infantum complex in China through microsatellite analysis

Infection, Genetics and Evolution 22 (2014) 112–119

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Genetic diversity of Leishmania donovani/infantum complex in China through microsatellite analysis Mohammad Zahangir Alam a,b, Ryo Nakao c, Tatsuya Sakurai a, Hirotomo Kato a, Jing-Qi Qu d, Jun-Jie Chai e, Kwang Poo Chang f, Gabriele Schönian g, Ken Katakura a,⇑ a

Laboratory of Parasitology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan Department of Parasitology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh Department of Collaboration and Education, Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan d National Institute of Parasitic Diseases, Center for Diseases Control and Prevention, Shanghai, PR China e Center for Diseases Control and Prevention, Uygur Autonomous Region, Urumqi, Xinjiang, PR China f Department of Microbiology/Immunology, Chicago Medical School/RFUMS, North Chicago, IL 60064, USA g Institut für Mikrobiologie und Hygiene, Charité Universitätsmedizin Berlin, Berlin, Germany b c

a r t i c l e

i n f o

Article history: Received 10 October 2013 Received in revised form 23 December 2013 Accepted 18 January 2014 Available online 27 January 2014 Keywords: Genetic diversity Leishmania infantum Microsatellite typing China

a b s t r a c t The Leishmania strains from different epidemic areas in China were assessed for their genetic relationship. Twenty-nine strains of Leishmania infantum isolated from 1950 to 2001 were subjected to multilocus microsatellite typing (MLMT) using 14 highly polymorphic microsatellite markers. Twenty-two MLMT profiles were recognized among the 29 L. infantum strains, which differed from one another in 13 loci. Bayesian model-based and distance-based analysis of the data inferred two main populations in China. Sixteen strains belonged to one population, which also comprised previously characterized strains of L. infantum non-MON1 and Leishmania donovani. The parasites within this population are assignable to a distinct cluster that is clearly separable from the populations of L. donovani elsewhere, i.e. India, Sri Lanka and East Africa, and L. infantum non-MON1 from Europe. The remaining 13 Chinese strains grouped together with strains of L. infantum MON1 into another population, but formed a separate cluster which genetically differs from the populations of L. infantum MON1 from Europe, the Middle East, Central Asia and North Africa. The existence of distinct groups of L. infantum MON1 and non-MON1/L. donovani suggests that the extant parasites in China may have been restricted there, but not recently introduced from elsewhere. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Visceral leishmaniasis (VL) is a vector-borne serious systemic disease caused by protozoan parasites of the Leishmania donovani complex (Alvar et al., 2006).The disease is endemic in Asia, Africa, Europe and south America, and approximately 0.2–0.4 million VL cases occur per year worldwide (Alvar et al., 2012). VL is still an important public health problem in China. By implementing an effective national control program in the 1950’s, the disease has been largely brought under control and virtually eradicated in the eastern regions of the country. However, the disease remains endemic in more than 50 counties in ⇑ Corresponding author. Address: Laboratory of Parasitology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Kita 18, Nishi 9, Kita-ku, Sapporo 060-0818, Japan. Tel.: +81 11 706 5198; fax: +81 11 706 5196. E-mail address: [email protected] (K. Katakura). http://dx.doi.org/10.1016/j.meegid.2014.01.019 1567-1348/Ó 2014 Elsevier B.V. All rights reserved.

six provinces or autonomous regions in western China, including Xinjiang, Gansu, Sichuan, Shaanxi, Shanxi and inner Mongolia (Guan et al., 2003; Jiang et al., 2008). Two types of VL have been described in western/northwestern China based on epidemiological parameters, i.e. the causative Leishmania, vector species and reservoirs (Lu et al., 1994; Zhou et al., 2009). The first type is an anthroponotic VL caused by L. donovani and currently endemic mainly in the plains of Kashi prefecture, Xinjiang. The widely distributed peridomestic sand fly Phlebotomus longiductus, indigenous to this area is the vector (Wang et al., 1966). The second is zoonotic type caused by Leishmania infantum with an animal reservoir, consisting of two subtypes (Guan, 1991). One is in the western hilly regions of the provinces of Gansu, Sichuan, Shaanxi and Shanxi. Dogs are the known reservoir and the vector of this form is Polistes chinensis (Wang et al., 2010a). The other subtype is in the deserts of the northwestern regions of China, including Xinjiang, northern Gansu and

