and BAG1-LACA were 70.0%, 80.0%, 80.0% and 30.0% with specificity 87.0%, 81.5%, 74.1% and 50.0%, respectively. The cocktail LACA using a mixture of rNluc-GRA6, rNluc-GRA7 and rNluc-GRA8 indicated higher sensitivity (90.0%) and a similar specificity (96.3%) in comparison with the commercial ELISA kit. Compared to the Dye-Test as a reference test, cocktail LACA showed strong agreement (kappa value=0.811) when we assessed pig sera collected at the slaughterhouse. In addition, we also successfully established the rapid LACA format for the detection of Toxoplasma infection in pigs (called Rapid-LACA) in which the test could be performed within 30 min. In Rapid-LACA, the protein A pre-coated/blocked plates could be preserved at -30°C, 4°C or room temperature conditions for at least two months without compromising on the quality of assay.

Sporadic endemicity of zoonotic Paragonimus in raccoon dogs and Japanese badgers from Miyazaki Prefecture, Japan.

Paragonimiasis is a zoonotic trematode infection caused by Paragonimus spp. To determine the recent status of Paragonimus infections in wild animals, this study investigated Paragonimus spp. in 39 raccoon dogs and 54 Japanese badgers from March 2019 to January 2021 in Miyazaki Prefecture, and examined metacercariae in freshwater crabs. Triploid P. westermani was found in one raccoon dog (2.6%), and metacercariae were recovered from Eriocheir japonica captured near the infected animal collected. One Japanese badger (1.9%) harbored P. skrjabini miyazakii; this prevalence was lower than the approximately 30% that was reported in the 1970s. Results indicated that zoonotic Paragonimus was sporadically prevalent in wild animals. Further investigation in various animals is awaited to elucidate current wildlife reservoirs for those Paragonimus.

Cotton rats (Sigmodon hispidus) with a high prevalence of hydrocephalus without clinical symptoms.

Normal-pressure hydrocephalus (NPH) is a condition in which the ventricle is enlarged without elevated cerebrospinal fluid pressure, and it generally develops in later life and progresses slowly. A complete animal model that mimics human idiopathic NPH has not yet been established, and the onset mechanisms and detailed pathomechanisms of NPH are not fully understood. Here, we demonstrate a high spontaneous prevalence (34.6%) of hydrocephalus without clinical symptoms in inbred cotton rats (Sigmodon hispidus). In all 46 hydrocephalic cotton rats, the severity was mild or moderate and not severe. The dilation was limited to the lateral ventricles, and none of the hemorrhage, ventriculitis, meningitis, or tumor formation was found in hydrocephalic cotton rats. These findings indicate that the type of hydrocephalus in cotton rats is similar to that of communicating idiopathic NPH. Histopathological examinations revealed that the inner granular and pyramidal layers (layers IV and V) of the neocortex became thinner in hydrocephalic brains. A small number of pyramidal cells were positive for Fluoro-Jade C (a degenerating neuron marker) and ionized calcium-binding adaptor molecule 1 (Iba1)-immunoreactive microglia were in contact with the degenerating neurons in the hydrocephalic neocortex, suggesting that hydrocephalic cotton rats are more or less impaired projections from the neocortex. This study highlights cotton rats as a candidate for novel models to elucidate the pathomechanism of idiopathic NPH. Additionally, cotton rats have some noticeable systemic pathological phenotypes, such as chronic kidney disease and metabolic disorders. Thus, this model might also be useful for researching the comorbidities of NPH to other diseases.

Sensitivity comparison between Mini-FLOTAC and conventional techniques for the detection of Echinococcus multilocularis eggs.

