Species identification and phylogenetic analysis of Leishmania isolated from patients, vectors and hares in the Xinjiang Autonomous Region, The People's Republic of China.

BACKGROUND: Visceral leishmaniasis (VL) has been declared as one of the six major tropical diseases by the World Health Organization. This disease has been successfully controlled in China, except for some areas in the western region, such as the Xinjiang Autonomous Region, where both anthroponotic VL (AVL) and desert type zoonotic VL (DT-ZVL) remain endemic with sporadic epidemics. METHODOLOGY/PRINCIPAL
FINDINGS: Here, an eleven-year survey (2004-2014) of Leishmania species, encompassing both VL types isolated from patients, sand-fly vectors and Tarim hares (Lepus yarkandensis) from the Xinjiang Autonomous Region was conducted, with a special emphasis on the hares as a potential reservoir animal for DT-ZVL. Key diagnostic genes, ITS1, hsp70 and nagt (encoding N-acetylglucosamine-1-phosphate transferase) were used for phylogenetic analyses, placing all Xinjiang isolates into one clade of the L. donovani complex. Unexpectedly, AVL isolates were found to be closely related to L. infantum, while DT-ZVL isolates were closer to L. donovani. Unrooted parsimony networks of haplotypes for these isolates also revealed their relationship.
CONCLUSIONS/SIGNIFICANCE: The above analyses of the DT-ZVL isolates suggested their geographic isolation and independent evolution. The sequence identity of isolates from patients, vectors and the Tarim hares in a single DT-ZVL site provides strong evidence in support of this species as an animal reservoir.

Introduction

Visceral leishmaniasis (VL), caused by species of the Leishmania donovani complex, is a potentially fatal disease if not treated [1]. In China, before the implementation of the national infectious diseases control programs in 1951, it was one of the major parasitic diseases. A total of approximately half a million VL cases were then reported in 16 provinces north of the Yangtze River [2]. The control programs have successfully eliminated VL in the northeastern plain, but not in the west and northwest regions, where this disease has persisted as three different types: mountain type zoonotic VL (MT-ZVL), anthroponotic VL (AVL) and desert type zoonotic VL (DT-ZVL) [2]. The total number of cases reported from these areas between 2002 and 2011 were 3,169 VL cases, ranging from 140 to 509 cases per year. This study considers the causative agents of AVL and DT-ZVL from the Xinjiang Autonomous Region in the northwest of China.

One old endemic site in Xinjiang is the Kashgar alluvial plain and the Aksu oasis, where the AVL is an endemic disease, whose pathogen is transmitted by the bites of the peridomestic vector Phlebotomus longiductus. Most patients are over 6 years old (70%) [3]. This type of VL has been considered as anthroponotic on account of its familial clustering and the uncertainty about the existence of potential animal reservoirs [4]. As such, the causative agent has long been referred to as L. donovani. More recently, isolates collected from this endemic region, have been identified as L. infantum based on the nagt and other single-copy gene analyses (isolates HOM/CN/91/911, HOM/CN/92/921, HOM/CN/97/9701) [5] and the multilocus sequence typing (MLST) of five enzyme-coding genes (e.g. isolate MHOM/CN/80/801) [6].

DT-ZVL is endemic in the northwestern China, mainly in the Bachu and Jiashi counties of Xinjiang, but also in the western part of Inner Mongolia and northern Gansu [3], with wild sand fly species Phlebotomus wui and P. alexandri as vectors specific for the sandy desert and pebble desert subtypes, respectively. Most patients are infants of 2 years-old or younger (94%). DT-ZVL is considered zoonotic and its pathogen is transmitted by wild sand fly species, Phlebotomus wui and P. alexandri, which live in rodent burrows as their natural habitats. This is further supported by the lack of familial clustering. Extensive studies in desert rodents, e. g. the great gerbil, have failed to establish their role as a reservoir animal for DT-ZVL, but have implicated the Tarim hare (Lepus yarkandensis) as a potential reservoir for DT-ZVL [7]. These wild hares are unique to the Tarim basin in the Taklamakan desert. They were found to be seropositive for Leishmania antigens (rk39) at a high prevalence (25%), microscopically positive for amastigotes in infected tissues and to suffer from the typical VL symptom of splenomegaly, hepatomegaly and sometimes ulcerative lesions of the ears. Moreover, promastigotes were isolated from the spleens of infected hares and found to be virulent for the laboratory-reared susceptible steppe rodent, Lagurus lagurus. The DNA (nagt locus) sequence identity of hare-, vector- and patient-derived isolates provided the preliminary evidence that the hare could be a potential reservoir of DT-ZVL [7]. The causative agent was considered to be L. infantum, since all the nagt sequences were identical to those of the reference L. infantum isolates from other endemic areas in China and elsewhere [5]. This was further confirmed by the ITS1 locus analysis [8]. In contrast, investigation of similar isolates with the same designated code names (MHOM/CN/00/Wangjie1 as a reference strain) classified them as L. donovani based on the ITS1 sequence [9] and MLST analyses [6].

The heterogeneity of Chinese Leishmania isolates was first observed by isoenzyme analysis and by DNA hybridization (kinetoplast and nuclear DNA) [10-12]. More recently, the diversity of the Chinese Leishmania isolates has been addressed by several other studies using various molecular markers [6,9,13-16]. By analysis of the molecular fingerprints, it was found that the isolates from the three epidemiological disease types were distinguishable (reviewed by [17]). However, there were limitations in these studies. Firstly, only a limited number of isolates were available for each given disease type. Secondly, the parasite samples did not represent the complete transmission cycle. Thirdly, only one isolate (IPHL/CN/77/XJ771) from the sand fly vector found in the DT-ZVL region has been studied thus far. Therefore, the species and their relationship with the epidemiological cycles in the regions of Xinjiang remained unclear. This was especially the case for DT-ZVL which until now had an unknown animal reservoir.

