Detection of Leishmania DNA in Phlebotomine Sand Flies in Tsatee, a Community in the Volta Region, Ghana

Leishmania parasites, which are spread by infected female sand flies, are the cause of the disease leishmaniasis. Although cutaneous leishmaniasis has been found to occur in the Volta Region, there is limited data on vector species and reservoirs. This study focused on the Tsatee community, in the South Dayi District of the Volta Region, and is aimed at identifying the sand fly fauna and detecting the presence of Leishmania DNA by the use of primers that target the conserved region of Leishmania spp. minicircle DNA of the parasite kinetoplast. The miniature light traps and hand aspirators provided by the Centers for Disease Control and Prevention (CDC) were used to collect outdoor and indoor sand flies for five months in a guinea woodland and semideciduous forest area. From the collections, 4,580 phlebotomine sand flies were obtained and identified, and females were examined for Leishmania DNA using PCR. The male flies were 1,202 (26.24%), non-blood-fed females were 3,321 (72.51%), and 57 (1.25%) were blood-fed females. It was observed that Sergentomyia species constituted 99.91% of the total collected sand flies with S. africana (76.77%) as the predominant species. Phlebotomus rodhaini (0.09%) was the only Phlebotomus species identified from the study area. From 283 non-blood-fed sand fly pools and 57 individual blood-fed species screened, Leishmania DNA was detected in 12 (4.24%) pools and 8 (14.04%) individuals, respectively. It was observed that Leishmania DNA was detected in all the sand fly species identified except S. collarti. This study reports the first detection of Leishmania DNA in P. rodhaini in Ghana, with an infection rate of 33.33% (95% CI, 1.23-88.32). The findings suggest that the role of Phlebotomus in disease transmission in the study area cannot be discounted. Future studies should include continuous surveillance, blood meal preferences, and vector competence of the various infected phlebotomine sand flies to create effective control measures.


Introduction
The disease cutaneous leishmaniasis (CL) causes skin lesions, mainly ulcers on exposed parts of the host, leaving scars.The transmission of most Leishmania responsible for CL is zoonotic [1].About 350 million people globally are thought to be at risk of contracting the illness, which is thought to occur in 98 tropical, subtropical, and temperate regions [2].Reported cases of CL in endemic communities of the Ho District suggest the presence of multiple Leishmania parasites including Leishmania aethiopica in humans [3], Leishmania tropica, and Leishmania major in Sergentomyia sand flies [4].More recently, a new species causing infections in inhabitants of the Volta Region [5] has been characterised and named Leishmania (Mundinia) chancei [6].With the spread of infections in communities within the Volta Region, it is not clear which specific Leishmania parasite is primarily responsible for infections and which vector is responsible for transmission.
Although Phlebotomus species are culprit vectors of Leishmania parasites, there is some evidence to suggest that some species of the genus Sergentomyia may also transmit the parasite [7][8][9][10].The situation seems to be similar in Ghana as L. tropica and L. major DNA have been identified in pools of Sergentomyia ingrami and Sergentomyia hamoni [4].Leishmania major DNA in pools of Sergentomyia species was closely related to that found in the human population of the same study area [11] suggesting S. ingrami as a possible vector due to its anthropophilic characteristics and abundance [12].
Suspected CL cases have been reported in Tsatee, South Dayi District of the Volta Region (personal communication).Tsatee shares similar climatic and environmental conditions with the Ho Municipality and is thus capable of supporting sand fly populations and transmission of Leishmania.In Ghana, the detection of a novel Leishmania species causing human infections [5,6] further highlights a need to collect and identify potential vector(s) to determine whether the species previously considered as nonvectors can play a role in parasite transmission.Thus, this study focused on Tsatee, a community with suspected cases of CL, to identify sand fly species potentially involved in Leishmania transmission.The data obtained will be essential in selecting sand fly species for experimental studies to establish their possible roles in disease transmission in Ghana.