Canines serve as the definitive host of Echinococcus multilocularis. This study evaluated the sensitivity of the Mini-FLOTAC technique (MF) for the detection of E. multilocularis eggs in definitive hosts. First, we investigated the effects of heat inactivation and preservative conditions on the detection rate of eggs obtained from experimentally infected dogs. The sensitivity of MF was compared with that of eight other techniques: the centrifugal flotation with sucrose or zinc sulfate, MGL, AMS III, and a combination of MF and flotation/sedimentation techniques. Finally, we compared the sensitivity of MF and the centrifugal flotation with sucrose for the feces of E. multilocularis-infected foxes. The detection rate reached a plateau level with a specific gravity (s.g.) 1.22 for fresh eggs, but the highest rates were obtained with s.g. greater than 1.32 for heat-inactivated eggs. There was no significant difference in the detection rate among the preservative conditions. MF showed significantly higher EPG than the other techniques. Moreover, it showed higher diagnostic sensitivity for the fox feces than the centrifugal flotation technique. These results suggest that heat inactivation may alter s.g. of E. multilocularis eggs and that MF with zinc sulfate (s.g. = 1.32) would be effective for detecting heat-inactivated E. multilocularis eggs.

Novel polychrome staining distinguishing osteochondral tissue and bone cells in decalcified paraffin sections.

The bone is a dynamic and metabolically active organ in which growth and resorption of the osteochondral matrix is orchestrated by osteoblasts and osteoclasts. For decalcified paraffin-embedded specimens, decalcifying agents alter the staining intensity, and excess decalcification interferes with bone staining. Robust bone staining methods independent of the decalcification conditions and animal species are lacking. In this study, we have developed a novel polychrome staining method, named JFRL staining, which stains the components of osteochondral tissue in different colors. With this staining we could visualize the hyaline cartilage as blue by alcian blue, osteoid as red by picrosirius red, and mineralized bone as green by picro-light green SF or picro-naphthol green B and easily distinguished osteoblasts, osteocytes, and osteoclasts. In mineralized bone, this staining revealed the obvious lamellar structures and woven bone. Notably, this staining was independent of the decalcification conditions and experimental animal species examined. To verify the usefulness of JFRL staining, we observed cotton rat tail which has shorter length and shows a false autotomy. The caudal vertebrae were normally developed via endochondral ossification without a fracture plane. At 6 months of age, the number of chondrocytes declined and the hypertrophic zone was absent at the epiphyseal plate, which might reflect the shorter tail. In conclusion, JFRL staining is the first method to simultaneously distinguish osteochondral matrix and bone cells in one section regardless of decalcifying conditions. This robust staining will provide new information for a wide number of biomedical fields, including bone development, physiology, and pathology.

Comparison of Ovarian Morphology and Follicular Disturbances between Two Inbred Strains of Cotton Rats (Sigmodon hispidus).

Most mammalian ovarian follicles contain only a single oocyte having a single nucleus. However, two or more oocytes and nuclei are observed within one follicle and one oocyte, respectively, in several species, including cotton rat (CR, Sigmodon hispidus). The present study compared ovarian histology, focusing on folliculogenesis, between two inbred CR strains, HIS/Hiph and HIS/Mz. At 4 weeks of age, ovarian sections from both the strains were analyzed histologically. Multi-oocyte follicles (MOFs) and double-nucleated oocytes (DNOs) were observed in all stages of developing follicles in HIS/Hiph, whereas HIS/Mz had MOFs up to secondary stages and lacked DNOs. The estimated total follicles in HIS/Mz were almost half that of HIS/Hiph, but interstitial cells were well developed in HIS/Mz. Furthermore, immunostaining revealed no clear strain differences in the appearance of oocytes positive for Ki67, PCNA, and p63 in MOF or DNOs; no cell death was observed in these oocytes. Ultrastructural analysis revealed more abundant mitochondrial clouds in oocytes of HIS/Hiph than HIS/Mz. Thus, we clarified the strain differences in the CR ovary. These findings indicate that early events during folliculogenesis affect the unique ovarian phenotypes found in CRs, including MOFs or DNOs, and their strain differences.

Early-phase migration dynamics of Echinococcus multilocularis in two mouse strains showing different infection susceptibilities.

