Increasing the Sensitivity of Leishmania RNA Virus 2 (LRV2) Detection with a Modification in cDNA Synthesis

Objective: Leishmania RNA virus was detected the first time in the New World Leishmania species. Recent studies were also showed the presence of Leishmania RNA virus 2 (LRV2) in Old Word Leishmania species including Turkish L. major and L. tropica isolates. This study aimed to increase the sensitivity of qPCR with a modification in the denaturation step of cDNA preparation protocol. Methods: In this study, LRV2+ three L. major, two L. tropica strains and L. major control strain (MHOM/SU/73/5-ASKH) were included. Total RNA isolation was done using different numbers of Leishmania promastigotes (108, 105 and 103). Before cDNA synthesis, samples were denatured at 95 °C for 2 min, as a modification of the kit procedure. qPCR was undertaken using 0.5 mM primers (LRV F-HR/LRV R-HR) diluted in SYBR Green Master mix. Results: We observed lower Ct values in amplicons with the modified version than with the classical kit protocol for cDNA synthesis, in all of the strains used in the study. The addition of pre-denaturation step at 95 °C showed lower Ct values meaning the sensitivity increased. Different parasite dilutions showed similar results. Conclusion: It is important to increase the sensitivity especially with the aim for detecting LRV in clinical samples obtained from patients probably have less number of parasites. The presence and burden of the virus can help to understand the relationship between the clinical findings and the pathogenicity of the parasite which may lead to changes in the course of treatment.

Blood Meal Analysis and Molecular Detection of Leishmania DNA in Wild-Caught Sand Flies in Leishmaniasis Endemic Areas of Turkey and Northern Cyprus.

INTRODUCTION: Phlebotomine sand flies (Diptera: Psychodidae) are known as the vector of diseases such as leishmaniasis, bartonellosis and viral diseases. The aim of this study is to detect the host feeding pattern of sand flies in the endemic areas for leishmaniasis in Turkey (Antalya, Kayseri) and Northern Cyprus (TRNC) as well as the presence of Leishmania DNA in the specimens. METHODS: One-hundred seventy-six blood-fed sand fly specimens were examined for blood meal analysis. A SYBR Green-PCR assay was performed with specific forward primers for each host and a universal reverse primer. Primers of human and goat were used together in multiplex PCR while goat and cow were studied separately. ITS-1 qPCR assay was also performed on both blood-fed and non-blood-fed females to detect Leishmania parasites. RESULTS: Blood sources could be detected in 69 out of 176 blood-fed sand fly specimens. The results of blood meal analysis showed that specimens were fed mostly on cows (22.2%) followed by humans (5.7%), goats (2.8%) and dogs (0.6%). Multiple feeding patterns were also detected as human + cow (3.4%), cow + goat (2.8%) and human + goat (1.7%). Five of the blood-fed specimens were Leishmania spp. positive: P. major s.l. (n = 1), P. tobbi (n = 2) were L. tropica positive from Antalya, P. simici was positive for L. infantum from Kayseri and P. papatasi (n = 1) was positive for L. major from Cyprus. Leishmania infection rates were determined as 3.79%, 1.69% and 2.63% among the blood-fed sand fly specimens in Antalya, Kayseri and TRNC, respectively. CONCLUSION: The SYBR-Green-based multiplex PCR assay is a cost-effective and promising tool for blood meal identification of wild-caught sand flies as well as other blood-sucking arthropods. Feeding patterns of important vector species detected in the present study show the high risk in these endemic areas. As a next step, to identify the blood source in a shorter time and to make the test more sensitive, development of this assay to probe-based and multiplex PCR will be also planned.

Investigation of Phlebotominae (Diptera: Psychodidae) Fauna, Seasonal Dynamics, and Natural Leishmania spp. Infection in Mugla, Southwest of Turkey.

Due to its geographical location, Mugla province is one of the most frequently used places by refugees. Although leishmaniasis have been previously reported in this region, there is a lack of information on the etiological agent and possible vectors. The main objectives of this study were; i) to investigate the sand fly fauna, ii) to reveal the natural Leishmania spp. infection in wild caught sand flies using molecular tools, and iii) to determine the annual seasonal dynamics of the sand flies in Mugla region. Totally, 2093 specimens belonging to 15 species [12 Phlebotomus, three Sergentomyia; 51 unidentified] were collected during the one-year (June 2016- June 2017) period. Of the collected sand flies, 1928 (92.12%) were caught by the Centers for Disease Control (CDC) light traps, while 165 (7.88%) of them were caught by sticky traps. Phlebotomus major sensu lato (s.l.), the potential vector of visceral leishmaniasis (VL) and canine leishmaniasis (CanL) in the Mediterranean and Aegean region, was detected in all sampling locations and found as the dominant taxon (n=1035; 49.45%) of the study area and followed by Phlebotomus tobbi (n=371; 17.72%). During the sampling period, sand fly activity was started in March and peaked in August. Sand fly population size reduced dramatically between mid-September and early October. The number of collected specimens was peaked in August, while there is only one sample collected both in November and March. The majority of the sand flies (78.66%) were collected at an altitude range of 200-400 m. Seventy-two monospecific pools were screened for the presence of Leishmania DNA by real time ITS1 PCR and 24 (nine P. major s.l., eight P. tobbi, two P. papatasi, two S. minuta, one P. alexandri, one P. similis, and one Phlebotomus (Transphlebotomus spp.) of them (33.8%) were found positive (L. infantum, L. tropica, and L. major). To the best of our knowledge, the presence of fifteen sand fly species and their distribution, seasonal dynamics, molecular detection of Leishmania parasites in Mugla province was reported for the first time. The presence of vector species in the study area, appropriate temperature and humidity conditions, long sand fly activity season, and presence of Leishmania parasite suggests that there is a serious risk in the transmission of leishmaniasis in Mugla.

