Detection of Leishmania donovani in Wild-Caught Phlebotomine Sand Flies in Endemic Focus of Leishmaniasis in Himachal Pradesh, India

Abstract An endemic focus of cutaneous leishmaniasis (CL) is located in the Sutlej River Valley in Himachal Pradesh (India) in the north-western Himalaya where CL co-exists with visceral leishmaniasis (VL). In areas of the Indian subcontinent such as Rajasthan, cutaneous leishmaniasis is transmitted by Phlebotomus papatasi (Scopoli) and Phlebotomus salehi. In Himachal Pradesh, Phlebotomus longiductus (Parrot) is suspected to be the vector for CL. In the current study, sand flies were collected and tested for Leishmania infection and to confirm the vector species causing CL. Sand flies were collected during April and September of 2017–2019 from CL endemic villages of Rampur (Shimla), Nirmand (Kullu) and Nichar (Kinnaur) districts of Himachal Pradesh. The sand flies were identified as Phlebotomus (adlerius) longiductus (Parrot) and Phlebotomus (larrousisus) major (Annandale). The density of P. longiductus was found highest. The elevation of villages ranged from 947 m to 2,130 m and were far from the presence of subsoil water. Field collected sand flies tested positive (7.69%) for Leishmania donovani by PCR-RFLP. The L. donovani sequences detected from P. longiductus were 97% similar to L. donovani sequences reported from the cases of CL in Himachal Pradesh.The Leishmania positive sand flies were morphologically identified as Phlebotomus adlerious longiductus providing one step further evidence towards the vector status of CL in Himachal Pradesh. The findings of the study are of epidemiological significance for strategic planning of vector control for leishmaniasis in India.

The leishmaniases are a group of diseases caused by the protozoan parasite Leishmania. Over 20 species of Leishmania known to be infective to humans, are transmitted by the bite of infected female phlebotomine sand flies. Of 200 countries and territories reporting to WHO, 97 countries are endemic exclusively for visceral leishmaniasis (VL), 65 for both VL and cutaneous leishmaniasis (CL) while 22 countries are endemic for CL alone (WHO 2017). Historically, the focus of CL in India was reported from Bikaner district in the Thar desert of Rajasthan in 1973. Desert gerbil, Meriones hurriane is the reservoir for zoonotic CL and Phlebotomus salehi Meshghali is the vector species. Dog has been reported as a reservoir for anthroponotic CL and Phlebotomus papatasi as the vector (WHO 2010). After a gap of around 30 years, a new focus of CL was reported along the River Sutlej in Himachal Pradesh from 2003 to 2005, (Sharma et al 2003, Sharma et al 2005. While investigating the focus of CL in Himachal was, therefore, undertaken in reported foci of Shimla, Kinnaur, and Kullu districts of Himachal Pradesh to determine the vector of CL.

Study Area
The present study was undertaken in Rampur, Nirmand, and Nichar subdivisions of Shimla, Kullu, and Kinnaur districts of Himachal Pradesh located along the Sutlej River Valley between 31°05′0″N and 31°42′0″N latitude and 76°52′E and 78°24′0″E latitude. Altogether 16 villages were selected for collection of sand flies (Lata et al. 2021) based on accessibility.

Collection of Sand Flies
The collection of sand flies was made during April and September 2017-2019. Sand flies were collected between 06:00-09:00 h.from human dwellings and cattle shades using aspirator, sticky trap, and CDC light trap. The density of sand flies collected from indoor resting sites was expressed as Man Hour Density (MHD) (Srivastava and Dhariwal 2016). After collection, the sand flies were stored in 70% ethanol for identification. Head and last three abdominal segments with external genitalia were used for morphological identification and remaining abdominal segments were used for DNA extraction. The specimens were kept in 10% Potassium hydroxide solution at room temperature for 2h before mounting on slides. Identification of sand flies was done following the identification key by Lewis (1978) and Kalra and Bang (Kalra et al. 1988).

DNA Extraction, Polymerase Chain Reaction-Restrictiction Fragment Length Polymorphism (PCR-RFLP) and DNA Sequencing
Total DNA was extracted from 52 bloodfed sand flies individually using the Qiagen DNA mini kit (Qiagen), as per manufacturer's instructions, and eluted in 20 µl buffer. The details of DNA extraction, PCR-RFLP, and DNA sequencing are given in Lata et al. (2021)

Man Hour Density of Sand Flies
The sand flies collected from various villages were morphologically identified as Phlebotomus (adlerious) longiductus and Phlebotomus (larrousisus) major. The altitude of villages ranged from 947 to 2,130 m in Shimla, Kullu, and Kinnaur districts respectively ( Table  1). The highest MHD was found in Kinnaur district (80), followed by Kullu (54), and Shimla district (15).

Detection of Leishmania in Sand Flies
Total DNA extracted from 52 bloodfed sand flies were subjected to PCR for detection of Leishmania, of which four sand flies tested positive for Leishmania with both JW11/12 and LITSR/L5.8S Leishmania genus-specific set of primers ( Fig. 1). By PCR -RFLP, it was confirmed that all four Leishmania positive sand flies showed L. donovani fragments with bands of size 54 bp, 75 bp and 187 bp (Ranasinghe et al. 2015) (Fig. 2).

