Identification of Phlebotomus chinensis(Diptera:Psychodidae) inferred by morphological characters and molecular markers

To clarify the taxonomic status of Phlebotomus chinensb, the morphological characters of Ph. chinensis from Shaanxi, Henan, Sichuan and Gansu Provinces, and Ph. wui and Ph. longiductus from Xinjiang were examined and the rDNA-ITS2 region and mtDNA Cyt b gene sequences were analyzed simultaneously. It was found that morphological variation was very low in Ph. chinensis among the different localities; the wings of adult samples distributed in high-altitude areas were merely longer. The rDNA-ITS2 and mtDNA Cyt b sequences had an intraspecific variability. The relationships among species in the genus Phlebotomus can be inferred by molecular markers. These results suggest that there is an obvious genetic divergence at the molecular level among the Ph. chinensis populations in China.

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.

Sandfly fauna and ecological analysis of Phlebotomus orientalis and Phlebotomus martini in the lowland foci of visceral leishmaniasis in Somali Regional State, southeast Ethiopia

Objective:To identify the sandfly fauna and analyze ecology of sandfly vector(s)of visceral leishmaniasis in three districts of Somali Regional State,southeast Ethiopia.Methods:Sandflies were collected from four sampling habitats,including indoor,peri-domestic,farm field and mixed forest using light and sticky traps in July 2016,and February and April 2017 in Liben and Dawa zones in the Somali Regional State,southeastern Ethiopia.Results:In total,4367 sandfly specimens,belonging to 12 species(three Phlebotomus spp.and nine Sergentomyia spp.)were identified.Phlebotomus(P.)heischi,P.orientalis,and P.martini constituted 45.7%,31.1%,and 23.1%of the sandfly collection,respectively.There were significant differences in the median number of P.orientalis,and P.martini captured per CDC trap/night between the three sampling districts(P<0.05).In light trap capture,collection habitats had significant effects on the abundance of P.orientalis,and P.martini(P<0.05).More median numbers of P.orientalis,and P.martini species were collected in agricultural fields followed by mixed forest and peri-domestic habitats.P.orientalis and P.martini were caught more in outdoor than indoor habitats,suggesting exophilic behaviour.Conclusions:The study demonstrated that the presence of P.orientalis and P.martini are probable vectors of visceral leishmaniasis in this new focus.The findings of our study will improve the understanding of the dynamics of visceral leishmaniasis transmission and will facilitate the implementation of integrated disease control measures based on ecological knowledge of visceral leishmaniasis vector in Liben and Dawa zones and its surrounding regions.

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

Objective: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)μg 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.

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^(th)antennal segment and one ascoid on segments 9^(th)to 15^(th), aedeagus with normal obtuseangled sub-terminal notch and coxite with 27–50 groups of hairs on the distal half of its body.Results: 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.

Preliminary study on investigation of zoonotic visceral leishmaniasis in endemic foci of Ethiopia by detecting Leishmania infections in rodents

Objective:To investigate the zoonotic visceral leishmaniasis(ZVL) by identification of the most probable reservoir hosts using parasite isolation and analysis of a possible transmission dynamics of the disease in extra-domestic agricultural fields and rural villages.Methods:Rodents were collected from selected study sites in kala-azar endemic areas based on information for localities of kala-azar cases for screening of Leshmania infections using parasitological,serological and polymerase chain reaction(PCR) from March.2013 to January,2014.Ketamine(Clorketam Veterinary) was used to anaesthesize the rodents according the prescribed dosage(average 2 mg/kg for intra-venous route).The blood obtained using sterile needle was dropped into sterile filter paper and allowed to air dry before sealing in plastic bags.The tissues from liver,spleen and skin were macerated in Locke's solution before transferring them into NNN medium.Blood and touch smears of liver,spleen,skin and bone marrow were prepared for fixing using methanol and staining by Giemsa stain for microscopy.These tissues were also ascd for DNA extractions and PCR amplification of Leishmania infection.Results:A total of 335 rodents(13 species) were analyzed by sampling internal organs.The infection rate by PCR was 11.1%(6/54) for Arvicanthis nilothicas compared to 17.6%(3/17) and 12.5%(2/16) for Acomys cahirinus and Tarera(C) robustus respectively.Almost all the infections were found from bone marrow samples(8/48 or 16.7%) compared with 1/91(1.1%) liver,2/87(2.2%) spleen and 0/87(0%) skin.In all study sites with past human VL cases,rodents and proved vectors shared similar habitats.Conclusions:Leishmania donovani might circulate among different species of rodents in kala-azar endemic lowlands and valleys of Ethiopia by Phlebotamus orientalis and Phlebotomus martini.Detailed studies to substantiate the preliminary data on the possible role of these rodents arc urgently needed.

