Molecular Detection and Genetic Diversity of Leishmania donovani in Naturally Infected Phlebotomus chinensi from Southwestern China

Abstract

Zoonotic visceral leishmaniasis is an important vector-borne infectious disease in western China. In this study, an epidemiological study was carried out on the vector of zoonotic visceral leishmaniasis in rural areas from Sichuan Province, southwestern China. In the 1263 phlebotomine sandflies captured, 859 (68.01%) were females and 404 (31.99%) males, belonging to Phlebotomus chinensis (83.37%), Sergentomyia koloshanensis (6.57%), Sergentomyia squamirostris (4.04%), and Sergentomyia barraudi (6.02%), respectively. The average prevalence of Leishmania parasites in P. chinensis females was 1.98%, which was detected by real-time quantitative PCR. Phylogenetic analysis based on ITS2-rDNA revealed that Leishmania parasites detected in sandflies belonged to the L. donovani group and formed a novel haplotype. This was the first report on molecular detection of L. donovani in naturally infected P. chinensi from China.

Introduction

T he leishmaniases are severe vector-borne diseases caused by genus Leishmania parasites. Zoonotic visceral leishmaniasis (ZVL) occurring in 51 countries worldwide with an estimated annual incidence of 500,000 clinical cases due to infection with the Leishmania donovani complex, including L. donovani and L. infantum, is the most severe form of the disease transmitted by sandflies to humans and domestic and wild animals and is frequently fatal if left untreated (Ashford 2000, Quinnell and Courtenay 2009). In China, ZVL had been effectively controlled in the east and north in 1950s via killing of sandflies, elimination of infected dogs, and treatment of patients. However, there are natural foci of ZVL in the west, including Sichuan, Gansu, Xingjiang, and Inner Mongolia Provinces (Wang et al. 2000, Li et al. 2010). Because of the mobility of human population, economic development, and change of ecological environment, incidence of ZVL has been on the rise in some areas in the western China (Gu et al. 2006). For example, at least 222 leishmaniasis cases were reported from years 2004 to 2007 in Sichuan Province (Zhang et al. 2007), and the disease has been becoming a serious public health and veterinary problem in southwestern China.

ZVL is associated with the distribution and abundance of phlebotomine sandfly vectors, and identification of the vectors is very important to determine the life cycle of the parasite and the epidemiology of the disease (Killick-Kendrick 1990, Sharma and Singh 2008). Phlebotomus chinensis is the main vector of ZVL in China with the exception of P. longiductus, P. wui, and P. alexandri as vectors in Xinjiang Uyghur Autonomous Region (Gu et al. 2006). The vector of ZVL in Sichuan Province has been investigated, indicating that P. chinensi is responsible for transmission of the disease from animals to humans (Jiang et al. 2003, Ji and Li 2005). However, the infected sandflies have been never found in this region.

In this study, we conducted molecular detection and genetic diversity of Leishmania parasite in naturally infected P. chinensi from southwestern China, using real-time quantitative PCR and phylogenetic analysis based on internal transcribed spacer 2 of ribosomal DNA (ITS2-rDNA) of Leishmania.

Materials and Methods

Study area and sandflies collection

The study was performed in rural areas of Dujiangyan City (103.63°E, 30.99°N), Mianyang City (103.45°E, 30.42°N), and Deyang City (104.37°E, 31.26°N), located in eastern Sichuan Province, southwestern China, where the sampling sites were determined according to the epidemiology references of human ZVL infection in the region. Sandflies were captured from 4 pm to 7 am, from June 5 to 28, 2008, during the active season of adult sandflies, using Center for Disease Control (CDC) light traps with a UV bulb inside the houses, in animal shelters, and in house yards (Alexander 2000).

All sandflies were identified according to the morphological characters of the head and abdominal terminalia, followed by dissection and DNA extraction.

