Analysis of antibody responses by commercial western blot assay in horses with alveolar echinococcosis.

Commercial western blot (WB) assay was used to detect serum antibodies specific to Echinococcus multilocularis in 23 horses in which infection was confirmed by postmortem inspection at a slaughterhouse. Livers contained from 1 to >20 nodular lesions; foci diameter ranged from 1 to 25 mm. Antibody tests of serum from all 23 animals were negative for antigen bands at 7, 16, 18, and 26-28 kDa, which show specificity in the serum of human patients. However, sera from two infected horses with the largest nodules (diameter, 25 mm) showed positive response to one of the 22-kDa and 30-kDa antigen bands. It may be possible to diagnose E. multilocularis infection in horses based on the detection of these bands on commercial WB assay.

[Prevalence of intestinal canine and feline parasites in Saitama Prefecture, Japan].

We studied the prevalence of intestinal parasites in animal companions in Saitama Prefecture, Japan, where no detailed data is currently available. Between May 1999 and December 2007, fecal samples were collected from 906 dogs and 1,079 cats in public animal shelters and examined by microscopy. Overall, prevalence of intestinal parasites in dogs was 38.6% and cats 43.1%. Trichuris vulpis was the most prevalent canine parasite species (22.3%), followed by Toxocara canis (12.5%), Ancylostoma caninum (10.4%), Isospora ohioensis (2.1%), Spirometra erinaceieuropaei (1.0%), Crypstosporidium sp. (0.9%), Giardia intestinalis (0.9%), I. canis (0.6%), Taeniidae (0.3%), Dipylidium caninum (0.2%), Echinostoma sp. (0.1%), and Pentatrichomonas hominis (0.1%). T. cati was the most prevalent feline parasite species (21.8%), followed by A. tubaeforme (13.2%), S. erinaceieuropaei (8.3%), I. felis (4.5%), Cryptosporidium sp. (2.8%), I. rivolta (2.2%), Pharyngostomum cordatum (1.6%), D. caninum (1.4%), Eimeria sp. (0.3%), Taeniidae (0.2%), Trichuris sp. (0.2%), Capillaria sp. (0.1%), Diphyllobothrium nihonkaiense (0.1%), and Metagonimus yokogawai (0.1%). Further molecular analysis to identify canine Taeniidae species and canine and feline Cryptosporidium species identified one canine taeniid positive species as Echinococcus multilocularis. Cryptosporidium species were identified as C. canis and C. felis. Parasites E. multilocularis and Cryptosporidium spp. in animal hosts were the first to be recorded in this prefecture. Compared to previous surveys conducted in the same area, the endemicity of some parasites appeared to have decreased, but some others remain. Given that most of these parasites have zoonotic potential, indicates the importance of having current data on parasite dissemination among animal companions. Government public health agencies should be responsible for educating pet owners about the control and prevention of zoonotic risk from such parasites.

Cystic and alveolar echinococcosis: an epidemiological survey in a Tibetan population in southeast Qinghai, China.

An epidemiological investigation on echinococcosis was made in Jiuzhi County of Qinghai Province, western China. Ultrasonography and an indirect hemagglutination test revealed a morbidity of 8.0% (124/1,549) and a seroprevalence of 25.8% (287/1,113), respectively, in the Tibetan population. The morbidity in herdsmen (16.6%) and Buddhist priests (15%) was significantly higher than that in other occupation groups (3.2%), and it was higher in females (9.8%) than in males (6.2%). The ultrasound images showed a coexistence of cystic echinococcosis (CE) and alveolar echinococcosis (AE), occupying 69 and 31% of the cases, respectively. An Echinococcus Western blot assay was performed as a serological backup test for differentiating CE and AE. The assay revealed that serum samples from most cases with a positive AE image showed a specific antibody against antigen bands at 16/18 kDa. Autopsy proved that 9 out of 12 stray dogs were infected with Echinococcus granulosus (n = 8) and E. multilocularis (n = 1). Inspection at the abattoirs demonstrated a hydatid rate of 78.5% in yaks and 82.6% in sheep. The data indicate that Jiuzhi County is an important endemic area for both CE and AE, in both human and animal populations.

Application of multiplex pcr for species discrimination using individual metacercariae of Paragonimus occurring in Thailand.

A total of 6 lung fluke species have been documented in Thailand, of which P. heterotremus is the most important, since it affects humans. Although P. westermani is found as metacercariae in the same crab species as P. heterotremus in Thailand, human infections with P. westermani have not been confirmed. To accurately discriminate between the individual metacercariae of these two species, we established a multiplex PCR method. Using this method, two products each were amplified from the metacercarial DNA samples of P. heterotremus (ca. 310 and 520 bp) and P. westermani (ca. 140 and 520 bp). In contrast, 520-bp products alone were found to be generated from the DNA samples of P. siamensis, P. bangkokensis and P. harinasutai, 3 other species of lung flukes known to occur in Thailand. Digestion of these 520-bp products with the restriction enzyme ScrFI could unequivocally discriminate species by the number and size of the produced band(s): 3 bands (ca. 60, 210 and 250 bp) for P. harinasutai, 2 bands (ca. 250 and 270 bp) for P. bangkokensis, and an uncut band (520 bp) for P. siamensis. The established multiplex PCR used in combination with restriction enzyme digestion (PCR-RFLP with ScrFI) is effective for discriminating the 5 different species of lung flukes occurring in Thailand, even at the metacercarial stage.

Investigation on the epidemiological factors of Clonorchis sinensis infection in an area of south China.

To detect the epidemiological factors of Clonorchis sinensis infection in Hengxian County, one of counties in Guangxi Zhuang Autonomous Region where Schistosoma japonicum was endemic but eliminated in the late 1980s, a questionnaire was designed with 37 questions covering socioeconomic conditions and human behavior, contamination of the environment and fish ponds, inadequate farming/fishery practices; and the formalin-ether sedimentation technique was used to examine the feces of cats, dogs and pigs for the eggs of C. sinensis. Fresh fish was sliced and digested to detect the metacercariae of C. sinensis under a stereomicroscope. Of 1,521 people interviewed, 64% of the interviewees did not know about fluke disease or its transmission route, 46% of those who knew about the fluke believed that the infection caused no harm or only slight harm to their health. More than half of the interviewees (51%) ate raw fish at least 1-2 times per month, more among the middle-aged males. Eight percent of them used the same utensils for both raw fish and cooked food. When advice was given not to eat raw fish, 73% of the interviewees thought it was not feasible. In relation to pisciculture, 25% and 9% of the owners of fish ponds fed their fish with feces of domestic animals and human feces, respectively. The prevalences of C. sinensis infection in cats, dogs and pigs were 70, 50 and 27%, respectively, and the infection rate in fish was 40%. These results indicate the poor knowledge, and beliefs, and unhealthy behaviors in the human population. They also show poor environmental hygiene and inappropriate farming/fishery practices are important in the increase of C. sinensis prevalence in humans. Combined interventions, including health education, environmental modification, reform of traditional farming/fishery practices, mass screening and chemotherapy for humans, and the management of domestic animals are needed in developing control strategies to decrease C. sinensis infection in the human population.

Molecular discrimination between individual metacercariae of Paragonimus heterotremus and P. westermani occurring in Thailand.

To accurately discriminate between individual metacercariae of Paragonimus heterotremus and P. westermani occurring in Thailand, polymerase chain reaction (PCR)-based molecular methods were established and subjected to an evaluation. We first amplified and sequenced the second internal transcribed spacer (ITS2) region of the nuclear ribosomal DNA of the two species. Based on their nucleotide differences, P. heterotremus and P. westermani were unequivocally discriminated from each other. These nucleotide differences were further utilized to select the ApaL1 endonuclease site for PCR-restriction fragment length polymorphism (PCR-RFLP) analyses and to design species-specific primers for multiplex PCR reactions. Both PCR-RFLP and multiplex PCR methods allowed a more rapid and labor-effective species discrimination. Furthermore, the multiplex PCR method enabled the most efficient discrimination because species identification involved a single round of PCR in a single tube. In Thailand, P. heterotremus is the only species affecting humans. Thus, the methods established in the present study can be used as reliable tools to identify the lung fluke metacercariae that cause human disease.

Paragonimus ohirai metacercariae in crabs collected along the Arakawa River in Tokyo, Japan.

Brackish water crabs infected with Paragonimus ohirai metacercariae have been reported in various regions in Japan. However, infected crabs have not been identified in Tokyo. We therefore collected the crab, Chiromantes dehaani, between August 2002 and July 2003 from 12 locations along the Arakawa River that flows through Tokyo. Of the 922 captured crabs, 177 (19%) from 6 locations were infected with Paragonimus metacercariae. The prevalence of metacercariae at these 6 locations ranged from 5 to 89%. The number of metacercariae per infected crab ranged from 1 to 190, with an average of 13.1. The morphological features of the metacercariae and of adult worms recovered from test rats infected with metacercariae showed that the metacercariae in the infected crabs were P. ohirai Miyazaki, 1939. The ITS2 sequence data support this conclusion. This paper is the first description of P. ohirai infection of crabs in Tokyo.

[Laboratory evaluation of commercial immunoblot assay kit for serodiagnosis of Echinococcus infections using sera from patients with alveolar hydatidosis in Hokkaido].

Using serum specimens from patients with alveolar hydatidosis (AH) in Hokkaido, we assessed the usefulness of "Echinococcus Western Blot IgG" (the French immunoblot assay, FIA), which has recently been launched from Ldbio Diagnostics (Lyon, France) as new commercial immunoblot assay kit of immunodiagnosis of Echinococcus infections. Eighty serum specimens were used for the present study: 64 preoperative sera and nine postoperative sera, which were taken from AH patients in Hokkaido, and seven sera from persons who were ELISA (enzyme-linked immunosorbent assay)--positive in mass screening which was conducted for checking on Echinococcus infections in Hokkaido since 1982. When the 64 preoperative sera were examined by the Western blotting method (the Hokkaido method of Western blotting, HWB) which had been carried out at Hokkaido Institute of Public Health between 1987 and 1993, it was found that 53 cases were positive and six cases were quasi-positive, i.e. the rate of the positive cases including quasi-positive cases was 92.2%. From immunostaining patterns, HWB-positive sera could be grouped in two types: the complete type, which showed a pattern of multiple bands containing the 55 and 66 kDa bands, and the incomplete type, which showed patterns of only few bands containing the AH-specific polysaccharide antigen named C antigen. Forty-three of the 53 HWB-positive sera were of the complete type and the residue was of the incomplete type. On the other hand, when the 64 preoperative sera were examined by FIA, 60 sera (93.8%) were judged to be positive and the others as negative sera. On the basis of the interpretation of immunostaining patterns described in the instruction manual, 47 (78.3%) of the 60 positive sera were regarded as pattern P3, five (8.3%) as pattern P4, and eight (13.3%) as pattern P5. All of the complete-type sera were regarded as P3, indicating high antibody titers. Contrarily, most of the incomplete-type or quasi-positive sera resulted in other patterns such as P4 and P5, indicating low antibody titers. Of 5 HWB-negative sera, two were FIA-positive (which showed P3 and P5 patterns respectively), however their immunoreactions were significantly low. Therefore, apart from interpretation of pathological conditions of cases with exceedingly low antibody titers, FIA may be able to give a serologically clear interpretation to HWB-quasi-positive cases, indicating that it is a highly sensitive and useful method for immunodiagnosis of Echinococcus infections.

Epidemiological investigation on Clonorchis sinensis in human population in an area of South China.

To detect the prevalence of Clonorchis sinensis, one of the important helminthes in the human population of the Guangxi Region where Schistosoma japonicum was endemic but eliminated in the late 1980s, the Kato-Katz thick smear technique was used for examining fecal samples from selected townships in Hengxian County. Among 1,552 people examined, 491 (31.6%) were found infected with C. sinensis. By counting eggs per gram feces (EPG), it was found that the light, moderate, and heavy intensities of infection occupied 55.4, 33.0, and 11.6%, respectively, with an average EPG of 4,845 in the infected subjects. The survey revealed that the prevalence in the age groups of 0 - 9 and 10 -19 years old was less than 10% but was 45 - 50% in the groups between 30 - 39 and 60 - 69 years old. A much higher prevalence was demonstrated in the male population (41.9%) than in the females (20.5%), and heavier intensity of infection was also found in the males than in the females. These results indicated that the prevalence of this liver fluke is increasing in the past decade in that region, and there is an urgent need to further assess the epidemiological factors in reference to the area's changing socioeconomic conditions and human behavior, contamination of the environment and fish ponds, inadequate farming/fishery practices, and the infection of domestic animals.