2.1 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.
2.2 Sand fly collection and species identification
The sand fly samples were collected at three sites located in the extension region of the Loess Plateau, China (Table 1; Fig. 1): Xizhang Village (111.22°E/34.62°N, 985m) in Shanxian County (SX), Henan Province in July 2015, Hedi Village (113.56°E/38.00°N, 895m) in Yangquan City (YQ) and Moyu Village (113.09°E/36.79°N, 1050m) in Wuxiang County (WX), Shanxi Province, China in June 2017.
Light traps (MYFS-HJY-1, Houji Dianzi, Dongguan, China) were used to catch sand flies. 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 flies were sorted and counted by male and female, respectively.
Some fresh female sand fly adults were randomly selected to dissect. The species were identified in according with the morphology of pharyngeal armature and spermatheca . The rest of the specimens were preserved in RNAfixer (Aidlab Biotechnologies, China) and brought back to the laboratory. All samples used in this study were identified by DNA sequences. Blood source analysis was conducted on those female specimens with visible blood residues, whereas all female sand flies were used to detect Leishmania infection.
2.3 The ecological niches of the sand flies
The ecological niches of the sand flies in the extension region of the Loess Plateau China were described in our published article. 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, dogs, 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.
2.4 DNA extraction and molecular identification of sand flies species
Genomic DNA of sand fly samples was extracted using DNAzol (Life Technologies, USA) following the manufacturer’s instructions. The fragment of the mitochondrial cytochrome b (mtDNA cyt b) gene of sand flies was amplified according to the method reported by Esseghir . 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 fluorescently labeled dideoxy termination method in Boshang Biotech Co., Ltd. (Shanghai, China). The sequences were Blast aligned in GenBank on the NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine the sand fly species.
2.5 Blood sources identification
The female sand flies 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 amplified by PCR [19-22]. According to the main animal species in the collection site, PCR assay was developed with primers specific to human, chicken, goat, pig, cattle and dog. The information of primer sets was listed in Table 2, and separate PCRs were performed for each pair of primers. 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 final 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 confirm.
2.6 Leishmania spp. detection in sand flies
Leishmania infection was identified in all female sand flies. The ribosomal DNA internal transcribed spacer 1 (ITS 1) fragment of Leishmania was amplified 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' . 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 profile 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 final 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.
2.7 Detection of anti-Leishmania antibody in the sera of dogs
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 and consented to the blood collection. The sera were separated by centrifugation at 3,000 rpm for 10 minutes after blood collection in the field. Then the sera were refrigerated and transported to the laboratory for testing. The Leishmania specific antibodies were detected using the commercialized Dogs Leishmania Ab ELISA kit (Fusheng Industrial Co., Ltd. Shanghai, China). A 96-well ELISA plate coated with Leishmania antigen, positive and negative control sera were provided in the kit. The serum samples were diluted and assayed according to the manufacturers’ instructions. Briefly, diluted serum samples, positive and negative control sera were added to the 96-well ELISA plate coated with antigen. After incubation and subsequent washing, the HRP-conjugate antigen was added to the wells. The plate was washed again after incubation. Then the HRP enzyme substrate was added for chromogenic reaction. The optical density (OD) value was recorded at 450 nm using a photometer (BioTek, Gene Company Ltd., USA). Each sample was tested in triplicate. The samples were considered positive if the mean OD value was higher than the positive cut-off value.