Acta Trop. 2016 Apr;156:95-9. doi: 10.1016/j.actatropica.2016.01.010.
Detection of natural infection of Leishmania donovani (Kinetoplastida: Trypanosomatidae) in Phlebotomus argentipes (Diptera: Psychodidae) from a forest ecosystem in the Western Ghats, India, endemic for cutaneous leishmaniasis.
- 1Vector Control Research Centre (ICMR), Puducherry 605 006, India.
- 2Vector Control Research Centre Field Station (ICMR), Kottayam, Kerala 686002, India. Electronic address: firstname.lastname@example.org.
A new focus of transmission of Leishmania donovani causing cutaneous manifestations (CL) was reported by us earlier, in the Western Ghats region of Thiruvananthapuram district, Kerala, India. 12,253 sand fly specimens, comprising of three species belonging to the genus Phlebotomus (24.7%) and 16 species belonging to the genus Sergentomyia (57.3%) were collected from the region during 2012-2014. Among Phlebotomus species, Phlebotomus argentipes was found predominant (77.3%), followed by Phlebotomus colabaensis (21.7%) and Phlebotomus stantoni (1.6%). From these collections, 793 P. argentipes (88 pools), 123 P. colabaensis (31 pools) and three P. stantoni (three pools) female specimens were processed for detection of natural infection with L. donovani parasites using a multiple genetic marker (kinetoplast DNA; 3’UTR of HSP70 gene & HSP70 gene) approach. Five pools of P. argentipes specimens (Unfed (one), Fulfed (one) and Gravid (two)) among these, were found positive for L. donovani infection. HSP70 gene sequences of the parasites in the vector species was found genetically identical with the human isolates reported earlier, evincing the role of P. argentipes in the transmission of CL in this region. This is the first finding of natural infection of P. argentipes with L. donovani (causing CL) from India. Copyright © 2016 Elsevier
KEYWORDS: Cutaneous Leishmaniasis; India; Kerala; Leishmania donovani; Phlebotomus argentipes; Western Ghats; natural infection
Leishmaniasis is a neglected tropical disease, reportedly affecting 98 countries globally. About 310 million people live at the risk of this disease in these countries. It is caused by a group of protozoan parasites belonging to the Genus Leishmania. Based on the symptomology, Leishmaniasis could be classified as Cutaneous (CL), Visceral (VL) and Mucosal/Mucocutaneous forms. CL is the most common form of Leishmaniasis: in the old world, this disease is caused by Leishmania tropica, Leishmania major & Leishmania ethiopica and in the new world by L. mexicana, L. (Viannia) amazonensis, L. venezuelensis, L. (Viannia) braziliensis, L. (Viannia) panamensis, L. (Viannia) guyanensis & L. (Viannia) peruviana. 70-75% cases of CL incidences occur in about 10 countries: Afghanistan, Algeria, Colombia, Costa Rica, Ethiopia, Iran, Peru, Sudan and Syria. It is estimated that 0.7-1.2 million cases of CL occur annually. VL (Kala Azar) is the most severe form, which cause tremendous morbidity among the patients and is fatal, if untreated. The parasite species involved are Leishmania donovani and Leishmania infantum. These two parasites had been recorded to cause CL also in few localities (Sharma et al., 2005). About 90.0% of VL cases are restricted to 6 Countries: Brazil, Bangladesh, Ethiopia, India, South Sudan and Sudan and approximately 20,000 people die due to Kala azar every year. Mucocutaneous form (Espundia) is generally restricted to the new world countries. The parasite species involved belong to Viannia subgenus and the species involved are Leishmania (V) braziliensis, Leishmania (V) panamensis, Leishmania (V) amazonensis and Leishmania (V) guyanensis.
Fig. 1. An acute case of Cutaneous Leishmaniasis.
Fig. 2. A typical human dwelling in the tribal villages.
