bioRxiv preprint doi: https://doi.org/10.1101/511162; this version posted January 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Multi-locus characterization and phylogenetic 2 inference of Leishmania spp. in snakes from 3 Northwest China 4 5 Han Chen1¶, Jiao Li1¶, Junrong Zhang1, Jinlei He1, Jianhui Zhang1, Zhiwan Zheng1, Dali 6 Chen1*, Jianping Chen1* 7 8 1 Department of Parasitology, West China School of Basic Medical Sciences and 9 Forensic Medicine, Sichuan University, Chengdu, Sichuan, China 10 11 * Corresponding author 12 E-mail: [email protected] (DLC); [email protected] (JPC) 13 14 ¶ These authors contributed equally to this work. 15 bioRxiv preprint doi: https://doi.org/10.1101/511162; this version posted January 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 16 Abstract 17 Background: Leishmaniasis caused by protozoan parasite Leishmania is a neglected 18 disease which is endemic in the northwest of China. Reptiles were considered to be the 19 potential reservoir hosts for mammalian Leishmaniasis, and Leishmania had been 20 detected in lizards from the epidemic area in the northwest of China. To date, few 21 studies are focused on the natural infection of snakes with Leishmania. 22 Methods: In this study, 15 snakes captured from 10 endemic foci in the northwest of 23 China were detected Leishmania spp. on the base of mitochondrial cytochrome b, heat 24 shock protein 70 gene and ribosomal internal transcribed spacer 1 regions, and 25 identified with phylogenetic and network analyses. 26 Result: In total, Leishmania gene was found in 7 snakes. The phylogenetic inference 27 trees and network analysis suggests that the species identification was confirmed as 28 Leishmania donovani, Leishmania turanica and Leishmania sp. 29 Conclusion: Our work is the first time to investigate the natural Leishmania spp. 30 infection of snakes in the northwest of China. Mammalian Leishmania was discovered 31 in snakes and the reptilian Leishmania was closely related to the clinical strains both 32 prompt the importance of snakes in the disease cycle. To indicate the epidemiological 33 involvement of snakes, a wide sample size in epidemic area and the pathogenic features 34 of reptilian Leishmania promastigotes are recommended in the future research. 35 Keywords: Leishmania; snake; cyt b; Hsp70; ITS1; phylogeny; northwest China 36 bioRxiv preprint doi: https://doi.org/10.1101/511162; this version posted January 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 37 Introduction 38 Leishmaniasis is a neglected disease caused by infection with flagellate protozoan 39 parasite Leishmania of the family Trypanosomatidae. There are three main forms of 40 leishmaniasis: visceral or kala-azar (VL), cutaneous (CL) and mucocutaneous (MCL). 41 Over 20 Leishmania species known to be infective to humans are transmitted by the 42 bite of infected sand flies of which at least 98 species were proven or probable vectors 43 worldwide [1]. An estimated 2 million new cases and 50 000 deaths occur in over 98 44 countries annually [2]. In China, which is one of 14 high-burden countries of VL [3], 45 there exists three epidemiological types: anthroponotic VL (AVL), mountain-type 46 zoonotic VL (MT-ZVL), and desert-type ZVL (DT-ZVL) [4]. Acknowledging the 47 information on the epidemiology including the vector and animal reservoir hosts of the 48 disease is better to understand the disease and its control. 49 In view of the topography in the northwest of China, DT-ZVL is found largely in 50 the oases and deserts including the southern and eastern of Xinjiang Uygur 51 Autonomous Region, the western part of the Inner Mongolia Autonomous Region, and 52 northern Gansu province [5]. Surveillance of the disease reflects that DT-ZVL is 53 persistence with sporadic outbreaks and still not under control now [5]. Sand flies 54 (Phlebotominae) were recognized as the transmission vector in the traditional sense, 55 while ticks (Ixodoidea) had been reported to be the vector in the transmission of canine 56 VL [6–9]. In addition to humans, there are various kinds of hosts, mainly mammals 57 such as canids, hyraxes and rodents, which could be the source of infection as the 58 reservoirs. Because transmission occurs in a complex biological system involving the bioRxiv preprint doi: https://doi.org/10.1101/511162; this version posted January 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 59 human host, parasite, vectors and in some cases an animal reservoir host, the control of 60 Leishmaniasis is pretty complex. As for the desert in the northwest of China, there is 61 no consistent agreement regarding dissemination of the actors playing the key roles in 62 leishmaniasis. 