To resolve this dilemma, 20 Leishmania isolates were collected from endemic foci of both AVL and DT-ZVL in Xinjiang and analyzed using three different phylogenetic markers (nagt, ITS1 and hsp70). Our analyses of representative isolates cultured from captured hares, patients and the vector P. wui strongly supports the conclusion that the Tarim hare serves as a unique reservoir of DT-ZVL.

Materials and methods

Ethics statement

Samples collected from patients were approved by The Ethical Committee of Xinjiang Uighur Autonomous Region Center for Disease Control and Prevention under license of #30460120. Formal verbal consent was obtained from each patient or parent of child patient.

Parasite collection and cultivation

Samples investigated in this study were collected in Bachu, Jiashi, Minfeng and Shufu counties of Xinjiang province, northwestern China, during an 11 year survey of the VL in the endemic regions of the Tarim Basin (Fig 1 and Table 1). In order to isolate and maintain parasites, bone marrow aspirates from patients, dissected gut of infected sand-flies and homogenates of the lesion or spleen tissue from infected Tarim hares were inoculated into the Lagurus lagurus or the grey hamsters. Parasites were recovered from spleen homogenates of infected animals and cultured in modified medium LLM at 27°C [7]. Parasites or spleen samples were stored at 70% ethanol before DNA extraction. Protocols for the use of animals were approved by The Center for Laboratory Animal Research of Xinjiang and Institutional Review Board for Animal Care at Sun Yat-Sen University.

Fig 1

The sampling areas in the Xinjiang Autonomous Region.

Leishmania samples were collected from oases (grey area) or desert (spotted area) (Upper Panel). SF, Shufu county; JS, eastern Jiashi county; BC, Bachu county; MF, Minfeng county. Black square: Patient, Black circle: Tarim hare, Black triangles: P.wui (vector). The Tarim basin is surrounded by the Tianshan mountains and Kunlun mountains which forms a barrier separating it from surrounding areas. https://usgs.maps.arcgis.com/apps/webappviewer/index.html?id=2b7d5dbc0be340b9b17f6d94aac5b713 (42.8–35.0°N, 71.4–87.6°E).

Table 1

Leishmania isolates from Xinjiang.

Name	WHO code	Source/Host (Age)	Time of isolation	Location*	Type	Haplotype		Genbank accession No. (ITS1, hsp70, nagt)
						ITS1	hsp70
934	MHOM/CN/2004/KBC-1	Patient (11 months)	Dec. 2004	Bachu	DT-ZVL	I2	H2	KU975140.1, KX150477.1, KX150463.1
2703	IMJW/CN/2005/KBC-2	P.wui	Jun. 2005	Bachu	DT-ZVL	I2	-	KU975141.1, NA, NA
2681	MLEP/CN/2005/KBC-1	Tarim hare	Dec. 2005	Bachu	DT-ZVL	I2	-	KU975142.1, NA, NA
418	MLEP/CN/2004/KBC-2	Tarim hare	Dec. 2004	Bachu	DT-ZVL	I2	H2	KU975143.1, KX150478.1, KX150464.1
432	MLEP/CN/2004/KBC-3	Tarim hare	Dec. 2004	Bachu	DT-ZVL	I2	H2	KU975144.1, KX150479.1, NA
3153	MHOM/CN/2009/KJS-1	Patient (1 year)	Jan. 2009	Jashi	DT-ZVL	I2	H2	KU975145.1, KX150480.1, KX150465.1
3081	MHOM/CN/2009/KJS-3	Patient (11 months)	Jan. 2009	Jashi	DT-ZVL	I2	H2	KU975146.1, KX150481.1, NA
3208	MHOM/CN/2009/KJS-4	Patient (7 months)	Feb. 2009	Jashi	DT-ZVL	I2	-	KU975147.1, NA, NA
3227	MHOM/CN/2009/KJS-5	Patient (7 months)	Nov. 2009	Jashi	DT-ZVL	I2	H2	KU975148.1, KX150482.1, KX150466.1
3228	MHOM/CN/2009/KJS-6	Patient (6 months)	Nov. 2009	Jashi	DT-ZVL	I2	H2	KU975149.1, KX150483.1, KX150467.1
2693	IMJW/CN/2008/KJS-1	P.wui	Jul. 2008	Jashi	DT-ZVL	I2	H2	KU975150.1, KX150484.1, KX150468.1
3044	MLEP/CN/2007/KJS-1	Tarim hare	Dec. 2007	Jashi	DT-ZVL	I2	H2	KU975151.1, KX150485.1, KX150469.1
3410	IMJW/CN/2014/HMF-1	P.wui	Sep. 2014	Minfeng	DT-ZVL	I2	H2	KU975152.1, KX150486.1, KX150470.1
3416	IMJW/CN/2014/HMF-2	P.wui	Sep. 2014	Minfeng	DT-ZVL	I2	H2	KU975153.1, KX150487.1, KX150471.1
3009	MHOM/CN/2009/KSF-1	Patient (2 years)	Jan. 2009	Shufu	AVL	I1	H1	KU975154.1, KX150488.1,NA
2616	MHOM/CN/2009/KSF-2	Patient (24 years)	Feb. 2009	Shufu	AVL	I1	H1	KU975155.1, KX150489.1, KX150472.1
3149	MHOM/CN/2009/KSF-3	Patient (10 years)	Mar. 2009	Shufu	AVL	I1	H1	KU975156.1, KX150490.1, KX150473.1
2618	MHOM/CN/2009/KSF-4	Patient (53 years)	Mar. 2009	Shufu	AVL	I1	H1	KU975157.1, KX150491.1, KX150474.1
3219	MHOM/CN/2009/KSF-6	Patient (4 years)	May. 2009	Shufu	AVL	I1	H1	KU975158.1, KX150492.1, KX150475.1
3344	MHOM/CN/2009/KSF-7	Patient (3 years)	Feb. 2009	Shufu	AVL	I1	-	KU975159.1, NA, NA

* Bachu, Jashi, Minfeng and Shufu are counties in Xinjiang Autonomous Region, the People’s Republic of China.

