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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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).
110
111 Table 1 List of sampling localities, snake species and sample size in this study
Site Label Locality Species sample
size
P1 Shawan county, Tarbagatay Psammophis lineolatus 1
Prefecture, Xinjiang
P2 Tekes County, Ili Kazak Gloydius halys 1
Autonomous Prefecture, Xinjiang
P3 Tekes County, Ili Kazak Natrix tessellate 1
Autonomous Prefecture, Xinjiang
P4 Yiwu County, Hami, Xinjiang Psammophis lineolatus 1
P5 Yizhou Area, Hami, Xinjiang Psammophis lineolatus 5
P6 Dunhuang, Jiuquan City, Gansu Psammophis lineolatus 1
Province
P7 Heshuo, Bayingol Mongolian Psammophis lineolatus 2 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.
Autonomous Prefecture, Xinjiang
P8 Makit County, Kashgar Prefecture, Psammophis lineolatus 2
Xinjiang
P9 Yopurga County, Kashgar Psammophis lineolatus 1
Prefecture, Xinjiang
P10 Yengisar County, Kashgar Psammophis lineolatus 2
Prefecture, Xinjiang
total 15
112
113 DNA extraction, amplification, cloning and sequencing
114 protocols
115 Total genomic DNA was extracted from liver tissues using the commercial kit,
116 TIANamp® Genomic DNA Kit (TIANGEN Bio, Beijing, China) following the
117 protocols of the manufacturer. PCR primers specific for the Leishmania synthesized by
118 Tsingke Biological Technology Co., Ltd (Chengdu, China) were used to amplify cyt b
119 [35], Hsp70 [36] and ITS1 [37] gene fragments by PrimeSTAR Max DNA polymerase
120 (TaKaRa Bio, Shiga, Japan) according to the manufacturer’s instruction. We picked
121 Leishmania strain MHOM/CN/90/SC10H2 as the positive control while preparing the
122 mixture, and the negative control was treated without template DNA. The PCR products
123 were purified by excision of the band from agarose gel using the Universal DNA
124 Purification Kit (TIANGEN Bio, Beijing, China), and ligated into a pGEM®-T vector
125 (Promega Co., Madison, USA) with added the poly (A) tails. After transformation of 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.
126 Escherichia coli DH5α competent cells with the ligation product and X-gal blue-white
127 screening, 8 positive colonies were selected by PCR using the plasmid primers T7 and
128 SP6, and grown in LB/ampicillin medium. DNA was extracted and sequenced at
129 Tsingke Biological Technology Co., Ltd (Chengdu, China). The chromatograms were
130 validated and assembled in BioEdit v7.2.6 [38].
131
132 Sequence alignment and phylogenetic analyses
133 GenBank searches were performed to initially identify the species of the original
134 DNA sequences using BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi). And all the
135 nucleotide sequences generated in this study have been submitted to the GenBank
136 database. The sequences were multiple-aligned with a set of Leishmania strains of each
137 locus examined in this study retrieved from the GenBank using ClustalW of MEGA
138 (Molecular Evolutionary Genetic Analysis v7.0.26 [39]) with its default option and
139 refined manually. The alignments were further trimmed to exclude regions with missing
140 data and then distinct haplotypes were defined by DAMBE v7.0.1 [40,41]. The
141 evolutionary history was inferred by phylogenetic tree construction using Bayesian
142 inference (BI). Gaps were treated as missing data and each haplotype was treated as a
143 taxon in the analyses. The program PartitionFinder v2.1.1 [42] was used to select the
144 most appropriated substitution model of all phylogenetic analyses. Bayesian analyses
145 were carried out using the program MrBayes v3.2 [43] which the trees were started
146 randomly. 2 parallel sets of 4 simultaneous Monte Carlo Markov chains (3 hot and 1
147 cold) were run for 10,000,000 generations until convergence was reached (stopval = 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.
148 0.01) and the trees were sampled for every 1000 generations. Temperature heating
149 parameter was set to 0.2 (temp = 0.2) and burn-in was set to 25% (burninfrac = 0.25).