63 Moreover, reptiles, mainly lizards, were found harboring Leishmania parasites 64 with controversies in the part of spreading the disease [10]. Blood cells of lizards 65 containing amastigotes were first found by Chatton and Blanc in Tarentola mauritanica 66 from southern Tunisia [11] and then several cases from the same lizard species were 67 reported in the next decades [12]. Wenyon provisionally named the leptomonad 68 flagellates in gecko as Leishmania tarentolae [13]. Killick-Kendrick recognized 69 Sauroleishmania as a separate genus for the leishmanial parasites of reptiles [14], later 70 DNA sequence-based phylogenies had clearly placed it within the Leishmania genus as 71 a secondarily derived development from the mammalian species [15–17]. Most studies 72 were focused on the infection of Leishmania parasites in lizards, but few were in snakes 73 except Belova and Bogdanov cultured promastigotes from the blood of five species of 74 snakes in the Turkmen S.S.R. [14,18]. 75 In reality, the detection of reptilian Leishmania is rare in China. Zhang et al. was 76 the first team to detect Leishmania via molecular methods from the lizards captured in 77 the northwest of China [19]. Interestingly, besides reptilian Leishmania, mammalian 78 Leishmania species (L. donovani and L. tropica) were involved despite 79 Sauroleishmania was considered not to infect humans or other mammals [20,21]. This 80 may support the potential reservoir role of lizards for human leishmaniasis. bioRxiv preprint doi: https://doi.org/10.1101/511162; this version posted January 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 81 Similarly, we employed the highly sensitive polymerase chain reaction (PCR) to 82 detect the presence of Leishmania spp. in snakes. The sequences of various genetic 83 markers have been used successfully to infer the phylogenetic relationships within 84 Leishmania including the sequences of DNA polymerase α [15], RNA polymerase II 85 [15], 7SL RNA [22–24], ribosomal internal transcribed spacer [25,26], mitochondrial 86 cytochrome b gene [27,28], heat shock protein 70 gene [29,30] and the N- 87 acetylglucosamine-1-phosphate transferase gene [31]. Therefore, this study was based 88 on three genetic loci, i.e., cytochrome b (cyt b), heat shock protein 70 (Hsp70), and 89 internal transcribed spacer 1 (ITS1) region to genetically characterize Leishmania spp. 90 detected in 15 snakes, and conduct tree-based species delimitation to infer the 91 phylogenetic positions by comparison with some representative Leishmania sequences 92 retrieved from GenBank. It was the first time to survey the infected snakes of 93 Leishmania in the northwest of China and try to reveal the primary evidence on the 94 epidemic role of snakes for human leishmaniasis. 95 96 Materials and methods 97 Study area and sampling 98 15 snakes identified as 3 species by morphological characters were captured alive 99 by hand and snake hooks at 10 sites across the endemic foci of VL in the northwest of 100 China (Table 1). These sites are located in arid desert areas with the altitude ranging 101 from 537 to 1222 m above sea level. Franchini [32,33] seems to have observed rare 102 amastigotes from the liver of lizards, and Shortt & Swaminath [34] cultured a strain bioRxiv preprint doi: https://doi.org/10.1101/511162; this version posted January 3, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 103 Leishmania on NNN (Novy-MacNeal-Nicolle) medium from the liver of lizards. 104 Consequently, a part of the liver was taken from the abdominal cavity of each snake to 105 detect the presence of Leishmania via PCR. In total, 15 liver specimens were collected 106 in Eppendorf tube and stored at -20°. 107 All surgery was performed under sodium pentobarbital anesthesia, and all efforts 108 were made to minimize suffering. The protocol was approved by the ethics committee 109 of Sichuan University (Protocol Number: K2018056).