Comparison of Toxoplasma gondii Seroprevalence in Shelter Cats and Dogs during 1999-2001 and 2009-2011 in Tokyo, Japan.

Toxoplasma gondii is an important human health concern with respect to abortion, congenital hydrocephalus, and encephalitis in immunocompromised people. Cats and dogs both are potential sources of T. gondii because they have close contact with humans. However, no epidemiological surveys have been conducted in Tokyo over the past decade. Therefore, the present study investigated and compared the seroprevalence of T. gondii infection in shelter cats and dogs during 1999-2001 and 2009-2011 in Tokyo, Japan. Serum samples were collected from 337 shelter cats and 325 shelter dogs in urban and suburban areas of Tokyo, during 1999-2001 (233 cats and 219 dogs) and 2009-2011 (104 cats and 106 dogs). T. gondii antibodies were measured in the serum samples using a commercial latex agglutination test. Data were compared using the Fisher's exact test, and significance was indicated at P < 0.05. The overall seroprevalence of T. gondii infection in cats was 5.6% (13 of 233) in 1999-2001 and 6.7% (7 of 104) in 2009-2011, and that in dogs was 1.8% (4 of 219) and 1.9% (2 of 106), respectively. Significantly higher seroprevalence was observed in cats from suburban areas compared with cats in urban areas during both periods (P < 0.05). These results reveal that there has been little change in the feline and canine seroprevalence over the past decade, indicating that the risk of T. gondii exposure for cats and dogs in Tokyo is considerably low as the seroprevalence has reached a steady state.

Detection of Dirofilaria immitis DNA in host serum by nested PCR.

The heartworm Dirofilaria immitis is the causative agent of dirofilariasis in dogs. Studies have shown that parasite-derived cell-free DNA (cfDNA) can be detected in host blood and may be a promising diagnostic marker for parasitic infections. Thus, our aim was to detect D. immitis-derived cfDNA in host serum by nested PCR. Sera were collected from 12 dogs with natural D. immitis infections; eight were microfilaria (mf)-positive, and the remaining four were mf-negative. Culture fluids derived from single-sex adult D. immitis worms (mf-producing females and males) were also tested for cfDNA. All mf-positive sera were positive by nested PCR, whereas no amplification products were detected in mf-negative sera. The culture fluid of mf-producing females was positive by nested PCR but that of males was negative. All products amplified by nested PCR were sequenced to confirm that the amplicons were those of D. immitis. These results indicate that D. immitis DNA circulates freely in dog serum, except in mf-negative dogs. Additionally, D. immitis cfDNA may primarily be derived from the mf, and adult worms appeared to be minor contributors of cfDNA concentrations in serum; however, the contribution of D. immitis cfDNA derived from larvae of other developmental stages is unclear. An evaluation of the kinetics of D. immitis cfDNA in host serum throughout the parasite life cycle could facilitate the development of early molecular diagnostic techniques. To the best of our knowledge, this is the first report of the detection of mitochondrial DNA from a filarial parasite in host serum.

Prevalence of Dirofilaria immitis among shelter dogs in Tokyo, Japan, after a decade: comparison of 1999-2001 and 2009-2011.

Changes in the seroprevalence of Dirofilaria immitis infection among shelter dogs between a decade ago and the present were evaluated. Serum samples were collected from 200 adult dogs in urban and suburban areas in Tokyo, Japan, during two 2-year periods (April 1999 to March 2001 and April 2009 to March 2011). Sera were tested for the presence of D. immitis antigen using a specific commercialized kit. The seroprevalence of D. immitis infection was 46% in 1999-2001 and 23% in 2009-2011. A decrease was observed in the prevalence of infection between 1999-2001 and 2009-2011; in particular, the prevalence in urban areas decreased significantly compared with that in suburban areas (P < 0.01). There was no significant difference in prevalence between the sexes in each period, but there was a significant difference between mixed-breed and purebred dogs (P < 0.01). The decrease in prevalence of canine heartworm disease in urban areas could be related to better veterinary care.

Case report: First confirmed case of canine peritoneal larval cestodiasis caused by Mesocestoides vogae (syn. M. corti) in Japan.

