Development of genomic resources for Wenchengia alternifolia (Lamiaceae) based on genome skimming data.

Wenchengia alternifolia (Lamiaceae), the sole species of the genus Wenchengia is extremely rare and is currently listed as Critically Endangered (CR) on the IUCN Red List. The species had long been considered endemic to Hainan Island, China and was once believed to be extinct until a small remnant population was rediscovered at the type locality in 2010. Four more populations were later found on Hainan and in Vietnam. In order to develop genomic resources for further studies on population genetics and conservation biology of this rare species, we identified infraspecific molecular markers in the present study, using genome skimming data of five individuals collected from two populations on Hainan Island and three populations in Vietnam respectively. The length of plastome of the five individuals varied from 152,961 bp to 150,204 bp, and exhibited a typical angiosperm quadripartite structure. Six plastid hotspot regions with the Pi > 0.01 (trnH-psbA, psbA-trnK, rpl22, ndhE, ndhG-ndhI and rps15-ycf1), 1621 polymorphic gSSRs, as well as 1657 candidate SNPs in 237 variant nuclear genes were identified, thereby providing important information for further genetic studies.

[An Epidemiological Survey on Echinococcosis in Yushu Prefecture of Qinghai Province].

Objective: To investigate the prevalence of echinococcosis in Yushu Prefecture of Qinghai Province in 2012. Methods: Two to three towns were selected in each of Chengduo, Nangqian, Qu malai, Yushu, Zaduo and Zhiduo Counties from June to August in 2012. Ultrasound examination was conducted for residents aged over 1 year, and ELISA was performed to detect serum antibody against Echinococcus. Visceral dissection was performed to detect hydatid infection in rodents and livestock. ELISA was used to detect Echinococcus antigen in collected dog feces. Results: A total of 7 025 residents received ultrasound examination, of whom 319 showed hydatid cysts with a morbidity rate of 4.54%. ELISA showed a serum antibody positive rate of 16.38% （457/2 790）. The mobidity of hydatid disease was highest in Chengduo County （7.41%, 181/2 444）, and the rate of serum antibody was highest in Yushu County （23.18%, 127/548）. The morbidity and serum antibody in males were 3.91% （118/3 018） and 13.93% （172/1 235） respectively, and those in females were 5.02% （201/4 007） and 18.33% （285/1 555）. In terms of age distribution, the morbidity was relatively higher in residents of 60- （8.39%, 38/453） and 40- years （6.61%, 67/1 014）; and the rate of serum antibody was highest in residents over 70 years （33.93%, 19/56）. In terms of occupation, the morbidity was relatively higher in herdsmen （5.28%, 252/4 777）, Herdsmen-peasants （6.52%, 24/368）, and religious workers（3.37%, 11/326）, while the rate of serum antibody was relatively higher in children（24%, 6/25）, religious workers （18.79%, 31/165） and herdsmen（18.34%, 328/1 788）. In terms of education level, the morbidity and the rate of serum antibody were both highest in the uneducated（5.04%, 41/4 779; 18.34%, 359/1 958, respectively）. In terms of residential pattern, the morbidity and the rate of serum antibody were both highest in those who were settled in winter and nomadic in summer （8.25%, 227/2 753; 19.48%, 158/811, respectively）. There were significant differences in the morbidity and the rate of serum antibody in aspects of residential region, sex, age, occupation, education level and residential pattern (P<0.05 or P<0.01). In 872 rodents detected, the Echinococcus hydatid rate was 0.46% （4/872）, while in 809 cattle and sheep detected, the Echinococcus hydatid rate was 10.14% （82/809）. The fecal antigen positive rate in 838 samples of dog feces was 10.74%（90/838）. Conclusion: It shows a high morbidity of hydatid diesease and serum antibody positive rate in residents, a high Echinococcus hydatid rate in cattle and sheep, and a high fecal antigen positive rate in dogs in Yushu Prefecture.

Molecular identification of Taenia mustelae cysts in subterranean rodent plateau zokors (Eospalax baileyi).

Cestode larvae spend one phase of their two-phase life cycle in the viscera of rodents, but cases of cestodes infecting subterranean rodents have only been rarely observed. To experimentally gain some insight into this phenomenon, we captured approximately 300 plateau zokors (Eospalax baileyi), a typical subterranean rodent inhabiting the Qinghai-Tibet Plateau, and examined their livers for the presence of cysts. Totally, we collected five cysts, and using a mitochondrial gene (cox1) and two nuclear genes (pepck and pold) as genetic markers, we were able to analyze the taxonomy of the cysts. Both the maximum likelihood and Bayesian methods showed that the cysts share a monophyly with Taenia mustelae, while Kimura 2-parameter distances and number of different sites between our sequences and T. mustelae were far less than those found between the examined sequences and other Taeniidae species. These results, alongside supporting paraffin section histology, imply that the cysts found in plateau zokors can be regarded as larvae of T. mustelae, illustrating that zokors are a newly discovered intermediate host record of this parasite.