150 The results of Bayesian analyses were visualized in FigTree v1.4.3 (available at
151 http://tree.bio.ed.ac.uk/software/figtree/).
152
153 Network reconstructions
154 Due to the limit of bifurcating tree to represent intraspecific gene evolution,
155 haplotype networks may more effectively portray the relationships among haplotypes
156 within species. The phylogenetic networks were constructed by using the Median
157 Joining (MJ) network reconstruction method in NETWORK v5.0.0.3 (available at
158 http://www.fluxus-engineering.com/sharenet.htm) [44,45].
159
160 Results
161 Leishmania infection of the captured snakes in the study area
162 Through preliminary determination of identity using BLASTn, the total
163 prevalence of Leishmania DNA detected via PCR was 46.7% (7/15). 5 (33.3%) snakes
164 were detected as positive for cyt b, 4 (26.7%) snakes for Hsp70 and 2 (13.3%) for ITS1.
165 Considering the sensitivity of different loci, two samples from P1 and P2 were
166 amplified successfully in all three loci, while other samples were detected by only one
167 locus in this study. The snakes from P1, 2, 3, 4, 5, 6, 9 were Leishmania-positive of all
168 the 10 foci as shown in Fig 1.
169 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.
170 Figure 1 Sampling sites of snakes in Northwest China, along with the current
171 foci of endemicity of anthroponotic visceral leishmaniasis (AVL) and desert-type
172 zoonotic visceral leishmaniasis (DT-ZVL) in China. The site numbers P1-P10
173 correspond to those in Table 1. Solid five-pointed star (★) represents the endemic focus
174 of AVL and solid triangle (▲) represents the endemic focus of DT-ZVL [4,5]. The
175 species of Leishmania detected from the samples are shown for different colors (yellow:
176 L. donovani complex, blue: L. turanica, pink: Leishmania sp.)
177
178 Sequence characteristics
179 After the haplotypes were defined, 10 cyt b sequences, 7 Hsp70 sequences and 10
180 ITS1 sequences were obtained in this study and deposited in the GenBank database
181 under Accession No. MK330198-MK330207, MK330208-MK330214 and
182 MK300708-MK300717, respectively (Table 2). PCR amplification of each target locus
183 resulted in amplicons of the expected sizes as follows: cyt b (543 bp), Hsp70 (738-767
184 bp), and ITS1 (328-334 bp). The GC contents were 23.4% for cyt b, 64.9% for Hsp70
185 and 41.8% for ITS1, respectively. Thus, both their lengths and their GC contents are
186 within the range of corresponding sequences of other Leishmania species.
187
188 Table 2 List of the lizard samples, origin, isolated Leishmania spp. and GenBank
189 accession numbers.
Snake Voucher Origi Isolated Haplotyp GenBank Sequenc
species number n Leishmani e number accession e length 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.
a spp. (bp)
(A)cyt b
Psammophi Guo470 P1 L. C1 MK33019 543
s lineolatus 7 donovani 8
L. sp. C6 MK33019 543
9
Gloydius Guo470 P2 L. C1 MK33020 543
halys 8 donovani 0
Natrix Guo470 P3 L. C2 MK33020 543
tessellata 9 donovani 1
L. turanica C4 MK33020 543
2
L. sp C5 MK33020 543
3
L. sp. C6 MK33020 543
4
Psammophi Guo471 P4 L. C3 MK33020 543
s lineolatus 0 donovani 5
L. turanica C4 MK33020 543
6
Psammophi Guo699 P9 L. sp. C6 MK33020 543
s lineolatus 5 7 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.
(B) Hsp70
Psammophi Guo470 P1 L. H1 MK33020 738
s lineolatus 7 donovani 8
L. infantum H2 MK33020 738
9
Gloydius Guo470 P2 L. infantum H2 MK33021 738
halys 8 0
Psammophi Guo402 P6 L. sp. H3 MK33021 767
s lineolatus 3 1
L. sp. H4 MK33021 766
2
L. sp. H5 MK33021 767
3
Psammophi Guo402 P5 L. sp. H5 MK33021 767
s lineolatus 6 4
(C)ITS1
Psammophi Guo470 P1 L. sp. I1 MK30070 334
s lineolatus 7 8
L. sp. I2 MK30070 328
9
L. sp. I3 MK30071 332
0 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.
