Investigation of Leishmania infection and blood sources analysis in Phlebotomus chinensis (Diptera: Psychodidae) along extension region of the Loess Plateau, China


 Background Visceral leishmaniasis (VL) was one of the most important parasitic diseases in China, caused by Leishmania protozoans and transmitted by sand flies. Recently VL cases have reappeared in China, including the extension region of the Loess Plateau . The purpose of this study was trying to reveal the reason of reappearance to the developing the prevention and control measures. Methods Sand flies were collected by light traps from rural areas in Shanxian, Henan, China in 2015, as well as in Wuxiang and Yangquan, Shanxi, China in 2017. The blood sources of sand flies were analyzed by PCR detecting the host-specific mitochondrial cytochrome b (mtDNA Cyt b ) gene fragments. Leishmania infection in sand flies was detected by amplifying and sequencing ribosomal DNA internal transcribed spacer 1 ( ITS1 ). The Leishmania specific antibodies in the sera of local dogs were detected by ELISA kit. Results Blood sources showed diversity in the extension region of the Loess Plateau, including human, chicken, dog, cattle, pig and goat. Multiple blood sources within a sand fly were observed in samples from Yangquan (17/118, 14.4%) and Wuxiang (12/108, 11.1%). Leishmania DNA was detected in sand flies collected from Yangquan with minimum infection rate of 1.00%. The ITS1 sequences were conserved with the Leishmania donovani complex. The positive rate of Leishmania specific antibodies in dogs was 5.97%. Conclusion This study detected the blood sources and Leishmania parasites infection of sand flies by molecular methods in the extension region of Loess Plateau, China. A high epidemic risk of leishmaniasis is currently indicated by the results as the infection of Leishmania in sand flies, the extensive blood sources of sand flies including humans, and positive antibody of Leishmania in local dog sera. Given the recent increase of VL cases, asymptomatic patients, dogs and other potential infected animals should be screened and treated. Furthermore, the density of sand flies needs to be controlled and personal protection should be strengthened.


Introduction
Visceral leishmaniasis (VL), also known as kala-azar, is a sand y borne disease caused by Leishmania protozoans. VL was one of the most important parasitic diseases in China [1][2][3][4]. At present, VL is mainly endemic in western China, and focal and sporadic cases occurred in Xinjiang, Inner Mongolia, Gansu, Sichuan, Shaanxi, and Shanxi [2,3]. A total of 3,337 cases were reported from 2004 to 2012, of which 97.03% were distributed in Xinjiang, Gansu and Sichuan [5]. In 2018, 180 cases of VL were reported in 78 counties of 11 provinces in China, mainly distributed in Gansu, Shanxi and Shaanxi Province, and the epidemic area has expanded [6].
There are three epidemic types of VL described in China, the anthroponotic type, the zoonotic mountain type and the zoonotic desert type [7]. The zoonotic desert type VL was dominated in Xinjiang and Inner Mongolia, China. The extension region of Loess Plateau in Henan, Shaanxi and Shanxi Provinces has been a representative of the zoonotic mountain type VL endemic region. The characteristics of the zoonotic mountain type VL in China include: the pathogen was mainly Leishmania infantum, the vector was Phlebotomus chinensis, and the zoonotic hosts were dog (Canis familiaris) as well as raccoon dog (Nyctereutes procyonoides) [8][9][10][11]. VL was eliminated in most endemic areas after stringent implementation of control programs by the government in the 1950s [8]. No VL case was reported in Henan Province from 1983 to 2013 [4]. However, local VL cases began to reappear after that [12,13]. In 2018, 38 VL cases were reported in Shanxi Province, mainly in Yangquan City (15 cases) and Pingding County (8 cases); 3 and 27 cases reported in Henan and Shaanxi Provinces [6].
Blood meal identi cation is important evidence for the determination of the host preferences of hematophagous arthropods. Detection of Leishmania infection in sand ies and animal hosts could provide critical information to estimate the vector competence and assess the epidemic risk of VL in the endemic areas [14,15]. However, there was no report on Leishmania infection and blood source animals of sand ies in the extension region of Loess Plateau.
In order to explore the reasons for the VL recurrence in the area, and provide a scienti c basis for the development of prevention and control measures, we collected sand ies from Shanxian in Henan Province, Yangquan and Wuxiang in Shanxi Province, which are located in the extension region of the Loess Plateau, China. Leishmania infection and blood sources were detected, and the Leishmania speci c antibodies were determined in the sera of local dogs.