Age-related infection with Cryptosporidium species and genotype in pigs in China.

OBJECTIVE: Pigs, as hosts of zoonotic Cryptosporidium species/genotypes, are domestic animals with public health significance. The present study was to characterize the infection rate and species/genotype of Cryptosporidium in pre-weaned and post-weaned pigs from Shanghai and Shaoxing, China. METHODS: A total of 208 fecal samples (42 from pre-weaned piglets, and 166 from post-weaned pigs) were examined by nested PCR of the 18S rRNA gene and analyzed by phylogenetic DNA fragment sequencing of secondary PCR products. RESULTS: Infection was detected in 79 samples (19/42 pre-weaned piglets, and 60/166 post-weaned pigs). C. suis (14/79) and Cryptosporidium pig genotype II (65/79) were identified; piglets were more susceptible to the former (13/14) and post-weaned pigs to the latter (59/65). CONCLUSION: Infection of Cryptosporidium spp. in pigs was age-specific; piglets were more susceptible to C. suis while pigs were more susceptible to Cryptosporidium pig genotype II. These findings combined with the isolation of the two Cryptosporidium from water suggest that pigs may be a source of zoonotic Cryptosporidium water pollution. Improvements in pig feeding practices, sewage discharge, feces disposal and field worker protection are therefore important to prevent potential public health problems.

[Survey on a new case of visceral leishmaniasis in Shanxi Province].

A new case of visceral leishmaniasis (kala-azar) was reported by Changzhi CDC of Shanxi Province in September 2011. The case was investigated clinically and epidemiologically. The patient was a two-year-old boy who lived in Huangnian Town of Changzhi County in Shanxi Province. Clinical examination showed hepatosplenomegaly, consistent decrease of blood cells and Leishman-Donovan body in the bone marrow smear. The rK39 immune diagnosis test showed strongly positive. The case was diagnosed as kala-azar. After one course treatment of sodium stibogluconate, the patient's condition improved markedly. There were no cases of kala-azar in this region historically. Blood samples of 17 individuals and 5 domestic animals including 3 dogs were all negative in the rK39 immunodiagnostic test. It is speculated that the potential risk of kala-azar transmission exists in this region.

[Geographical distribution of echinococcosis among children in Qinghai Province].

OBJECTIVE: To analyze the epidemiological status of echinococcosis among children in three different zones of Qinghai Province. METHODS: B-ultrasound and ELISA were used in the survey to evaluate echinococcus infection among 6 to 15 year old children in the three zones, namely, Qinghai southern plateau, Qilian mountain-Hehuang valley and Chaidamu basin. RESULTS: The prevalence of echinococcus infection scanned by B-ultrasound and sera positive rate detected by ELISA in children were 1.5% (320/20 730) and 6.5% (1024/15 762) respectively, and the prevalence of cystic echinococcosis and alveolar echinococcosis by ultrasound were 1.0% (206/20730) and 0.6% (114/20730), respectively. The prevalence and sera positive rate were 9.5% (707/7 453) and 2.3% (269/11 618) in Qinghai southern plateau, 3.8%(289/7 544) and 0.6% (50/8 275) in Qilian mountain-Hehuang valley, and 3.7% (28/765) and 0.1% (1/837) in Chaidamu basin, respectively. The southern plateau showed the highest prevalence, with a significant statistical difference among the three areas (P < 0.01). The prevalence of alveolar echinococcosis was 1.0% (114/11 618) in the southern plateau, but no alveolar echinococcosis patients were found in other two areas. Correlation analysis showed that the prevalence increased with the altitude (r(s-e) = 0.96, P < 0.05, R2 = 0.93; r(p-e) = 0.82, P < 0.05, R2 = 0.67). CONCLUSION: The prevalence of echinococcosis among children shows an apparent geographical difference in Qinghai Province.

[Cloning, expression and immunodiagnostic evaluation of antigen EPC1 from Echinococcus granulosus].

OBJECTIVE: To clone and express EPCl gene of Echinococcus granulosus, and investigate its immunogenicity and diagnostic value. METHODS: Total RNA was extracted from hydatid cyst protoscoleces and EPC1 gene of Echinococcus granulosus was amplified by RT-PCR. The PCR product was cloned into pGEM-T vector, and then subcloned into the prokaryotic expression vector PET28a(+). The positive recombinants were transformed into Escherichia coli BL21 (DE3), and followed by expression of the protein induced by IPTG. The recombinant protein was identified by SDS-PAGE and Western blotting, and used to establish ELISA. Serum samples from patients with cystic echinococcosis (60 cases), alve-olar echinococcosis (37 cases), cysticercosis (16 cases), clonorchiasis sinensis (7 cases), schistosomiasis japonica (4 cases) and healthy persons (33 cases) were examined. RESULTS: The recombinant plasmid PET28a-EgEPC1 was identified by restriction enzyme digestion and sequencing. SDS-PAGE result showed that the recombinant containing recombinant plasmid PET28a-EgEPC1 expressed a soluble fission protein of EgEPC1 (about M, 11 000). The protein was recognized by pool sera of cystic echinococcosis patients. The overall sensitivity and specificity of diagnosis by ELISA for cystic echinococcosis were 78.3% (47/60), and 98.3% (59/60), respectively. The cross reaction with sera of alveolar echinococcosis was 40.5% (15/37). CONCLUSION: The recombinant EgEPC1 antigen has diagnostic value in cystic echinococcosis.

[Epidemiological survey on echinococcosis in Darlag County of Qinghai Province].

OBJECTIVE: To make an epidemiological investigation on echinococcosis in Darlag County, Guoluo Tibetan Autonomous Prefecture of Qinghai Province. METHODS: Local participants were screened for echinococcosis using B ultrasound from August to September in 2007. IHA was carried out to detect antibody in human sera. Indirect ELISA using recombinant-AgB and Em18 antigen was conducted to further diagnose the human alveolar echinococcosis (AE) and cystic echinococcosis (CE) . The prevalence of echinococcosis in wild rodents, yaks, sheep and dogs was examined either by dissection or through slaughterhouses. Adult worms and hydatid cysts were detected by PCR-RFLP to discriminate 3 Echinococcus species and genotype of Eg. Dog feces were tested by double antibody sandwich ELISA method to confirm the coproantigen positive rate. RESULTS: 1723 people were examined by ultrasound and 236 were clinically diagnosed as hydatid diseases with an overall morbidity of 13.7%, of which 5.5% were CE and 8.2% were AE. Prevalence in males and females was 11.6% and 15.6% respectively with a statistically significant difference (chi2=7.0, P< 0.05) . Prevalence in herdsmen and monks/nuns was as high as 23.0% and 13.2%, respectively. Adult worms were found in 5 out of 9 stray dogs by necropsy. 27 of 239 pikas (Ochotona curzoniae) were infected by E. shiquicus (11.3%). CE prevalence in sheep and yaks was 26.4% and 5/16 respectively. The Eg genotype was G1 (sheep strain). CONCLUSION: Three species of Echinococcus, e.g. E. granulosus, E. multilocularis and E. shiquicus are all prevalent in Darlag County. The prevalence of both AE and CE in human population is high in the area. Domestic dogs are the primary definitive host for both E. granulosus and E. multilocularis.

[An epidemiological survey on echinococcosis in Zhiduo County of Qinghai Province].

The survey was carried out in July, 2006 in Zhiduo County. The IHA and ELISA positive rate in human population was 4.5% (42/933) and 8.2% (76/931) respectively. Ultrasonography revealed a morbidity of 3.4% (33/979) with 3.2% Echinococcus granulosus and 0.2% of E. multilocularis respectively. Animal dissection showed an infection rate of 15.1% (14/93) in pikas with one infected by E. shiquicus proved by molecular biology. Coproantigen rate by ELISA was 62% (12/193) in dogs and 35.7% (5/14) in wolves. The results indicated that Zhiduo County is a mixed endemic area for echinococcosis.

Simultaneous determination of 109 pesticides in unpolished rice by a combination of gel permeation chromatography and Florisil column purification, and gas chromatography/mass spectrometry.

A multi-residue method is described for the simultaneous analysis of 109 pesticides with different properties in unpolished rice. The range covers organophosphorus, organochlorine, carbamate, and synthetic pyrethroid pesticides. The pesticides were extracted from the sample using ethyl acetate. Most higher molecular weight components such as lipids in the co-extractives were removed by gel permeation chromatography (GPC) with a Bio-bead SX-3 column. A Florisil column with ethyl acetate/hexane as the eluting solvents was used for further cleanup. The pesticides were finally simultaneously determined by gas chromatography/mass spectrometry (GC/MS) in selective ion monitoring (SIM) mode. The average recoveries for most pesticides (spiked level 0.02, 0.1 and 1 microg/g) ranged from 70% to 110%, the relative standard deviation (RSD) was below 20% in every case, and the limit of detection (LOD) varied from 1 to 20 ng/g.