A multiplex PCR for differential detection of Echinococcus granulosus sensu stricto, Echinococcus multilocularis and Echinococcus canadensis in China.

BACKGROUND: Echinococcosis caused by Echinococcus is one of the most major infectious diseases in north-west highland of China. E. granulosus sensu strict, E. multilocularis, and E. canadensis are known to be the only three species related to human health transmitting in the areas. To achieve targeted treatment and control of echinococcosis, the accurate identification and discrimination of the species are important. However, currently the available diagnostic approaches do not present ideal results either in accuracy or efficiency. METHODS: In the study, a set of primers were designed to aim at the three human-pathogenic Echinococcus species in China. The one-step multiplex PCR assay was developed and evaluated for the specificity and sensitivity. A total of 73 parasitic lesions and 41 fecal materials obtained from human and various animals collected in the clinic and the field were tested to assess the applicability of this method. RESULTS: The multiplex PCR effectively detected the individual DNA from the targeted species and their random mixtures generating with distinguishable expected size of products. The detection limit of the assay for each of the three species was 5 pg/µl when they were tested separately. When DNA mixtures of the targeted species containing the same concentration were used as templates, the lowest amount of DNA which can be detected was 50 pg/µl, 10 pg/µl and 5 pg/µl for E. granulosus s. s., E. multilocularis, and E. canadensis respectively. No cross-reactivity was observed when DNA from eight genetically close species was used as control templates. The multiplex PCR identifications of all samples were in line with the original sequencing results except for those infected with E. shiquicus, which showed negative signals in the developed assay. Of all the tested stool materials, 16 were previously found positive for Echinococcus by visual and microscopic examination. Among these 16 samples, 13 were confirmed by the multiplex PCR, and the other three tested negative. Additionally, the multiplex PCR identified another 14 positive feces from the remained 25 stool samples which absence of worms. CONCLUSIONS: The developed multiplex PCR shows advantages in fast diagnosis and large-scale epidemiological investigation, which proven to be a promising tool utilized in clinic and surveillance system.

[A Social Network Perspective on the Transmission of Cysticercuscellulose Infections in Tibetan School Children in Sichuan].

OBJECTIVE: To determine the transmission characteristics of Cysticercuscellulose infections from a social network perspective in Tibetan school children in Sichuan. METHODS: A cluster sampling strategy was adopted to select two primary schools with high level of Cysticercuscellulose infections in the Tibetan agriculture areas of Liangshan prefecture, Sichuan province. All of the students from the selected schools were enrolled in the study. Their social network data, including classroom seating, dormitory roommates, best playmates, and those who shared meals and snacks etc, were collected by trained investigators. Stool and blood samples of the students were collected for parasite detection. The transmission network of Cysticercuscellulose infections and the overall social network of school children were analysed. RESULTS: A total of 644 children participated in the study. Taenia solium were found in 6.11% of the stool samples and 13.25% blood samples returned with seropositive. The transmission was centered around the sources of infections: dormitory-clustering in the boarding school and playmate-clustering in the day school. The overall social network analysis revealed "core people" (more relationships), "information disseminators" (closer to other nodes) and "information hubs" (between two nodes) in both schools. CONCLUSION: Close contacts in dormitories and playgrounds are the main sources of transmission of cysticercosis in the Tibetan schools. The "core people" "information disseminators" and "information hubs" are critical for the prevention and control of cysticercosis in the future.

[Endemic situation and control progress of taeniasis in western China].

Taeniasis, caused by Taenia species, is one of the common zoonoses in China, particularly in the western region of China. Up to now, not enough attention has been given in the high prevalence and high burden of the diseases. In order to study the endemic patterns and control strategies of taeniasis, a series of epidemiological investigations, molecular researches and pilot control activities have been conducted in recent years. This paper reviews the relevant publications in taeniasis research over the last 10 years.

[Production of a monoclonal antibody specific to protoscolex of Echinococcus multilocularis].