Ethics statement
This study was carried out in strict accordance with the National Natural Science Foundation of China ethical guidelines for biomedical research involving living animals and human subjects.

Sand y collection and species identi cation
The sand y samples were collected at three sites located in the extension region of the Loess Plateau China ( Light traps (MYFS-HJY-1, Houji Dianzi, Dongguan China) were used to catch sand ies. With the consent of the owners, light traps were set up in utility room, cave dwelling, courtyard and chicken farm from 5:30 pm to 8:30 am, and collected manually in the evening by mouth aspirators. The captured sand ies were sorted and counted by male and female, respectively.
The fresh female sand y adults were randomly dissected and the pharyngeal armature and spermatheca were observed under microscope. The species were identi ed according to the morphology [16]. The rest of the specimens were preserved in RNA xer (Aidlab Biotechnologies, China) and brought back to the laboratory. Blood source analysis was conducted on those female specimens with visible blood residues, whereas all female sand ies were used to detect Leishmania infection.

The ecological niches of the sand ies
The ecological niches of the sand ies in the extension region of the Loess Plateau China were described in our published article [17]. In brief, the collection sites are located in hilly lands with altitude ranges from 895m to 1050m, with similar geographical features and typical northern temperate climate. The buildings are cave dwellings or brick houses with tile roof. There are a variety of domesticated animals in the villages, including chickens, goats, pigs, cattle and goats. Most animals were kept in caves or semi-closed livestock circles adjacent to the houses, and some animals are kept open in the courtyard..

DNA extraction and molecular identi cation of species
Genomic DNA of sand y samples was extracted using DNAzol (Life Technologies, USA) following the manufacturer's instructions. The fragment of the mitochondrial cytochrome b (mtDNA cyt b) genewas ampli ed according to the method reported by Esseghir [18]. The primers were forward CB1 (5'-TAT GTA CTA CCA TGA GGA CAA ATA TC-3') and reverse CB3-R3A (5'-GCT AAT TAC TCC TCC TAA CTT ATT-3'). The positive PCR products were sequenced using four-color uorescently labeled dideoxy termination method in Boshang Biotech Co., Ltd. (Shanghai, China). The sequences were Blast aligned in GenBank on the NCBI website to determine the sand y species.

Blood sources identi cation
The female sand ies with visible blood residues were used for blood sources analysis, including 31 pooled and 216 individual samples. There were 12 pools from SX, 9 pools from YQ and 10 pools from WX. Every pooled sample contained 10 individuals. The individual samples include 118 from YQ and 108 from WX. The mtDNA cyt b fragments of different animals and human were ampli ed by PCR [19][20][21][22]. According to the main animal species in the collection site, PCR assay was developed with primers speci c to human, chicken, goat, pig, cattle and dog. The information of primer sets was listed in Table 2. The PCR reaction was carried out in 25μl containing 1.5μl DNA template, 0.2μmol/L primers and 12.5μl 2×PCR mix reagents (Aidlab Biotechnologies, China). The PCR running parameters were starting at 94 °C for 2 min; continuing with 35 cycles of 94°C for 15s, 51°C for 30s, and 72°C for 1min; and a nal extension with 72°C for 8min. The PCR products were electrophoresed on a 1.5% agarose gel to determine the size and were sequenced to con rm.

Leishmania spp. detection in sand ies
Leishmania infection was identi ed in all female sand ies. The ribosomal DNA internal transcribed spacer 1 (ITS 1) fragment of Leishmania was ampli ed using the following primers: forward LITSR 5' -CTG GAT CAT TTT CCG ATG-3', reverse L5.8S 5' -TGA TAC CAC TCG CAC TT-3' [23]. The PCR reaction was performed in 25μl contained 1.5μl DNA template, 0.2μmol/L primers and 12.5μl 2×PCR mix reagents. The PCR temperature pro le was as follows: starting at 94°C for 2min; continuing with 35 cycles of 94°C for 30s, 52°C for 30s, and 72°C for 30s; and a nal extension with 72°C for 8min. A positive control containing Leishmania donovani DNA (provided by National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention) and a negative control without DNA template were utilized.