Real-time quantitative PCR

Leishmania DNA in sandflies was detected by real-time quantitative PCR (qPCR) using SYBR^® Green Real-time PCR Master Mix-Plus (Toyobo Bio-Technology Co., Ltd.) as previously described (Nicolas et al. 2002). Briefly, DNA from a single sandfly female was extracted using Genomic DNA Extraction Kit Ver 3.0 (Takara Biotechnology Co., Ltd.) according to the manufacturer's instruction. The qPCR was carried out with the primers (forward: 5′-CCTATTTTACA CCAACCCCCAGT-3′; reverse: 5′-GGGTAGGGGCGTTCTG CGAAA-3′) that amplify a 120-bp fragment of the minicircle kinetoplast DNA (kDNA) of Leishmania. The limit of detection was 0.1 parasites per qPCR. Genomic DNA from L. donovani reference strain MHOM/CN/92/SC10H2 was used as positive control, and the negative control was established with deionized water instead of DNA extract.

PCR assay

The positive DNA samples were used to identify the parasites at the species level by PCR of ITS2 rDNA. The PCR was performed using the forward primer L5.8SR (5′-AAGTGCG ATAAGTGGTA-3′) and the reverse primer LITSV (5′-ACAC TCAGGTCTGTAAAC-3′), according to the method previously described (El Tai et al. 2000, 2001). The L. donovani reference strain MHOM/CN/92/SC10H2 was also amplified under the same condition.

PCR products were directly sequenced to identify Leishmania haplotypes infecting individual sandfly, and all haplotypes were identified to species by phylogenetic analysis. For this, the multiple alignments of new DNA haplotypes and homologous GenBank sequences were analyzed with the Editseq program of DNAStar software. Phylogenetic tree was constructed using the neighbor-joining algorithm of Phylip program with Kimura 2-parameter model.

Statistical analysis

Chi-square test was used to compare Leishmania prevalences in P. chinensis from different sampling sites. The difference was considered statistically significant when p < 0.05. Statistical analysis was performed using SPSS v. 11.0 software.

Results

Sandflies identification

In the 1263 phlebotomine specimens captured, 859 (68.01%) were females and 404 (31.99%) males, and four sandflies species P. chinensis, Sergentomyia koloshanensis, Sergentomyia Squamirostris, and Sergentomyia barraudi were identified based on the morphological characters. P. chinensis predominated in Sichuan Province, China, accounting for 83.37% of the phlebotomines captured. Species and sex distribution of sandflies are shown in Table 1.

Table 1.

Phlebotomine Sandflies Collected in Sichuan Province, China

	Dujiangyan		Mianyang		Deyang
Species	No. of females (positive) ^a	No. of males	No. of females (positive) ^a	No. of males	No. of females (positive) ^a	No. of males	Total
Phlebotomus chinensis	336 (9)	76	158 (5)	107	214 (2)	162	1053
Sergentomyia koloshanensis	45	12	0	0	17	9	83
Sergentomyia squamirostris	16	8	0	0	18	9	51
Sergentomyia barraudi	34	15	0	0	21	6	76
Total	431	111	158	107	270	186	1263

Real-time quantitative PCR was used to detect the presence of Leishmania DNA in a single sandfly.

Real-time qPCR

Of 859 females, 16 (1.98%) were found to be Leishmania positive, in which 9 of 336 P. chinensis were from Dujiangyan, 5 of 158 P. chinensis from Mianang, and 2 of 214 P. chinensis from Deyang, which was detected by real-time qPCR. However, no Leishmania DNA was detected in S. koloshanensis, S. squamirostris, and S. barraudi females from the three sampling sites. All infected females belonged to P. chinensis. There was no statistical difference in Leishmania prevalences among the three sampling sites (p > 0.05), and the average prevalence of Leishmania in P. chinensis was 1.98%.