India is one of worst affected countries of ‘Kala Azar’ (means Black Fever in Hindi language). L. donovani is the parasite involved and Phlebotomus argentipes acts as the sole vector species in the Country. 54 Districts distributed to four states, viz: Bihar, Uttar Pradesh, Jharkhand and West Bengal remains affected. CL forms had been restricted to the Thar Desert region of North Western India. Localized cases had been reported from Uttar Pradesh, Himachal Pradesh and Kerala. The parasite species involved are L. major and L. tropica. Involvement of L. donovani complex in CL form had been described from Himachal Pradesh (Sharma et al., 2005) and from Kerala (Kumar et al., 2015) recently.
The present study attempted to characterize the vector species involved in the transmission of cutaneous manifestations of leishmaniasis, endemic to the tribal villages in Thiruvananthapuram District, Kerala State, reported way back during 2010 (Simi et al., 2010). The parasite involved in the human infections was identified to be L. donovani. (Kumar et al., 2015). The endemic villages are located deep in the thickly forested region of Western Ghats, the second largest mountainous region in India. Prevalence of acute cases of CL were recorded in the study villages during the study period (Fig. 1) (Kumar et al., 2015). The villages lie scattered and are isolated hamlets with about 10-30 houses each in a locality. The main occupation of the population is apiculture and agriculture of forest crops. Also rubber plantations were introduced into this region by Rubber Board, Govt. of India. Most of the houses/huts where people dwell are made up of mud and shrubs/ leaves of different trees which grow there (Fig. 2). The population in the regions belongs to Kanikkars (Kani), one of the oldest tribal settlements of south India, who live in Thiruvananthapuram & Kollam Districts of Kerala State and in Kanyakumari and Tirunelveli Districts of the adjacent state of Tamil Nadu, India.
Fig. 3. Entomological collection of resting sandfly specimens.
Sandfly population in 10 villages were sampled following standard procedures (Fig. 3) for a period of 3 years (2012-2015) and were subjected to morphological identification following standard taxonomic keys. 19 species of sandflies belonging to two genera: Sergentomyia and Phlebotomus were collected from the region. Among the Phlebotomus collected, Phlebotomus argentipes was found to be the predominant species. The Plebotomus specimens were pooled and processed further for determining their infection status with amastigotes of Leishmania parasites. The procedure followed was briefly: extraction of total DNA from sandfly specimens followed by the PCR protocol proposed earlier by Kumar et al., 2015. A multiple (four) genetic marker approach involving mini-circle of kinetoplast DNA to detect the infection status followed by the Restriction Fragment Length Polymorphism analysis 3’UTR of hsp70 gene and sequencing of a larger fragment (~1400bp) of the heat shock protein 70 (hsp70), to characterize the species involved. Another genetic marker, 6 phospho-gluconate dehydrogenase (6-PGDH) sequences were analyzed to characterize the strain of the species involved.
The results of the study showed five pools of Ph. argentipes specimens (n=9), among 88 pools (n=793 specimens), processed were found to be infected with Leishmania parasites. This included all the three different (Unfed, Full-fed and Semi-Gravid/Gravid) abdominal stages of the sandflies. To confirm the species identity of the naturally infected specimens, their DNA were subjected to DNA Barcoding (Kumar et al., 2012) and the amplified COI sequences of the vector species confirmed their morphological identification as Ph. argentipes. The positivity recorded in the gravid developmental stages of the vector clearly indicate the role of Ph. argentipes as the vector of L. donovani (cutaneous form) in this region. This is the first report from India where this species act as a vector of cutaneous leishmaniasis, in India. A similar observation was made from Sri Lanka earlier (Siriwardana, et al., 2007). The study on the genetic lineage of the Indian parasite indicated its close genetic relatedness to this Sri Lankan strain.
Ph. argentipes had been proposed as a sibling species complex (Ilango et al., 2010; Gajapathy et al., 2013). Based on DNA Barcodes the specimens from Western Ghats were found to be belonging to more than one sibling species. We propose carrying out further studies to taxonomically characterize the sibling species complex, following a multiple genetic marker approach, among samples collected from different geographical localities.
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