A survey of argasid ticks and tick-associated pathogens in the Peripheral Oases around Tarim Basin and the first record of Argas japonicus in Xinjiang, China.

Argasid ticks (Acari: Argasidae) carry and transmit a variety of pathogens of animals and humans, including viruses, bacteria and parasites. There are several studies reporting ixodid ticks (Acari: Ixodidae) and associated tick-borne pathogens in Xinjiang, China. However, little is known about the argasid ticks and argasid tick-associated pathogens in this area. In this study, a total of 3829 adult argasid ticks infesting livestock were collected at 12 sampling sites of 10 counties in the Peripheral Oases, which carry 90% of the livestock and humans population, around the Tarim Basin (southern Xinjiang) from 2013 to 2016. Tick specimens were identified to two species from different genera by morphology and sequences of mitochondrial 16S rRNA and 12S rRNA were derived to confirm the species designation. The results showed that the dominant argasid ticks infesting livestock in southern Xinjiang were Ornithodoros lahorensis (87.86%, 3364/3829). Ornithodoros lahorensis was distributed widely and were collected from 10 counties of southern Xinjiang. Argas japonicus was collected from Xinjiang for the first time. In addition, we screened these ticks for tick-associated pathogens and showed the presence of DNA sequences of Rickettsia spp. of Spotted fever group and Anaplasma spp. in the argasid ticks. This finding suggests the potential role for Argas japonicus as a vector of pathogens to livestock and humans.

Establishment and optimization of a liquid bead array for the simultaneous detection of ten insect-borne pathogens.

BACKGROUND: Insect-borne diseases could induce severe symptoms in human and clinical signs in animals, such as febrility, erythra, arthralgia and hemorrhagic fever, and cause significant economic losses and pose public health threat all over the world. The significant advantages of Luminex xMAP technology are high-throughput, high parallel and automation. This study aimed to establish a liquid bead array based on Luminex xMAP technology that was able to simultaneously detect multiple insect-borne pathogens. METHODS: Specific probes and primers to detect the nucleic acid of 10 insect-borne pathogens were designed. Probes were coupled with fluorescent carboxylated microspheres. The parameters of the system were optimized, including ratio of forward/reverse primers (1:2), hybridization temperature (50 °C) and duration (30 min) and quantity of PCR product (2 µl). The sensitivity and specificity of the system were also evaluated. Moreover mixed nucleic acid of 10 insect-borne pathogens, including Bluetongue virus, Epizootic hemorrhagic disease virus of deer, Coxiella burnetii, African swine fever virus, West Nile fever virus, Borrelia burgdorferi, vesicular stomatitis virus, Rift Valley fever virus, Ebola virus and Schmalenberg's disease virus, and 3000 clinical samples were tested for practicability. RESULTS: The optimized detection system showed high sensitivity, specificity and reproducibility. Each probe showed specific fluorescence signal intensity without any cross-hybridization for the other insect-borne pathogens tested, which included dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus, Xinjiang hemorrhagic fever virus, spotted fever group rickettsiae, ehrlichiae and chikungunya virus. The limit of detection was 10 copies of target gene. Insect-borne pathogens were successfully detected among the 3000 clinical samples, and the results were consistent with those obtained using gold-standard assays or commercial nucleic acid detection kits. CONCLUSIONS: This optimized liquid array detection system was high-throughput and highly specific and sensitive in screening of the insect-borne pathogens. It was promising in detection of these pathogens for molecular epidemiological studies.

[Development of an IMS-qPCR method for detection of Cryptosporidium parvum in water].

