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

Date Published: September 22, 2015

Publisher: Public Library of Science

Author(s): Cong-Nuan Liu, Zhong-Zi Lou, Li Li, Hong-Bin Yan, David Blair, Meng-Tong Lei, Jin-Zhong Cai, Yan-Lei Fan, Jian-Qiu Li, Bao-Quan Fu, Yu-Rong Yang, Donald P. McManus, Wan-Zhong Jia, Aaron R. Jex.

Abstract: BackgroundInfections 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 FindingsA 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/SignificanceThe 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.

Partial Text: In the most recent taxonomic revision, nine species were recognized in the genus Echinococcus [1]. Of these, the most important and widespread are E. granulosus sensu stricto (genotypes G1-G3) and E. multilocularis, which cause cystic echinococcosis (CE) and alveolar echinococcosis (AE), respectively. The former is commonly associated with livestock and human infections worldwide whereas the latter is primarily found in voles and humans and is geographically limited to the northern hemisphere [2]. To date, E. granulosus s.s., E. canadensis (G6), E. multilocularis and E. shiquicus have been identified in China [3–5]. Both E. multilocularis and E. granulosus s.s. are particularly widespread in western China, including Qinghai, Ningxia, Gansu, Xinjiang and Sichuan provinces/autonomous regions, and are well known as major public health and medical threats. Unlike the other species, E. shiquicus has a very restricted distribution, being reported only from Qinghai Province, China. This species is not known to cause human echinococcosis. The intermediate hosts are plateau pikas (Ochotona curzoniae), in which unilocular cysts occur.

China is the most severe pandemic country for cystic echinococcosis (CE), in humans and livestock, due mainly to E. granulosus s.s., and for alveolar echinococcosis (AE) due to E. multilocularis in humans and small wild mammals. E. shiquicus is also endemic although it has not been reported to infect humans. Dual infections of animal hosts with different Echinococcus spp have been reported in the eastern Qinghai-Tibet plateau region of China [4, 25]. The very close relationship between dogs and humans can lead readily to human infection. The increasing number of human AE and CE cases in northwestern China, where considerable numbers of dogs are present, places a heavy burden on public health and veterinary services. To aid surveillance, management and diagnosis, effective methods are needed for rapid and accurate detection and identification of different life cycle stages of the three Echinococcus spp. simultaneously. The multiplex PCR assay developed in this study provides such a method.



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