L. sp. I4 MK30071 330
1
L. sp. I5 MK30071 330
2
L. sp. I6 MK30071 330
3
L. sp. I7 MK30071 330
4
Gloydius Guo470 P2 L. sp. I8 MK30071 330
halys 8 5
L. sp. I9 MK30071 332
6
L. sp. I10 MK30071 328
7
190
191 Phylogenetic relationship
192 Prior to the phylogenetic analyses, the most adequate models of nucleotide
193 substitution were selected by PartitionFinder v2.1.1: GTR +G and GTR+I+G for cyt b,
194 HKY+I for Hsp70, K80+G for ITS1. Using Bayesian inference method, the trees
195 showed similar phylogenetic topology for all three loci supported by posterior
196 probability (PP) values.
197 Regarding the BI tree inferred from each locus, the phylogenetic analyses inferred 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.
198 from the aligned matrix of cyt b (Fig 2 (A)) separated two distance clades, the Viannia
199 isolates apparently formed an independent clade (PP=1.0) outside the clusters of those
200 other species (PP=1.0), while the subgenera Leishmania and Sauroleishmania were still
201 separated into different branches (PP=1.0 and 0.56, respectively). The isolates C1, C2
202 and C3 were clustered with L. donovani and L. infantum (PP=1.0). The isolate C4 was
203 shared an identical cyt b sequence to a L. turanica isolate and they were grouped with
204 L.arabica, L. major, L. tropica, L. aethiopica and L. killicki in a subclade (PP=0.91)
205 separated with L. donovani complex. The isolate C6 shared identical sequences with
206 Chinese Leishmania sp. isolates from Yang et al. [26] was clustered in a separated clade
207 (PP=0.96) together with C5 and other Leishmania sp.
208
209 Figure 2 The majority-rule consensus tree (midpoint rooted) of three genetic loci
210 inferred from Bayesian inference by using MrBayes v.3.2, and the corresponding
211 median-joining networks were implemented by NETWORK v5.0.0.3: (A) Cyt b, (B)
212 Hsp70, (C)ITS1.
213
214 The Hsp70 analysis (Fig 2 (B)) showed a similar structure to the phylogenetic tree
215 of cyt b: Viannia (PP=1.0), Sauroleishmania (PP=1.0) and Leishmania (PP=0.6). The
216 isolates H1 and H2 searching by BLASTn (Megablast) revealed over 98% identity with
217 L. donovani and L. infantum respectively, while both of them were nested within L.
218 donovani complex. In this subclade, the isolates of other species of Leishmania
219 subgenus (except L. mexicana clustered by the PP 1.0) were not in reciprocally 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.
220 monophyletic groups. The isolates H3, H4 and H5 were clustered in a separated clade
221 (PP=1.0) with Leishmania sp. from China.
222 For the ITS1-5.8S region, the phylogenetic tree as Fig 2 (C) shown was constructed
223 by three clades with high support (PP=1.0, 0.97 and 1.0, respectively), corresponding
224 to the three subgenera Viannia, Leishmania, and Sauroleishmania. All the 10 fragments
225 from snakes formed a strongly supported cluster with Leishmania sp. isolated from
226 lizards [19] (PP=1.0), and revealed a close relationship of isolates from Sergentomyia
227 minuta in Europe.
228
229 Median Joining network
230 To get additional insight into the relationships among the strains, we analyzed our
231 data sets using the Median Joining algorithm network approach. The MJ analysis of
232 three regions was congruent with the topology described in the BI trees, and no
233 haplotype was shared among the species groups.
234 The network of cyt b in Fig 2 (A) showed that the isolate C6 and a L. donovani
235 strain from Xinjiang, China (HQ908267) seemed to be the central haplotypes. C1
236 (MK330198 isolated from two snakes captured at P1 and MK330200 from P2), together
237 with C2 (MK330201 from P3) and C3 (MK330205 from P4) were most closely related
238 to the central HQ908267, within 2 mutational steps. C4 (from P3 and P4) shared the
239 same haplotype with one strain of L. turanica from Russia, and only 2 and 3 steps away
240 from the Chinese strains L. turanica (from Xinjiang) and L. gerbilli (from Gansu)
241 respectively. The Leishmania sp. isolate C6 (from P1 and P9) as the central haplotype 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.
242 shared the same sequences to Leishmania sp. from China with a wide geographical
243 distribution, including Shandong (HQ908255), Sichuan (HQ908260, HQ908264),
244 Gansu (HQ908263, HQ908271).
245 For the Hsp70 region, the haplotype network of Fig 2 (B) could intuitively reflect
246 the genetically small distances between the obtained sequences H1 (MK330208
247 isolated from P1), H2 (MK330209 and MK330210 from P1 and P2, respectively) and
248 the reference L. donovani and L. infantum strains from India, Sudan and the northwest
249 of China. The haplotype H5 shared by the isolates from P5 (MK330213) and P6
250 (MK330214) was the interior haplotype of Leishmania sp. and may be older than other
251 tip haplotypes H3 (MK330211) or H4 (MK330212) which were both from P6). It
252 reflected a close distance between JX021439 (Xinjiang, China) and the interior
253 haplotype as three mutational steps.
254 As shown in the haplotype network of ITS1-5.8S in Fig 2 (C), the Leishmania sp.
255 group was centered around haplotype KU194967 (Dunhuang, China). All haplotypes
256 found in this group of Sauroleishmania isolated from lizards of the northwest of China
257 were very closely related to each other. One strain from Tuokexun (KU194973) shared
258 the same haplotype with I4 (MK300711 from P1). Similarly, two strains from
259 Dunhuang (KU194969, KU194971) and another one from Lukchun (KU194939)
260 shared the same haplotype with I10 (MK300709 from P2). In addition, three strains
261 isolated from Sergentomyia minuta of Europe shared a close distance with the
262 Sauroleishmania group as less than 3 steps.
263 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.
264 Discussion
265 This study was the first time to detect the prevalence of Leishmania infection in
266 snakes by DNA sequencing. Our result showed that the snakes from 7 loci of the 10
267 study areas were detected positive for Leishmania. Yopurga county, Hami and
268 Dunhuang are the DT-ZVL epidemic foci [5], while Shawan and Texes county are lack
269 of data currently.
270 Besides one snake (Natrix tessellate) from Texes were detected three Leishmania
271 species (including L. donovani, L. turanica and Leishmania sp.), the snakes from other
272 positive foci were detected 1-2 species. Leishmania sp. was the most widely distributed,
273 while the distribution of the other two species was lack of the significant geographic
274 specificity in this study owing to the limit of the sample size.
275 The effect of many factors (e.g., the condition of specimen, the DNA extraction
276 methodology, the length of amplified gene sequences and the PCR protocols) would
277 cause different positive rates. Amplification of multiple gene sequences could
278 significantly increase the comprehensiveness of the Leishmania infections to be
279 investigated in snakes. Various genetic loci had been developed to detect Leishmania,
280 such as kinetoplast DNA (kDNA) genes, nuclear DNA (nDNA) genes and ribosomal
281 RNA (rRNA) genes [25,46,47]. Cyt b, one of the cytochromes encoded on the
282 kinetoplast DNA maxicircle, is considered one of the most useful markers that had
283 delimitated most tested Leishmania species for phylogenetic work. Hsp70 gene, an
284 antigen gene on the chromosome, is the most optimal marker for the species
285 discrimination and phylogenetic inference. ITS1, one of the highly variable regions of 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.
286 rRNA gene, have been successfully used to resolve taxonomical questions and to
287 determine phylogenetic affinities among closely related Leishmania species. Therefore,
288 three genetic loci were carried out in this study and the positive rates were different
289 indeed. Two samples from Shawan and Tekes were detected Leishmania sp. and L.
290 donovani by all the three genetic loci while other samples were detected by only one
291 genetic locus. It was suggested that different Leishmania primers might have different
292 selection preferences for the gene amplification. At the same time, the use of different
293 primers to amplify the corresponding gene fragments did significantly improve the
294 detection rate of infected snake samples.