Sequence Analysis
In Silico Analysis MN861106.1 and MN861107.1 accession having sequences of L. donovani identified from P. longiductus showed close similarity with the L. donovani sequences from cases of CL in Himachal Pradesh with accession numbers MT423519-21 (Lata et al. 2021).
Only a single "T" nucleotide deletion was observed at the 265 nt position in both the sequences (MN861106.1 and MN861107.1) in comparison to the L. donovani sequences from the cases of CL in HP ( MT423519-21.) In the tree obtained by the Neighborhood-joining method ( Fig.  3), it has also been revealed that both the accessions MN861106.1 and MN861107.1 were found 97% similar to L. donovani. Both of these sequences showed ~45-97% of similarity with the other species included in the analysis. The distantly related species Leishmania tropica (MT423522 MH763643), Leishmania major (MT423523 and MH347926), and Leishmania infantum (AJ000289 and KY973656) were grouped under a distant clade (Fig. 3), indicating about the sequence variability. These species showed similarity of 45% (L. major), 75% (L. tropica), and 96% (L. infantum) with L. donovani species. The results of detection of natural infection of L. donovani by PCR and DNA sequencing techniques are given in Table 2.
In order to know both the genetic and geographic relatedness of identified isolates among themselves and with standard reference strains, the results of ITS1 polymorphic microsatellite repeat analysis indicated that Leishmania sequences isolated from P. longiductus are similar to only L. donovani isolates identified from different locations. However, the polymorphism was detected in the microsatellite markers in the sequences derived from either L. infantum, L. tropica, or L. major species (Fig.  4, Table 3). By sequence analysis it can be concluded that the L. donovani sequence detected from CL cases in HP are similar to L. donovani sequences detected from P. longiductus which provide the one-step further evidence towards the vectorial status of P. longiductus for CL in HP and also confirming that the L. donovani is the predominant Leishmania species detected.

Discussion
The findings of the present study reveal that phlebotomine sand flies are found up to the altitude of 2,130 m and their distribution is wide spread in villages in Shimla, Kullu, and Kinnaur districts of Himachal Pradesh. In China, the distribution of P. longiductus has been reported from 2,100 m elevation (Guan et al. 2016), however, we found its distribution ranging from 947 to 2,130 m. In study villages close to river Sutlej, the maximum MHD of sand flies was found up to 54 and 16 in villages of Kullu and Shimla district respectively. On the other hand, in village Punang, which is located at an altitude of 1,775 m in Kinnaur district and far away from Sutlej river, the highest MHD was 80. Earlier studies from Himachal Pradesh reported only the number of sand flies collected (did not report MHD) and the distribution of sand flies was also not such widespread as observed in the present study. Sharma et al. (2009) reported one P. longiductus as suspected positive for L. tropica. The present study confirms the detection of L. donovani from field-collected P. longiductus (confirmed by both PCR-RFLP and sequencing methods) from the foci of CL in Himachal Pradesh, India which provide one step further evidence towards vectorial status of P. longiductus. Further work on identification of blood meal sources of P. longiductus and transmission through artificially infected sand flies to a susceptible host is desired to confirm it as vector of CL.
As per World Health Organization (1990), P. longiductus has been listed as a proven vector of VL in Kazakhstan, Kyrgyzstan, and in China, however, in these countries, P. longiductus was not found naturally infected with L. donovani. The present study areas being close to China border reflect the epidemiological characteristics of CL similar to the China. The endemic areas of VL and CL in India are diverse, i.e. VL is in eastern part of the country while CL is mainly in Rajasthan and Himachal Pradesh. In the present study area, the parasite species, i.e., L. donovani have been detected (similar to Mediterranean region).
It is indeed intriguing that L. donovani has been detected from the cases of CL from several parts of the world including neighboring country Sri Lanka (Siriwardana et al. 2007;Ranasinghe et al. 2015, Thakur et al. 2020. A recent study by Thakur et al. (2020) provided evidence that L. donovani isolated from CL patients in Himachal Pradesh is L. donovani zymodeme MON37 strain which does not cause VL.
The distribution of P. longiductus has also been reported from Banihal and Punch district of Jammu & Kashmir (Lewis 1978), where cases of CL have been reported, however, the parasite and vector species still remain unknown, warranting further studies (Rather et al. 2021). In the geographic belt ranging from Jammu & Kashmir to Himachal Pradesh, a few cases of VL have been reported along the Ravi River in Chamba district but the vector species is unknown (Raina et al. 2016). It is expected that P. longiductus may be the vector of VL also as the same species has been reported from Chamba and Kangra districts of Himachal Pradesh (Lewis 1978). The question that still remains unresolved is whether the so-called CL cases detected in Himachal Pradesh are of Dermal leishamanoid (a form of VL) or the pathogenicity of L. donovani and L. tropica are interchangeable between visceral to cutaneous and vice versa. The sand flies were identified based on morphology. Molecular tools like sequencing and DNA barcoding may be employed to find out the lineage of the species.
The state of Himachal Pradesh had reported cases of VL during 1979 (Naik et al. 1979), 1985(Dutta et al. 1984) and 2016 (Raina

Conclusion
The detection of L. donovani in field-collected P. longiductus from the villages along Sutlej River in Shimla and Kullu districts of Himachal Pradesh provides one-step further evidence towards the vectorial status of P. longiductus for cutaneous leishmaniasis in Himachal Pradesh. The findings are of epidemiological significance and strategic planning of vector control for leishmaniasis in India. The need of the hour is to protect the population from the bites of sand flies, therefore, communities in the affected areas should be educated to prevent themselves from the bites of sand flies. S/Shri M.C Sharma, V.P Singh, and Surendra Kumar is duly acknowledged. This work was supported by the Indian Council of Medical Research New Delhi, India. (Grant 68/10/2014-NCD-1). The study does not require any Ethical approval. The study does not required consent of participants for publication

Data Availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.