Salivary gland antigens of laboratory-bred Phlebotomus sergenti and their immunogenicity in human volunteers in laboratory condition

To investigate Phlebotomus (P.) sergenti Parrot, 1917 (Diptera: Psychodidae) salivary gland antigens and their immune response in human. Methods: Human volunteers were exposed to sand flies' bites in the laboratory, and following each exposure the size of induration was recorded. The mean protein concentration of salivary gland lysate and specific anti-P. sergenti saliva IgG was measured. Sand fly salivary proteins were separated by SDS-PAGE and their immunoreactivity was examined by Western blotting assays. Results: Individuals exposed to P. sergenti salivary gland lysate for 8 months showed both antibody and delayed type hypersensitivity responses, although exposure for one month did not provoke any immune responses. The trend of antibody fluctuated during the exposure time and dropped by the end of antigen loading. The mean protein content was (0.36?0.08) ug in each pair salivary glands. Salivary gland lysate showed 11 to 12 major protein bands and 3 to 6 of them were immunoreactive. Conclusions: Our study showed that the salivary gland components of P. sergenti provoked both cellular and humoral immune responses in human. Furthermore, there are some immunogenic proteins in P. sergenti saliva which could be subjected for further investigation as vector-based vaccine candidate/s against anthroponotic cutaneous leishmaniasis.

Diversity of Phlebotomine sand flies (Diptera: Psychodidae) in mountainous and plain areas of an endemic focus of anthroponotic cutaneous leishmaniasis in Iran

Objective: To determine the diversity of sand flies in different biotopes of mountainous and plain areas of Bam County as the most infected focus of anthroponotic cutaneous leishmaniasis in southeast Iran, and synanthropic index of Phlebotomus sergenti Parrot, and Phlebotomus papatasi Scopoli as the main vectors of cutaneous leishmaniasis in Iran. Methods: Sand flies were captured once a month using sticky traps in domestic, peri-domestic, agricultural, and sylvatic biotopes in the plain and mountainous areas. Alpha diversity indices, including richness, evenness, Shannon-Wiener; beta diversity indices (Jaccard's and Sorensen's similarity indices) and synanthropic index were calculated. Results: A total of 2 664 specimens of 9 sand fly species were collected from mountainous (47%) and plain (53%) areas. Species richness, species evenness, and Shannon-Wiener indices were obtained as 9, 0.637, and 1.399, respectively in the mountainous area. Phlebotomus sergenti and Phlebotomus papatasi were constant species with the synanthropic index of-18.463 and-29.412, respectively. In addition, species richness, species evenness, and Shannon-Wiener indices were 4, 0.690, and 0.956, respectively in the plain area. Phlebotomus sergenti and Phlebotomus papatasi were dominant species with the synanthropic index of +9.695 and +36.207, respectively. Similarity indices were low among different biotopes of plain and mountainous areas. Conclusions: A basic knowledge about the diversity of sand flies in various biotopes is essential to design sound control programs. Biodiversity and synanthropic indices of sand flies are different in plain and mountainous areas due to the difference in biotic and abiotic factors between the two areas.

Morphometric discrimination between females of two isomorphic sand fly species, Phlebotomus caucasicus and Phlebotomus mongolensis (Diptera: Phlebotominae) in endemic and non-endemic foci of zoonotic cutaneous leishmaniasis in Iran