-, not available; NA, no amplicon.

DNA extraction, PCR amplification and sequencing of the ITS1, hsp70 and nagt region

Sample DNA was extracted as described elsewhere [18]. The ITS1, hsp70 and nagt fragments were amplified by using the following primer sets, ITS1: L5.8S (5’ TGA TAC CAC TTA TCG CAC TT 3’) and LITSR: (5’ CTG GAT CAT TTT CCG ATG 3’) [19]; hsp70: F25 (5’ GGA CGC CGG CAC GAT TKC T 3’) and R1310 (5’ CCT GGT TGT TGT TCA GCC ACTC 3’) [20]; nagt: L1(5’ TCA TGA CTC TTG GCC TGG TAG 3’) and L4 (5’ CTC TAG CGC ACT TCA TCG TAG 3’) using standard conditions as published [5]. PCR products were sequenced by Invitrogen (Life technology) and all sequences were deposited in GenBank under the accession numbers provided in .

Sequence alignment and phylogenetic analysis

The sequences were checked manually and aligned with a set of Leishmania strains retrieved from GenBank (Table 2) using MEGA, version 5.0 [21] The unaligned 5’ and 3’ ends were removed before phylogenetic analysis. An imported Iranian L. major strain (MHOM/CN/2015/CPOLM-1) collected from a patient found in Guangzhou was used as an outgroup in the ITS1 analysis [22]. The phylogenetic relationships among the isolates were inferred from the phylogenetic tree reconstruction by the Neighbor Joining (NJ) using MEGA 5.0 by default setting and the reliability of the internal branches was tested by 1,000 bootstrap replications.

Table 2

List of reference strains used in this study.

Species	WHO code	Origin	Gene	Haplotype number	Accession
L. donovani	MHOM/CN/00/Wangjie1	China	ITS1	I2	AJ000294
L. donovani	IPHL/CN/77/XJ771	Bachu, China	ITS1	I2	HM130608
L. donovani	MCAN/CN/60/GS1	Gansu, China	ITS1	I5	HQ830354
L. donovani	MHOM/ET/67/HU3	Ethiopia	ITS1	I3	AJ634373
L. donovani	MHOM/IN/80/DD8	India	ITS1	I4	AJ000292
L. donovani	MHOM/SD/93/9S	Sudan	ITS1	I3	AJ634372
L. donovani	MHOM/IQ/1981/SUKKAR2	Iraq	ITS1	I6	AM901452
L. donovani	MHOM/KE/83/NLB189	Kenya	ITS1	I4	AJ634374
L. infantum*	MHOM/CN/08/Jiashi-1	Jiashi, China	ITS1	I2	GQ367486
L. infantum	MHOM/CN/54/Peking	Beijing, China	ITS1	I1	AJ634345
L. infantum	MHOM/CN/78/D2	Xinjiang, China	ITS1	I1	AJ000303
L. infantum	MHOM/TN/80/IPT1	Tunisia	ITS1	I1	AJ000289
L. infantum	MHOM/FR/78/LEM75	France	ITS1	I1	AJ634339
L. infantum	MCAN/ES/86/LEM935	Spain	ITS1	I1	AJ634355
L. infantum	MHOM/IT/94/ISS1036	Italy	ITS1	I1	AJ634353
L. infantum	MHOM/IR/2012/Savodjbolagh11	Iran	ITS1	I1	KC347299
L. infatum	MHOM/UZ/2007/KU	Uzbekistan	ITS1	I1	FM164420
L. major	MHOM/CN/2015/CPOLM-1	China, imported	ITS1	-	KU975160
L. donovani	MHOM/CN/00/Wangjie1	China	hsp70	H2	HF586394
L. donovani	MHOM/CN/90/9044	Shandong, China	hsp70	H1	JX021428
L. donovani	MHOM/CN/86/SC6	Sichuan, China	hsp70	H1	JX021429
L. donovani	IWUI/CN/77/771	Bachu, China	hsp70	H2	JX021425
L. donovani	MHOM/NP/2003/BPK282	Nepal	hsp70	H3	XM_003862348
L. donovani	MHOM/ET/67/HU3	Ethiopia	hsp70	H4	X52314
L. donovani	MHOM/IN/00/DEVI	India	hsp70	H3	FN395028
L. donovani	MHOM/MA/95/CRE72	Morocco	hsp70	H3	HF586352
L. donovani	MHOM/SD/87/UGX-MARROW	Sudan	hsp70	H3	HF586386
L. infantum	MHOM/EG/87/RTC2	Egypt	hsp70	H1	HF586350
L. infantum	MCAN/IL/97/LRC-L720	Israel	hsp70	H1	HF586393
L. infantum	MHOM/MA/67/ITMAP263	Morocco	hsp70	H1	FN395033
L. infantum	MHOM/PT/00/IMT260	Portugal	hsp70	H1	FN395032
L. infantum	MHOM/MT/85/Buck	Malta	hsp70	H1	FN395031
L. infantum	MHOM/MA/67/ITMAP263	Morocco	hsp70	H1	FN395033
L. infantum	MHOM/BR/07/ARL	Brazil	hsp70	H1	FN395037
L. infantum	MCAN/ES/98/LLM-877	Spain	hsp70	H1	XM_001470287
L. donovani*	MHOM/CN/80/801	Kashi, China	hsp70	H1	JX970993
L. donovani*	MHOM/CN/86/SC9	Sichuan, China	hsp70	H1	JX021430
L. donovani*	MCAN/CN/97/WDD23	Gansu, China	hsp70	H1	JX970994
L. donovani*	MHOM/CN/96/KS6	Kashgar, China	hsp70	H1	JX970996
L. braziliensis	MHOM/CO/90/LEM2216	Colombia	hsp70	H5	FN395043
L. guyanensis	MHOM/PE/02/LH2372	Peru	hsp70	H6	FN395051
L. aethiopica	MHOM/ET/72/L100	Ethiopia	hsp70	H7	FN395021
L. tropica	MHOM/IN/79/DD7	India	hsp70	H8	FN395025
L. major	MHOM/IL/67/LRC-L137	Israel	hsp70	H9	FN395023
L. amazonensis	MHOM/BR/73/M2269	Brazil	hsp70	H10	EU599090
L. mexicana	MNYC/BZ/62/M379	Belize	hsp70	H11	EU599091
L. infantum	MHOM/KE/84/NLB_323	Kenya	nagt	-	DQ836148
L. infantum	unknown	Turkey	nagt	-	AF205934
L. infantum	MHOM/CN/50/Bman	China	nagt	-	DQ836147
L. infantum	MHOM/IR/11/Kazeroun	Iran	nagt	-	KF701211
L. infantum	MHOM/IR/11/Lamerd1	Iran	nagt	-	KF701212
L. donovani	HOM/IN/97/JD	India	nagt	-	DQ836150
L. major	MHOM/IR/11/Farashband	Iran	nagt	-	KF701209
L. tropica	MHOM/IR/11/Ghir-Karzin3	Iran	nagt	-	KF701206
L. mexicana	HOM/CO/94/1182	Colombia	nagt	-	DQ836161
L. braziliensis	HOM/BR/75/M2903	Brazil	nagt	-	DQ836162
L. turanica	MRHO/IR/11/Gol-6	Iran	nagt	-	JX103553
L. gerbilli	MRHO/IR/10/Gol-9	Iran	nagt	-	JX103531