Phylogenetic analysis

Sequence analysis of ITS2-rDNA from Leishmania-positive sandfly females showed identical sequence, but it was different from the reference strain L. donovani strain MHOM/CN/92/SC10H2 isolated from Sichuan Province, China, in 1992. The sequences were submitted to GenBank (GenBank accession nos. HM452148 and HM452147). Phylogenetic analysis indicated that Leishmania parasites detected in the sandfies belonged to the L. donovani group, which was distinguished from the Sichuan strain (MHOM/CN/92/SC10H2). The two haplotypes of L. donovani and three other haplotypes from India (GenBank accession nos. EU753215, EU753224, and EU753219) formed a clade distinct from the other clades (Fig. 1). The haplotype of L. donovani from sandflies in southwestern China was found to be novel.

FIG. 1.

Phylogenetic tree for ITS2-rDNA sequences of Leishmania parasites constructed by the neighbor-joining method using Phylip program. ITS2-rDNA sequences were retrieved from GenBank and L. mexicana venezuelensis was included as an outgroup. Neighbor-joining consensus tree used 1000 bootstrap replicates. The number represents bootstrap values. The parasite name, GenBank accession number, and the country are indicated.

Discussion

As we have known, leishmaniasis is a kind of diverse and complicated disease caused by many species of Leishmania, and correct identification of the parasite species in the vectors is very important to make control programs. It is clear that molecular techniques are more sensitive and have greater specificity than the cellular methods, such as morphology and in vitro culture. Because of the high sensitivity and specificity, several methodologies based on PCR have been used for Leishmania detection in sandflies in the endemic areas (van der Meide et al. 2008). For example, 4% Migonemyia migonei was found infected with L. infantum in Brazil (de Carvalho et al. 2010), 0.47% P. perniciosus infected with L. infantum in Portugal (Maia et al. 2009), and 30.0% P. sergenti infected with L. tropica in southern Iran (Oshaghi et al. 2010).

Sichuan Province is located in southwestern China, and ZVL is endemic in the eastern Sichuan, including Lixian, Wenchuan, Beichuan, Maoxian, Heishui, and Jiuzhaigou. There was no case reported in Lixian, Wenchuan, and Beichuan in the recent years. However, the incidence has shown increasing tendency in Maoxian and Heishui (Zhang 2006). ZVL involves a number of animal reservoirs, making the control of both vector and reservoirs of ZVL difficult (Sacks and Kamhawi 2001).

Because of the high similarity of rRNA genes of Leishmania and other trypanosomatids such as Leptomonas, the presence of Leishmania as well as other trypanosomatids in the sandflies captured from Nepal, based on PCR products to target the rRNA genes of Leishmania, has been reported (Bhattarai et al. 2009). In this study, Leishmania-specific primers targeting kDNA minicircle were used to detect Leishmania DNA from sandflies, so the qPCR assay only detected Leishmania from sandflies (Nicolas et al. 2002).

In conclusion, we found four species of sandflies, P. chinensis (83.37%), S. koloshanensis (6.57%), S. squamirostris (4.04%), and S. barraudi (6.02%), and the average prevalence of 1.98% for Leishmania parasites in female P. chinensis. These results indicated that P. chinensis is the principal vector for Leishmania parasites in southwestern China. The other three sandflies species were negative for the presence of Leishmania parasites. This could be attributed to the few number of sandflies available or the fact that these species are not vectors of Leishmania found in this region. Phylogenetic analysis indicated that Leishmania parasite from sandflies in southwestern China is a novel L. donovani haplotype. This was the first report on molecular detection of L. donovani in naturally infected P. chinensi from southwestern China.

Footnotes

Acknowledgments

This work was supported by the National High Technology and Development Program of China (“863” program) (grant no. 2009AA10Z402) and the “Gold Idea” Foundation of Institute of Military Veterinary, Academy of Military Medical Sciences (grant no. YCX0901).

Disclosure Statement

No competing financial interests exist.

References

Alexander

. Sampling methods for phlebotomine sandflies. Med Vet Entomol, 2000; 14:109–122.

Ashford

. The leishmaniases as emerging and reemerging zoonoses. Int J Parasitol, 2000; 30:1269–1281.