The early-phase migration dynamics of Echinococcus multilocularis in the intermediate hosts remain largely unknown. We compared the parasite burden in the intestine, liver and faeces of DBA/2 and C57BL/6 mouse strains using parasite-specific quantitative PCR. Our results indicated that the parasites invaded mainly from the middle segments of the small intestine and completed migration to the liver within 24 h p.i. C57BL/6 mice had lower parasite DNA burdens in the intestine and liver but higher in the faeces than DBA/2 mice, suggesting that parasite invasion of the intestine may be a critical stage regulating susceptibility to E. multilocularis infection in mice.

In vivo efficacy of combination therapy with albendazole and atovaquone against primary hydatid cysts in mice.

Alveolar echinococcosis (AE) is caused by the larval stage of Echinococcus multilocularis. Chemotherapy for AE involves albendazole (ABZ), which has shown insufficient efficacy. More effective chemotherapy for AE is needed. Previously, we have demonstrated that atovaquone (ATV), an antimalarial, inhibits mitochondrial complex III of E. multilocularis and restricts the development of larval cysts in in vivo experiments. Therefore, in this study, we evaluated the efficacy of ABZ and ATV combination therapy on E. multilocularis in culture and in vivo experiments. Protoscoleces were treated with 50 µM ABZ and/or ATV in the medium; the duration of parasite elimination was determined under aerobic and anaerobic culture. In the in vivo experiment, the effects of ABZ and ATV combination treatment in BALB/c mice infected orally with eggs from the feces of an adult-stage E. multilocularis-infected dog were compared with those of standard oral ABZ therapy. In the culture assay, the duration of elimination associated with ABZ and ATV combination treatment was shorter than that associated with ATV alone under aerobic conditions. Protoscolex viability progressively reduced owing to the combination treatment under anaerobic conditions; however, either drug used singly did not exhibit antiparasitic effects under hypoxia. Furthermore, compared with ABZ alone, the combination treatment significantly reduced the growth of the primary cyst in the liver of mice infected orally with parasite eggs (P = .011). ATV enhances the effect of ABZ in the treatment of AE in mice.

Unique histological features of the tail skin of cotton rat (Sigmodon hispidus) related to caudal autotomy.

Caudal autotomy in rodents is an evolutionarily acquired phenomenon enabling escape from predators, by discarding the tail skin after traumatic injuries. The histological mechanisms underlying caudal autotomy seem to differ among species. Cotton rats (Sigmodon hispidus), which are important laboratory rodents for human infectious diseases, possess a fragile tail. In this study, we compared the tail histology of cotton rats with that of laboratory rats (Rattus norvegicus), which have no fragility on their tail, to elucidate the process of rodent caudal autotomy. First, the cotton rats developed a false autotomy characterized by loss of the tail sheath with the caudal vertebrae remaining without tail regeneration. Second, we found the fracture plane was continuous from the interscale of the tail epidermis to the dermis, which was lined with an alignment of E-cadherin+ cells. Third, we found an obvious cleavage plane between the dermis and subjacent tissues of the cotton-rat tail, where the subcutis was composed of looser, finer, and fragmented collagen fibers compared with those of the rat. Additionally, the cotton-rat tail was easily torn, with minimum bleeding. The median coccygeal artery of the cotton rat had a thick smooth muscle layer, and its lumen was filled with the peeled intima with fibrin coagulation, which might be associated with reduced bleeding following caudal autotomy. Taken together, we reveal the unique histological features of the tail relating to the caudal autotomy process in the cotton rat, and provide novel insights to help clarify the rodent caudal autotomy mechanism.

Effect of the anti-parasitic compounds pyrvinium pamoate and artemisinin in enzymatic and culture assays: Data on the search for new anti-echinococcal drugs.