Canine peritoneal larval cestodiasis (CPLC) is an unusual parasitic disease in dogs that is caused by asexual proliferation of larval Mesocestoides. A 12 year-old spayed Shetland sheepdog with abdominal distension was referred to the Animal Medical Center at Nihon University, Japan. The presence of ascites was confirmed by abdominal ultrasonography and X-ray imaging. In addition, a number of parasites were observed in the ascitic fluid collected by abdominal paracentesis. Each of the whitish colored parasites was less than 1mm in size. The parasites were morphologically identified as Mesocestoides sp. tetrathyridia. The parasites had four suckers and calcareous corpuscles, but no hooks or rostellum. Mitochondrial (mt) 12S rDNA and mt cytochrome c oxidase subunit 1 DNA amplified from the tetrathyridia were used for molecular identification to species level. DNA sequence analysis showed that the tetrathyridia shared more than 99% identity with M. vogae (syn. M. corti) for each gene. The patient was treated with a standard dose (5mg/kg) of praziquantel, which was administered subcutaneously twice at an interval of 14 days. This resulted in successful deworming. This is the first case that CPLC was diagnosed in a dog that had never been taken outside of Japan, indicating that M. vogae is distributed in this country.

Screening of novel malaria DNA vaccine candidates using full-length cDNA library.

No licensed malaria vaccine exists, in spite of intensive development efforts. We have been investigating development of a DNA vaccine to prevent malaria infection. To date, we have established a full-length cDNA expression library from the erythrocytic-stage murine malaria parasite, Plasmodium berghei. We found that immunization of mice with combined 2000 clones significantly prolonged survival after challenge infection and that splenocytes from the immunized mice showed parasite-specific cytokine production. We determined the 5'-end one-pass sequence of these clones and mapped a draft genomic sequence for P. berghei for use in screening vaccine candidates for efficacy. In this study, we annotated these cDNA clones by comparing them with the genomic sequence of Plasmodium falciparum. We then divided them into several subsets based on their characteristics and examined their protective effects against malaria infection. Consequently, we selected 104 clones that strongly induced specific IgG production and decreased the mortality rate in the early phase. Most of these 104 clones coded for unknown proteins. The results suggest that these clones represent potential novel malaria vaccine candidates.

Successful resolution of esophageal granulomas in a dog infected with Spirocerca lupi.

A 13-year-old female Labrador Retriever presented with chronic regurgitation. Radiography and computed tomography (CT) revealed nodules in the caudal esophagus. Upper gastrointestinal endoscopy followed by histopathological examination revealed esophageal granulomas caused by Spirocerca lupi. The infection was treated with milbemycin oxime. The therapy was successful, and a remarkable reduction in granulomas was observed. This case report describes the diagnostic imaging findings and treatment outcome of a dog with S. lupi infection in Japan.

Coelacanth genomes reveal signatures for evolutionary transition from water to land.

Coelacanths are known as "living fossils," as they show remarkable morphological resemblance to the fossil record and belong to the most primitive lineage of living Sarcopterygii (lobe-finned fishes and tetrapods). Coelacanths may be key to elucidating the tempo and mode of evolution from fish to tetrapods. Here, we report the genome sequences of five coelacanths, including four Latimeria chalumnae individuals (three specimens from Tanzania and one from Comoros) and one L. menadoensis individual from Indonesia. These sequences cover two African breeding populations and two known extant coelacanth species. The genome is ∼2.74 Gbp and contains a high proportion (∼60%) of repetitive elements. The genetic diversity among the individuals was extremely low, suggesting a small population size and/or a slow rate of evolution. We found a substantial number of genes that encode olfactory and pheromone receptors with features characteristic of tetrapod receptors for the detection of airborne ligands. We also found that limb enhancers of bmp7 and gli3, both of which are essential for limb formation, are conserved between coelacanth and tetrapods, but not ray-finned fishes. We expect that some tetrapod-like genes may have existed early in the evolution of primitive Sarcopterygii and were later co-opted to adapt to terrestrial environments. These coelacanth genomes will provide a cornerstone for studies to elucidate how ancestral aquatic vertebrates evolved into terrestrial animals.

Echinostoma hortense infection with enteritis diagnosed by upper gastrointestinal endoscopy in a dog.

An 8-year-old male Shiba dog presented with chronic vomiting and diarrhea. Upper gastrointestinal endoscopy revealed severe enteritis and infection of the duodenal mucosa with Echinostoma hortense. We performed therapy for parasites and enteritis. The therapy was successful for deworming and temporarily improved the symptoms, but the dog died soon thereafter. To the authors' knowledge, this is the first case report of an antemortem diagnosis of E. hortense infection in a dog.

Feline demodicosis caused by an unnamed species.

A case of feline demodicosis is described in this report. A 13-year-old spayed female domestic short hair cat weighing 4.5 kg was being treated with cefovecin and alternately with prednisone or methylprednisolone. On further physical examination, the cat showed mild erythema and hair loss on the bridge of the nose, around the eyes, on the chin, on the side part of the breast and on the abdomen. A large number of Demodex mites were found in deep skin scrapings from the affected areas. The cat was then treated with ivermectin at 600 µg/kg administered SC daily. After 4 weeks of treatment, the cat was clinically normal with no mites detected in the skin scrapings from the face or breast areas. The mite responsible may represent a previously seen but as yet unnamed new species. This is third report that describes a case of feline demodicosis caused by a different, unnamed mite species that has different morphological characteristics to those of known Demodex mites and may represent a previously seen but as yet unnamed species.

N-acetylglucosaminyltransferase V-deficiency increases susceptibility to murine malaria.

It is considered that several glycoproteins on erythrocytes in mammalian species are involved in malaria parasite infection. To elucidate the role of N-glycans on malaria parasite infection, we induced experimental murine malaria infection (using Plasmodium berghei ANKA) in mice deficient in N-acetylglucosaminyltransferase V (Mgat5), which is one of the enzymes involved in ß1,6-GlcNAc N-glycan biosynthesis. After infection, Mgat5(-/-) mice showed severe body weight loss and parasitemia compared with wild-type mice. The Mgat5(-/-) mice, but not wild-type mice, also showed severe pathology accompanied by marked infiltration of plasma cells into the lungs and liver. These results suggest that ß1,6-GlcNAc N-glycans on/in host erythrocytes may interfere with invasion of the parasites and progression to severe malaria.

Seroprevalence of Toxoplasma gondii in wild boars (Sus scrofa leucomystax) and wild sika deer (Cervus nippon) in Gunma Prefecture, Japan.

The ingestion of undercooked meat from wild animals can be a source of Toxoplasma gondii infection in humans and other animals. In this study, we determined the seroprevalence of T. gondii infection in 175 wild boars (Sus scrofa leucomystax) and 107 wild sika deer (Cervus nippon) hunted in 2004-2007 in Gunma Prefecture, Japan, by using a commercial latex agglutination test (LAT). Antibodies (LAT, 1:64 or higher) to T. gondii were found in 6.3% of wild boars and 1.9% of sika deer. This is the first record of T. gondii infection in wild deer in Japan, and deer and wild boar meat should be cooked well before human consumption.

Seroprevalence of Toxoplasma gondii infection in feral raccoons (Procyon lotor) in Japan.

Antibodies to Toxoplasma gondii were found in 92 (9.9%) of 929 feral raccoons ( Procyon lotor ) in Japan with the use of the latex agglutination test (LAT, 1∶64 or higher). Seropositivity varied by geographic location, season, and weight of raccoons trapped. Seroprevalences in the northern, central, and western areas of Japan were found to be 7.9% (39/492), 16.5% (47/285), and 3.9% (6/152), respectively. The seroprevalence by season varied from 8.5% (13/153) in spring to 18.9% (14/74) in winter, which was significantly higher than those in other seasons (P < 0.05). Seroprevalence of T. gondii was elevated in accordance with the increase in body weight of raccoons ( r(s)  =  0.9), suggesting that the animals acquired the infection postnatally. The results suggest that the raccoon may serve as a useful indicator for the distribution of T. gondii in peridomestic environments in Japan.

Serological survey of five zoonoses, scrub typhus, Japanese spotted fever, tularemia, Lyme disease, and Q fever, in feral raccoons (Procyon lotor) in Japan.

We investigated the seroprevalence of five tick- or mite-borne zoonoses, scrub typhus (Orientia tsutsugamushi), Japanese spotted fever (Rickettsia japonica), tularemia (Francisella tularensis), Lyme disease (Borrelia afzelii and Borrelia garinii), and Q fever (Coxiella burnetii), in feral raccoons (Procyon lotor) captured in Hokkaido and Kanagawa Prefectures in Japan. Of the 559 raccoons captured in Hokkaido, 8 (1.4%), 3 (0.5%), 1 (0.2%), and 1 (0.2%) carried antibodies against O. tsutsugamushi (Gilliam type), F. tularensis, B. afzelii, and B. garinii, respectively. Of the 193 animals investigated in Kanagawa, 31 (16.1%) and 14 (7.3%) carried antibodies against O. tsutsugamushi and R. japonica, respectively, and the major serotype (27/31) of O. tsutsugamushi was Kuroki. No antibodies against C. burnetii were detected in either area examined. Therefore, feral raccoons could be an indicator of the prevalence of these four tick- or mite-borne zoonoses in the peridomestic environment in Japan.