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western Inner Mongolia. The transmission is sylvatic, but the reservoir(s) have not been identified. The sand fly species Paralycoptera wui and Phlebotomus alexandri serve as the vectors (Guan et al., 2003). Of relevance to mention is a variant of L. infantum, which is also transmitted by P. wui, but causes a largely self-healing cutaneous leishmaniasis (CL) in the desert settlement of Karamay in northern Xinjiang. Thus, the species composition of Leishmania is complicated in China. Though the human pathogenic species are designated as L. infantum and L. donovani, they cause various clinical symptoms ranging from asymptomatic (Wang et al., 2007), CL (Guan et al., 1997) to VL. The complexity of Leishmania strains is further compounded by the presence of different leishmaniasis in distant foci, which are marked geographically by very different landscapes. On the basis of multilocus enzyme electrophoresis (MLEE) of six isolates from the plain, mountainous, and desert regions, L. donovani sensu lato and L. infantum were initially identified as causative agents for VL in China (Xu et al., 1984). Interestingly, MLEE analysis of two isolates from the VL cases in Kashi city of Xinjiang was unable to identify them definitely as L. infantum or L. donovani (Xu et al., 1989). On the basis of kDNA (kinetoplast DNA) and nDNA (nuclear DNA) heterogeneity, 19 Leishmania isolates from epidemiologically different foci in China were classified into five genotypes (group I–V) (Lu et al., 1994). These authors found that members of group II, tentatively designated as L. infantum sensulato, displayed much heterogeneity in both kDNA and nDNAs. This subspecies heterogeneity is however not reflected in the subsequent sequence analysis of nagt plus 4 additional housekeeping genes: 30 independent isolates are separated only into 4 variants, i.e. L. infantum, L. infantum-var. 2, -var. 4 and -var. 7; of which the first 3 that account for almost all the VL are undistinguishable from other geographic isolates of L. infantum/L. donovani, except those responsible for the Indian kala-azar (Waki et al., 2007). Alternative markers are thus needed for population structure and gene diversity analyses among strains of L. infantum in China. Several molecular markers, capable of resolving genetic differences to species and strain levels have been used to address key epidemiological and population genetic questions. MLEE is still considered and used by some as the ‘gold standard’ for identification and classification of species and strains of Leishmania (Rioux et al., 1990). The limitation of MLEE for discriminating Leishmania below species level is indicated by its classification of most of the L. infantum parasites in the Mediterranean and South America to the same zymodeme MON-1. Other drawbacks of MLEE include the requirements of parasite cultivation, which is not always possible, and the procedure can be laborious, time-consuming and doable only in specialized laboratories. Of the genotyping methods that have been developed to overcome the disadvantages of MLEE, MLMT was most discriminating and adequate for typing strains of the Leishmania donovani/infantum complex, even within the zymodeme like MON-1. Data obtained by MLMT are highly informative in an eco-geographical context (Botilde et al., 2006; Kuhls et al., 2008, 2007; Schönian et al., 2008). MLMT has the advantage of providing reproducible results that can be stored as databases for sharing among different laboratories, including its use for predicting evolutionary origin of the L. donovani/infantum complex (Kuhls et al., 2007; Lukes et al., 2007). Here, a panel of previously described microsatellite markers (Kuhls et al., 2007; Ochsenreither et al., 2006) was used to infer the genetic variation among Chinese strains of L. infantum to determine their population structure and to compare it with strains from VL foci in different countries.