The dataset presented herein is related to a previous research article titled "Mitochondrial Complex III in Larval Stage of Echinococcus multilocularis as a Potential Chemotherapeutic Target and in vivo Efficacy of Atovaquone Against Primary Hydatid Cysts" [1]. In this report, data were collected by screening drugs for echinococcosis. We investigated the inhibitory activities of artemisinin and pyrvinium pamoate against the mitochondrial respiratory enzymes in E. multilocularis protoscoleces. Artemisinin did not inhibit mitochondrial complexes I, II, and III. However, pyrvinium pamoate inhibited complex I at 11 µM, although complexes II and III were not inhibited. In the culture assay, E. multilocularis protoscoleces were treated with atovaquone (ATV), rotenone, praziquantel, artemisinin, and pyrvinium pamoate at a final concentration of 50 µM in different culture media. The viability of protoscoleces was compared under aerobic and anaerobic conditions via culture experiments. The survival days of E. multilocularis protoscoleces were evaluated in the drug-treated group compared with those in the non-treated group. The results of these culture assays revealed that praziquantel and artemisinin did not eliminate the protoscoleces under both aerobic and anaerobic conditions. However, a stronger elimination ability was observed with the co-administration of praziquantel or artemisinin with ATV than with ATV alone under aerobic conditions. Pyrvinium pamoate completely killed protoscoleces at 5 and 7 days under aerobic and anaerobic conditions, respectively. Pyrvinium pamoate behaved identically to rotenone, the complex I inhibitor, in the culture treatment assay. The data serve as a reference for the development of novel anti-echinococcal drugs.

Seroprevalence of fasciolosis in Hokkaido sika deer (Cervus nippon yesoensis) from Hokkaido Prefecture, Japan revealed by ELISA using recombinant cathepsin L1.

Fasciolosis, a zoonotic disease caused by liver flukes of the genus Fasciola, has been reported in Hokkaido (Yezo) sika deer (Cervus nippon yesoensis) in Hokkaido Prefecture, Japan; however, the actual seroprevalence in the animal has not been adequately evaluated. The objective of the present study was to analyze the seroprevalence of the disease among Hokkaido sika deer. Recombinant cathepsin L1 (rCatL1) was used as an antigen for an indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against Fasciola flukes. The sensitivity and specificity of the ELISA were 84.6% and 100%, respectively. The average seroprevalence in 1109 Hokkaido sika deer from 20 locations in Hokkaido Prefecture was 43.9%. Mature deer showed higher seroprevalence than younger individuals; however, even younger animals may act as a reservoir for the disease. Monitoring infection levels in the Hokkaido sika deer population is important not only for the livestock industry, but also for preventing human fasciolosis.

Characterization of microRNAs expressed in the cystic legion of the liver of Mus musculus perorally infected with Echinococcus multilocularis Nemuro strain.

Alveolar echinococcosis (AE) is a zoonosis caused by the metacestode of Echinococcus multilocularis. The published genome of E. multilocularis showed that approximately 86% of its genome is non-coding. Micro RNAs (miRNAs) are small non-coding regulatory RNAs, and recent studies on parasitic helminths expect miRNAs as a promising target for drug development and diagnostic markers. Prior to this study, only a few studies reported the E. multilocularis miRNA profiles in the intermediate host. The primary objective of this study was to characterize miRNA profiles via small RNA-seq in E. multilocularis Nemuro strain, a laboratory strain of Asian genotype, using mice perorally infected with the parasite eggs. The data were then compared with two previously published small RNA-seq data. We identified 44 mature miRNAs as E. multilocularis origin out of the 68 mature miRNA sequences registered in the miRNA database miRbase. The highest quantities of miRNAs detected were miR-10-5p, followed by bantam-3p, let-7-5p, miR-61-3p, and miR-71-5p. The top two most abundant miRNAs (miR-10-5p and bantam-3p) accounted for approximately 80.9% of the total parasite miRNAs. The highly expressed miRNA repertoire is mostly comparable to that obtained from the previous experiment using secondary echinococcosis created by an intraperitoneal administration of metacestodes. A detailed characterization and functional annotations of these shared miRNAs will lead to a better understanding of parasitic dynamics, which could provide a basis for the development of novel diagnostic and treatment methods for AE.

