Morphological and molecular phylogenetical identification of Tricodectes pinguis from Japanese black bears (Ursus thibetanus japonicus) in Aomori Prefecture, Japan.

Trichodectes pinguis, referred to commonly as the bear-biting louse, has been reported in several bear species. However, graphical (blurred or coarse) and genetic information on the louse is limited. In this study, we identified T. pinguis collected from Japanese black bears in the Aomori Prefecture, Japan. We confirmed 12S rDNA sequences derived from the collected T. pinguis and performed molecular phylogenetic analysis based on 12S rDNA. The analysis revealed the parasitic louse to be T. pinguis. Interestingly, the body size of T. pinguis found in this study was smaller than the previous recorded body size of them in Japan and Turkey. To better understand the biting louse infesting bears, morphometric and genetic information from other bear hosts needs to be accumulated.

Raccoon dogs (Nyctereutes procyonoides) are the new natural definitive hosts of Metagonimus hakubaensis.

The definitive hosts of Metagonimus hakubaensis are reported to be hamsters, rats, mice, dogs, cats, chickens, and quails in experimental infection and Japanese water shrews in natural infection. Here we report that raccoon dogs are new natural definitive hosts of M. hakubaensis, based on morphological and molecular analyses of Metagonimus flukes collected from the host species from Aomori Prefecture, Japan. Moreover, M. hakubaensis recovered from raccoon dogs showed higher fecundity than those recovered from Japanese water shrews. Therefore, raccoon dogs were considered as a more suitable natural definitive host of M. hakubaensis than Japanese water shrews.

Survey of Francisella tularensis in Wild Animals in Japan in Areas Where Tularemia is Endemic.

Samples taken from 428 wild animals and 126 ticks, collected from a tularemia-endemic area in Japan between 2005 and 2013, were analyzed for the presence of Francisella tularensis. F. tularensis was isolated from a Japanese hare carcass whereas the samples from live animals and ticks were negative for F. tularensis by real-time PCR. Our results suggest that F. tularensis is still present in Japan although its prevalence is considerably low even in areas where tularemia is endemic.

Efficacy of levamisole alone and in combination with mebendazole against Gongylonema pulchrum infection in rabbits.

Gongylonema pulchrum is an important parasite of captive primates. Twelve rabbits were infected with 30 third-stage larvae of G. pulchrum. At 4-7 months post-infection, animals were administered levamisole at a single dose of 12 mg/kg, levamisole at 8 mg/kg three times at 2-day intervals, levamisole at a single dose of 8 mg/kg after administration of mebendazole at 70 mg/kg for 3 days or 8 ml of distilled water for 3 days (control). Necropsy at 14 days after treatment revealed that single and multiple dosages of levamisole reduced nematode burdens by 68.4% and 89.5%, respectively. The combined regimen of mebendazole and levamisole exhibited high efficacy for treating G. pulchrum located widely within the upper digestive tract, with a reduction of 98.2%. These results suggest that this combined chemotherapy treatment may be effective against G. pulchrum infection, including buccal and lingual gongylonemiasis in primates.

Discovery of natural infection by Metagonimus hakubaensis Shimazu, 1999 (Trematoda: Heterophyidae) in Japanese water shrews (Chimarrogale platycephala) in Japan.

A total of 611 preserved adult Metagonimus spp. specimens recovered from 32 of 53 Japanese water shrews (Chimarrogale platycephala) that had previously been collected in Aomori Prefecture between June 1994 and August 1996, were examined in this study. Morphological examination revealed that 603 of these flukes were identical to M. hakubaensis Shimazu, 1999, and that the others were M. takahashii Suzuki, 1930 (n=4), M. otsurui Saito et Shimizu, 1968 (n=2), and M. miyatai Saito et al., 1997 (n=2). Each of the 32 Japanese water shrews infected with M. hakubaensis contained between 1 and 83 flukes. This is the first record of the natural final host for M. hakubaensis, since this fluke species was described.

Reduced efficacy of ivermectin treatments in gastrointestinal nematode infections of grazing cattle in Japan.