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2. Materials and methods 2.1. Parasite strains Twenty-nine strains of L. infantum isolated from China during the period of 1950–2001 were analyzed in this study. Of the local strains, 15 were from Xinjiang, 5 from Gansu, 4 from Beijing (Hebei), 2 from Shandong, one each from Henan, Sichuan provinces, and one strain of unknown origin (Fig. 1). Eighteen of the 29 strains were isolated from human VL cases, three from CL cases, one from PKDL, four from sand flies, one each from a dog and a rodent (Nyctomus spp.), and one had an unknown pathology. Leishmania DNA was extracted from cultured promastigotes as described previously (Lu et al., 1994; Schönian et al., 1996). The isolates previously named as L. donovani/infantum were subsequently considered as L. infantum based on phylogenetic analysis of their sequences N-acetylglucosamine-L-phosphate transferase (nagt) plus additional 4 different genes (Waki et al., 2007). The source, designation and geographic origin of these studied strains from China are listed in Table 1.

2.2. Microsatellite typing The 14 variable microsatellite markers Li 22-35, Li 23-41, Li 41-56, Li 45-24, Li 46-67, Li 71-5/2, Li 71-7, Li 71-33, Lm2TG, Lm4TA, TubCA, CS20, kLIST 7031 and kLIST 7039 (Supplementary Table 1) were used in the present study as previously described (Kuhls et al., 2007; Ochsenreither et al., 2006). Fluorescence labeled forward primers were used for the amplification of microsatellite containing sequences applying the PCR condition described earlier (Kuhls et al., 2007). The size of the amplicons was determined by capillary electrophoresis with an automated ABI PRISM Gene Mapper sequencer (Applied Biosystem). In each run, a reference strain of L. infantum (MHOM/ES/93/PMI) was included for which the microsatellite sizes for the 14 loci had been determined by sequencing (Ochsenreither et al., 2006). MLMType for each strain was obtained by compiling all alleles at each locus. Multilocus microsatellite profiles of 132 L. donovani complex strains (Alam et al., 2009a,b; Chargui et al., 2009; Kuhls et al., 2008, 2007; Leblois et al., 2011; Seridi et al., 2008) originated from different continents, including Africa, Europe, the Middle East, Central Asia and Indian subcontinent were included for comparison (Supplementary Table 2). One L. donovani strain from China (MHOM/CN/00/Wangjie 1) was also included.

2.3. Inference of population structures Population structure was investigated by a Bayesian modelbased clustering approach implemented in STRUCURE software (Pritchard et al., 2000), which determines genetically distinct population on the basis of allele frequencies and estimates the individual’s membership co-efficient in each probabilistic population. The following parameters were used: burn in period of 20,000 iterations, 200,000 Markov Chain Monte Carlo iterations, admixture model. The most probable number of population (K) was identified by plotting DK values of K for 1–10 calculated with 10 replicate runs for each K. The peak of the DK graph corresponds to the most probable number of populations in the data set (Evanno et al., 2005). Microsatellite-based genetic distances were calculated with the software packages MSA (Dieringer and Schlötterer, 2002) and POPULATIONS (http://www.legs.cnrs-gif.fr/bioinfo/populations) by applying the proportion of shared alleles distance measure (Dps).

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Fig. 1. Map of China, showing the geographical origins of the 29 L. infantum strains investigated in this study.

