Leishmania infection in Phlebotomus species in Mehran city, Ilam province, Iran, Iran

1. Introduction

Leishmania is a protozoan of the genus Leishmania, which is transmitted by sandflies ( 1 ) and is the causative agent of cutaneous Leishmaniasis. Leishmania is a protozoan parasite that can infect both humans and animals. There are two forms of the parasite: the first is a small, round form called an amastigote, which lives inside the cells of the vertebrate host. The second form is elongated and has flagella, and is called a promastigote ( 2 ). This form lives inside the body of the insect that transmits the disease. To date, approximately 30 species of parasites have been documented, of which a mere 20 have been determined to be pathogenic to humans ( 3 , 4 ). The protozoan parasite Leishmania is transmitted through the bite of infected female Phlebotomine mosquitoes, which feed on blood to produce eggs. The epidemiology of Leishmaniasis is contingent upon the characteristics of the parasite and the mosquito species, the environmental characteristics of the transmission sites, the exposure of the human population to the parasite, and the behavior and habits of humans. It is estimated that approximately 70 species of animal, in addition to humans, act as natural reservoir hosts for Leishmania parasites ( 5 ). The feeding habits of sandflies are diverse, with some species exhibiting a wide range of hosts, including canids, rodents, and blood-sucking reptiles. In contrast, other species have a pronounced anthropophilia, feeding primarily on humans. Consequently, human Leishmaniasis exhibits comparable patterns of disease transmission between animals and humans or between humans ( 6 ). It is important to note that Phlebotomus may be infected with different species of Leishmania during the process of blood feeding, whether from humans or animals. During the process of blood feeding, the parasite may transfer Leishmania to a new host. The epidemiological and clinical manifestations of Leishmaniasis vary depending on the specific Phlebotomus species. It is evident that two cutaneous anthroponotic (ACL) and zoonotic (ZCL) forms of human Leishmaniasis are prevalent in Iran. In the anthropontic form, Ph. sergenti and Phlebotomus tropica and humans usually play the main role, and in the zoonotic form of Leis hmaniasis, Ph. papatasi and Phlebotomus major and rodents play the main role. The frequency of these forms varies across different regions of Iran. In the province of Ilam, the zoonic form is typically documented. In the following section, a number of recent studies conducted in Ilam province regarding Leishmaniasis will be mentioned. These include the studies of Asgari Nezhad et al. (2012); Yazdanpanah & Rostamianpur (2013); Roghani et al. (2012); Gholami Parizad et al. (2015); Kassiri et al. (2012); and Kermanjani et al. (2017) ( 7 - 12 ). To date, no research has been conducted on the Phlebotomus population of Mehran city, Ilam province, with regard to its potential for transmitting Leishmania. The objective of the present study was twofold: firstly, to ascertain the prevalence of Leishmania infection in Phlebotomus in Mehran city; and secondly, to determine the species of said infection by molecular method.

2. Materials and Methods

2.1. Area of Study

Ilam province is located in western Iran, adjacent to the following provinces: Kermanshah to the north, Khuzestan to the south, Lorestan to the east, and Iraq to the west (Figure 1). The most prominent cities of Ilam province are as follows: The subjects in this study were Ivan, Dehlran, Mehran, and Shirvan. Mehran city is located on the left bank of the Kanjan Cham river and is less than a few kilometers away from the Iraqi border. The city's population is 46,981 individuals, and it is divided into three districts. Mehran, Saleh Abad, and Malekshahi. Mehran city is located at an altitude of 155 meters above sea level. Mehran is considered one of the most fertile areas of the province during years with high precipitation. ( 13 ).

Figure 1. Geographical location of Mehran city, Ilam province, Iran.

2.2. Sampling

The sticky papers method was utilised for the collection of sandflies. The papers were divided into two seasons, with 100 published in the summer and 300 in the autumn. The installation of 400 sticky paper traps resulted in the collection of 2,860 sandflies (950 females and 1,910 males) over the course of two seasons. The sticky papers were installed in indoor locations, such as houses and stables, as well as in outdoor and open areas in the vicinity of these structures, spanning a total of 17 regions during both the summer and autumn seasons. Subsequently, the sand flies were extracted from the adhesive papers using entomological needles or fine brushes. Thereafter, they were washed multiple times with 75% ethanol to eliminate any residual oil. Following this step, the flies were preserved in 70% ethanol and stored in micro tubes prior to identification.

