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.

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.

The protective effect of melatonin on the in vitro development of yak embryos against hydrogen peroxide-induced oxidative injury.

SummaryMelatonin plays a critical role in several types of cells as an antioxidant to protect intracellular molecules from oxidative stress. The anti-oxidation effect of melatonin in yak embryos is largely unknown. We report that melatonin can protect the development of yak preimplantation embryos against oxidative stress induced by hydrogen peroxide (H2O2). Therefore, the quality of blastocysts developed from zygotes exposed to H2O2 was promoted. In addition, we observed that melatonin reduced H2O2-induced intracellular reactive oxygen species (ROS) levels and prevented mitochondrial dysfunction in zygotes. These phenomena revealed the effective antioxidant activity of melatonin to prevent oxidative stress in yak embryos. To determine the underlying mechanism, we further demonstrated that melatonin protected preimplantation embryos from oxidative damage by preserving antioxidative enzymes. Collectively, these results confirmed the anti-oxidation effect of melatonin in yak embryos that significantly improved the quantity and quality of blastocysts in the in vitro production of embryos in yaks.

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.

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.

A comparison of loop-mediated isothermal amplification (LAMP) with other surveillance tools for Echinococcus granulosus diagnosis in canine definitive hosts.

BACKGROUND: Cystic echinococcosis is highly prevalent in northwest China. A cost-effective, easy to operate diagnostic tool with high sensitivity and specificity would greatly facilitate the monitoring of Echinococcus infections in canine definitive hosts. METHODS: The primers used in the LAMP assay were based on the mitochondrial nad5 gene of E. granulosus sensu stricto (E. granulosus s.s., or E.g.s.s.) and were designed using Primer Explorer V4 software. The developed LAMP assay was compared with a conventional PCR method, copro-ELISA and microscopy, using the faeces of dogs experimentally infected with E.g.s.s., and field-collected faeces of domestic dogs including 190 from Qinghai province highly endemic for E.g.s.s. and 30 controls from an area in Gansu, where a domestic dog de-worming program was in operation. RESULTS: The positivity rates obtained for the field-collected faecal samples were 12.6%, 1.6% and 2.1% by the LAMP, PCR and copro-ELISA assays, respectively. All samples obtained from the control dogs were negative. Compared with the conventional PCR, the LAMP assay provided 88.8% specificity and 100% sensitivity. The higher sensitivity of the LAMP method was also shown by the fact that it could detect the presence of laboratory challenge dog infections of E. granulsous s.s. four days earlier than the PCR method. Three copro-samples shown positive by the commercial copro-ELISA were all negative by LAMP, PCR and microscopy, which suggests these samples may have originated from another infection rather than E. granulsous s.s., possibly E. shiquicus or E. Canadensis, which is also present in China. CONCLUSIONS: We have developed a potentially useful surveillance tool for determining the prevalence of canine E. granulosus s.s. infections in the field. The LAMP assay may lead to a more cost-effective and practicable way of tracking Echinococcus infections in canids, especially when combined with the copro-ELISA.

Loop-mediated isothermal amplification (LAMP) assay for the identification of Echinococcus multilocularis infections in canine definitive hosts.

BACKGROUND: Alveolar echinococcosis, caused by the metacestode larval stage of Echinococcus multilocularis, is a zoonosis of public health significance and is highly prevalent in northwest China. To effectively monitor its transmission, we developed a new rapid and cheap diagnostic assay, based on loop-mediated isothermal amplification (LAMP), to identify canine definitive hosts infected with E. multilocularis. METHODS: The primers used in the LAMP assay were based on the mitochondrial nad5 gene of E. multilocularis and were designed using Primer Explorer V4 software. The developed LAMP assay was compared with a conventional PCR assay, using DNA extracted from the feces of dogs experimentally infected with E. multilocularis, on 189 dog fecal samples collected from three E. multilocularis-endemic regions in Qinghai province, the People's Republic of China, and 30 negative control copro-samples from dogs from an area in Gansu province that had been subjected to an intensive de-worming program. Light microscopy was also used to examine the experimentally obtained and field collected dog copro-samples for the presence of E. multilocularis eggs. RESULTS: The E. multilocularis-positivity rates obtained for the field-collected fecal samples were 16.4% and 5.3% by the LAMP and PCR assays, respectively, and all samples obtained from the control dogs were negative. The LAMP assay was able to detect E. multilocularis DNA in the feces of experimentally infected dogs at 12 days post-infection, whereas the PCR assay was positive on the 17th day and eggs were first detectable by light microscopy at day 44 post-challenge. CONCLUSION: The earlier specific detection of an E. multilocularis infection in dog copro-samples indicates that the LAMP assay we developed is a realistic alternative method for the field surveillance of canines in echinococcosis-endemic areas.

Prevalence and genetic characterization of Cryptosporidium in yaks in Qinghai Province of China.

The objective of this study was to determine the prevalence, species and subtypes of Cryptosporidium infecting yaks in the Qinghai Province of Northwestern China. The prevalence of Cryptosporidium spp. was detected by microscopy and nested-PCR. A total of 586 fecal samples were collected from yaks in 6 counties, of which 142 (24.2%) samples tested positive for Cryptosporidium. The small subunit (SSU) rRNA gene of fifty-five samples were amplified and sequenced successfully and demonstrated that Cryptosporidium bovis (31/55, 56.4%) was the most common species, followed by C. parvum (16/55, 29.1%) and C. ryanae (5/55, 9.0%). Mixed infections of C. parvum and C. bovis (n = 2), C. ryanae and C. bovis (n = 1) were also detected. All three species were found in yaks ranging in age from <1 year, 1-2 years, to >2 years. Cryptosporidium was most commonly detected in spring (28.4%), followed by summer (20.9%), then winter (17.5%). Cryptosporidium parvum positive samples were subtyped using the 60 kDa glycoprotein (gp60) gene. Subtypes IIaA15G2R1 (n = 8), IIaA16G2R1 (n = 2), IIaA14G1R1 (n = 1), IIaA14G2R1 (n = 1) and IIaA16G3R1 (n = 1) were detected. All of these subtypes are zoonotic, and may pose a potential threat to human health.

The mitochondrial genome of Paramphistomum cervi (Digenea), the first representative for the family Paramphistomidae.

We determined the complete mitochondrial DNA (mtDNA) sequence of a fluke, Paramphistomum cervi (Digenea: Paramphistomidae). This genome (14,014 bp) is slightly larger than that of Clonorchis sinensis (13,875 bp), but smaller than those of other digenean species. The mt genome of P. cervi contains 12 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 2 non-coding regions (NCRs), a complement consistent with those of other digeneans. The arrangement of protein-coding and ribosomal RNA genes in the P. cervi mitochondrial genome is identical to that of other digeneans except for a group of Schistosoma species that exhibit a derived arrangement. The positions of some transfer RNA genes differ. Bayesian phylogenetic analyses, based on concatenated nucleotide sequences and amino-acid sequences of the 12 protein-coding genes, placed P. cervi within the Order Plagiorchiida, but relationships depicted within that order were not quite as expected from previous studies. The complete mtDNA sequence of P. cervi provides important genetic markers for diagnostics, ecological and evolutionary studies of digeneans.