Neighbour-Joining (NJ) trees, including the test of confidence intervals by bootstrapping (1000 replicates) based on the resulting distance matrix, were constructed with the programs POPULATIONS and MEGA (Kumar et al., 2004). The software GDA (http://www.hydrodictyon.eeb.uconn.edu/ people/plewis/software.php) was applied to analyze the microsatellite data with respect to allelic diversity (A), mean number of alleles (MNA), expected heterozygosity (He), observed heterozygosity (Ho) and inbreeding coefficient (Fis). Genetic differentiation was assessed by calculating Fst values with comparing p-values using the MSA software (Dieringer and Schlötterer, 2002). Fst values higher than 0.25 indicate strong genetic differentiation (Wright, 1978). 3. Results 3.1. Multilocus microsatellite typing- genotypes In total, 22 different multilocus microsatellite profiles summarizing the repeat numbers obtained for the 14 microsatellite markers were assigned to 29 L. infantum strains originating from China (Table 1). Eighteen of the 29 strains had their own specific microsatellite profiles, and the remaining 11 presented 4 different profiles. Seventeen percent of the allelic combinations of the 22 profiles were di-allelic presenting two alleles of different sizes and considered to be heterozygous. Heterozygosity was seen in 11 markers. Most profiles were predominantly homozygous, but four strains (MHOM/CN/50/Bman, MHOM/CN/60/Gman, MHOM/ CN/86/Gansu and MHOM/CN/99/CAI) were heterozygous at 5 loci. 3.2. Population structures Bayesian statistic model-based analysis of the 29 Chinese strains using STRUCTURE showed that the optimal number of populations was 2 (Fig. 2). One population (China Pop 1) consisted of 13 strains, of which three were from Beijing (Hebei), two from Gansu, five from Kashi and Aksu (Xinjiang), one each from Henan and Shandong, and one was of unknown origin. The second population (China Pop 2) comprised 16 strains, of which ten strains were from Xinjiang, three strains from Gansu and one strain each

from Beijing (Hebei), Shandong and Sichuan. Interestingly, four strains isolated from sand flies, three strains from human CL cases from Xinjiang and one rodent strain from Beijing were grouped in China Pop 2. Simultaneously, a genetic distance method based on the Dps measure was used to analyze the MLMT data. The resulting Neighbor-Joining (NJ) tree (Fig. 3) assigned the 29 Chinese strains to two clusters that were in complete agreement with the populations identified by STRUCTURE analysis. Confidence intervals were obtained by bootstrapping (1000 replicates). Two major clusters in the tree were supported by bootstrap analysis (>50%). When merging the 29 Chinese strains with 132 strains of L. infantum and L. donovani from different countries in Africa, Europe, central Asia, Middle East and the Indian subcontinent, STRUCTURE analysis assign 161 strains to 2 main populations, the L. infantum MON-1 group and the L. donovani/L. infantum non MON-1 group. The 16 strains of China Pop 2 belonged to the L. donovani/L. infantum non MON-1 population, whereas the 13 Chinese strains of China Pop 1 grouped together with the L. infantum MON-1 population. These two main populations were re-analyzed independently by STRUCTURE. Calculation of DK for the L. infantum MON-1 population confirmed the existence of 5 cluster which were well defined and stable at K = 5: Algeria + Tunisia; Uzbekistan + Tajikistan; Spain + France + Italy + Portugal; Greece + Turkey; and China Pop 1 (Fig. 4). A second run of STRUCTURE analysis was also performed for the strains of the L. donovani/L. infantum non MON-1 population, in which the first split, at K = 2, separated the strains of L. infantum non MON-1 from those L. donovani/China Pop 2 (Fig. 4). Calculation of DK revealed 4 well-defined clusters: India + Kenya + Sri Lanka (L. donovani); L. infantum non MON-1; Sudan + Ethiopia (L. donovani); and China Pop 2.The L. donovani strain (MHOM/CN/00/Wangjie 1) from China was grouped with China Pop 2 rather than L. donovani clusters. The construction of the NJ unrooted tree for the 161 strains of L. donovani complex using POPULATIONS and MEGA gave the same clustering pattern (Fig. 5), which were in full congruence with the results of STRUCTURE analysis. 3.3. Population characteristics F statistics revealed a significant population structure among the clusters in the L. infantum MON-1 and the L. donovani/L. infantum

Table 1 Strains of L. infantum analysed in this study and their multilocus microsatellite (MLMT) profiles. Origin

Clinical condition Genotype Cluster

Li 41-56 Li 46-67 Li 22-35 Li 23-41 Li 45-24 Li 71-33 Li 71-5/2 Li 71-7 Lm4TA Lm2TG TubCA CS20 kLIST7031 kLIST7039