Detection of anti-Leishmania antibody
The sera of 67 dogs (Canis familiaris) from YQ were provided by Yangquan Center for Disease Control and Prevention. The dogs were (0.5-14 years old). The owners of the dogs had been informed in advance and consented to the blood collection. The sera were separated by centrifugation at 3,000 rpm for 10 minutes after blood collection in the eld. Then the sera were refrigerated and transported to the laboratory for testing. The Leishmania speci c antibodies were detected using the commercialized Dogs Leishmania Ab ELISA kit (Fusheng Industrial Co., Ltd. Shanghai, China) in accordance with the manufacturer's instructions.

Sand y species identi cation
Thirty species were identi ed by morphology as Ph. chinensis. All other samples were identi ed as Ph. chinensis by molecular marker,which mtDNA cyt b gene sequence conserved with the HM747267 sequences in GenBank database. The Ph. chinensis is the absolutely dominant sand y species inthe extension region of the Loess Plateau China.

Blood sources identi cation
The blood sources of the fed sand ies include human, pig, chicken, goat, dog and cattle (Table 3). In SX, chicken blood was positive in all 12 pooled samples (100%), followed by cattle (66.7%), human (33.3%), dog (33.3%) and pig (16.7%), whereas goat was negative in all samples. In YQ, chicken and human were the most common blood sources. Chicken blood was identi ed in 88.9% of pooled samples and 59.3% of individuals, and human blood was positive in 66.7% pooled samples and 26.3% individuals. However, goat and human were the most common blood sources in WX. Goat blood was found in all pooled samples (100%) and 34.3% individuals; human blood was detected in 70.0% pooled samples and 64.8% individuals. Chicken blood was positive in 10.0% pooled samples and 21.3% individuals. Dog blood was positive in 10.0% pooled samples and 0.9% individuals.
In general, the sand y has a wide range of blood sources, and multiple blood sources means the sand ies take a variety of blood meal from different hosts in a short period.

Leishmania infection in sand ies
Out of the 59 pooled samples (10 individuals in a pooled sample) and the 108 individuals from YQ, Leishmania DNA was detected in ve pooled samples and two individual specimens. The minimum infection rate of Leishmania in the sand y population of YQ was 1.00% (7/698). All sequence of Leishmania amplicons were conserved with L. donovani complex (L. donovani/L. infantum) (MH200624) [24]. Of the two positive individuals, one had human blood meal while the other had chicken blood meal. No sample of SX and WX populations was positive in Leishmania DNA detection.

Detectionof Leishmania speci c antibodies in dog sera
Out of the 67 dog sera samples, four were identi ed as positive (5.97%) in the detection of Leishmania speci c antibodies using Dogs Leishmania IgG ELISA. The four positive dogs had no obvious pathological manifestations such as hair removal, desquamation, mental wilting, etc.