OBJECTIVE: To develop a detection method for Cryptosporidium parvum oocysts from water samples, which combined immunomagnetic separation (IMS) with Taqman real-time PCR (qPCR). METHODS: Conditions of separation and enrichment of IMS method by using specific streptavidin magnetic beads coated with monoclonal antibodies Cp23 directed against C. parvum oocysts were optimized. Special primers of PCR and Taqman probes were designed referring to the 18S rDNA gene of C. parvum (GenBank Accession No. AB513881.1). The conserved genes were amplified from genomic DNA of C.parvum, and then cloned into Peasy-T1 vector. Tenfold dilutions of positive plasmids (10(4)-10(8) copy/microl) were used to construct a standard curves by Taqman qPCR. The specificity of the assay was determined using genomic DNA of C. baileyi, Toxoplasma gondii, C. canis and Escherichia coli. The sensitivity of this assay was evaluated by analyzing 10-fold serially dilutions of plasmids ranging from 10(0) to 10(8) copy/microl. Both IMS-qPCR assay and indirect immunofluorescent-antibody assay (IFA) were applied to detect 50 water samples collected from the dairy cattle farms in Hebei. RESULTS: The optimal incubation concentration and time of antibody Cp23 were 20 ng/ml and 30 min, respectively, and the catching time was 30 min, the recovery rate was more than 95%. PCR product was 272 bp, and identified by restriction enzyme digestion and nucleotide sequencing. There was a good linear relationship between the standard plasmids and Ct value (correlation r2 = 0.996 1) of the Taqman qPCR. No cross-reactivity was observed with C. baileyi, T. gondii, C. canis and E. coli. The sensitivity of C. parvum-specific assay was 10 copy/microl. Compared with IFA as golden standard method, the specificity and sensitivity of IMS-qPCR for 50 water samples was 100%(18/18) and 93.8% (30/32), respectively. CONCLUSION: The IMS-qPCR assay can be used to specifically detect C. parvum oocysts in water samples.

Infection prevalence and phylogenetic analysis of Perkinsus olseni in Ruditapes philippinarum from East China.

The prevalence of Perkinsus sp. infection in Manila clam Ruditapes philippinarum was investigated in the coastal areas of east China. Thirteen groups of clams were collected from 5 sites: Dandong and Qingdao Bays (Yellow Sea), Weifang Bay (Bohai Sea), and Ningbo and Fuzhou Bays (East China Sea). The clams were tested for perkinsosis infection using Ray's fluid thioglycollate medium culture assay. Perkinsus sp. was found in samples from all 5 sites from May 2008 to May 2009. Infection prevalence ranged from 43.75 to 95.83%, and was significantly higher in October than in May. The only 3 uninfected groups of clams were collected from Weifang Bay, the site farthest from the ocean. There was no difference in the prevalence of infection among the remaining 4 sites. The conserved internal transcribed spacer regions of the ribosomal RNA gene complex in each of the Perkinsus sp. isolates were amplified by PCR. The resulting amplicons were sequenced and phylogenetically analyzed. All the Perkinsus isolates were identified as Perkinsus olseni.

Construction of DNA vaccines and their induced protective immunity against experimental Eimeria tenella infection.

In an attempt to construct a DNA vaccine against chicken coccidiosis, the TA4 gene of Eimeria tenella strain BJ was ligated to the mammalian expression vector pcDNA3.1/Zeo(+) to give pcDNA3.1-TA4 (pcDT). Then, Et1A (E. tenella refractile body gene) was ligated to it, upstream, aiming to be expressed in fusion with TA4, giving pcDNA3.1-Et1A-TA4 (pcDET). The constructed DNA vaccines were given to broilers intramuscularly 10-15 min after the breasts had been pre-treated with 25% sucrose solution. At 7 days after the second vaccination, chickens were challenged with 3 x 10(4) sporulated oocysts of E. tenella BJ. The chickens were killed and the lesion scores of the ceca, the relative body-weight gains and the numbers of oocysts in the ceca of each group of chickens were calculated at day 8 post-inoculation. Results indicated that both pcDT and pcDET could induce protective immunity against coccidial challenge. Their use could obviously reduce oocyst output and alleviate chicken body-weight decrease due to coccidial infection. An anti-coccidial index of 160 was achieved with a treatment of 50 microg pcDET and 100 microg pcDT.