MHOM/CN/1978/D2 MHOM/CN/1954/Peking MHOM/CN/1980/StrainA MHOM/CN/54/#3 MNYC/CN/80/RD MHOM/CN/50/Bman MHOM/CN/78/SmanB MCAN/CN/76/#2 MHOM/CN/89/Shandong MHOM/CN/83/Henan MHOM/CN/88/801 MHOM/CN/86/SC6 MHOM/CN/60/Gman MHOM/CN/86/Gansu MHOM/CN/99/CAI MHOM/CN/97/9701 MHOM/CN/90/901 IWUI/CN/77/771 MHOM/CN/81/811 MHOM/CN/80/801 MHOM/CN/05/Kuche-2 MHOM/CN/05/Kuche-3 MHOM/CN/93/KXG-Lu MHOM/CN/93/KXG-LIU IWUI/CN/87/KXG65 IAND/CN/91/KXG918 IWUI/CN/92/KXG927 MHOM/CN/94/Xu MHOM/CN/87?/XJ872

Xinjiang Beijing, Hebei Unknown Beijing, Hebei Beijing, Hebei Beijing, Hebei Shandong Wenxian, Gansu Shandong Henan Gansu Jiuzhaigou, Sichuan Lanzhou, Gansu Gansu Gansu Kashi, Xinjiang Shanshan, Xinjiang Bachu, Xinjiang Xinjiang Kashi, Xinjiang Aksu, Xinjiang Aksu, Xinjiang Karamay, Xinjiang Karamay, Xinjiang Karamay, Xinjiang Karamay, Xinjiang Karamay, Xinjiang Karamay, Xinjiang Xinjiang

VL VL Unknown VL VL VL PKDL CVL VL? VL VL? VL VL VL? VL VL VL N/A VL VL VL VL CL CL N/A N/A N/A CL VL

10 7 10 7 7 7 10 7 10 10 10 11 12 12 12 10 11 11,12 11 10 10 10 10 10 10 10 10 12 11

A B C D E F G H I J J K L L L M N O P Q Q Q R R R S T U V

China China China China China China China China China China China China China China China China China China China China China China China China China China China China China

Pop1 Pop1 Pop1 Pop1 Pop1 Pop2 Pop1 Pop1 Pop2 Pop1 Pop1 Pop2 Pop2 Pop2 Pop2 Pop1 Pop2 Pop2 Pop2 Pop1 Pop1 Pop1 Pop2 Pop2 Pop2 Pop2 Pop2 Pop2 Pop2

9 9 9 9 9 9 9 9 7 9 9 7 9 9 9 9 7 9 7 9 9 9 7 7 7 7 7 7 7

17 14 17 14 14 14,15 17 14 15 16,17 16,17 16 14,15 14,15 14,15 17 19 15 16 17 17 17 15 15 15 15 15 14,15 19

17 16 17 16 16 7 17 18 7 17 17 19 7 7 7 17 18,19 7 7 17 17 17 7 7 7 7 7 7 19

15 16 15 16 16 10 15 16 10 15 15 10 10 10 10 15 10 10 9,10 15 15 15 10,11 10,11 10,11 10 10,11 10 10

14 12 11 12 12 18,21 11 11 9 11 11 9 18,21 18,21 18,21 11 9 18,19 9 11 11 11 17 17 17 9,18 17 15 9

8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8

13 13 13 13 13 19,21 13 13 12 13 13 15 19,21 19,21 19,21 13 15 14,16 15 13 13 13 12 12 12 12 12 2 15

10 16 11 16 16 9 11 15 8 11,12 11,12 10 9 9 9 11 10 9 9 11 11 11 9 9 9 8 9 9 10

20 21 19,20 20,21 21 12 20 21 9 16,20 16,20 8 12 12 12 19,20 9 12 9 20 20 20 9 9 9 9 9 9 9

9 9 9 9 9 12 9 9 11 9 9 12 12 12 12 9 12 12 12 9 9 9 11 11 11 11 11 11 12

19 19 19 19 19 20,21 19 19 25,27 19 19 23 20,21 20,21 20,21 19 23 20,21 23 19 19 19 26 26 26 25,27 26 26 23

11 11 9 11 11 9 9 11 9 11 11 9 9 9 9 11 9 9 9 11 11 11 9,10 9,10 9,10 9 10 9 9

17 17 17 17 17 21,28 17 18 19 17 17 18,19 22,28 22,28 22,28 21,22 19 23 18 17 17 17 18,19 18,19 18,19 19 19 18,19 19

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WHO code

VL = visceral leishmaniasis; PKDL = post kala-azar leishmaniasis; CL = cutaneous leishmaniasis; CVL = canine visceral leishmaniasis; N/A = not applicable.