Fecal egg count reduction tests (FECRT) and larval migration inhibition tests (LMIT) were conducted to assess the efficacy of ivermectin (IVM) against gastrointestinal nematodes on 2 cattle farms in northern Japan in 2009 and 2010. Twelve to 20 calves on each farm were treated topically with 0.5 mg IVM/kg 2 (Farm 2) or 4 times (Farm 1) during the grazing season (May-October). On Farm 1, fecal egg count (FEC) reduction at 14 days post-treatment ranged from 16 to 87% in 2009 and from 24 to 96% in 2010, with relatively low reductions in August and October (16-53%). Conversely, IVM treatment on Farm 2 reduced FEC by 97% in September 2009. Larvae obtained from fecal cultures and identified by PCR-RFLP analysis revealed that the dominant species on both farms prior to IVM administration was Cooperia oncophora. In 2009, the FEC reduction of C. oncophora on Farm 1 decreased from 85% in May to 56% in August. In 2010, the reduction in C. oncophora in August was 28%. In the LMIT using larvae collected from the fecal cultures on Farm 1 in May and August 2009, the EC50 value of IVM in C. oncophora in August (0.892 µg/ml) was 3 times higher than that in May (0.296 µg/ml). The results of the LMIT corroborated the FECRT data, indicating the presence of IVM-resistant C. oncophora on Farm 1, at least in August. This is the first report of IVM-resistant nematodes in Japanese cattle.

Serosurveillance for Francisella tularensis among wild animals in Japan using a newly developed competitive enzyme-linked immunosorbent assay.

Tularemia, a highly infectious zoonotic disease caused by Francisella tularensis, occurs sporadically in Japan. However, little is known about the prevalence of the disease in wild animals. A total of 632 samples obtained from 150 Japanese black bears, 142 Japanese hares, 120 small rodents, 97 rats, 53 raptors, 26 Japanese monkeys, 21 Japanese raccoon dogs, 20 masked palm civets, and three Japanese red foxes between 2002 and 2010 were investigated for the presence of antibodies to F. tularensis by competitive enzyme-linked immunosorbent assay (cELISA) and the commonly used microagglutination (MA) test. Seropositive cELISA and MA results were obtained in 23 and 18 Japanese black bears, three and two Japanese raccoon dogs, and two and one small rodents, respectively. All MA-positive samples (n=21) were also positive by cELISA. Six of seven samples that were only positive by cELISA were confirmed to be antibody-positive by western blot analysis. These findings suggest that cELISA is a highly sensitive and useful test for serosurveillance of tularemia among various species of wild animals. Because this is the first study to detect F. tularensis-seropositive Japanese raccoon dogs, these could join Japanese black bears as sentinel animals for tularemia in the wild in Japan. Further continuous serosurveillance for F. tularensis in various species of wild animals using appropriate methods such as cELISA is important to assess the risks of human exposure and to improve our understanding of the ecology of F. tularensis in the wild.

Experimental final hosts of Metagonimus hakubaensis (Trematoda: Heterophyidae) and their suitability to the fluke.

Seven laboratory mammal and bird species were orally inoculated with 200-1,000 encysted Metagonimus hakubaensis metacercariae that had been isolated from naturally infected lampreys (Lethenteron reissneri) captured in Aomori Prefecture. At 8 and 15 days post-infection, adult flukes were recovered from all of the laboratory animals tested, and therefore, hamster, rat, mouse, dog, cat, chicken and quail were considered as final hosts of M. hakubaensis. Recovery rates of the fluke were higher in dogs and hamsters than in cats, rats, mice, chickens and quails. The flukes recovered from dogs and hamsters showed increased body length and higher fecundity than those recovered from the other hosts. These results indicate that the suitability of dogs and hamsters for M. hakubaensis infection is higher than that of the other laboratory animals.

Encephalitozoon infections in Rodentia and Soricomorpha in Japan.

Encephalitozoon is an obligate intracellular microsporidian parasite that infects a wide range of mammalian hosts. In this study, we used nested PCR to investigate the presence of Encephalitozoon infection in Rodentia and Soricomorpha in Japan. We attempted to amplify and sequence Encephalitozoon-specific DNA from brain and viscera samples of 180 animals collected between 2008 and 2010. Forty-three samples (23.9%) from the orders Rodentia and Soricomorpha were positive for Encephalitozoon. This study is the first report of Encephalitozoon infection in Rodentia and Soricomorpha in Japan, and our findings suggest that these hosts may play a role in the spread of microsporidian spores in the environment.

Pathological and microbiological studies of Japanese Hare (Lepus brachyurus angustidens) naturally infected with Francisella tularensis subsp. holarctica.