First molecular detection and identification of Leishmania species in small wild rodents from Turkey.

Leishmaniasis is a parasitic disease infecting animals and humans. Two clinical forms (Visceral and cutaneous leishmaniasis) and four species are reported to be present in Turkey. Several studies have investigated canine and human leishmaniasis in Turkey but no study was performed to screen the infection among wild rodents, so far. The present study aims to investigate the role of small wild rodents as reservoir animals for Leishmania spp. in different regions of Turkey. Formalin-preserved tissue samples (spleen, liver, lung) of 712 rodents from 30 provinces were screened for the presence of Leishmania spp. DNA. Before DNA extraction, tissues were dried, rehydrated, and homogenated. Leishmania screening in rodent tissues and species determination was performed with a combination of real-time kDNA and ITS1 polymerase chain reaction protocols. Eight (1.12%) out of 712 animals were found to be positive for Leishmania spp. DNA and species typing revealed five L. infantum, two L. tropica and one L. major among positives. Leishmania major and L. infantum DNA were detected in Apodemus spp. from Zonguldak province located in the Western Black Sea Region, while L. tropica DNA was found in Meriones sp. and Gerbillus dasyurus from Adana and Hatay provinces located in Eastern Mediterranean Region of Turkey. The present study is first to report natural infection of L. infantum, L. major and L. tropica in small wild rodents in Turkey, suggesting their possible roles as reservoirs. Further studies are needed for planning epidemiological studies and also for developing rodent control measures in risky endemic areas to break the transmission cycle.

Molecular investigation of Rickettsia spp. and Francisella tularensis in ticks from three provinces of Turkey.

Ticks are obligate hematophagous ectoparasites as well as mechanical and biological vectors of a wide variety of microbial pathogens. To date, 19 tick-borne diseases have been reported from Turkey. In this study, ticks collected from Aydin, Izmir and Sanliurfa provinces of Turkey were identified using morphological and molecular methods. After the presence of bacterial DNA was checked, Rickettsia spp. and Francisella tularensis were investigated in bacterial DNA-positive tick specimens by PCR. Furthermore, amplicons belonging to tick specimens and positive bacterial samples were sequenced and processed for BLAST, alignment and phylogenetic analysis. As a result, seven tick species were identified: Rhipicephalus sanguineus, Rh. bursa, Rh. turanicus, Hyalomma marginatum, Hy. aegyptium, Hy. anatolicum and Haemaphysalis erinacei. Fifty-five tick specimens tested positive for bacterial DNA and among them, rickettsial DNA was found in five ticks (infection rate = 9.1%) belonging to Hy. marginatum, Hy. aegyptium, Rh. bursa and Rh. turanicus. Of the five Rickettsia-positive ticks, three contained Rickettsia aeschlimannii, one Ri. massiliae and one an unidentified Rickettsia sp. No Francisella tularensis DNA was detected. Sequence analysis of the ompB gene indicated two novel single nucleotide polymorphisms (SNP) in two different Ri. aeschlimannii strains and two novel SNPs as well as a novel insertion (GACGGT) were found in Rickettsia sp. This study indicated the presence of polymorphic Rickettsia species in ticks from Turkey.

Detection of Leishmania RNA virus 2 in Leishmania species from Turkey.

BACKGROUND: Leishmania RNA virus (LRV) is a double-stranded RNA (dsRNA) virus infecting some Leishmania strains and triggering a destructive hyperinflammatory response in mammalian hosts in the New World. There is limited knowledge of the presence of this virus in Old World Leishmania species and its role in the outcome of the disease. We aimed to investigate the presence of LRV in Leishmania species/strains from Turkey. METHODS: Twenty-nine previously identified Leishmania isolates (24 L. tropica, 2 L. infantum, 3 L. major) were examined for LRV positivity using dsRNA visualization in agarose gel after total nucleic acid extraction and RQ-deoxyribonuclease treatment and amplification of a 526 bp fragment of the LRV2-specific RNA-dependent RNA polymerase gene by reverse transcription polymerase chain reaction. RESULTS: Ten (7 L. tropica [24.13%], 3 L. major [10.34%]) of the 29 Leishmania strains gave positive results for LRV. Basic Local Alignment Search Tool analysis showed that all these viruses are LRV2-1. LRV2 was detected for the first time in L. tropica strains in the present study. CONCLUSIONS: The clinical manifestation and resistance status of the disease can be different depending on the host and parasite species/strains. The presence of LRV2 may be one of the factors contributing the course of disease. Further studies are needed to elucidate the specific role of LRV2, as it may be a potential target for effective treatment strategies.