*, designation of the strain does not represent the genotype; -, not available.

Haplotype networks

Unrooted parsimony networks of haplotypes for L. donovani complex were constructed using TCS v.1.21 [23], with gaps treated as a fifth state.

Results

Sample collection

Samples were collected from 2004 to 2014. In total, 20 Leishmania isolates were obtained from patients, sand fly vector (P. wui) and Tarim hares (). The collection was carried out during the annual surveys for VL by the Xinjiang Center for Disease Control and Prevention in the AVL-endemic Shufu county in the alluvial plain and in the DT-ZVL-endemic regions in the Bachu, Jiashi and Minfeng counties in the desert area (. A total of 12 samples were collected from the patients, of which six were under 1 year old from the DT-ZVL-endemic Bachu or Jiashi counties and six were 2 years old or older from the AVL-endemic Shufu county. The demographic data are consistent with the designation of the VL as the two different types indicated. The remaining eight samples were isolated from the vector P. wui and Tarim hares in the DT-ZVL-endemic Bachu, Jiashi and Minfeng counties.

PCR amplification of nagt, ITS1 and hsp70 sequences and phylogenetic analyses

DNA isolated from cultured promastigotes of all 20 samples were subjected to PCR amplification. PCR amplification of the DNA samples with the primer set for nagt yielded a single product of the expected size (~1.4 kb) from 13 of the 20 isolates. The reason for the lack of amplification might be caused by several reasons. For instance, nagt is a single copy gene which may not have been successfully amplified due to poor sample quality in some cases. These nagt sequences were subjected to phylogenetic analysis together with those from representative sequences of other strains/species (Tables 1 and 2). The results clearly showed the segregation of all samples analyzed here into two groups according to the two VL types in the same clade with the reference sequences of the L. donovani/L. infantum complex. They were all distant from the other species complexes of the subgenus Leishmania (e. g. L. major, L. tropica, L. mexicana) and the subgenus Viannia (L. braziliensis) (.

PCR amplification of the same DNAs for the ITS1 locus yielded ~320 bp products from all 20 samples. Sequence analyses of these products separated the 20 samples again into Type 1 and Type 2, in accordance with the two VL types (.

Alignment of Types 1 and 2 ITS1 sequences revealed C/T-substitution at position 78 and A-deletion at location 119 in the latter (. Type 1 includes all isolates from the 6 patients in the AVL-endemic Shufu county, while Type 2 includes the remaining 14 isolates, from patients, the vector P. wui and Tarim hares in the DT-ZVL-endemic Bachu, Jiashi and Minfeng counties.

Multiple sequence alignment of the ITS1 sequences from different haplotypes of Xinjiang Leishmania isolates.