Comprehensive results of Yangquan
Comprehensive results were obtained in the samples collected from Yangquan Shanxi Province, China, which were summarized as below: Total of 3,599 sand ies were collected, Ph. chinensis was absolutely dominant species [23]. Chicken and human were the most common blood sources. Leishmania DNA was detected in ve pooled samples and two individual specimens, which sequences were conserved with L. donovani complex (L. donovani/ L. infantum).. The positive rate of dogs was 5.97% (4/67) in the detection of Leishmania speci c antibodies.
VL cases along the extension region of Loess Plateau in China belong to zoonotic mountain type, which patients were mostly children under 10 years of age [2,3]. High Leishmania spp. infection rate was detected in local dogs, so the number of human VL cases can be reduced by the elimination and suppression of local dogs [11,25,26]. The dog was the principal reservoir host in this epidemic area. The primary vector of the zoonotic mountainous type VL was Ph. chinensis, a zoophilic species that also feeds on human. Recently, VL cases in extension region of the Loess Plateau, China were recurrence after the eradication of the disease for 20 years, which posed a challenge for exploration and control.
Sand ies have a broad range of hosts as blood sources [19,20,[27][28][29]. They take blood mostly from mammals [20,[30][31][32][33] and cold-blooded animals [21]. In Jiuzhaigou Sichuan, China, swine was the dominant blood source of sand ies, followed by chickens and dogs [22]. However, there has been no report concerning the blood sources of sand ies in extension region of the Loess Plateau, China. In this study, chickens and humans were the most common blood sources of sand ies in Yangquan, Wuxiang Shanxi and Shanxian Henan from China, while dogs, goats, cattle and pigs were blood sources of sand ies as well. The proportion of blood sources was different with the locations of collection and the environment. As a good and su cient blood source in the region, chickens may contribute to the sustainability of a large sand y population, which was similar to what has been documented in other investigations [34,35]. Although chickens attract sand ies in Brazil and in this study, the role of chickens in the epidemiology of the sand y-borne diseases has not been de ned yet [35].
There are also many reports on the detection of Leishmania spp. in animals by PCR assay, such as in bovines (5%), buffaloes (4%) and goats (16%) [36], even in desert lizards [37]. In another case, goats were believed to constitute a reservoir host of L. donovani in Nepal [7]. Therefore, other reservoir animal hosts of Leishmania should be further investigated. In Yangquan Shanxi, China, Leishmania was found in sand ies andthe speci c antibody was also positive in local dogs. However, there was no dog blood in the sand y samples, probably because the collection sites were far away from the dogs' haunts in Yangquan. In Shanxian and Wuxiang, where the ecologic niches of sand ies were similar to that in Yangquan, dog was one of the blood sources animals. Consequently, we believed that dogs were still the blood source of sand ies in Yangquan, which frequently moving around sand ies' habitats. Multiple blood sources were found in individual fed specimens, suggesting a complex feeding behavior, which was critical for sand ies to transmit zoonotic diseases to humans.
In China, vector sand ies with Leishmania infection have been reported in Sichuan and Shaanxi Province [16,38]. The natural infection rate of a new haplotype of L. donovani was 1.98% in some villages in Sichuan Province [39], consistent with the results of sand y infection rate in this study. To the best of our knowledge, this study was the rst report of sand iesinfected with Leishmania spp. in the extension region of the Loess Plateau, China. Monitoring natural Leishmania infection in sand ies would provide critical information to estimate the vector competence and assess the local epidemic risk of VL situation. Of the seven positive samples, the ampli ed sequences were all conserved with L. donovani complex (L. donovani/L. infantum).. Multiple lines of evidence suggested that there were heterogeneous Leishmania strains in China. These strains were distinct from but phylogenetically related to L. donovani/L. infantum complex [24,26,37,39,40]. Our results would be a complement for the heterogeneity information of Leishmania in China.
Dogs were con rmed as the reservoir host of Leishmania spp. in mountainous type of zoonotic VL in the extension region of Loess Plateau, China [2,3]. It has been reported that the positive rate of Leishmania spp. in dogs was above 50% in Jiuzhaigou Sichuan, 41.9% in Heishui Sichuan, and 77.21% in Wenxian Gansu, China [11,25,41]. In Shanxi Province, there was only one investigation in 1959, in which the Leishmania positive rate in dogs was 0.01% [16]. In this study, the positive rate of serum antibodies in dogs from Yangquan Shanxi was 5.97%, and there was no disease manifestation in these dogs. Among the seven positive sand y samples tested, at least three contained human blood. Based on the preceding investigation, local asymptomatic patients and dogs should the important sources of VL infection, and the screening and treatment of the disease need to be strengthened.
Although we have systematically detected the blood sources, Leishmania infection and the antibody of Leishmania in dog sera in this study, there were still some limitations in this study. First, the primers for PCR detection of blood sources were designed based on the observation of the environment of the collection sites, but some blood sources animals may not be observed, so they may be missed. Moreover, the pooled samples were used for blood sources and Leishmania infection detection, making the positive rates not accurate. In addition, the investigation of potential reservoir hosts was insu cient in the study. In the future, we will collect more animal blood samples to detect Leishmania infection and antibodies, so as to provide more accurate guidance on the prevention and control of VL in the extension region of Loess Plateau in China.

Conclusions
This study detected the blood sources and Leishmania parasites infection of sand ies by molecular methods in the extension region of Loess Plateau, China. A high epidemic risk of leishmaniasis is currently indicated by the comprehensive information as below: high density of Ph. chinensis vector, the infection of Leishmania in sand ies, the extensive blood sources of sand ies including humans and dogs, and positive antibody of Leishmania in local dog sera. Given the recent increase of VL cases, asymptomatic patients, dogs and other potential infected animals should be screened and treated. Furthermore, the density of sand ies needs to be controlled and personal protection should be strengthened.