An adult male hare (Lepus brachyurus angustidens) was discovered in a moribund condition in the bush in the mountains of Aomori prefecture in Japan. Upon gross inspection, many ticks were found on the neck and the external ear regions, and more than half the ticks contained blood in the intestine. The skin around the tick bite wounds was alopecic and mildly thickened. At necropsy, enlargement of the cervical lymph nodes and spleen were observed. Histologically, acute necrotizing splenitis, lymphadenitis, hepatitis, pneumonia, myelitis, adrenalitis, and encephalitis with bacterial organisms were observed. The cutaneous lesions were chronic and cysts had formed in the areas marked by tick bites. Immunohistochemically, the organisms in the skin, liver, spleen, lymph nodes, lungs, adrenal glands, brain, bone marrow, and ticks were positive for F. tularensis antigen. Microbiological and polymerase chain reaction results were consistent with F. tularensis subsp. holarctica. Because the cutaneous lesions were more chronic than those in the visceral organs and F. tularensis was detected in the ticks, we inferred that F. tularensis was transmitted to the hare via tick bites.

Efficacy of thiabendazole, mebendazole, levamisole and ivermectin against gullet worm, Gongylonema pulchrum: in vitro and in vivo studies.

In vitro and in vivo studies were conducted to evaluate the effects of thiabendazole, mebendazole, levamisole and ivermectin against Gongylonema pulchrum. For in vitro assays, third-stage larvae (L3) incubated with the drugs were administered orally to mice and the ability of larvae to invade the gastric mucosa of the animals was examined. After incubation, only those larvae treated with high concentrations of levamisole (1 and 10 microg/ml) were tightly coiled with intestines exhibiting morphological abnormalities. Good dose-response data for the drugs tested was observed at the time of worm recovery from mice, with no worms recovered at the two highest concentrations of levamisole. In vivo efficacy of the drugs against adult worms was evaluated in six groups of three rabbits, each of which was infected with 30 L3 of G. pulchrum and treated with thiabendazole at 100 mg/kg for 3 days, mebendazole at 70 mg/kg for 3 days, levamisole as a single dose of 8 mg/kg, and subcutaneously injected ivermectin as a single dose of 0.2 mg/kg or vehicles of the drugs (control) at 4 months post-infection. Necropsy 14 days after treatment revealed that levamisole, mebendazole and ivermectin reduced worm burdens by 63.2%, 22.8% and 25.8%, respectively, with no reductions in worms observed with thiabendazole. The surviving worms were principally found in the esophagus with the remainder distributed among the buccal mucosa, the tongue, and/or pharyngeal mucosa in all groups. A number of morphologically abnormal eggs were observed within the uterus and ovijector in female worms recovered from the thiabendazole-treated group. These findings suggest that levamisole exhibits in vivo efficacy against G. pulchrum infection and that the larval invasion tests using mice could be used to screen for anthelmintic susceptibility of nematodes.

Morphological survey of bovine Setaria in the abdominal cavities of cattle in Aomori and Kumamoto Prefectures, Japan.

An epidemiological survey of bovine Setaria collected from the abdominal cavities was performed morphologically on the cattle in Aomori and Kumamoto Prefectures, Japan, between August 2005 and July 2006. Fifty Setaria worms were collected from the cattle in Aomori Prefecture and 847 from those in Kumamoto Prefecture. Of these worms, 35 were identified as Setaria digitata, 14 as S. marshalli, and one as S. labiatopapillosa in Aomori Prefecture, while 816 were identified as S. digitata and 31 as S. marshalli in Kumamoto Prefecture.

Short report: molecular cloning and characterization of a putative binding protein of Babesia caballi.

A composite 2,206 nucleotide DNA sequence encoding a putative immunoglobulin-binding protein (BiP) was constructed from a sequence obtained from Babesia caballi cDNA library clones. The 1,962 nucleotide open reading frame predicts a 72 kD protein with extensive homology with BiPs from Apicomplexa parasites. The BiP gene had a predicted N-terminal signal sequence of 18 amino acids and a C-terminal tetrapeptide sequence (Ser-Asp-Glu-Leu) for signaling in the endoplasmic reticulum lumen. The recombinant protein expressed in baculovirus showed an apparent mass of 72 kD, which is identical to that of the native B. caballi protein. Monoclonal antibodies (MAbs) against B. caballi BiP reacted strongly with extracellular merozoites, but not in early intraerythrocytic stage. Detailed observation showed that the reaction of MAbs against pear-shaped forms was markedly irregular, with either no reaction, or reaction with one or two brightly fluorescent pear-shaped forms (two parasites) of B. caballi.

Inhibitory effect of lactoferrin on in vitro growth of Babesia caballi.

Lactoferrin (LF) is an important biologic molecule with many functions, one of which is antimicrobial defense. We evaluated the growth-inhibiting effects of four types of LF (native LF, Fe(+3)-bound [holo] LF, Fe(+3)-free [apo] LF, and LF hydrolyzate) on the in vitro growth of Babesia caballi and B. equi. The growth of B. caballi was significantly suppressed in media containing apo LF, but was not inhibited in media containing native LF, holo LF, or LF hydrolyzate. The growth of B. equi was not inhibited by media containing native LF, holo LF, or apo LF. These data indicate that apo LF had the strongest inhibitory effect on B. caballi. This may have been caused by inactivation or inhibition of a growth factor in the culture medium.

Further observations on the development of Gongylonema pulchrum in rabbits.

Third-stage larvae of Gongylonema pulchrum from naturally infected dung beetles were inoculated orally into 24 rabbits. Worm recovery ranged from 54 to 91% (mean = 67.5%) during the period from 24 hr to 52 wk postinoculation (PI). Two hours PI, the larvae entered the mucosa at the junction of the stomach and esophagus and migrated upward. Early development occurred primarily in pharyngeal mucosa, tongue, and buccal mucosa. The third molt took place 11 days PI and the final molt at 36 days PI. Male worms reached sexual maturity at 7 wk PI and females at 9 wk PI. Adult worms were found mainly in the esophagus but also occurred in the tongue and the wall of the oral cavity after 30 wk PI. Embryonated eggs appeared in the feces of 3 rabbits inoculated with 50 or 100 larvae on days 72-81 PI. Morphologically, the cuticle in young fourth-stage larvae exhibited bosses on the anterior portion on day 11 PI, and the left spicule length : total body length exhibited no remarkable change between 9 and 52 wk PI. The latter finding confirms the utility of the ratio for identification of the nematode.

Molecular evidence of infections with Babesia gibsoni parasites in Japan and evaluation of the diagnostic potential of a loop-mediated isothermal amplification method.

Detection and analysis of Babesia gibsoni infection were performed with whole-blood samples collected between July 2002 and July 2003 from 945 and 137 dogs from the Aomori and Okinawa Prefectures of Japan, respectively, by PCR and loop-mediated isothermal amplification (LAMP). On the basis of the criterion for positivity by PCR, 3.9% (37 of 945) and 10.9% (15 of 137) of the dogs had B. gibsoni DNA. All 37 positive animals from Aomori Prefecture were male Tosa dogs (Japanese mastiff). The 15 dogs from Okinawa Prefecture with positive PCR assay results were of various breeds, ages, and sexes. The 18S ribosomal DNA (18S rDNA) sequences from all samples showed 100% homology to each other and to published B. gibsoni sequences. The limits of detection of B. gibsoni parasitemia by the PCR and LAMP methods with an 18S rDNA-based primer set were 0.0005% each. A comparison of the PCR and LAMP methods with microscopic examination for the detection of B. gibsoni infections in blood samples from 945 field dogs in Aomori Prefecture and 137 field dogs in Okinawa Prefecture showed that 37 and 15 dogs, respectively, were positive by the PCR and LAMP methods and that 16 and 12 dogs, respectively, were positive by light microscopic examination. All samples found to be positive by microscopic examination were also positive by the PCR and LAMP methods. The results of the PCR and LAMP methods agreed for samples with positive results by either method. Moreover, nonspecific reactions were not observed by the LAMP method. These results suggest that the LAMP method provides a useful tool for the detection of B. gibsoni infections in dogs.

Experimental infection of laboratory animals and sheep with Gongylonema pulchrum in Japan.

Japanese White rabbits, Wistar rats, ddY mice, Suffolk sheep, and a domestic cat were each orally inoculated with 20-140 third-stage larvae (L3) of Gongylonema pulchrum, isolated from naturally infected dung beetles captured in Aomori Prefecture. Worm recovery rates were 40.0-72.0% in rabbits at 7, 14, and 19 weeks post-infection (PI) and 3.3-25.0% in rats at 19 weeks PI. Those in 2 sheep at 7 weeks PI showed 53.6% and 29.3%. No worms were recovered from the mice and the cat. In the susceptible animals, many worms were found in the esophagus, and a few were present in the pharyngeal mucosa, tongue, buccal mucosa, and cardiac portion of the stomach wall. No distinct morphological differences were observed in the worms from rabbits and sheep. These results indicate that rabbits are very suitable experimental definitive hosts for G. pulchrum.