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Fig. 2. Estimated population structure for L. infantum from China as inferred by STRUCTURE software on the basis of data for 14 microsatellite markers obtained for the 29 strains studied herein. Each strain is represented by a single vertical line divided into K colors, where K is the number of populations assumed. Each color represents one population, and the length of the colors segment shows the strain’s estimated proportion of membership in that population. The derived graph for DK shows at K = 2, indicating the existence of two populations in the investigated strain set.

non MON-1 groups. All Fst values (Table 2) were significant and higher than 0.25 indicating very great genetic differentiation between the subpopulations in the L. infantum MON-1 group. There is also significant genetic differentiation (Fst = 0.23, p = 0.0001) between the subpopulations of L. donovani/L. infantum non MON-1 group (Table 3), which proved that four populations obtained were genetically isolated. The genetic diversity within the two main Chinese populations, China Pop1 and China Pop 2,was estimated by calculating the mean number of alleles per locus (MNA), proportion of polymorphic loci (P), expected heterozygosity (He) and observed heterozygosity (Ho) (Table 4). MNA, which is considered to be an indicator of genetic variation within a population, varied between 2.42 for China Pop 1 and 3.78 for China Pop 2. P was relatively lower for China Pop 1 (0.642) and higher for China Pop 2 (0.928). In China Pop 2, both observed and expected heterozygosity was higher than China Pop 1. In China Pop 1, homozygous allele combinations were predominating where as China Pop 2 represented higher degree of heterozygosity. All these findings confirmed that China Pop 2 was more heterogeneous than China Pop 1.

4. Discussion

Fig. 3. Neighbour-Joining tree (midpoint rooted) inferred from Dps distance calculated for the data of 14 microsatellite markers for the 29 L. infantum strains from China. Only bootstrap values P50% are shown.

The genetic structure and diversity of strains of L. infantum from China was investigated, compared, and correlated with their geographical sources. To the best of our knowledge, this study is the first one that has investigated the population structure of L. infantum from different foci of endemicity in China using the MLMT approach. Considerable genetic variation was revealed for the 29 Chinese L. infantum strains presenting 22 different microsatellite profiles. This demonstrated a much higher heterogeneity of Chinese L. infantum as previously suggested when 3 different RFLP-nagt patterns were found for the same set of strains (Waki et al., 2007). Using different types of population genetic analysis, namely Bayesian statistics and distance-based models as well as F-statistics, we exposed two populations (China Pop1 and China Pop 2) among the 29 Chinese strains of L. infantum which were clearly separate from the populations comprising strains from Europe, Africa, the Middle East, Central Asia and India. The two Chinese populations did not correlate exactly with the geographical origin of the strains that fell into them. Their analysis was hampered owing to the small number of strains available from some provinces and their very wide geographical dispersal. So, three strains from Beijing were

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Fig. 4. Estimated population structure and substructure of the L. donovani complex strains. MLMT profiles based on 14 microsatellite markers for 161 strains including 29 L. infantum strains and one L. donovani strain from China were analyzed by Bayesian statistics implemented in the STRUCTURE Software. In the bar plots each strain is represented by a single line divided into K colors, where K is the number of population. Isolates are organized by membership coefficients. According to DK the most probable number of populations in the data set is two corresponding to L. infantum MON-1(red) and L. donovani/L. infantum non Mon-1 (green) isolates. DK calculations suggest five and four sub-populations in the main populations of L. infantum MON-1 and L. donovani/L. infantum non MON-1, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. Neighbour-Joining tree (unrooted) inferred from the Dps distances calculated for the data of 14 microsatellite markers for 161 strains of L. donovani complex, of which 29 L. infantum strains and one L. donovani strain from China and the rest from Europe, Africa, the Middle East, Central Asia and India.

