New insights on the Taenia solium tapeworm using molecular tools: age-based human definitive host prevalence and deliberation on parasite life span.

Information on age-based Taenia solium taeniasis prevalence is crucial for control of cysticercosis. T. solium taeniasis prevalence was determined for a village in Liangshan Prefecture, Sichuan Province, China that was co-endemic for T. solium, Taenia saginata asiatica, and Taenia saginata. Individuals who were Taenia egg-positive by stool microscopy and/or expelled tapeworms or proglottids post-treatment were diagnosed as having taeniasis. Infecting species was identified via multiplex PCR on tapeworm specimens or coproPCR followed by sequencing. In addition, initial stool samples from 10 children with taeniasis suspected of having spontaneous expulsion of tapeworms within the period between diagnosis and treatment were subject to species confirmation via coproPCR and sequencing. Of the 389 study subjects, 194 (49.9%) were diagnosed with taeniasis. Children (< 16 years of age) had a higher T. solium taeniasis prevalence (8.8%) than older individuals (2.5%) (P = 0.0127). Molecular analysis of initial stool samples from 7 of 10 children suspected of spontaneously passing tapeworms indicated 6 infections due to T. solium and 1 infection due to T. saginata. This study found that young children had a higher T. solium taeniasis prevalence than older individuals, providing additional support for the belief that adult T. solium likely has a relatively short lifespan compared to other Taenia species with human definitive hosts.

Low genetic variation in Echinococcus multilocularis from the Western Sichuan Plateau of China revealed by microsatellite and mitochondrial DNA markers.

The prevalence of E. multilocularis is a major public health problem in China. To better understand the molecular epidemiology and evolutionary patterns of E. multilocularis, an adequate dataset regarding the genetic variance of this parasite is necessary. However, for now, available genetic data of E. multilocularis is still insufficient. In the study, the EmsB microsatellite and the partial mitochondrial cox1 gene were combined to investigate the genetic diversity of 64 E. multilocularis samples from human, dogs and voles. These samples were collected in the Western Sichuan Plateau, where the highest village-based human prevalence of alveolar echinococcosis was recorded worldwide. The aim of the study is to gather more informative genetic data of E. multilocularis in the areas, especially those obtained using the EmsB marker. The microsatellite analysis revealed 7 different EmsB profiles, 1 of which was found in 90.63% of the total samples collected from all 3 hosts. This major profile was identical to the one detected in the same area 16 years ago. The rest of the 6 profiles, each represented by only 1 isolate, did not correspond to any of the profiles previously reported. All the profiles detected in the study belonged to the Asian cluster. Meanwhile, according to sequence analysis of the 758 bp cox1 region, 4 haplotypes all assigned to the Asian clade were detected among the isolates. A star-like haplotype network was exhibited with a centrally positioned haplotype found in 93.75% of the samples. The overall haplotype and nucleotide diversities were both low. These findings provided evidence for a founder event or bottleneck and subsequent population expansion in E. multilocularis. The EmsB profiles were not fully consistent with the cox1 haplotypes. The same correspondence relationship was mainly observed in samples with the major profile P5 and the main haplotype EmHa1. A total of 54 isolates assigned to profile P5 were classified to the EmHa1 haplotype. In conclusion, both the microsatellite and mtDNA markers showed low variability within the Tibetan population of E. multilocularis. An EmsB profile and a cox1 haplotype were found to be predominant in the study area, which appears to remain steady for over a decade. The results reinforce the higher potential of the microsatellite DNA marker with high discriminative power to identify the very low genetic polymorphism of E. multilocularis than that of the partial cox1 sequencing. The data obtained in the study would be helpful to enlarge the data pool to further probe the possible origins and dispersal of E. multilocularis in China.

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.

Molecular characterization of human echinococcosis in Sichuan, Western China.