The detailed information for each haplotype (I1 to I6) are provided in Tables 1 and 2. The first nucleotide of the ITS1 region that follows the end of the 18s rRNA (NC_007268.2 of the L. major strain Friedlin complete genome) was designated as the starting site. Haplotype I6 was chosen as a reference (GenbankAccession: AM901452) and the aligned fragments between position 3 and 121 are shown. Areas shaded in grey show identical sequences while the variable sites were marked with the location of the nucleotide shown above. I1, indicates isolates of AVL foci and L. infantum strains. I2, indicates isolates from DT-ZVL foci and L. donovani.

The types 1 and 2 ITS1 sequences were subjected to phylogenetic analyses together with those of the reference strains in the L. donovani/infantum complex from other geographical origins (). L. major sequence was used as the outgroup (. All 20 sequences in question were found to group with members of the L. donovani/infantum complex in a primary clade, which was then separated into three subclades. One subclade contains all six samples from the AVL foci (Shufu county) that were identical in sequence with those of the L. infantum strains that are widely distributed in Mediterranean regions (France, Spain, and Italy), Middle East (Iran), Central Asia (Uzbekistan), and China (Beijing, Xinjiang). Another subclade contains the 14 samples from the DT-ZVL foci that were identical in sequence to those of some Chinese isolates, which were previously typed either as L. infantum or L. donovani. The third subclade, more distant from the first two, contains samples of mixed origins, ranging from Africa to the Middle East to Asia (China and India).

Statistical parsimony haplotype network and phylogenetic tree based on the ITS1 sequence of the L. donovani complex.

The network is generated with TCS software (version 1.21). The haplotypes, I1 to I6, as given in Fig 2, Tables 1 and 2, are represented by an oval circle. Small hollow circles represent unsampled haplotypes and each line represents one mutational step. The phylogenetic tree was constructed using the Neighbor-Joining (NJ), Maximum likelihood (ML) and Minimum Evolution (ME) methods, bootstrap values were provided next to nodes (NJ/ML/ME). The origins and hosts of the isolates are provided. Terrains where isolates were collected in this study are indicated with a triangle (▲, DT-ZVL foci) or a square (■, AVL foci). don, L. donovani; inf, L. infantum; maj, L. major; *, the species is suggested to be L. donovani.

Fig 2

Phylogenetic tree based on the alignment of amplified section of the nagt sequences.

The phylogenetic tree was constructed using the Neighbor-Joining (NJ), Maximum likelihood (ML) and Minimum Evolution (ME) methods, bootstrap values were provided next to nodes (NJ/ML/ME), a value lower than 50 was indicated as (--). Information on the origins and hosts of isolates are provided. The terrain where isolates were collected in this study is indicated with triangle (▲, DT-ZVL foci) or square (■, AVL foci). don, L. donovani; inf, L. infantum; maj, L. major; aet, L. aethiopia; tro, L. tropica; tur, L. turanica; ger, L. gerbilli; mex, L. mexicana; bra, L. braziliensis; *, the species is suggested to be L. donovani.

We successfully PCR- amplified the hsp70 (~1,286 bp) from 16 of the 20 samples. Alignment of their sequences also revealed differences according to their origin from AVL and DT-ZVL (see . Isolates from the AVL foci had the identical hsp70 sequence haplotype H1, while isolates from the DT-ZVL foci were all H2. A set of hsp70 sequences from reference strains, representing both Old and New world species, was retrieved from GenBank (. Phylogenetic analyses of the hsp70 sequences also support the conclusion that all 20 isolates belong to the L. donovani/infantum complex (. Consistent with the ITS1 analysis, isolates from the AVL foci clustered with L. infantum reference strains. In contrast, isolates from the DT-ZVL foci were separated from the isolates of the AVL foci.

Collectively, Leishmania isolates from AVL and DT-ZVL in Xinjiang clearly separate into two genetically distinct groups, as determined by sequence analysis of the three different genetic markers. The potential of the Tarim hare as a reservoir for DT-ZVL is strongly suggested by the sequence identity of its isolates with those from patients and vectors for all three phylogenetic markers examined.

Discussion

In the past, most of the work on leishmaniasis in China has been focused on prevention and control programs. Meanwhile the biological characteristics of the persisting Leishmania spp. remains unclear, especially in some of the northwestern regions where VL is still endemic. In this study, two major findings were made from analyses of more recent isolates. Firstly, we demonstrated a clear separation of the AVL and DT-ZVL isolates into 2 different groups in the same L. donovani/infantum clade. Secondly, the sequence identity of patient-, vector- and Tarim hare-derived isolates strongly suggests they have a zoonotic transmission cycle and that the Tarim hare acts as a potential reservoir of DT-ZVL. The role of Tarim hares as a reservoir is further supported by the fact that other lagomorphs have been reported in this role for zoonotic VL in Spain [24-26]. This also raises the question as to whether lagomorphs should be studied, at the global level, as a potential reservoir for Leishmania spp. The pathogen is thought to be transmitted among the natural hosts by sand flies. Humans are occasionally infected when they enter the region where sylvatic infected sand flies exist.