grouped with China Pop 1, one strain from Beijing and one strain from Shandong with China Pop 2. The overlap between two populations might be due to human migration for travelling or seeking

employment or other reasons as there is no epidemiological information about these strains available. In a previous study, strain (MHOM/CN/90/9044) isolated from VL patient in Shandong province grouped with the isolates from CL patients in Karamay, Xinjiang (Wang et al., 2010b). There is no correlation to either geographic origin or clinical symptoms for the isolates of Shandong and Karamay. The genetic similarity between the strains causing VL or CL indicates that there is no association between the parasite’s genotypes and the clinical forms of the diseases. The change of surface molecules of the pathogens could affect host tissue and organ localization and causes variable severity of diseases (Deitsch et al., 1997). Strains from Gansu provinces were grouped with both China Pop 1 and China Pop 2. This indicates the co-existence of different eco-epidemiological situations, different sand fly vectors and different reservoir hosts in this geographical area. Two types of zoonotic VL (desert and mountainous) exist in Gansu provinces. The desert type is endemic in north-western desert regions of China, including Xinjiang, northern Gansu and western Inner Mongolia. This region is considered to be the natural nidus of VL infecting wild animals that are presumably the source of human infection. The sand fly species, P. wui and P. alexandri, are the vectors infesting different landscapes, the dry desert and the stony desert regions, respectively (Guan, 1991; Wang et al., 2010a). The mountainous type of VL occurs in the western mountainous and hilly regions of Gansu, Sichuan, Shaanxi and Shanxi provinces. The vector of this form is P. chinensis and dogs are likely the principal source of infection to human. It is clear, that L. donovani

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Table 2 Fst values and corresponding p-values for the five subpopulations of the L. infantum MON-1 population as assumed by STRUCTURE. Fst-values

China Pop1

Turkey/Greece

Spain/Portugal/France/Italy

Tunisia/Algeria

Uzbekistan/Tajikistan

China Pop1 Turkey/Greece Spain/Portugal/France/Italy Tunisia/Algeria Uzbekistan/Tajikistan

0 0.0001 0.0001 0.0001 0.0001

0.4689 0 0.0001 0.0001 0.0001

0.4982 0.3854 0 0.0001 0.0001

0.5406 0.4321 0.4356 0 0.0001

0.7035 0.6297 0.7035 0.6273 0

Table 3 Fst values and corresponding p-values for the four subpopulations of the L. infantum non MON-1/L. donovani population as assumed by STRUCTURE. Fst-values

India/Kenya/Sri Lanka

Sudan/Ethiopia

China Pop2

European non MON-1

India/Kenya/Sri Lanka Sudan/Ethiopia China Pop2 European non MON-1

0 0.0001 0.0001 0.0001

0.2368 0 0.0001 0.0001

0.3431 0.2777 0 0.0001

0.2944 0.2343 0.2984 0

Table 4 Population genetic characterization of the two main populations found for the analyzed 29 L. infantum strains from China. Population

N

P

MNA

Ho

He

Fis

China Pop1 China Pop2 Mean

13 16

0.642 0.928 0.785

2.42 3.78 3.10

0.054 0.187 0.121

0.307 0.525 0.416

0.827 0.650 0.715

N, number of strains; P, proportion of polymorphic loci; MNA, mean number of allele per locus; Ho, observed heterozygosity; He, expected heterozygosity; Fis, inbreeding coefficient.