295 It was curious about the spreading mechanism of Leishmania in reptiles. Wilson
296 & Southgate assumed that transmission was achieved by the reptile host eating an
297 infected sand fly [48,49]. Nevertheless, there were some reptile-feeding species of
298 phlebotomine as we known [50,51]. The same as lizards, it was reasonable that snakes
299 could be infected naturally. Furthermore, there were several reports of snake
300 trypanosome [52–54] which was very close to Leishmania in phylogeny. In view of the
301 small sample size of each location and species, the infection rate does not make much
302 sense. Nevertheless, all three detected snake species in the endemic foci of VL were
303 found Leishmania DNA even the sample size of both Gloydius halys and Natrix
304 tessellate was 1. Therefore, a wide range of sample size would be necessary for the
305 further study to enrich our understanding of the natural infections of Leishmania
306 parasites in snakes.
307 One of the most noteworthy findings in the present study was that two mammalian 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.
308 Leishmania taxa, i.e., L. donovani complex and L. turanica appeared to occur in snakes.
309 Previously, Zhang et al. had a similar discovery in lizards [19]. They believe lizards
310 played a reservoir role for human leishmaniasis because of the common ancestry of the
311 obtained haplotypes of L. tropica and clinical samples. At present, apart from the above
312 study, there are few studies of mammalian parasites natural infections in reptiles.
313 Belova [10] carried out intensive research on reptilian and mammalian Leishmania and
314 found that the mammalian parasites (such as L. donovani, L. tropica, and some other
315 promastigotes recovered from sandflies) were infective to lizards. To determine that the
316 reptile is reservoir host of human leishmaniasis, the Leishmania strain collection from
317 VL patients in the epidemic foci and phylogenetic analyses with the reptilian strains are
318 needed.
319 On the other hand, the Leishmania sp. isolated from snakes in this study shared
320 the same haplotypes or located in the close proximity to the strains from VL patients or
321 canines in China as the network shown. Indeed, the same Chinese Leishmania sp.
322 isolates were speculated to be closely related to the lizard-infected strains, L. tarentolae,
323 on the basis of COII [55] and cyt b [28] genes. Analogously, L. adleri was found to
324 cause transient CL in humans by Manson-Bahr & Heisch [56], and could infect rodents
325 which the strains were isolated by Adler [57] and Coughlan et al. [58]. L. tarentolae
326 was chosen as a candidate vaccine against VL in virtue of the character that the parasites
327 could invade in human macrophages and exist as amastigotes in mammals with slower
328 replication [59]. These studies seem to be contradictory with the former which indicated
329 that Sauroleishmania was not pathogenic to human [10]. For the reason that the Chinese 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.
330 strains from patients or canines were isolated in the 70s to 90s of last century, the
331 medical records were no longer available, and we could not confirm whether the
332 patients got leishmaniasis due to Sauroleishmania or co-infection with other
333 mammalian Leishmania. Thus, to further explore the pathogenicity of Sauroleishmania,
334 it is recommended to isolate and culture the promastigotes from liver or blood of
335 reptiles and study the virulence to mammals.
336 In conclusion, the present study is the first time to investigate the natural
337 Leishmania spp. infection of snakes in the northwest of China. The species
338 identification was primarily achieved by comparisons of the obtained sequences with
339 the GenBank database and determined the belongs with phylogenetic and network
340 analyses, including Leishmania sp., L. donovani, L. turanica. This is the first time to
341 discover mammalian Leishmania in snakes, after the similar finding in lizards. The
342 Sauroleishmania strains in this study closely related to the clinical isolates, which
343 suggests us to be cautious about the widely accepted view that Sauroleishmania is
344 nonpathogenic to human. These findings invite us to ponder the importance of snakes
345 in the disease cycle. However, more snake samples in epidemic foci and the pathogenic
346 features of Sauroleishmania promastigotes are required for indicating their
347 epidemiological involvement.
348
349 Acknowledgments
350 We are grateful to Xianguang Guo (Chengdu Institute of Biology, Chinese
351 Academy of Sciences) for his assistance in fieldwork. 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.
352
353 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.
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