Our study has provided preliminary evidence for the genetic difference in Leishmania isolates from the AVL and DT-ZVL regions in Xinjiang. In our study, we used genetic markers which were either single copy (nagt) or multicopy (ITS1 and hsp70). The latter proved more successful for amplification due to their multicopy nature when used for these challenging samples. However, discriminatory markers or typing methods with a higher resolution such as multilocus microsatellite typing (MLMT) or multilocus sequence typing (MLST) are encouraged to be considered for further analyses [6,27]. Ideally, whole genome sequencing would provide the ultimate solution to identify all possible genetic factors correlated with adaptation to different disease conditions [28-30]. Another possible limitation in this study was that only cultivable parasites were used for the analyses. These might represent a bias in the parasite population studied. Thus, phylogenomic analysis of lesion-derived amastigotes from mammalian hosts or promastigotes from sand-fly gut material will be a necessity for further study. When we compared published nagt, ITS1 and hsp70 sequences (MHOM/IN/1983/AG83; MHOM/IN/00/DEVI; MHOM/IN/80/DD8) from Indian L. donovani clinical samples, they were 100% identical to the Indian representative sequences we used in this study–we did not, therefore, include these in our phylogenetic study. To confirm the role of the Tarim hare as an animal reservoir, further detailed studies are required which need to involve collection of a larger sample set of infected animals (and human hosts) and analyse lesion-derived amastigotes with the multilocus markers or genomics analyses described above. However, due to the comprehensive control of leishmaniasis in China, both patient and animal infections are rare, and therefore some of the questions posed above may not be able to be answered.

There is little doubt that Leishmania spp. causing persistent VL in Xinjiang belongs to the L. donovani complex, but we found an atypical association between L. infantum/donovani and the epidemiology of the ZVL/AVL types. For the isolates from Shufu county, a well-known foci of AVL [3], sequence analysis indicated that they are, most likely, L. infantum. This is unusual, considering that the disease type is AVL. First, these isolates were mainly found in patients over 2 y.o. (Table 1), which is considered the main characteristic of L. donovani infection [31]. In addition, no animal reservoir has been found for the Xinjiang L. infantum species. A possibility that a hidden ZVL co-exists with AVL in the Shufu county was discounted, since all 6 Leishmania isolates in this region display the same ITS1 and hsp70 haplotype. The existence of L. infantum in AVL has also been reported in other studies. A previously investigated isolate from Kashi City (an AVL focus), MHOM/CN/80/801, with an identical hsp70 sequence to the L. infantum clade, was suggested to be L. infantum by MLST in another study [6]. In addition, several adult cases of L. infantum infection were reported in Spain where L. infantum was not considered to be a local species [32]. Thus, we conclude that the Xinjiang AVL isolates from the oases of the Kashgar county are L. infantum with atypical clinical manifestations and no identified animal reservoir.

On the other hand, all 14 isolates from the DT-ZVL foci, i.e. Bachu, Jiashi and Minfeng counties, are genetically close to L. donovani, even though they are responsible for zoonotic disease. The ITS1 phylogenetic tree showed a similar tree topology to a previous study [8], in which all the isolates formed a sub-clade within the L. infantum cluster. However, further analysis revealed that their ITS1 sequences were identical to the MLEE-typed strain L. donovani MHOM/CN/00/Wangjie1, and also the strain IPHL/CN/77/XJ771 from Bachu county, which were deemed previously to have identity with L. donovani [9,13]. Additionally, our hsp70 data also suggested that these isolates are Xinjiang-specific L. donovani strains that are genetically close to the common L. donovani strains (H3) from India, Sudan, Nepal and Morocco in the haplotype network (Fig 5).

Fig 5

Haplotype network and phylogenetic tree based on the hsp70 sequences of the L. donovani complex.

A haplotype network constructed using Median Joining and post processing with an MP calculation. The haplotypes of H1 to H11, as given in Fig 2, Tables 1 and 2, are represented by yellow circles. Small red solid circles represent median joining points, and mutational sites between two circles are shown next to the connecting line. The subgenera Leishmania and Viannia are separated by a thick black line. The phylogenetic tree was constructed using the Neighbor-Joining (NJ), Maximum likelihood (ML) and Minimum Evolution (ME) methods, bootstrap values were provided next to nodes (NJ/ML/ME), values lower than 50 were indicated as (--). The origins and hosts of isolates are provided. The terrains where isolates were collected in this study are indicated with triangle (▲, DT-ZVL foci) or square (■, AVL foci). don, L. donovani; inf, L. infantum; maj, L. major; aet, L. aethiopia; tro, L. tropica; ama, L. amazonensis; mex, L. mexicana; bra, L. braziliensis; guy, L. guyanensis; *, the species is suggested to be L. donovani. (A total of 1047 NT between sites 590–1636 were analyzed in the final data set and are numbered from the start codon of the hsp70 gene). Note: MHOM/CN/00/Wangjie1 and IWUI/CN/77/771 are heterozygous and only H2 was analyzed. H1: Leishmania infantum and isolates from AVL; H2: isolates from DT-ZVL. Other details of each haplotype are provided in Tables 1 and 2.

L. donovani is considered to be mainly anthroponotic while L. infantum is zoonotic with dogs serving as a primary reservoir. There have been no reports of AVL caused by L. infantum, while ZVL caused by L. donovani has been documented before. Thus, all the Xinjiang Leishmania isolates we have studied display atypical epidemiological features. This suggests that more attention needs to be paid when classifying these species on clinical grounds, since there might also be underreporting or mis-reporting occurrences elsewhere.

In conclusion, species of the L. donovani complex are responsible for AVL and DT-ZVL in Xinjiang Autonomous Region of China. We consider that the two types of VL are caused by two different groups of parasite. Epidemiological conditions have a great impact on shaping the endemic area occupied by these parasites. Our results further support that the Tarim hare is most likely the reservoir for L. donovani and the source of infection in the desert region. Further control measures targeting these wild animals may be needed for the effective control of this disease. More discriminatory methods, particularly direct whole genome sequencing of parasites from host tissues, will be the preferred approach to clearly dissect the complicated situation in these Chinese leishmania parasites.