complex in China has distinct epidemiological and biological characteristics. Interestingly, ten strains, including four from sand flies and three from human CL cases from different counties of Xinjiang grouped with China Pop 2, whereas four strains from Kashi and Aksu prefectures of Xinjiang were clustered with China Pop 1. These findings indicate the presence of two different sand fly vectors (P. wui and P. alexandri) in these endemic foci. However, we cannot rule out that other factors such as population migration, ecological variation, varying host backgrounds etc., or a combination of these and other factors might be responsible for observed genetic differentiation. Variation among the strains of L. infantum from the same endemic area leading to assignment to different populations could be attributed to differences in sand fly vector populations (Hamarsheh et al., 2007) and reservoir hosts (Githure et al., 1986). The degree of polymorphism and the number of di-allelic loci was much higher in China Pop 2 than in China Pop 1 (Tables 1 and 4). At the moment it is unclear whether the presence of two different alleles at a locus reflects true heterozygosity or is due to mixed infections or eventual bias occurring during cultivation of parasites as shown for several Leishmania species (Antoniou et al., 2004; Barrios et al., 1994Cortés et al., 1997; Pratlong et al., 1989). For resolving this problem it would be needed to clone isolates and re-type a representative number of clones. Di-allelic loci can also occur because of chromosomal copy number variations. Recent whole genome sequencing and FISH analyses have revealed significant chromosomal copy number variations for different species of Leishmania, including L. donovani (Downing et al., 2011; Mannaert et al., 2012; Rogers et al., 2011; Sterkers et al., 2012, 2011). In L. major, the chromosome numbers differed not only from strain to strain but also from cell to cell creating ‘mosaic aneuploidy’ (Sterkers et al., 2012, 2011), resulting in a high karyotypic diversity and conserved intra-strain genetic heterogeneity, but in a loss of heterogeneity per cell. The total number of alleles can, however, be maintained in a strain. At the moment, DNA-based typing methods, including the microsatellite typing approach used

herein, cannot distinguish between cell populations (or strains) consisting of heterozygous cells or of homozygous cells differing in allelic and ploidy content (Sterkers et al., 2012). Resolution of this and other uncertainties will ultimately depend on whole sequencing of leishmanial parasites, preferentially of amastigotes directly in the field-collected samples. By comparing, the MLMT profiles of L. infantum strains obtained from China to strains of the L. donovani complex from Europe, Africa, Central Asia, Middle East and the Indian subcontinent assigned the parasite to two distinct clusters (Figs. 4 and 5). The strains from China Pop1 grouped together with strains of L. infantum MON-1, but formed a cluster which was genetically clearly distinct from all other populations of L. infantum MON-1 from Europe, Middle East, Central Asia and North Africa. The strains belonging to China Pop 2 were clustered with previously characterized strains of L. donovani/L. infantum non MON-1. Although the strains of China Pop2were assigned to a distinct group clearly separate from the populations of L. donovani from India, Sri Lanka and East Africa, and L. infantum non MON-1 from Europe, they seem to be closer to Indian and African L. donovani rather than to L. infantum non MON-1group. The L. donovani strain MHOM/CN/00/Wangjie1 clustered with China Pop 2 instead of the Indian and African L. donovani groups suggesting that strains of China Pop 2 could be L. donovani rather than L. infantum. To answer this question methods based on genomic sequencing should be applied to these samples. Since microsatellite markers are prone to homoplasy, they are not well suited for phylogenetic analyses. Indeed, the existence of distinct groups of Chinese L. infantum MON-1 and L. donovani/L. infantum non MON-1 indicate that the parasites circulating in China have been restricted there for a long time rather than having been recently introduced from elsewhere by human or animal reservoir migration. In conclusions, this study showed that multilocus microsatellite typing is suited for discerning polymorphisms among closely related strains of the L. donovani complex and also sheds some light on the population structures within this species complex. Two genetically isolated populations were identified for the Chinese L. donovani complex strains studied. For future studies we suggest: (i) extending this approach to other regions endemic for VL by including strains from Sichuan, Shaanxi and Shanxi of China; (ii) investigating possible differences in reservoirs and sand fly vectors in different foci of China. Acknowledgements This study was supported in part by Grants-in-Aid for Japan Society for the Promotion of Science (JSPS) Postdoctoral Fellows

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