Methodology
2.1.Sand Fly Collection.The study was conducted in Tsatee (N 06 °36.206 ′ , E 000 °14.689 ′ ), a community within the South Dayi District, in the Volta Region of Ghana.The district has a mix of semideciduous forest and guinea woodland as its vegetation.Human and animal shelters were randomly selected for sand fly collections using hand aspirators and CDC light traps, respectively, from June to August and October 2015.A total of 20 CDC light traps were used to collect outdoor sand flies while hand aspirators were used for collecting indoor resting sand flies.The collected sand flies were freeze killed, sorted by sex with the female and male sand flies preserved with silica gel and 70% ethanol, respectively, and sent to the laboratory for morphological identification using taxonomic keys [13] and further analysis (Supplementary Data 1).
2.2.Detection of Leishmania DNA in Sand Fly Samples.The identified female sand flies were pooled (1-20) based on the species, for DNA extraction using Qiagen DNA Mini Kit (Qiagen Inc., Hilden, Germany) following the manufacturer's instructions.The obtained genomic DNA was used as a template in PCR for the detection of Leishmania DNA, using oligonucleotide primers Mincr2-F (5 ′ GGG GAG GGG CGT TCT GCG AA 3 ′ ) and Mincr3-R (5 ′ CGC CCC CTA TTT TAC ACA ACC CC 3′).At a band size of 120 bp, these primers target the Leishmania spp.minicircle DNA of the parasite kinetoplast, conserved region [14,15].Positive controls (L.major and L. tropica DNA) obtained from culture and negative control (deionized water) were included in each PCR reaction.Each reaction was performed in a final volume of 25 μl and contained 10× buffer, MgCl 2 (25 mM), dNTP (0.2 mM of each), Taq DNA polymerase (1.25 units), nuclease-free water, and about 25 ng template DNA.The PCR was performed in a Gene Amp PCR system 9700 (Applied Biosystems, USA) with the cycling parameters as follows: 5 min at 94 °C, 35 cycles for 30 sec at 94 °C, 30 sec at 60 °C, 1 min at 72 °C, and a final extension for 3 min at 72 °C.Ten microlitres of the PCR product was run on a 2% agarose gel for approximately 1 hour and 30 minutes at 100 volts.The gel was stained using ethidium bromide and visualised using Benchtop UVP Transilluminator (BioDoc-It® 210 Imaging System, Upland, California).

Data Analysis.
The results obtained were documented using Microsoft Word and Excel 2013 and analysed to determine infections and infection rates in sand fly pools by making use of PoolScreen 2.0 (version 2.0.1,January 2002) [16].

Distribution of Phlebotomine Sand Flies.
A total of 4,580 phlebotomine sand flies were collected during the fourmonth sampling period which comprised of 1,202 (26.24%) males, 3,321 (72.51%) non-blood-fed females, and 57 (1.25%) blood-fed females.The majority of the sand flies were caught using the CDC light traps as opposed to the hand aspirators (Table 1).

Detection of Leishmania DNA in the Identified Sand Fly
Species.A total of 283 pools of non-blood-fed sand flies were analysed, from which Leishmania DNA was detected in 12 (4.24%)pools (Figure 2).Phlebotomus rodhaini had an infection rate of 33.33% (95% CI, 1.23-88.32)(Table 2).