29 Aug 2021

Dear Mr Lai,

Thank you very much for submitting your manuscript "Species identification and phylogenetic analysis of Leishmania isolated from patients, vectors and hares in the Xinjiang Autonomous Region, The People's Republic of China" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

All reviewers came to the conclusion that this is an very interessant study, which has however some restrictions e.g. small sample size and limited number of loci studied. I think the authors should try to address these comments in the revised manuscript. For instance comparison of their data to data from Indian L. donovani as suggested by reviewer 2 seems to be feasible. I also agree with reviewer 1 that it would be very interesting to perform whole-genome sequencing of these Chinese strains and to compare these data with the numerous L. donovani/ L. infantum sequences already available . But this could be perhaps w be published in a next manuscript. The present study could be considered as a pilot study. The authors need however to discuss the restrictions and future prospects of their work in this submission.

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***********************

All reviewers came to the conclusion that this is an very interessant study, which has however some restrictions e.g. small sample size and limited number of loci studied. I think the authors should try to address these comments in the revised manuscript. For instance comparison of their data to data from Indian L. donovani as suggested by reviewer 2 seems to be feasible. I also agree with reviewer 1 that it would be very interesting to perform whole-genome sequencing of these Chinese strains and to compare these data with the numerous L. donovani/ L. infantum sequences already available . But this could be perhaps w be published in a next manuscript. The present study could be considered as a pilot study. The authors need however to discuss the restrictions and future prospects of their work in this submission.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: With cultures available, it may be relatively easy to sequence genomes of all these 20 isolates. The downstream analysis would make all the conclusions stronger. The presented analyses (based on 3 loci, some of which were not successfully amplified across the dataset - f.e. nagt) should be considered preliminary.

Reviewer #2: Objective is clear as stated

The sample size is a bit small

Sequence phylogenetic analysis based on MEGA version 5

Met ethical/regulatory requirements

Reviewer #3: The manuscript “Species identification and phylogenetic analysis of Leishmania isolated from patients, vectors and hares in the Xinjiang Autonomous Region, The People's Republic of China” applies a molecular epidemiological approach on 20 new field isolates obtained from two endemic sites, which are analysed for the three diagnostic genes ITS1, HSP70 and NAGT by PCR amplification, sequencing and phylogenetic mapping. The manuscript provides important new epidemiological information by (i) distinguishing both L. infantum and L. donovani infection in the Xinjiang Autonomous Region, (ii) demonstrating a non-classical, anthroponotic transmission cycle for L. infantum and a non-classical, zoonotic transmission cycle for L. donovani isolates, and (iii) involving hares as novel reservoir for L. donovani. Overall, the paper is very well written, and the drawn conclusions are fully supported by the results. Thus, I do not have any major criticism. One minor comment is that often the authors write that leishmaniasis is transmitted by sand flies, yet it is the parasite and not the disease that is transmitted. Also, the authors may consider combining the haplotype networks with the corresponding phylogenetic analyses rather than showing the first ones in separate figures.

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The presented results should be further complemented (or replaced altogether) by the analyses based on whole genome sequences.

Reviewer #2: Phylogenetic analyses are standard

Figures and Tables are acceptable

Reviewer #3: The manuscript “Species identification and phylogenetic analysis of Leishmania isolated from patients, vectors and hares in the Xinjiang Autonomous Region, The People's Republic of China” applies a molecular epidemiological approach on 20 new field isolates obtained from two endemic sites, which are analysed for the three diagnostic genes ITS1, HSP70 and NAGT by PCR amplification, sequencing and phylogenetic mapping. The manuscript provides important new epidemiological information by (i) distinguishing both L. infantum and L. donovani infection in the Xinjiang Autonomous Region, (ii) demonstrating a non-classical, anthroponotic transmission cycle for L. infantum and a non-classical, zoonotic transmission cycle for L. donovani isolates, and (iii) involving hares as novel reservoir for L. donovani. Overall, the paper is very well written, and the drawn conclusions are fully supported by the results. Thus, I do not have any major criticism. One minor comment is that often the authors write that leishmaniasis is transmitted by sand flies, yet it is the parasite and not the disease that is transmitted. Also, the authors may consider combining the haplotype networks with the corresponding phylogenetic analyses rather than showing the first ones in separate figures.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: See above. I believe the phylogenomic analyses will make the manuscript much stronger.

Reviewer #2: Conclusions acceptable, but weak due to small sample size

Discussion includes statements for the limitations of the analyses

Significance of the data obtained was discussed

Public health is not part of the work, but relevant points mentioned

Reviewer #3: The manuscript “Species identification and phylogenetic analysis of Leishmania isolated from patients, vectors and hares in the Xinjiang Autonomous Region, The People's Republic of China” applies a molecular epidemiological approach on 20 new field isolates obtained from two endemic sites, which are analysed for the three diagnostic genes ITS1, HSP70 and NAGT by PCR amplification, sequencing and phylogenetic mapping. The manuscript provides important new epidemiological information by (i) distinguishing both L. infantum and L. donovani infection in the Xinjiang Autonomous Region, (ii) demonstrating a non-classical, anthroponotic transmission cycle for L. infantum and a non-classical, zoonotic transmission cycle for L. donovani isolates, and (iii) involving hares as novel reservoir for L. donovani. Overall, the paper is very well written, and the drawn conclusions are fully supported by the results. Thus, I do not have any major criticism. One minor comment is that often the authors write that leishmaniasis is transmitted by sand flies, yet it is the parasite and not the disease that is transmitted. Also, the authors may consider combining the haplotype networks with the corresponding phylogenetic analyses rather than showing the first ones in separate figures.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: N/A

Reviewer #2: P. 3, last para., 1st sentence: Clarify the description of DT-ZVL, which has been categorized into sandy desert type and pebble desert type with different sand fly vectors in Xinjiang.