Unique Running Pattern and Mucosal Morphology Found in the Colon of Cotton Rats.

Cotton rats are one of the experimental rodents used for testing different infectious and non-infectious diseases, including gastrointestinal tract pathology. However, their intestinal morphological characteristics are still poorly understood. Here, we clarified the anatomical and histological characteristics of the cecum and ascending colon (AC) of young (1-3-month old), adult (4-6-month old), and old (10-12-month old) cotton rats. The large intestine (LI) in cotton rats is composed of the cecum, AC, transverse and descending colons, and rectum, and is similar to that of other mammals. The AC begins with a double or triple spiral loop-like flexure (SLLF) and ends with a coupled horseshoe-like flexure (HSLF). A single longitudinal mucosal fold (SLMF) was found at the beginning of the AC along the mesentery line and developed with age. Furthermore, the SLMF contained several lymphatic nodules (LNs), indicating their role in digestive and immunological functions. Small and large protuberant LNs were found in the cecum and SLLF, respectively, whereas thin and flat LNs were observed in the HSLF and transverse colon, respectively. Regarding sex-related differences, adult females had a significantly longer AC with a higher number of SLLFs compared to males. The SLMF length and LN number were also longer and higher, respectively, in adult females compared to adult males. These are crucial findings, indicating the presence of sex-related differences in the morphology of the LI in cotton rats, and ours is the first study to discover a sex difference in the mammalian LI lining. Our study clarified the unique morphology of the LI in cotton rats, which could serve as the principal model for elucidating species-specific digestive tract functions and gastrointestinal disorders.

Unique morphological characteristics in the ovary of cotton rat (Sigmodon hispidus).

Cotton rats (Sigmodon hispidus, CRs) are commonly used as animal models in biomedical research. However, the reproductive characteristics and ovarian development in the CRs has not been widely investigated. We have previously shown that female CRs, in particular, show several unique phenotypes associated with the urogenital system, such as chronic kidney disease and pyometra. Our investigation revealed unique morphologies in CR ovaries, particularly in oocytes. Cotton rat ovaries at 6-8 weeks of age were obtained from the Hokkaido Institute of Public Health, and their sections analyzed by light microscopy and transmission electron microscopy. Although the general histology and folliculogenesis of CR ovaries were similar to those of other experimental rodents, multi-oocyte follicles (MOFs) and double nucleated oocytes (DNOs) were also observed. Although MOFs were found at all stages of follicular development, a greater frequency of MOFs was observed in the primary and secondary stages. However, DNOs tended to be frequently observed in primordial follicles. Almost all MOF oocytes and a few DNOs possessed a clear zona pellucida, expressed DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 and Forkhead box protein 2, a representative marker of oocytes and follicular epithelial cells. Thus, our investigations revealed the unique phenotypes of the CR ovary. As MOFs and DNOs are occasionally observed in human patients with infertility, the CR would be a useful animal model to study for gaining a better understanding of folliculogenesis and oocytogenesis, as well as their abnormalities in humans and other animals.

Adult worm exclusion and histological data of dogs repeatedly infected with the cestode Echinococcus multilocularis.

The data presented in this article are related to a previously published research article titled "The timing of worm exclusion in dogs repeatedly infected with the cestode Echinococcus multilocularis" (2016) [1]. This data describe a comparison of worm exclusion in the early stage of infection (1 day and 6 days post-infection) between dogs infected for the first time (control group) and dogs repeatedly infected with the parasite 4 times (repeated infection groups). We observed that 6 days post reinfection, the number of adult worms in repeated-infection groups decreased by 88.7% compared with the control group. Histological analysis comparison of the small intestinal mucosa from healthy, first infected, and repeatedly infected dogs are also reported. We observed no clear pathological abnormality, except the shortening of microvillus in reinfected dogs. However, eosinophil accumulation and eosinophilic ulcers were observed in some reinfected dogs. This data could be useful as preliminary data to develop a final host vaccine for this parasite.