Bhattarai

, Das

, Rijal

, van der Auwera

et al. Natural infection of Phlebotomus argentipes with Leishmania and other trypanosomatids in a visceral leishmaniasis endemic region of Nepal. Trans R Soc Trop Med Hyg, 2009; 103:1087–1092.

de Carvalho

, Valenca

, da Silva

, de Pita-Pereira

et al. Natural Leishmania infantum infection in Migonemyia migonei (Franca, 1920) (Diptera: Psychodidae: Phlebotominae) the putative vector of visceral leishmaniasis in Pernambuco State, Brazil. Acta Trop, 2010; 116:108–110.

El Tai

, El Fari

, Mauricio

, Miles

et al. Leishmania donovani: intraspecific polymorphisms of Sudanese isolates revealed by PCR-based analyses and DNA sequencing. Exp Parasitol, 2001; 97:35–44.

El Tai

, Osman

, el Fari

, Presber

et al. Genetic heterogeneity of ribosomal internal transcribed spacer in clinical samples of Leishmania donovani spotted on filter paper as revealed by single-strand conformation polymorphisms and sequencing. Trans R Soc Trop Med Hyg, 2000; 94:575–579.

, Jin

, Zhang

. Present situation and perspective of leishmaniasis and its vector sandfly control in China. Int J Med Parasit Dis, 2006; 33:236–238.

, Li

. The epidemiology and control of visceral leishmaniasis in Li county, Sichuan, China. Parasit Infect Dis, 2005; 3:44–45.

Jiang

, Shang

, Xu

. The epidemiology and control situation of visceral leishmaniasis in Wenchuan county, Sichuan, China. Parasit Infect Dis, 2003; 1:185–186.

10.

Killick-Kendrick

. Phlebotomine vectors of the leishmaniases: a review. Med Vet Entomol, 1990; 4:1–24.

11.

, He

, Zhao

et al. Major trends in human parasitic diseases in China. Trends Parasitol, 2010; 26:264–270.

12.

Maia

, Afonso

, Neto

, Dionisio

et al. Molecular detection of Leishmania infantum in naturally infected Phlebotomus perniciosus from Algarve region, Portugal. J Vector Borne Dis, 2009; 46:268–272.

13.

Nicolas

, Prina

, Lang

, Milon

. Real-time PCR for detection and quantitation of Leishmania in mouse tissues. J Clin Microbiol, 2002; 40:1666–1669.

14.

Oshaghi

, Rasolian

, Shirzadi

, Mohtarami

et al. First report on isolation of Leishmania tropica from sandflies of a classical urban Cutaneous leishmaniasis focus in southern Iran. Exp Parasitol, 2010; 126:445–450.

15.

Quinnell

, Courtenay

. Transmission, reservoir hosts and control of zoonotic visceral leishmaniasis. Parasitology, 2009; 136:1915–1934.

16.

Sacks

, Kamhawi

. Molecular aspects of parasite-vector and vector-host interactions in leishmaniasis. Annu Rev Microbiol, 2001; 55:453–483.

17.

Sharma

, Singh

. Insect vectors of Leishmania: distribution, physiology and their control. J Vector Borne Dis, 2008; 45:255–272.

18.

van der Meide

, Guerra

, Schoone

, Farenhorst

et al. Comparison between quantitative nucleic acid sequence-based amplification, real-time reverse transcriptase PCR, and real-time PCR for quantification of Leishmania parasites. J Clin Microbiol, 2008; 46:73–78.

19.

Wang

, Xiong

, Guan

. The epidemiology and control achievements of leishmaniasis in new China. Chin J Epidemiol, 2000; 21:51–54.

20.

Zhang

. Prevalence and control of the status of visceral leishmaniasis in Sichuan, China. Parasit Infect Dis, 2006; 4:126–128.

21.

Zhang

, Li

, Lei

, Chen

. Analysis of leishmaniasis in Sichuan Province, China during the latest years. J Pathol Biol, 2007; 2:79–80.