2.3. Microscopic Study

In order to identify the species of sand fly, the head and the posterior two abdominal segments of female sand flies were detached, mounted in Puri's media, and the species were identified using a valid morphological identification key for adult sandflies. ( 14 , 15 ). In order to ascertain whether the sandfly had been infected by the Leishmania parasite, the remainder of the sandfly's body was preserved in 85% ethanol for the purpose of DNA extraction.

2.4. DNA Extraction

The phenol-chloroform method was utilized for the extraction of deoxyribonucleic acid (DNA) from the body of Phlebotomus. The Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) technique was utilized in order to conduct an investigation into the infection of female Phlebotomus and to ascertain the parasite species.

2.5. Polymerase Chain Reaction

The ITS1 region of the Leishmania parasite was amplified using the following primers: forward, 5'-CTGGATCATTTTCCGATGT-3'; reverse, 5'-TGATACCACTTATCGCACTT-3'. ( 12 , 15 ). The reaction mixtures were then adjusted to a final volume of 20 µL, consisting of Taq Master Mix (9.5 µL), 10 pmol of each primer (forward primer: 1 µM; reverse primer: 1 µM), template DNA (4 µL), and sterile deionized water (4.5 µL). The initial denaturation process was conducted at a temperature of 94°C for a duration of 5 minutes. This was followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 54°C for 30 seconds, and extension at 72°C for 30 seconds. The final extension step at 72°C for 10 minutes was subsequently followed by cooling to 4°C. Subsequently, the final product from each reaction was subjected to electrophoresis and analysis on a 2% agarose gel with safe stain.

2.6. RFLP

In order to determine the species of Leishmania, HaeIII enzyme was used for cutting the bands in RFLP assay. The anticipated band pattern for L. major comprises two fragments of 132 and 203 base pairs, respectively, while for L. tropica, four fragments of 185, 53 and 57 base pairs are expected.

2.7. Sequencing

A total of 20 µl of PCR products, 10 µl of forward primer and 10 µl of reverse primer were dispatched to the Niagen Noor Company (Iran). To facilitate this, the sequences were compared to homologous sequences in GenBank using the nucleotide-nucleotide Basic Local Alignment Search Tool (BLAST: www.ncbi.nlm.nih.gov/BLAST). The identification of the parasite species was conducted through a meticulous comparison of their respective sequences with those already deposited in GenBank.

2.8. Statistical Analysis

The statistical analysis of the variables was conducted using SPSS software version 16. The data were subjected to a chi-squared test at a 95% confidence level, with a P value of less than or equal to 0.05. A result was deemed to be statistically significant if the P value was less than or equal to 0.05.

3. Results

3.1. General Result

During the course of two seasons, a total of 400 sticky papers were placed in 34 districts of Mehran city, and a total of 2,860 Phlebotomus were collected. The results obtained from two sampling seasons indicate that 950 samples are female, of which 617 are from the genus Phlebotomus and 333 are from the genus Sargentomia. As indicated in Table 1, the proportion of female Phlebotomus and Sergentomyia that had fed on blood was 8.75% and 3.30%, respectively. As indicated by the data presented in the table, 10.75%, 8.39%, and 8.75% of the Phlebotomus females, and 2.04%, 12.5%, and 3.30% of the female Sergentomyia, respectively, had blood-feeding activity during the summer, autumn, and throughout the year.

3.2. PCR-RFLP results

Among the 617 617 Phlebotomus female samples collected, 34 samples were found to be infected with the Leishmania parasite (Table 2). A total of 32 (5.18%) of Ph. papatasi and 2 (0.32%) of Ph. sergenti were identified as infected. After the secondary amplification of the ITS1 gene in PCR assay, the desired band was observed in the fragment ~320 base pairs on the gel (Figure 2). Following the analysis of the data, it was determined that the obtained results exhibited 99-100% homology with the isolates registered as L. major species in GenBank. The phylogenetic tree of the identified isolates is depicted in Figure 3. Preliminary analysis of the OMEGA CLUSTAL multiple alignment results from the EBI site indicates that the isolates designated t25 and t27 are likely to be L. major (Figure 3).

Figure 2. Electrophoresis results from PCR amplification of ITS1 fragment gene of positive Phlebotomus samples. From left to right: 0: 100 bp Ladder, Lane1 to 6: Phlebotomus samples.