Discussion
The first outbreak of cutaneous leishmaniasis (CL) occurred in the Ho Municipality of the Volta Region in 1999 which is further south of the West African CL belt [12].The disease has persisted since then is currently spreading to previously nonendemic districts.
This study was carried out outside the Ho District at Tsatee in the South Dayi District, where CDC light traps and hand aspirators were used.However, the results obtained collaborate with the higher preponderance of Sergentomyia species with few Phlebotomus species found by previous studies in the Ho District.Since 1999, various epidemiology, entomology, and parasitology studies have been carried out within the Ho District.These studies involved the identification of the possible vectors and the Leishmania species responsible for the disease in the affected communities.Three trapping methods (CDC light traps, sticky traps, and hand aspirators) used in these studies had previously caught Sergentomyia species in predominantly large numbers and very few Phlebotomus species suggesting that the latter may not be involved in the disease transmission [4,12].In Ghana, the specific vectors responsible for Leish-mania transmission remain uncertain, reservoir hosts are still unknown, and treatment for infections is not in place.Because vaccines are not available for the prevention of cutaneous leishmaniasis infection, intervention measures to control the infection should be put in place to prevent or reduce the rate of sand fly bites and thus reduce the burden of the disease [17].It is also essential to understand the vectors involved, taking into account factors such as their feeding habits and transmission periods to establish prevention and control strategies [18].
In this study, a total of 99.91% of the species identified were Sergentomyia which is similar to the species composition in the Ho District found by Fryauff et al. [11].Furthermore, Sergentomyia africana (76.69%) was the most predominant species.Phlebotomus species, specifically P. rodhaini (0.09%), identified were in low numbers which is also consistent with the results obtained by Fryauff et al. [11].However, the low occurrence does not minimize the importance of the vector species.For instance, in eastern Sudan, a study carried out indicated P. rodhaini to be a potential vector of L. donovani despite their occurrence in small numbers [19].The author further proposed that P. rodhaini was a possible vector responsible for L. donovani transmission between animal reservoir hosts but did not infect humans.Also in parts of West Africa, P. rodhaini is a vector for L. major [20].
All the blood-fed sand flies collected were Sergentomyia with S. africana (57.89%) being the dominant species.Man-biting species, S. schwetzi as described by Boakye et al. [21], was also identified but in low numbers.The predominance of Sergentomyia species suggests that they may play a role in the transmission of Leishmania parasites within the present study area and the Volta Region at large.Whether they transmit zoonotic leishmaniasis was not determined by the present study.
Sergentomyia species transmit reptilian Leishmania [22].However, the findings from certain studies suggest that Sergentomyia species could vector human Leishmania.For example, a study conducted in Kenya supports the role of Sergentomyia species in the transmission of mammalianinfecting Leishmania parasites [23].The authors found an infection rate of 1% in S. ingrami and also observed that L. major isolated from the guts of infected females produced lesions when inoculated into BALB/c mice.The findings, coupled with a large number of amastigotes found in prepared smears of the lesions, led the authors to suggest a role of S. ingrami in transmitting L. major in the study area [23].In Mali and Kenya, L. major DNA was detected in S. darlingi and S. garnhami, respectively [8,24].In this study, Leishmania DNA was detected in all the trapped non-blood-fed sand fly species except S. collarti unlike in the Ho District where parasite DNA was detected in only pools of S. ingrami and S. hamoni [4].Amongst the 57 individual blood-fed sand flies, Leishmania DNA was detected in all five identified species.S. ingrami and man-biting species S. schwetzi were trapped in the dwellings of inhabitants in the study area.Even though Leishmania DNA was detected in Sergentomyia species, that is not enough evidence to consider them vectors.Since infections from blood meals may last in   3 BioMed Research International nonvectors for a while but ultimately do not in established disease, harbouring the parasites does not inevitably make them vectors [25].Furthermore, a study showed that even though S. schwetzi was infected with L. major, complete development of the parasite did not occur and was not transmitted to a host postfeeding [26].Studies have also reported the refractoriness of Sergentomyia to Leishmania parasites [10,22,27].Establishing colonies of Sergentomyia species in Ghana will be necessary for infection experiments aimed at observing their potential to house the complete development of Leishmania parasites and further transmit them to a host.
For the first time in Ghana, Leishmania DNA was detected in a single unfed P. rodhaini, thus an infection rate of 33.33%.This suggests that although P. rodhaini occurs in low numbers, it can be highly infected, hence can, therefore, be considered as a potential vector of Leishmania parasite transmission in the study area.P. rodhaini were collected outdoors which makes it uncertain as to whether the parasite transmission is occurring from reservoir hosts to humans or human to human.
With the discovery of a new Leishmania species [5], more questions rather than answers are being raised.It is not clear which Leishmania species are responsible for infections in communities of the Volta Region.Further investigations will be essential to fully determine the different Leishmania parasites in the region and identify the vector(s) responsible for infections.

Conclusions
The present study has confirmed the dominance of Sergentomyia species in previous studies in CL areas of Volta Region and the relatively low occurrence of Phlebotomus species.Although Sergentomyia species are considered to be nonvectors for human pathogenic Leishmania species, some harboured Leishmania DNA indicating that they may be involved in the disease transmission.Further experiments are however required to demonstrate their ability to accommodate the complete development of Leishmania parasites and further transmit to a suitable host.This study also reports for the first time in Ghana the detection of Leishmania DNA in P. rodhaini.The findings suggest P. rodhaini as a potential vector of cutaneous leishmaniasis at Tsatee in the South Dayi District.
o l l a r t i P .r o d h a i n i

Figure 1 :
Figure 1: Distribution of sand fly species identified from the study area.

Table 1 :
Distribution of sand flies trapped using CDC light trap and hand aspirator.

Table 2 :
Infection rates in non-blood-fed sand flies.