Molecular Diagnosis of a Hybridized Tapeworm (Between Taenia Saginata and Taenia Asiatica) Infection Case in Yunnan, China, 2019

Yun-Zhi Zhang Dali University Ce-Heng Liao Hunan University Zhi You Hunan University Ye Qiu Hunan University Zuo-Shun He Dali University Juan Liu Dali University Wen Fang Dalian Center for Disease Control and Prevention Hong-Ying Li EcoHealth Alliance Peter Daszak EcoHealth Alliance Jun-Jie Hu Yunnan University Xing-Yi Ge (  xyge@hnu.edu.cn ) Hunan University https://orcid.org/0000-0003-3964-5140


Main Text
Human taeniasis is caused by three species of tapeworms in the genus Taenia, including Taenia asiatica, Taenia saginata, and Taenia solium. Pigs are the intermediate host for T. solium and T. asiatica (mostly found in pig liver), and T. saginata mainly establishes in bovine striated muscles [1]. As the de nitive host, humans are infected by ingesting raw or undercooked infected pork and beef [2]. T. asiatica is mainly con ned to Asian countries, including Korea, China, Thailand, etc. T. saginata and T. solium are distributed all over the world [3][4][5][6]. The genetic similarity between T. saginata and T. solium are high. Here, we report a rare case caused by a T. saginata-T. asiatica hybridized tapeworm in the Yunnan Province of China.
On February 3, 2019, an 18-year-old boy was hospitalized in E'Shan, Yunnan, China, due to a long-lasting abdominal distension, and white tapeworm segments in his feces. Complete blood count and biochemical analysis were performed and the results showed the high eosinophil count (5.3 × 10 8 /L).
Traditional Chinese medicine was prescribed for the treatment with oral administration of pumpkin (Cucurbita moschata) seed power rst; the extract of areca nut (Areca catechu) one hour after; followed by 30% hydrated magnesium sulfate (MgSO 4 ) solution half an hour later. A cestode of about 2.7 m long was expelled after 1.5 hours since the medicine was administrated.
The tapeworm specimen was collected and kept in a dish with phosphate buffer solution ( Figure,  The complete 1620 bp cox1 gene sequences showed 99.1-99.9% nucleotide (nt) identity to that of T. saginata, 95.7-96.1% to T. asiatica, and 88.8-89.1% to T. solium ( Figure, panel E). The complete 912 bp nadh1 gene sequences showed 99.2% nt identity to that of T. saginata, 95.7-95.9% to T. asiatica, and 87.1%-87.4% to T. solium ( Figure, panel D). However, 2 different sequences of 18S rRNA gene were identi ed. One sequence was 2604 bp which showed 99.4% nt identity to that of T. saginata, and 97.8% to that of T. asiatica. The other sequence was 2579 bp and showed 98.3% nt identity to that of T. asiatica, and 97.5% to that of T. saginata ( Figure, panel F). According the cox1 gene and nadh1 gene, this specimen was most close to T. saginata tapeworms reported in Thailand. But, the 18S rRNA gene of this specimen showed hybridization between T. saginata and T. asiatica, and indicated that the tapeworm was a heterozygote (Figure,  Morphological characteristics of the adult worms, larvae and ova have been used for cestodes identi cation. However, the phenotypic methods are time-consuming and require special expertise. Now, PCR ampli cation-sequencing and real-time-PCR have been employed to determine the species of cestodes [7]. The targets are conserved regions in mitochondrial genes (nadh1 and cox1) and ribosomal RNA genes (18S and 28S). Most of the previous reports used nadh1 and cox1. However, the present case indicates that sequencing the mitochondrial genes only may not able to identify the hybridization of close related species, like T. asiatica and T. saginata, because of the matrilineal inheritance of mitochondrial genes [8]. Thus, amplifying and sequencing mitochondrial and ribosomal genes together will be better for species determination.
The parents of this patient were examined and showed negative of tapeworms. The life history of this patient revealed his dietary preference of barbecue, which is suspected as the source of the tapeworm.
Even with the improvement of sanitation and dramatically changes in dietary habits. Taeniasis is still prevalent in some rural areas in Yunnan. A current investigation in 4 townships in Yunnan reported a 16.71% infection rate in the total population and all the cases were diagnosed as T. asiatica infection [5]. But this case suggested that T. saginata and even new types of hybridized taeniases may exist in the region and further investigation and research on the pathogens of human taeniases in Yunnan is needed.

Conclusion
The present study reported an investigation of a young male patient with taeniasis case in Yunnan, China, in 2019. The genetics and evolution of the tapeworm was identi ed by both of the mitochondrial genes cox1 and nadh1, and the nuclear 18S rRNA gene. The sequencing and phylogenetic analysis of the nuclear 18S rRNA gene indicated that the tapeworm was a heterozygote of T. saginata and T. asiatica. This case study gave the rst evidence of hybridization between T. saginata and T. asiatica in China. In addition to mitochondrial genes, nuclear gene information is very important to understand the genetic characteristics of pathogens in the future. Gene sequences obtained from this study were deposited in GenBank under accession numbers: MN452861-MN452864.

List Of Abbreviations
YZZ treated the patient and collected the sample; CHL, ZY, YQ, and JL did the gene ampli cation, cloning and sequencing; ZSH, WF, and JJH performed the morphological investigation; XYG coordinated and designed the experiment; XYG, HYL, PD, YQ, and YZZ wrote the manuscript.