OBJECTIVE: To prepare monoclonal antibody (McAb) specific to protoscolex of Echinococcus multilocularis. METHODS: BALB/c mice were immunized with crude antigen derived from E. multilocularis metacestodes. Spleen cells from immunized BALB/c mice were fused with SP2/0 myeloma cells by using hybridoma technique. ELISA and immunohistochemical staining were used to select hybridomas that secreted McAb P325 which especially against protoscolex. The number of metaphase chromosomes of hybridoma cells was counted. Characteristics of McAb P325 were identified by ELISA and immunohistochemical staining. RESULTS: One hybridoma cell clone secreting McAb against protoscolex was obtained. The number of metaphase chromosomes found in hybridoma cells was 98, which showed the characteristics of their parents. Immunohistochemical analysis showed that McAb P325 demonstrated binding activity to the germinal layer and protoscolex of E. multilocularis, especially to the hooklets and suckers, while did not bind with E. granulosus metacestodes and Cysticercus tenuicollis. CONCLUSION: The McAb is a valuable tool for immunohistochemical analysis, cell classification of E. multilocularis protoscolex, and study of specific antigen.

Epidemiology and risk factor analysis for canine echinococcosis in a Tibetan pastoral area of Sichuan.

OBJECTIVE: To determine the prevalence and evaluate the risk factors of canine echinococcosis based on a field survey of dog infections with Echinococcus granulosus and E. multilocularis in Chalong, Kalong, Dade and Chazha Townships in a district of Ganzi County, Sichuan Province, China. METHOD: Questionnaire associated with the acquisition of canine echinococcosis was administered to dog owners. Stray dogs were examined post-mortem and rectal faeces at necropsy were collected to validate a copro-antigen ELISA. Owned dogs were screened for Echinococcus spp. infection in faeces using the genus specific copro-ELISA and the effectiveness of dog treatment was assessed. Chi-square and one-way ANOVA were used for statistical analysis. RESULTS: The prevalence of Echinococcus spp. infection at necropsy in stray dogs was 60.9% (14/23) in 2000; E. multilocularis infection accounted for 34.8% (8/23) and E. granulosus for 26.1% (6/23). The specificity of the copro-ELISA was 80.0% and the sensitivity was 92.3%, compared with the results at necropsy. Fifty percent of owned dogs (290/580) tested was copro-antigen positive at the beginning of the project in 2000, which decreased to 17% (99/580) in the same cohort of owned dogs after praziquantel treatment (5 mg/ kg) at 6-monthly period from 2003 to 2005. Analysis for risk factors associated with copro-antigen positive dogs showed that the never tethered dogs had a higher rate (40.4%, 65/161) than dogs tethered during the day (32.3%, 109/337), or tethered at night [29.2% (21/72)], or those always tethered [20%(2/10)](P<0.01). Dogs that their owners lacked hydatid transmission knowledge [38.1% (121/318)] and did not have de-worming practice [47.7% (92/193)] had significantly higher copro-antigen positive rate than those dogs that their owners knew relevant knowledge [28.6% (75/262)] and were dewormed regularly [20.4% (79/387)](P<0.05 and P<0.01). There was no correlation between the prevalence and dog sex or age or the varieties of livestock that the owner raised. CONCLUSION: Local dogs show high prevalence with both E. granulosus and E. multilocularis. The copro-ELISA can be used to detect infection of Echinococcus in dogs. Allowing dogs to roam, lack of the basic knowledge of hydatid disease transmission and no de-worming practice for dogs are significant factors for the transmission of canine echinococcosis.

[Biological features of a new Echinococcus species (Echinococcus shiquicus) in the east of Qinghai-Tibet Plateau].

In the eastern Qinghai-Tibet Plateau region, a variety of domestic and wild mammals are involved in the transmission cycles of Echinococcus species. E. granulosus and E. multilocularis are known being sympatrically distributed in the plateau region. Recently, an unknown Echinococcus species was isolated from infected plateau pika (Ochotona curzoniae) and Tibetan fox (Vulpes ferrilata). The species shows quite distinct characteristics on morphology, genetics, host specificity and geographical distribution from others. It was therefore identified as a new Echinococcus species, Echinococcus shiquicus. This paper discussed the biological genetics and epidemiological features of the species, and proposed hypotheses and considerations for further exploration.

The Tibetan hare Lepus oiostolus: a novel intermediate host for Echinococcus multilocularis.

Multicystic hydatids have been found in the livers of hares ( Lepus oiostolus) examined from the Qinghai-Tibet plateau of China. In this study, the causative species was definitively identified as Echinococcus multilocularis by mitochondrial DNA sequencing. This is the first confirmation of larval E. multilocularis from hares. The hydatids contained protoscolices, suggesting that the hare may contribute to the transmission of E. multilocularis on the Tibetan plateau.