Figure 3. Phylogenetic tree inferred of ITS1 gene sequences of L. major isolates from Phelebotomus of the present study and other Leishmania species obtained from GenBank using MEGA software and maximum likelihood algorithm and bootstrap 500. The genotypes of this study are identified with isolates t25 and t27.

4. Discussion

The primary objective of the present study was to investigate the infection of Phlebotomus with the Leishmania parasite in the Mehran region. However, in this region, two species of Leishmania have been documented: L. major and L. tropica. In the present study, however, only L.major was identified from Phlebotomus samples collected from Mehran City. Earlier studies in Iran have reported the predominance of L. major as the causative agent of Leishmaniasis ( 12 , 16 , 17 ). In the study by Gholami Parizad et al. (2015), the molecular identification of Leishmania parasites in smears prepared from skin lesions of patients referred to health centers in Ilam province was performed using the PCR-RFLP method. The study detected L. major species ( 10 ). In this regard, the findings of the study by Saberi et al. (2018) also demonstrated that the primary cause of CL in Ilam L. major ( 18 ) was identified. However, it is noteworthy that other Iranian researchers have reported L. major based on molecular assay using various genes in Ilam province ( 9 , 11 , 12 ). Kassiri et al. (2012) conducted a study from 2000 to 2007 on Leishmaniasis in humans, rodents, and vectors. The study found L. major to be the dominant species in Ilam province, with a rate of 1.2 per 1,000 individuals ( 11 ). In the period spanning from 2011 to 2012, Roghani et al. (2012) undertook a descriptive study of individuals afflicted with Leishmaniasis in Ilam province. In this study, the cities of Dehloran and Mehran demonstrated the highest rate of infection and L. major as the dominant species ( 9 ). In a study conducted by Kermanjani et al. (2017) on cutaneous Leishmaniasis species in Ilam province, it was determined that among a total of 61 patient samples that exhibited clinical symptoms, 64% of them were found to be infected with Leishmania species. As indicated by the findings derived from molecular methods, the specified species were identified as L. major and L. tropica ( 12 ). According to certain reports, in areas where Leishmaniasis has been reported to be endemic for a considerable duration, there is a possibility of its abrupt transformation into an epidemic. In addition, the possibility exists of their appearance in an area where no case has been reported in the past. The difficulty of predicting the occurrence of an epidemic of this disease is well documented. The factors that may influence the epidemic include environmental changes in the area where the vector is present, mass migration of people, and weakened immunity (malnutrition). However, the life cycle of Leishmaniasis is of such complexity that effective control measures must be implemented across multiple domains. A pivotal action to consider is the investigation of the parasite's life cycle, the means by which it is transmitted, and the control of the vectors of this disease, which facilitate transmission between different reservoirs and from reservoirs to humans ( 19 ). In the present study, two species of Ph. papatasi and Ph. sergenti were collected, both of which are vectors of zoonotic and anthropogenic cutaneous Leishmaniasis (ZCL, ACL) in Iran. The abundance of these species, especially Ph. papatasi, which is known as the main vector of cutaneous leishmaniasis in Iran, can be a risk for the spread of the disease in Mehran city. Ph. papatasi was caught in most of the trapped areas, and this finding shows that there is a possibility of ZCL transmission in this city. In conclusion, the results of this study indicate that ZCL type of Leishmaniasis is prevalent in Mehran City. In order to corroborate the findings of preceding studies in this is a possibility of ZCL transmission in this city provided that attention to the results of this study by health officials of the province.

Acknowledgement

The authors express gratitude to Dr. Naseh Maleki Ravasan from Pasteur Institute for valuable advice and direction throughout this work. Additionally, the authors acknowledge the staff of the Department of Parasitology at Tarbiat Modares University, Iran for their contributions.

Authors' Contribution

Study concept and design: T.G. and A.D.

Acquisition of data: T.G.

Analysis and interpretation of data: A.D.

Drafting of the manuscript: A.D. and T.G.

Critical revision of the manuscript for important intellectual content: A.D.

Statistical analysis: A.D.

Administrative, technical, and material support: A.D.

Ethics

This study was confirmed by the Medical Ethics Committee of the Faculty of Medical Sciences of Tarbiat Modares University with code No. IR.MODARES.REC.1397.172.

Conflict of Interest

The authors do not have any conflict of interest.

Data Availability

The data that support the findings of this study are available on request from the corresponding author.

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