Cystic echinococcosis (CE) and alveolar echinococcosis (AE) are highly co-endemic in Sichuan, a part of Qinghai-Tibet Plateau where is a typical Tibetan nomadic community living area. In order to better understand the Echinococcus spp. of human being infected origins in this area, 140 lesions were collected from echinococcosis patients who were received operations during the period of 2014-2016 in different geographic districts in this region. Partial DNA sequences of the mitochondrial cox1 gene were analyzed. The genetic characterization of the isolates from 3 different places including Ganzi, Aba and Liangshan were assessed. Of all the 140 samples, the great majority was identified as Echinococcus granulosus sensu stricto (n = 108). Echinococcus multilocularis was confirmed to be another important pathogen of the human infections (n = 31). Additionally, one Echinococcus canadensis (G6/7) isolate from Ganzi was confirmed. Comparing the clinical diagnosis with the sequencing results, 6.4% (9/140) of the cases were misdiagnosed between AE and CE, and another 8.6% (12/140) were unclassified to sub-type in echinococcosis. Higher rates of misdiagnosis and unclassified diagnosis were found in AE cases (12.9%, 4/31 and 16.1%, 5/31 respectively) compared to CE (4.6%, 5/109 and 6.4%, 7/109 respectively). In E.granulosus s.s., a total of 34 haplotypes were detected, and 4 haplotypes were inferred from E.multilocularis. The haplotype networks of the 2 species exhibited a similar star-shaped feature with a dominant haplotype in the center. Geographically specific haplotypes were observed in Ganzi and Aba respectively. This study provides insight into the current species causing human echinococcosis in the Tibetan districts of Sichuan. E.granulosus s.s. and E.multilocularis are confirmed to be the main causative agents, and the existence of E.canadensis (G6/7) is also observed in the region. Molecular diagnosis was proven to be essential for the confirmation of human echinococcosis in the area.

Seasonal pattern of Echinococcus re-infection in owned dogs in Tibetan communities of Sichuan, China and its implications for control.

BACKGROUND: Human cystic echinococcosis (CE) and alveolar echinococcosis (AE) are highly endemic in Tibetan communities of Sichuan Province. Previous research in the region indicated that domestic dog was the major source of human infection, and observations indicated that domestic dog could have more access to intermediate hosts of Echinococcus spp.: both domestic livestock (CE) viscera and small mammals (AE), in early winter and again in spring. We hypothesized that there would therefore be a significant increase in the risk of canine infection with Echinococcus spp. in these two seasons and conducted a reinfection study to investigate this further. METHODS: Faecal samples were collected from owned dogs in seven townships in Ganze Tibetan Autonomous Prefecture (Sichuan Province, China), and Echinococcus spp. infection status was determined using copro-antigen ELISA. Dogs were sampled in April (spring), July (early summer), September/October (autumn/early winter) and December (winter) in 2009; and in April (spring) 2010. Dogs were treated with praziquantel following each of the five sample collections to eliminate any tapeworms. Information on dog sex, age and body weight was also collected. The t-test, Fisher's exact test, Poisson regression and logistic regression were used to compare means and prevalences, and to identify factors associated with infection status. RESULTS: The proportion of female dogs was significantly lower than that of male dogs; female dogs had significantly higher (22.78 %) baseline copro-ELISA prevalence than males (11.88 %). Dog body weight, sex, age, county and previous infection status at any sampling point had no influence on the re-infection prevalence in general. Poisson regression did not found a significant influence on the re-infection prevalence due to different deworming/sampling time spans. Dogs exhibited significantly higher re-infection prevalences in spring and early summer of 2009 and in early winter between September/October and December of 2009, suggesting a higher infection pressure in these seasons comparing with other seasons. CONCLUSION: Following praziquantel treatment, dog body weight, sex, age, county, deworming time span and previous infection status at any sampling point had no influence on the re-infection prevalence in the region in general. The differences between re-infection prevalences were probably due to the seasonality in Echinoccocus spp. infection pressure in the region. Early winter, spring and early summer should be important seasons for optimal dog deworming intervention in these Tibetan communities.

[Intervention effect of probiotic bifidobacterium on type 2 diabetes mellitus rats].

OBJECTIVE: To observe the influences of bifidobacterium on glucose and lipid metabolism and intestinal flora in type 2 diabetes mellitus rats. METHODS: Rats were randomly divided into 4 groups, the blank control group was fed with basic forage, the other 3 groups were fed with high fat diet. Meanwhile the high-dose and low-dose groups were gavage administrated with 12 and 4 ml/(kg x d) probiotic bifidobacterium for 6 weeks, then, injected with low dose of streptozotocin (20 mg/kg). All rats were sacrificed 2 weeks later, blood and feces samples were collected to analyse. RESULTS: Compared with rats of type 2 diabetes model, glucose concentrations and serum concentrations of FFA, TG, LDL-C of 2 experimental groups were significantly lower, serum concentrations of HDL-C were higher, insulin sensitivity index was higher, content of bifidobacterium was higher, but there were no significant differences in other 5 bacteria. CONCLUSION: The probiotic bifidobacterium could significantly ameliorate the disorder of glucose and lipid metabolism, increase the amount of bifidobacterium of type 2 diabetes rats.