Serological survey of arthropod-borne viruses among wild boars in Japan.

A total of 90 blood samples were collected from wild boars in the Kyushu region of Japan, and a seroepidemiological survey for 7 arthropod-borne viruses was performed using hemagglutination inhibition tests. The individual seropositive rates for each virus were 52.2% for Japanese encephalitis virus (JEV), 47.8% for Getah virus (GETV), 13.3% for Akabane virus, 10.0% for Aino virus and less than 5% for Bluetongue virus, Chuzan virus and Ibaraki virus. The results indicated that among the 7 viruses, JEV and GETV infections were prevalent among the wild boars and that the animals were involved in the natural transmission cycle of JEV and GETV in Japan. It is necessary to consider the participation of wild animals for the control of arthropod-borne virus infections.

The first report of phototaxis of fish ectoparasite, Argulus japonicus.

We discovered positive phototaxis in the fish ectoparasite, Argulus japonicus (Subclass Branchiura). Positive phototaxis of A. japonicus showed circadian rhythmicity. The phototaxis of A. japonicus has a unique circadian rhythm, which has been reported in no other crustacean. No phototaxis was observed at 4:00. After this time, however, A. japonicus gradually showed positive phototaxis until 16:00. There was no difference between male and female A. japonicus in this phototaxis. In addition, A. japonicus could distinguish between light of different colours, and showed positive phototaxis in the order blue light>yellow light>green light>red light.

Detection of DNA closely related to 'Candidatus Rickettsia principis' in Haemaphysalis danieli recovered from cattle in Xinjiang Uygur Autonomous Region Area, China.

Tick DNA samples from cattle in Xinjiang Uygur Autonomous Region Area, China, were examined for Rickettsia infection by citrate synthase gene-based PCR and sequencing. Four positive samples were detected from Haemaphysalis danieli and high levels of similarity were found with recently detected 'Candidatus Rickettsia principis.'

Serological survey of Ehrlichia and Anaplasma infection of feral raccoons (Procyon lotor) in Kanagawa Prefecture, Japan.

Numbers of feral raccoon; the possible reservoir animal of Ehrlichia and Anaplasma, are increasing in Japan. Thus serological methods were utilized to examine Ehrlichia and Anaplasma infection in raccoons from Kanagawa Prefecture, Japan. By using an indirect immunofluorescence assay, among 187 feral raccoons examined, 1 (0.5%) serologically reacted with Ehrlichia canis, 3 (1.6%) with Ehrlichia chaffeensis and 1 (0.5%) with Anaplasma phagocytophilum with the titers of 1:40 or more. Although screening PCR for Ehrlichia and Anaplasma species failed to detect the presence of ehrlichial DNA in serum samples, results of the serological tests suggested that the feral raccoons might be infected with some species of Ehrlichia and Anaplasma.

Two Eimeria species isolated from wild Japanese rock ptarmigans (Lagopus mutus japonicus) in Japan.

Fecal samples were collected from 64 Japanese rock ptarmigans (Lagopus mutus japonicus) at 13 locations in the alpine zone of Japan and examined for internal parasites. We found 2 morphologically different types of eimerian oocysts. Based on morphological characteristics, one was identified as Eimeria uekii, which had already been found from Japanese rock ptarmigans in 1981, and the other was likely a new type of Eimeria. The new type of eimerian oocysts required a longer sporulation time than E. uekii. The prevalence of both eimerian oocysts in Japanese rock ptarmigans was 62.5%, while that of E. uekii was 60.9% and the new type 29.7%. Mixed infection of both types was found in 28.1%.

First record of Leptospira borgpetersenii isolation in the Amami Islands, Japan.

In 2003, a Leptospira survey was performed on Yoroshima Island of the Amami Islands located in the southwestern part of Japan. Seven Leptospira strains were isolated from the field rat Rattus rattus, which were identified as L. borgpetersenii by flaB sequencing, 16S rDNA sequencing and gyrB sequencing, and serovar Javanica was determined by a microscopic agglutination test. NotI-long restriction fragment analysis indicated that these isolates were genetically indistinguishable from an isolate from the Okinawa Islands. The present results suggest that L. borgpetersenii is migrating into the Amami Islands in Japan.