Objective: To delineate reliable morphological characteristics for identifying and separating female Phlebotomus caucasicus and Phlebotomus mongolensis which exist sympatrically in the main foci of zoonotic cutaneous leishmaniasis in Iran. Methods: Sand flies were collected using sticky trap papers from active colonies of rodent burrows installed from 16 catching sites. Morphometric measurements were analyzed of 87 Phlebotomus caucasicus and 156 Phlebotomus mongolensis. Univariate and multivariate analysis were carried out to determine significant morphometric variables for discrimination of the two species. Finally, seven morphological characteristics of 65 female Phlebotomus caucasicus and 124 female Phlebotomus mongolensis were described. Results: Univariate and multivariate analyses of 10 morphometric variables via Discriminant Function Analysis (DFA) and Principal Component Analysis (PCA) showed that five morphometric variables had an accuracy of 100% for discriminating female Phlebotomus caucasicus and Phlebotomus mongolensis. Moreover, PCA revealed that the five morphometric variables with the highest loadings separated these two species. Morphological studies on antennal flagellum (and its associated structures) and mouth-parts of female specimens demonstrated significant differences in several structures. Conclusions: The results show that morphological and morphometrical features can be used to discriminate two female isomorphic species, Phlebotomus caucasicus and Phlebotomus mongolensis accurately.

Spatial distribution and infection rate of leishmaniasis vectors (Diptera: Psychodidae) in Ardabil Province, Northwest of Iran

To determine the spatial distribution and infection rate of sand flies as vectors of Leishmania parasite in Ardabil province, northwest of Iran. Methods: This was a descriptive cross-sectional study. The sand flies were collected from 30 areas in all 10 districts of Ardabil province during 2017. The specimens were caught using the sticky traps. The head and genitalia of sand flies were separated and mounted in Berlese solution for microscopic identification. The Geographical Information System ArcMap10.4.1 software was used to provide the spatial maps. Results: A total of 2 794 sand flies specimens were collected and 22 species of sand flies were identified from the two genera: Phlebotomus and Sergentomyia from Ardabil province. The highest frequency was found in Phlebotomus papatasi (23.7%) followed by Phlebotomus kandelakii (13.0%). The promastigote form of Leishmania infantum parasite has been reported from the three main vectors of visceral leishmaniasis (Phlebotomus kandelakii, Phlebotomus perfiliewi and Phlebotomus tobbi) from Ardabil province, where the spatial distribution map of these visceral leishmaniasis vectors was prepared. Some important species of sand flies such as Phlebotomus kandelakii, Phlebotomus perfiliewi and Phlebotomus tobbi were reported and identified as main and probable vectors of visceral leishmaniasis in Ardabil. Conclusions: According to the Geographic Information System based maps, the frequency of the sand flies as leishmaniasis vectors, the leishmania parasite infection rate and the prevalence of the disease in the central areas of Ardabil province are higher than in other areas in Ardabil province.

Distribution and ecological aspects of sand flies (Diptera: Psychodidae) species in Northeastern Iran

Objective:To determine both the distribution and the ecological characteristics of sand flies in Golestan Province, northeast of Iran in 2016.Methods:In this study, 34 villages were selected based on their geographical conditions. Sticky paper traps were used for collecting the sand flies. Sampling was carried out in each of villages from May to November. In each village, 60 traps for indoors and 60 for outdoors were monthly installed. The species of all collected sand flies were determined using approved morphological keys. Pearson coefficient correlation was used to find the relationship between the number of collected Phlebotomus papatasi from different villages and incidence rate of zoonotic cutaneous leishmaniasis as well as the number of positive cases of the disease. The altitude of the studied villages was extracted from digital elevation model of the area using GIS and vegetation cover density index of the province was extracted from Modis satellite imagery and distribution map of sand flies drown up.Results:Overall, 5 428 sand flies were collected and identified, belonging to 18 species. Phlebotomus wenyoni was reported for the first time from the area in this study. The frequency of sand flies in the villages located in northeast of the Golestan province (the plateau area, lower altitude, arid and semi-arid climates, and lower vegetation cover density), were more than other villages in this province. There was a significant correlation between the number of collected Phlebotomus papatasi and incidence rate of the zoonotic cutaneous leishmaniasis cases in different villages (r=0.837, P=0.019) as well as the number of positive cases of the disease (r=0.688, P<0.001).Conclusions:In the northeaster areas of Golestan Province which is known as the endemic foci of zoonotic cutaneous leishmaniasis, the abundance of sand flies were more and the conditions for their growth and development were more appropriate.