P. 4, lines 5-6: Define what are "Leishmania antigens" and "typical VL symptoms" besides splenomegaly ?

P. 5, last 3 lines: "unknown reasons" and poor "sample quality" are inconsistent statements

P. 6, 3rd para., line 1: "A/G substitution" is inconsistent with nts shown in position 78 in Fig. 3 ?

P. 6, last para., 1st line: Is hsp70 encoded by tandem-repeated genes in Leishmania ? If so, the negative PCR is surprising for 4 of the 20 samples examined.

P. 8, 2nd para., line 5-8: Several points here need attention for clarification. L. infantum has long been considered as the causative species of AVL in the Kashi alluvial plain based on DNA/sequence analyses. It is incorrect to say "no animal reservoir has been found for the Chinese L. infantum". Dog has been clearly shown as a reservoir and racoon dog considered as another one for infantum VL in eastern China. It may be acceptable to replace "Chinese" with "Xinjiang" in that statement.

P. 8, last sentence: The same problem as stated above. Negative finding in search for animal reservoirs also cannot be conclusive.

P. 9, 1st para. line 10 and 2nd para., line 4: Replace "China" and "Chinese" with Xinjiang in these two statement to avoid the same problem.

P. 10,Line 8: "bug" "Largus largus" ? Do you mean Lagurus lagurus ? If so, it is not a bug !?

Reviewer #3: The manuscript “Species identification and phylogenetic analysis of Leishmania isolated from patients, vectors and hares in the Xinjiang Autonomous Region, The People's Republic of China” applies a molecular epidemiological approach on 20 new field isolates obtained from two endemic sites, which are analysed for the three diagnostic genes ITS1, HSP70 and NAGT by PCR amplification, sequencing and phylogenetic mapping. The manuscript provides important new epidemiological information by (i) distinguishing both L. infantum and L. donovani infection in the Xinjiang Autonomous Region, (ii) demonstrating a non-classical, anthroponotic transmission cycle for L. infantum and a non-classical, zoonotic transmission cycle for L. donovani isolates, and (iii) involving hares as novel reservoir for L. donovani. Overall, the paper is very well written, and the drawn conclusions are fully supported by the results. Thus, I do not have any major criticism. One minor comment is that often the authors write that leishmaniasis is transmitted by sand flies, yet it is the parasite and not the disease that is transmitted. Also, the authors may consider combining the haplotype networks with the corresponding phylogenetic analyses rather than showing the first ones in separate figures.

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: In my opinion, this is a very interesting manuscript and this is a good fit for the PLoS Neglected Tropical Diseases Journal. I would strongly recommend to enforce it by whole-genome sequencing of the isolates under study and subsequent phylogenomic analysis. From the point of view of a classical parasitologist, the story is extremely important.

Reviewer #2: The work presented is highly significant by studying Leishmania rarely examined in an endemic area of VL with unique epidemiology. The authors correctly pointed out in the Discussion that amastigotes should be examined for genomic phylogenetic analyses. Such data from amastigotes of Indian L donovani clinical samples have been published. Their hsp70, ITS1 and nagt sequences should be included for phylogenetic analysis in this study. The relative merits of the three sequence markers used should be discussed with reference to their copy number per haploid genome. It is further suggested to include a brief discussion for future experimental study to verify the role of Tarim hare as a reservoir of DT-ZVL.

Reviewer #3: The manuscript “Species identification and phylogenetic analysis of Leishmania isolated from patients, vectors and hares in the Xinjiang Autonomous Region, The People's Republic of China” applies a molecular epidemiological approach on 20 new field isolates obtained from two endemic sites, which are analysed for the three diagnostic genes ITS1, HSP70 and NAGT by PCR amplification, sequencing and phylogenetic mapping. The manuscript provides important new epidemiological information by (i) distinguishing both L. infantum and L. donovani infection in the Xinjiang Autonomous Region, (ii) demonstrating a non-classical, anthroponotic transmission cycle for L. infantum and a non-classical, zoonotic transmission cycle for L. donovani isolates, and (iii) involving hares as novel reservoir for L. donovani. Overall, the paper is very well written, and the drawn conclusions are fully supported by the results. Thus, I do not have any major criticism. One minor comment is that often the authors write that leishmaniasis is transmitted by sand flies, yet it is the parasite and not the disease that is transmitted. Also, the authors may consider combining the haplotype networks with the corresponding phylogenetic analyses rather than showing the first ones in separate figures.

--------------------

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Reviewer #2: No

Reviewer #3: No

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23 Nov 2021

Submitted filename: reply to reviewers GH.docx

Click here for additional data file.

4 Dec 2021

Dear Dr Lai,

We are pleased to inform you that your manuscript 'Species identification and phylogenetic analysis of Leishmania isolated from patients, vectors and hares in the Xinjiang Autonomous Region, The People's Republic of China' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Gabriele Schönian

Associate Editor

PLOS Neglected Tropical Diseases

Michael Boshart

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

Ther are no further comments to the authors

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #3: The authors have responded to my queries.

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #3: The authors have responded to my queries.

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #3: The authors have responded to my queries.

**********

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #3: The authors have responded to my queries.

**********

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: (No Response)

Reviewer #3: The authors have responded to my queries.

**********

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Reviewer #1: No

Reviewer #3: No

10 Dec 2021

Dear Dr Lai,

We are delighted to inform you that your manuscript, "Species identification and phylogenetic analysis of Leishmania isolated from patients, vectors and hares in the Xinjiang Autonomous Region, The People's Republic of China," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases