Epidemiological Investigation of Yak (Bos grunniens) Fascioliasis in the Pastoral Area of Qinghai-Tibet Plateau, China.

The objective of this investigation was to understand the epidemiology of fascioliasis in yaks in the alpine pastoral areas of the Qinghai-Tibet Plateau, China. The prevalence of Fasciola hepatica infection was estimated by examining eggs in the feces of yaks and by autopsy after the slaughter. Yaks were sampled from a total of 16 representative counties in Qinghai province, and risk factors were assessed based on regional and age characteristics. Fecal samples were obtained from 1542 yaks aged 0-1 (<1 year old), 1-2 (≥1 year old and <3 years old), and over 3 years (≥3 years old). In addition, 242 yaks over 3 years old who had not undergone fecal examinations were randomly selected for autopsy. A total of 267 fecal samples were positive for Fasciola spp. eggs. The average infection rate was 17.32% (0-60.61%), and the average infection intensity was 51.9 eggs per gram (epg) of feces, with intensities ranging from 18 to 112 epg. In Maduo, Dari, Zhiduo, Chengduo, and Datong counties, the Fasciola spp. eggs infection rate was zero. Fasciola spp. adult flukes were detected in 66 out of 242 yaks at autopsy, with a total infection rate of 27.27% and an average infection intensity of 21.2 (adult worms), with intensities ranging from 3 to 46 worms. Logistic regression model analysis showed that age was a significant risk factor for yak infection with Fasciola spp. In addition, the risk varied between regions: Haiyan, Gangcha, Duran, and Wulan were all high-risk areas for yak infection with Fasciola spp. The spatial distribution of the Fasciola spp. infection rate in each region showed a very weak negative correlation (Moran's I = -0.062), Duran formed a spatial distribution of high-low clusters with surrounding areas, and Datong formed a low-high clustering distribution characteristic with the surrounding areas. This investigation revealed that the infection rate of Fasciola spp. in yaks was higher on the Qinghai-Tibet Plateau. Increasing age was a risk factor for infection with Fasciola spp.; different regions also have a different risk of Fasciola spp. infection. Only two regions showed clustering characteristics in the spatial distribution of infection rates. These findings extend the epidemiological information on Fasciola spp. infection in yaks and provide baseline data for the execution of control measures against Fasciola spp. infection.

Effects of chemotherapy and immunotherapy on microbial diversity in TME and engineered bacterial-mediated tumor therapy.

Tumor microbiota is a group of microorganisms located in tumor tissues with rich diversity that can promote tumorigenesis and development, and different types of tumors have different tumor microbiotas, which has important implications for tumor research, detection, and clinical treatment. In this review, we examine the diversity of the tumor microbiota, discuss the impact of chemotherapy and immunotherapy on tumor microbiota diversity, and summarize recent advances in the use of genetically engineered bacteria for the treatment of tumors. In addition, we propose key questions that need to be further addressed by the tumor microbiota.

Predictors of Improvement of Sarcopenia after Transjugular Intrahepatic Portosystemic Shunt Creation in Cirrhotic Patients.

To investigate the risk factors affecting the improvement of sarcopenia after transjugular intrahepatic portosystemic shunt (TIPS) in cirrhotic patients, this study retrospectively analyzed the data of 111 cirrhotic patients with sarcopenia who underwent TIPS creation. Computed tomography-based measurement of skeletal muscle area was used to calculate skeletal muscle index (SMI) in all patients at baseline and 6 months after TIPS creation. Multivariate logistic regression analysis was used to identify independent risk factors, which showed a significant increase in 6-month post-TIPS SMI compared with that at baseline in both men and women (for both, P < .001). Pre-TIPS SMI (odds ratio [OR], 0.93; 95% CI, 0.87-0.99; P = .031) and change in portal pressure gradient (OR, 1.13; 95% CI, 1.03-1.24; P = .009) were found to be independent risk factors for experiencing substantial improvement in post-TIPS SMI.

Ceramide synthase 3 affects invasion and metastasis of hepatocellular carcinoma via the SMAD6 gene. / ç¥žç»é °èƒºåˆæˆé ¶3通过SMAD6åŸºå› å½±å“è‚ç»†èƒžç™Œçš„ä¾µè¢­å’Œè½¬ç§».

OBJECTIVES: Patients with hepatocellular carcinoma (HCC) have poor prognosis due to lack of early diagnosis and effective treatment. Therefore, there is an urgent need to better understand the molecular mechanisms associated with HCC and to identify effective targets for early diagnosis and treatment. This study is to explore the expression and biological role of ceramide synthase 3 (CerS3) in HCC. METHODS: A total of 159 pairs of HCC tissues and adjacent non-tumor tissues were obtained from the patients underwent radical resection in Shenzhen People's Hospital, and the total RNA and proteins from HCC tissues and adjacent non-tumor tissues were obtained. The expression of CerS3 protein and mRNA in HCC was detected by immunohistochemistry, Western blotting and real-time PCR. In vitro experiments, Hep3B cells were divided into a control vector group and a CerS3 vector group, and the cells were transfected with retroviral vector containing control cDNA or CerS3 cDNA, respectively. HCCLM3 cells were divided into a normal control shRNA group and a CerS3 shRNA group, and the cells were transfected with lentiviral vectors containing normal control shRNA or CerS3 shRNA, respectively. MTT, EdU, Transwell and scratch method were used to detect cell proliferation, migration and invasion. RNA sequencing was performed to determine the downstream signal of CerS3. RESULTS: Compared with the corresponding adjacent tissues,the mRNA and protein levels of CerS3 were elevated in the HCC tissues, with significant difference (both P<0.05). The Univariate and multivariate analysis showed that the overall survival rate was significantly correlated with the presence of venous invasion (95% CI 1.8-9.2, P<0.01), TNM stage (95% CI 2.3-5.2, P<0.05), poor histological grade (95% CI 1.4-6.8, P<0.05), and CerS3 (95% CI 1.5-3.9, P<0.05). Furthermore, the high CerS3 expression levels in tumor tissues were significantly associated with shorter overall survival rates compared with the low CerS3 expression (P<0.05). Compared with the vector control group, the Hep3B cell viability, EdU positive cells, and migration and invasion cell numbers in the CerS3 vector group were significantly increased (all P<0.05). Compared with the shRNA normal control group, the HCCLM3 cell viability, EdU positive cells, and numbers of migrating and invasive cells in the CerS3 shRNA group were significantly lower (all P<0.05). The RNA sequencing confirmed that the small mothers against decapentaplegic family member 6 (SMAD6) gene as an oncogenic gene could promote the HCC metastasis. CONCLUSIONS: Clinically, the overexpression of CerS3 is closely related to poor clinical features and poor prognosis. Functionally, CerS3 participates in the proliferation, invasion and metastasis of liver cancer cells via activating SMAD6 gene.

Research progress of electrochemical oxidation and self-action of electric field for medical wastewater treatment.

A large number of pathogenic microorganisms exist in medical wastewater, which could invade the human body through the water and cause harm to human health. With the global pandemic coronavirus (COVID-19), public health safety become particularly important, and medical wastewater treatment is an important part of it. In particular, electrochemical disinfection technology has been widely studied in medical wastewater treatment due to its greenness, high efficiency, convenient operation, and other advantages. In this paper, the development status of electrochemical disinfection technology in the treatment of medical wastewater is reviewed, and an electrochemical three-stage disinfection system is proposed for the treatment of medical wastewater. Moreover, prospects for the electrochemical treatment of medical wastewater will be presented. It is hoped that this review could provide insight and guidance for the research and application of electrochemical disinfection technology to treat medical wastewater.GRAPHICAL ABSTRACT.

Advances in the study of selenium and human intestinal bacteria.

Selenium (Se) is an essential trace element for humans and has conveyed great a wide range of interests due to its contribution to health. Presently, the regulatory mechanisms of selenium on human health, especially the regulatory mechanisms of selenium on human intestinal (gut) microflora and its effects on diseases are receiving attention from academic circles. This review involves the effects of selenium on physical health, the relationship between selenium and intestinal microflora, and the progress of research between selenium, intestinal microflora, and diseases. Furthermore, the current status of research on the selenium, intestinal microflora, and diseases is also presented.

Krüppel-Like Factor 6 Silencing Prevents Oxidative Stress and Neurological Dysfunction Following Intracerebral Hemorrhage via Sirtuin 5/Nrf2/HO-1 Axis.

As a severe neurological deficit, intracerebral hemorrhage (ICH) is associated with overwhelming mortality. Subsequent oxidative stress and neurological dysfunction are likely to cause secondary brain injury. Therefore, this study sought to define the role of Krüppel-like factor 6 (KLF6) and underlying mechanism in oxidative stress and neurological dysfunction following ICH. An in vivo model of ICH was established in rats by injection of autologous blood, and an in vitro ICH cell model was developed in hippocampal neurons by oxyhemoglobin (OxyHb) exposure. Next, gain- and loss-of-function assays were performed in vivo and in vitro to clarify the effect of KLF6 on neurological dysfunction and oxidative stress in ICH rats and neuronal apoptosis and mitochondrial reactive oxygen species in OxyHb-induced hippocampal neurons. KLF6, nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase 1 (HO-1) were highly expressed in hippocampal tissues of ICH rats, whereas sirtuin 5 (SIRT5) presented a poor expression. Mechanistically, KLF6 bound to the SIRT5 promoter and transcriptionally repressed SIRT5 to activate the Nrf2/HO-1 signaling pathway. KLF6 silencing alleviated neurological dysfunction and oxidative stress in ICH rats and diminished oxidative stress and neuronal apoptosis in OxyHb-induced neurons, whereas SIRT5 overexpression negated its effect. To sum up, KLF6 silencing elevated SIRT5 expression to inactivate the Nrf2/HO-1 signaling pathway, thus attenuating oxidative stress and neurological dysfunction after ICH.

Seroprevalence and associated risk factors of Toxoplasma gondii infection in yaks (Bos grunniens) on the Qinghai-Tibetan Plateau of China.

Toxoplasma gondii is an intracellular parasite that is extensively prevalent globally. Studies have indicated the presence of T. gondii infection in animals in some provinces of China, but little is known about T. gondii infection in yaks (Bos grunniens) on the Qinghai-Tibetan Plateau. In the current study, to determine the seroprevalence and associated risk factors of T. gondii, a total of 2784 serum samples were collected from 18 different sampling sites in eight counties of the Qinghai and Tibet regions of China from 2018 to 2019. Serum antibodies against T. gondii were detected in 261 yaks (9.38%) via enzyme-linked immunosorbent assay (ELISA). We found that seroprevalence differed significantly among different counties (ranging from 5.41% in Gangcha to 19.79% in Datong), by year in the Tibet Autonomous Region (from 2.34% in 2018 to 13.24% in 2019), and by age (from 5.59% in 0 < year ≤ 1 to 11.76% in year > 7) (p < 0.05). Climate, geographical conditions, and age are the main factors influencing T. gondii infection in yaks in these regions. Therefore, our study provides a data reference for public health and prevention of yak toxoplasmosis.

TITLE: Séroprévalence et facteurs de risque associés à l'infection par Toxoplasma gondii chez les yaks (Bos grunniens) du plateau Qinghai­Tibet en Chine. ABSTRACT: Toxoplasma gondii est un parasite intracellulaire largement répandu dans le monde. Des études ont indiqué la présence d'une infection par T. gondii chez les animaux dans certaines provinces de Chine, mais on connaît peu l'infection par T. gondii chez les yaks (Bos grunniens) sur le plateau Qinghai­Tibet. Dans la présente étude, pour déterminer la séroprévalence et les facteurs de risque associés de T. gondii, un total de 2784 échantillons de sérum ont été prélevés sur 18 sites d'échantillonnage différents dans huit comtés des régions du Qinghai et du Tibet en Chine entre 2018 et 2019. Des anticorps sériques contre T. gondii ont été détectés par dosage immuno-enzymatique (ELISA) chez 261 yaks (9,38 %). Nous avons constaté que la séroprévalence différait considérablement entre les différents comtés (allant de 5,41 % à Gangcha à 19,79 % à Datong), d'une année à l'autre dans la région autonome du Tibet (de 2,34 % en 2018 à 13,24 % en 2019), et par âge (de 5,59 % pour les animaux de moins d'un an à 11,76 % pour ceux âgés de plus de 7 ans) (p < 0,05). Le climat, les conditions géographiques et l'âge sont les principaux facteurs influençant l'infection à T. gondii chez les yaks de ces régions. Par conséquent, notre étude fournit des données de référence pour la santé publique et la prévention de la toxoplasmose du yak.

The single-tube quantitative rapid detection of coliform bacteria based on enzyme-specific technology.

To detect coliform bacteria (CB) with simplicity and portability, a novel, single-tube quantitative rapid detection method was developed based on CB-specific enzymatic technology. This technology involved a simple multi-channel spectrometer which could detect 7 cfu mL-1 CB specifically, and the approach was applied to various food and water samples.

Preparation and properties of carboxymethyl chitosan/oxidized hydroxyethyl cellulose hydrogel.

Hydrogel wound dressing is a type of hydrophilic polymer, which has been widely studied and applied in biomedical field. In this study, a simple and non-toxic method was developed to prepare a new type of composite hydrogel, which was formed through the Schiff-base reaction between the aldehyde of Oxidized Hydroxyethyl Cellulose (OHEC) and the amino of Carboxymethyl Chitosan (CMCS). Hence, a series of tests toward this new composite hydrogel which contained its structure and performance was applied. Statistics achieved from those tests showed that this composite hydrogel comprised of some high-quality properties such as suitable gelation time, good swelling ability, suitable water evaporation rate, good blood compatibility and biocompatibility. Considering these properties, this hydrogel has a potential to be explored as wound dressing.

Prevalence and genetic diversity of Echinorhynchus gymnocyprii (Acanthocephala: Echinorhynchidae) in schizothoracine fishes (Cyprinidae: Schizothoracinae) in Qinghai-Tibetan Plateau, China.

BACKGROUND: The schizothoracine fishes, an excellent model for several studies, is a dominant fish group of the Qinghai-Tibet Plateau (QTP). However, species populations have rapidly declined due to various factors, and infection with Echinorhynchus gymnocyprii is cited as a possible factor. In the present study, the molecular characteristics of E. gymnocyprii in four species of schizothoracine fishes from the QTP were explored. METHODS: We investigated the infection status of E. gymnocyprii in 156 schizothoracine fishes from the upper Yangtze River, upper Yellow River, and Qinghai Lake in Qinghai Province, China. The complete internal transcribed spacer (ITS) of the ribosomal RNA (rRNA) gene and part of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of 35 E. gymnocyprii isolates from these fishes were sequenced and their characteristics analyzed. In addition, we inferred phylogenetic relationships of the E. gymnocyprii populations based on the rRNA-ITS and cox1 sequences. RESULTS: The total prevalence of E. gymnocyprii in schizothoracine fishes was 57.69% (90/156). However, the prevalence among different species as well as that across the geographical locations of the schizothoracine fishes was significantly different. The results of sequence analysis showed that the four E. gymnocyprii populations from different hosts and regions of Qinghai Province were conspecific, exhibiting rich genetic diversity. Phylogenetic analysis based on rRNA-ITS and cox1 sequences supported the coalescence of branches within E. gymnocyprii; the cox1 gene of E. gymnocyprii populations inferred some geographical associations with water systems. In addition, three species of schizothoracine fishes were recorded as new definitive hosts for E. gymnocyprii. CONCLUSIONS: To the best of our knowledge, this is the first molecular description of E. gymnocyprii populations in schizothoracine fishes from the Qinghai-Tibet Plateau that provides basic data for epidemiological surveillance and control of acanthocephaliasis to protect endemic fish stocks.

Extremely Low Program Current Memory Based on Self-Assembled All-Inorganic Perovskite Single Crystals.

Memory devices based on lead halide perovskite have attracted great interests because of their unique current-voltage hysteresis. However, current memory devices based on polycrystalline perovskites usually suffer from large intrinsic electronic current and parasitic leakage current due to the existence of grain boundaries, which further leads to high power consumption. Here, a low-power resistance switching random-access memory device is demonstrated by assembling single-crystalline CsPbBr3 on Ag electrodes. The assembled structure serves as a bipolar nonvolatile resistance switching memory device with a low program current (∼10 nA), good endurance, long data retention (>103 S), and big on/off ratio of ∼103. The low program current results in a power of ∼3 × 10-8 W, which is much lower than that of polycrystalline perovskite-based devices (10-1-10-6 W). It is found that the formation and annihilation of Ag and bromide vacancy conductive filaments contribute to the significant resistive switching effect. At a low resistive state, the conductive filaments originate from the accumulation of Br- ions at the drain. Furthermore, the conductive filaments are proved to be a cone shape, shrinking from the drain to the source.

Direct Growth of Perovskite Crystals on Metallic Electrodes for High-Performance Electronic and Optoelectronic Devices.

Metal halide perovskite has attracted enhanced interest for its diverse electronic and optoelectronic applications. However, the fabrication of micro- or nanoscale crystalline perovskite functional devices remains a great challenge due to the fragility, solvent, and heat sensitivity of perovskite crystals. Here, a strategy is proposed to fabricate electronic and optoelectronic devices by directly growing perovskite crystals on microscale metallic structures in liquid phase. The well-contacted perovskite/metal interfaces ensure these heterostructures serve as high-performance field effect transistors (FETs) and excellent photodetector devices. When serving as an FET, the on/off ratio is as large as 106 and the mobility reaches up to ≈2.3 cm2 V-1 s-1 . A photodetector is displayed with high photoconductive switching ratio of ≈106 and short response time of ≈4 ms. Furthermore, the photoconductive response is proved to be band-bending-assisted separation of photoexcited carriers at the Schottky barrier of the silver and p-type perovskites.

An efficient rearing system rapidly producing large quantities of poultry red mites, Dermanyssus gallinae (Acari: Dermanyssidae), under laboratory conditions.

The poultry red mite, Dermanyssus gallinae, is one of the most economically deleterious ectoparasites affecting egg-laying hens in many parts of the world. New approaches to control D. gallinae often require the maintenance of colonies of D. gallinae under laboratory conditions. In the present study, we present an efficient rearing system for D. gallinae, consisting of a metal cage, a plastic storage box and a tray filled with water. Chicks were raised in the cage as host animals. A novel trap was developed to monitor the dynamic changes of mite populations, made with a plastic centrifuge tube and a disposable breathing mask with folds. Mite parameters were analyzed, including number of mites and eggs, survival and feeding rates, oviposition, hatchability and the proportion of D. gallinae at different life stages. The results show that the rearing system had a 53.5-fold increase in the number of mites over a period of six weeks after the introduction of mites. The survival rates of mites were above 94%, and the mean feeding rates ranged from 22.57% to 37.30%. The mean number of eggs per female ranged from 3.42 to 3.50, with the hatchability of eggs above 97%. Nymphs made up most of the population, ranging from 71.46% to 81.37%, while the population of larvae was minor and ranging from 7.54% to 13.04%. The mask trap used in this study was an effective and convenient device to shelter D. gallinae and monitor the dynamic changes of the mite population. The rearing system proved very effective in maintaining and reproducing colonies of D. gallinae, with great potential for the evaluation of the efficacy of vaccines or compounds against D. gallinae under laboratory conditions. It would be a useful tool for close observations in studies on the biology, acology and physiology of poultry red mites.

Enterocytozoon bieneusi genotypes in Tibetan sheep and yaks.

Few studies have been conducted on the distribution of Enterocytozoon bieneusi genotypes in Tibetan sheep and yaks, which live outdoors in extreme climate with high altitude. In this study, fecal specimens from 312 Tibetan sheep and 554 yaks in Qinghai, China, were collected and examined for E. bieneusi by PCR-sequence analysis of the ribosomal internal transcribed spacer. Among them, 73 (23.4%) specimens from Tibetan sheep and 40 (7.2%) from yaks were positive for E. bieneusi. There were eight E. bieneusi genotypes in Tibetan sheep, including three known ones (BEB6, COS-I, and NESH5) and five novel ones (named as CHS13-CHS17). Similarly, seven E. bieneusi genotypes were found in yaks, including five known ones (J, BEB4, BEB6, COS-I, and NESH5) and two novel ones (named as CHN13 and CHN14). Most of the E. bieneusi genotypes and all frequent ones identified in the study belonged to group 2. One new subgroup of genotypes was identified within group 1. The distribution of E. bieneusi genotypes was different between Tibetan sheep and yaks, with BEB6 as the dominant one (42.5%) in Tibetan sheep and J as the dominant one (47.5%) in yaks. These data support the occurrence of host adaptation among E. bieneusi genotypes within group 2.

Amplitude response of conical multiwalled carbon nanotube probes for atomic force microscopy.

Carbon nanotubes are considered as great candidates for atomic force microscopy (AFM) probes because of their high aspect ratio and outstanding mechanical properties. In this work, we report that a conical AFM probe can be fabricated with arc discharge prepared multiwalled carbon nanotubes (MWCNTs) with an individual MWCNT at the apex by dielectrophoresis. The amplitude-displacement curve of the conical MWCNT probe demonstrates that this structure can remain stable until the force exerted on it increases to 14.0 ± 1.5 nN (nanonewton). Meanwhile, the conical MWCNT probes are able to resolve complex structure with high aspect ratio compared to commercial AFM probes, suggesting great potential for various AFM applications.

Multilocus genotyping of Giardia duodenalis in Tibetan sheep and yaks in Qinghai, China.

Giardia duodenalis is a common gastrointestinal protozoon in mammals. Although many studies have been reported on the distribution of G. duodenalis genotypes in sheep and cattle raised under intensive farming, few studies are available on the distribution of G. duodenalis in Tibetan sheep and yaks, which are raised free ranging in a continental plateau climate. In this study, 495 fecal specimens from Tibetan sheep and 605 from yaks were collected from eight counties in Qinghai, China and analyzed for G. duodenalis by PCR targeting the ß-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) genes. Based on PCR positivity at the bg locus, G. duodenalis occurrence rates were 13.1% (65/495) in Tibetan sheep and 10.4% (63/605) in yaks. DNA sequence analysis identified the presence of G. duodenalis Assemblages A (in 10 Tibetan sheep and 2 yaks) and E (in 51 Tibetan sheep and 60 yaks). In addition, mixed infections of the two were identified in four Tibetan sheep and one yak. Among the sequences obtained in this study, 1, 10, and 2 new subtypes of Assemblage E were detected at the bg, gdh and tpi loci, respectively. Based on sequences from the three loci, 28 multilocus genotypes (MLGs) were obtained, including 27 MLGs in Assemblage E and one MLG in Assemblage A. Each MLG was found in no more than seven animals, with most MLGs forming host-specific clusters in phylogenetic analysis except for one cluster including MLGs from both Tibetan sheep and yaks. Only two MLGs were found in both sheep and yaks. The above results demonstrate a high subtype diversity of G. duodenalis Assemblage E in Tibetan sheep and yaks raised in a traditional animal husbandry system and suggest that only limited cross-species transmission of G. duodenalis occurs between yaks and sheep sharing pastures.

Genetic diversity in Echinococcus shiquicus from the plateau pika (Ochotona curzoniae) in Darlag County, Qinghai, China.

The metacestode of Echinococcus shiquicus has been recorded previously in the lung and liver of its intermediate host, the plateau pika (Ochotona curzoniae), but there is limited information regarding other organ sites. There is also limited evidence of intra-specific genetic variation within E. shiquicus. A PCR-amplified mitochondrial (mt) nad1 gene fragment (approximately 1400bp in size), with unique EcoRI and SspI restriction sites, was used to distinguish cysts or cyst-like lesions of E. shiquicus from E. multilocularis. Then, the complete mt nad1 and cox1 genes for the E. shiquicus isolates were amplified and sequenced. Phylogenetic tree and haplotype network analyses for the isolates were then generated based on a concatenated dataset of the nad1 and cox1 genes using the neighbour-joining (NJ) method and TCS1.21 software. Nineteen of eighty trapped pikas were found to harbor cysts (71 in total) when dissected at the survey site. Seventeen animals had cysts (fertile) present only in the lungs, one animal had fertile cysts in the lungs and spleen, and one individual had an infertile kidney cyst. Restriction endonuclease analysis of a fragment of the nad1 gene indicated all the cysts were due to E. shiquicus. Genetic diversity analysis revealed that the nad1 and cox1 genes varied by 0.1-1.2% and 0.1-1.0%, respectively. Haplotype network analysis of the concatenated nad1 and cox1 sequences of the isolates showed they were classified into at least 6 haplotypes, and different haplotype percentages ranged from 4.2% to 29.6%. Although, high haplotype diversity was evident in the study area, the complete nad1 and cox1 gene sequences obtained indicated that all samples represented isolates of E. shiquicus. The study has also provided a new PCR-restriction endonuclease-based method to rapidly distinguish E. shiquicus from E. multilocularis which provides a useful tool for epidemiological investigations where the two species overlap.

Distribution of Cryptosporidium species in Tibetan sheep and yaks in Qinghai, China.

Few data are available on the distribution of Cryptosporidium species in Tibetan sheep and yaks, which are free-range animals living in a cold, low oxygen, and high ultraviolet radiation habitat. In this study, 904 fecal specimens were collected from 350 Tibetan sheep and 554 yaks in six counties. Cryptosporidium spp. were detected and differentiated by PCR and sequence analyses. Altogether, 43 (12.3%) Tibetan sheep and 158 (28.5%) yaks were positive for Cryptosporidium spp. In Tibetan sheep, Cryptosporidium xiaoi (39/43, 90.7%) was the dominant species, with the remaining cases (4/43, 9.3%) by Cryptosporidium ubiquitum. All C. ubiquitum specimens belonged to the subtype family XIIa. In contrast, Cryptosporidium andersoni (72/158, 45.6%), Cryptosporidium bovis (47/158, 29.7%), Cryptosporidium ryanae cattle type (35/158, 22.2%), C. ryanae buffalo type (2/158, 1.3%), and Cryptosporidium suis-like (2/158, 1.3%) were identified in yaks. Contradictory to previous observations, C. andersoni was one of the dominant Cryptosporidium species in yaks in this study. Despite sharing habitats, Tibetan sheep and yaks are evidently infected with different Cryptosporidium species.

Discrimination between E. granulosus sensu stricto, E. multilocularis and E. shiquicus Using a Multiplex PCR Assay.

BACKGROUND: Infections of Echinococcus granulosus sensu stricto (s.s), E. multilocularis and E. shiquicus are commonly found co-endemic on the Qinghai-Tibet plateau, China, and an efficient tool is needed to facilitate the detection of infected hosts and for species identification. METHODOLOGY/PRINCIPAL FINDINGS: A single-tube multiplex PCR assay was established to differentiate the Echinococcus species responsible for infections in intermediate and definitive hosts. Primers specific for E. granulosus, E. multilocularis and E. shiquicus were designed based on sequences of the mitochondrial NADH dehydrogenase subunit 1 (nad1), NADH dehydrogenase subunit 5 (nad5) and cytochrome c oxidase subunit 1 (cox1) genes, respectively. This multiplex PCR accurately detected Echinococcus DNA without generating nonspecific reaction products. PCR products were of the expected sizes of 219 (nad1), 584 (nad5) and 471 (cox1) bp. Furthermore, the multiplex PCR enabled diagnosis of multiple infections using DNA of protoscoleces and copro-DNA extracted from fecal samples of canine hosts. Specificity of the multiplex PCR was 100% when evaluated using DNA isolated from other cestodes. Sensitivity thresholds were determined for DNA from protoscoleces and from worm eggs, and were calculated as 20 pg of DNA for E. granulosus and E. shiquicus, 10 pg of DNA for E. multilocularis, 2 eggs for E. granulosus, and 1 egg for E. multilocularis. Positive results with copro-DNA could be obtained at day 17 and day 26 after experimental infection of dogs with larval E. multilocularis and E. granulosus, respectively. CONCLUSIONS/SIGNIFICANCE: The multiplex PCR developed in this study is an efficient tool for discriminating E. granulosus, E. multilocularis and E. shiquicus from each other and from other taeniid cestodes. It can be used for the detection of canids infected with E. granulosus s.s. and E. multilocularis using feces collected from these definitive hosts. It can also be used for the identification of the Echinococcus metacestode larva in intermediate hosts, a stage that often cannot be identified to species on visual inspection.