Phlebotomus (Adlerius) kabulensis (Diptera: Psychodidae) a new record sand fly species from Iran: Morphological and molecular aspects

Objective:To represent a new geographical record, Phlebotomus (Adlerius) kabulensis (P. kabulensis), which is suspected to be a potential vector of visceral leishmaniasis.Methods:For the first time, P. kabulensis specimens were collected using the sticky paper traps method in outdoor places in mountainous areas with vegetation coverage of three provinces in Iran. Identification of males was based on ecological, morphological, morphometric and molecular (mtDNA cytochrome b gene sequences) criteria. Generally, males have two ascoids on the 8Results:Morphometric measurement revealed that P. kabulensis specimens were the same as compared with seven other morphological characters in three provinces of the country but lengths of the coxite were significantly different. The PCR result of the cytochrome b (Cyt b)-mtDNA fragment shows 550-bp length, with its special nucleotide arrangement. The male and female of P. kabulensis were newly discovered members of the subgenus Adlerius from Iran. Initial DNA analysis indicated how distinct this species is.Conclusions:The results show that the P. kabulensis female will be identified by comparing with other Adlerius female groups regarding its morphometric characters and molecular sequencing.

Spatial distribution of sand flies (Diptera: Psychodidae; Larroussius group), the vectors of visceral leishmaniasis in Northwest of Iran

Objective: To determine spatial distribution of sand flies (Diptera: Psychodidae; Larroussius group), the vectors of visceral leishmaniasis in Ardabil province, Northwest of Iran. Methods: Sand flies were collected using sticky traps from the 30 selected points in Ardabil province, during May-November 2017. The MaxEnt model in GIS software was used for modeling. Results: A total of 2 794 specimens of sand flies were collected, of which 33% were Larroussius subgenus sand flies. Phlebotomus kandelakii and Phlebotomus wenyoni were the highest and lowest collected species respectively. Based on the modeling, four areas in the province were identified with more than 70% probability of the presence of Larroussius group vectors which were at risk of visceral leishmaniasis disease transmission. Conclusions: The distribution of Larroussius subgenus sand flies was observed in all parts of Ardabil. But the northern parts of the province (Germi and Bilesavar counties) as well as central part (Ardabil and Meshkinshahr counties) were of great importance in terms of the presence of Larroussius subgenus sand flies and the possibility of transmission of the visceral leishmaniasis.

Distribution and ecological aspects of sand flies (Diptera: Psychodidae) species in Northeastern Iran

Objective: To determine both the distribution and the ecological characteristics of sand flies in Golestan Province, northeast of Iran in 2016. Methods: In this study, 34 villages were selected based on their geographical conditions. Sticky paper traps were used for collecting the sand flies. Sampling was carried out in each of villages from May to November. In each village, 60 traps for indoors and 60 for outdoors were monthly installed. The species of all collected sand flies were determined using approved morphological keys. Pearson coefficient correlation was used to find the relationship between the number of collected Phlebotomus papatasi from different villages and incidence rate of zoonotic cutaneous leishmaniasis as well as the number of positive cases of the disease. The altitude of the studied villages was extracted from digital elevation model of the area using GIS and vegetation cover density index of the province was extracted from Modis satellite imagery and distribution map of sand flies drown up. Results: Overall, 5 428 sand flies were collected and identified, belonging to 18 species. Phlebotomus wenyoni was reported for the first time from the area in this study. The frequency of sand flies in the villages located in northeast of the Golestan province (the plateau area, lower altitude, arid and semi-arid climates, and lower vegetation cover density), were more than other villages in this province. There was a significant correlation between the number of collected Phlebotomus papatasi and incidence rate of the zoonotic cutaneous leishmaniasis cases in different villages (r=0.837, P=0.019) as well as the number of positive cases of the disease (r=0.688, P<0.001). Conclusions: In the northeaster areas of Golestan Province which is known as the endemic foci of zoonotic cutaneous leishmaniasis, the abundance of sand flies were more and the conditions for their growth and development were more appropriate.

Phlebotomus (Adlerius) kabulensis (Diptera: Psychodidae) a new record sand fly species from Iran: Morphological and molecular aspects

Objective: To represent a new geographical record, Phlebotomus (Adlerius) kabulensis (P. kabulensis), which is suspected to be a potential vector of visceral leishmaniasis. Methods: For the first time, P. kabulensis specimens were collected using the sticky paper traps method in outdoor places in mountainous areas with vegetation coverage of three provinces in Iran. Identification of males was based on ecological, morphological, morphometric and molecular (mtDNA cytochrome b gene sequences) criteria. Generally, males have two ascoids on the 8

Traumatic myiasis agents in Iran with introducing of new dominant species, Wohlfahrtia magnifica (Diptera: Sarcophagidae)

Objective: To study agents of animal wound myiasis in various geographical districts of Fars province. Methods: This study has been done in Fars province, located in the southern part of Iran. Sums of 10 358 domestic animals have been visited from April 2011 to March 2012. The infected wounds in any parts of animal body were sampled by means of forceps. Results: About 61% of all animal wound myiasis were caused by larvae of Wohlfahrtia magnifica. The most wound myiasis cases due to this species occurred in central part of Fars province. There wasn't any significant difference between sheep and goat in infestation with myiasis (P>0.05). The infestation rate of myiasis in cattle community was 0.86%. Conclusions: The infestation rate of livestock was lower than other works in Iran and some other countries like Saudi Arabia. Chrysomya bezziana has been mentioned as main myiasis agent in Iran. But in this study it cleared that similarly to some European countries, the common animal myiasis agent in Iran is Wohlfahrtia magnifica. Introducing new species as principal agent for myiasis can help public health and animal husbandry policy makers to prepare sufficient and effective control and/or preventive measures for this disease.

Traumatic myiasis agents in Iran with introducing of new dominant species, Wohlfahrtia magnifica (Diptera: Sarcophagidae)

<p><b>OBJECTIVE</b>To study agents of animal wound myiasis in various geographical districts of Fars province.</p><p><b>METHODS</b>This study has been done in Fars province, located in the southern part of Iran. Sums of 10 358 domestic animals have been visited from April 2011 to March 2012. The infected wounds in any parts of animal body were sampled by means of forceps.</p><p><b>RESULTS</b>About 61% of all animal wound myiasis were caused by larvae of Wohlfahrtia magnifica. The most wound myiasis cases due to this species occurred in central part of Fars province. There wasn't any significant difference between sheep and goat in infestation with myiasis (P>0.05). The infestation rate of myiasis in cattle community was 0.86%.</p><p><b>CONCLUSIONS</b>The infestation rate of livestock was lower than other works in Iran and some other countries like Saudi Arabia. Chrysomya bezziana has been mentioned as main myiasis agent in Iran. But in this study it cleared that similarly to some European countries, the common animal myiasis agent in Iran is Wohlfahrtia magnifica. Introducing new species as principal agent for myiasis can help public health and animal husbandry policy makers to prepare sufficient and effective control and/or preventive measures for this disease.</p>

First detection of Leishmania infantum DNA in wild caught Phlebotomus papatasi in endemic focus of cutaneous leishmaniasis, South of Iran.

OBJECTIVE: To identify the vectors and reservoirs of cutaneous leishmaniasis in the endemic focus of Farashband, Fars Province, South of Iran. METHODS: Sticky papers and Sherman trap were used for collection of sand flies and rodents, respectively. Polymerase chain reaction (PCR) of kDNA, ITS1-rDNA were used for identification of Leishmania parasite in sand flies as well as rodents. RESULTS: Totally 2 010 sand flies were collected and the species of Phlebotomus papatasi Scopoli was the common specimen in outdoors and indoors places. PCR technique was employed on 130 females of Phlebotomus papatasi. One of them (0.76%) was positive to parasite Leishmania major (L. major) and one specimen (0.76%) was positive to Leishmania infantum. Microscopic investigation on blood smear of the animal reservoirs for amastigote parasites revealed 16 (44%) infected Tatera indica. Infection of them to L. major was confirmed by PCR against kDNA loci of the parasite. CONCLUSIONS: The results indicated that Phlebotomus papatasi was the dominant species circulating two species of parasites including L. major and Leishmania infantum among human and reservoirs. Furthermore, Tatera indica is the only main host reservoir for maintenance of the parasite source in the area.

Phlebotomus papatasi and Meriones libycus as the vector and reservoir host of cutaneous leishmaniasis in Qomrood District, Qom Province, central Iran.

OBJECTIVE: To determine the sand flies species responsible for most transmission of Leishmania major (L. major) to human, as well as to determine the main reservoir hosts of the disease. METHODS: Sand flies were collected using sticky papers and mounted in Puri's medium for species identification. Rodents were trapped by live Sherman traps. Both sand flies and rodents were subjected to molecular methods for detection of leishmanial parasite. RESULTS: Phlebotomus papatasi (P. papatasi) was the common species in outdoor and indoor resting places. Employing PCR technique only three specimens of 150 P. papatasi (2%) were found naturally infected by parasites with a band of 350 bp which is equal to the L. major parasite. Forty six rodents were captured by Sherman traps and identified. Microscopic investigation on blood smear of the animals for amastigote parasites revealed 1 (3.22%) infected Meriones libycus (M. libycus). Infection of this animal to L. major was confirmed by PCR against rDNA loci of the parasite. CONCLUSIONS: This is the first molecular report of parasite infection of both vector (P. papatasi) and reservoir (M. libycus) to L. major in the region. The results indicated that P. papatasi was the primary vector of the disease and circulating the parasite between human and reservoirs and M. libycus was the most important host reservoir for maintenance of the parasite source in the area.

Phenology and population dynamics of sand flies in a new focus of visceral leishmaniasis in Eastern Azarbaijan Province, North western of Iran

OBJECTIVE@#To investigate species composition, density, accumulated degree-day and diversity of sand flies during April to October 2010 in Azarshahr district, a new focus of visceral leishmaniasis in north western Iran.@*METHODS@#Sand flies were collected using sticky traps biweekly and were stored in 96% ethanol. All specimens were mounted in Puri's medium for species identification using valid keys of sandflies. The density was calculated by the formula: number of specimens/m(2) of sticky traps and number of specimens/number of traps. Degree-day was calculated as follows: (Maximum temperature + Minimum temperature)/2-Minimum threshold. Diversity indices of the collected sand flies within different villages were estimated by the Shannon-weaver formula ( H'=∑i=1sPilog(e)Pi).@*RESULTS@#Totally 5 557 specimens comprising 16 Species (14 Phlebotomus, and 2 Sergentomyia) were indentified. The activity of the species extended from April to October. Common sand-flies in resting places were Phlebotomus papatasi, Phlebotomus sergenti and Phlebotomus mongolensis. The monthly average density was 37.6, 41.1, 40.23, 30.38 and 30.67 for Almalodash, Jaragil, Segaiesh, Amirdizaj and Germezgol villages, respectively. Accumulated degree-day from early January to late May was approximately 289 degree days. The minimum threshold temperature for calculating of accumulated degree-day was 17.32°. According on the Shannon-weaver (H'), diversity of sand flies within area study were estimated as 0.917, 1.867, 1.339, 1.673, and 1.562 in Almalodash, Jaragil, Segaiesh, Amirdizaj and Germezgol villages, respectively.@*CONCLUSIONS@#This study is the first detailed research in terms of species composition, density, accumulated degree-day and diversity of sand flies in an endemic focus of visceral leishamaniasis in Azarshahr district. The population dynamics of sand flies in Azarshahr district were greatly affected by climatic factors. According to this study the highest activity of the collected sand fly species occurs at the teritary week of August. It could help health authorities to predicate period of maximum risk of visceral leishamaniasis transmission and implement control program.