Climate change and potential distribution of zoonotic cutaneous leishmaniasis in Central Iran: Horizon 2030 and 2050

Objective: To investigate and predict the effects of climate change on the potential distribution of the main vector and reservoir hosts of the disease in Yazd province in the future.Methods: Distribution data for vector and reservoir hosts of zoonotic cutaneous leishmaniasis in Yazd province were obtained from earlier studies conducted in the area.MaxEnt ecological niche modeling was used to predict environmental suitability.BCC-CSM1-1(m) model and two climate change scenarios, RCP 4.5 and RCP 8.5 were used for horizons 2030 and 2050 climate projections.Future projections were based on data of a regional climate change model.Results: With both scenarios in 2030 and 2050, the results of jackknife test indicated that the mean temperature of wettest quarter and temperature annual range had the greatest effect on the model for the vector and the reservoir hosts, respectively.Conclusions: The climate conditions are the major determinants of zoonotic cutaneous leishmaniasis incidence rate in Yazd Province.These climate conditions provide favorable habitats for ease transmission of zoonotic cutaneous leishmaniasis in this endemic area.Habitats suitability for the vector and reservoir will be expanding in the coming years compared with the current conditions, such that, in horizon 2030 & 2050, the probability of the presence of the vector and reservoir within 38 580 and 37 949 km^2, respectively, from Yazd province is above 60%.Moreover, an increase is predicted in the presence of the vector in the western parts and the reservoir in the northern and central parts of the province in the future.Understanding the role of environmental and bioclimatic factors in zoonotic cutaneous leishmaniasis occurrence can provide a guide for policy-makers in the creation and implementation of more effective policies for prevention and control.

Visceral leishmaniasis among children in an endemic area of northwestern Iran between 2016 and 2017: An epidemiological study

Objective: To clarify the epidemiological aspects of visceral leishmaniasis in Kaleybar and Khoda-Afarin districts, north-west of Iran.Methods: A total of 1 420 human(children under 12 years) samples, 101 domestic dogs samples(Canis familiaris), and 577 female sand fly samples were collected. Sera of human and dogs were tested using the direct agglutination test, and sand flies were identified at species level using the microscopic method. Furthermore, a structured questionnaire was applied to evaluate the correlation between the potential risk factors and the related clinical signs/symptoms with the human and dogs’ seropositivity.Results: Totally, 2.18% of human samples were positive at titers≥1: 800;among them, 13 cases(41.94%) were above 1:3 200, and clinical symptoms were observed in all of them except for an 11-year old girl. Anti-Leishmania infantum antibodies were found at titer≥1: 320 in 9.90% of dogs’ samples, half of them had at least one sign of canine visceral leishmaniasis. Moreover, 10 Phlebotomus species were identified in the study areas, and Phlebotomus(Larroussius) major group was the predominant species. There are significant correlations between the presence of anti-Leishmania infantum antibodies and the fever(P<0.001), anemia(P=0.001) and weight loss(P=0.016) in children. On the other hand, significant correlations were revealed between the Leishmania infection and the shelter(P=0.039), cutaneous lesion(P=0.005), lymphadenopathy(P=0.001) and weight loss(P<0.001) in the infected dogs.Conclusions: Visceral Leishmania infection is prevalent in rural areas of Kaleybar and KhodaAfar districts located in East-Azerbaijan province, therefore active detection and treatment of visceral leishmaniasis cases should not be neglected.

Molecular characterization of sandflies and Leishmania detection in main vector of zoonotic cutaneous leishmaniasis in Abarkouh district of Yazd province,Iran

Ohjective:To assess molecular characterization,distribution,seasonal activities of sandfly species and Leishmania parasites infecting them for this zoonotic cutaneous leishmaniasis focus.Methods:The collections were carried out in 2009-2011 using CDC traps,Sticky Papers and manual aspirator in and around the villages in Abarkouh district.Individual sandflies were characterized by PCR amplification and sequencing of fragments of their mitochondrial cytochrome b gene.Leishmania parasite infections within sandflies were performed by targeting Cyt b,ITS-rDNA,k-DNA and microsatellite genes.Results:The PCR assays detected only Leishmania major(L.major).All infections(30)were found in the abundant and widespread vector Phlebolomus papalasi(P.papatasi).Small numbers of other sandfly species were also screened for infections,but none was found.Sergentomyia sinloni and P.papalasi were the predominant members in all locations of this district and in all habitats throughout the trapping season.Only five other sandfly species were found,namely Phlebolomus ansari,Phlebotomus caucasicus,Phlebotomus sergenti,Sergentomyia dentata and Sergentomyia merviney.Conclusions:In the current survey,the only infections detected are of L.major in females of P.papatasi(30 out of190).The rates of infection of P.papalasi by L.major are not significantly different in compare with other locations in Iran with no diversity of parasite strains.Zoonotic cutaneous leishmaniasis may have emerged only recently in Abarkouh district,and the reason may well be the instability of the transmission cycles there.

Emergence of Toscana virus in the mediterranean area

Toscana virus(TOSV) is an arthropod-borne virus, identified in 1971, from Phlebotomus perniciosus and Phlebotomus perfiliewi in central Italy. TOSV belongs to the Phlebovirus genus within the Bunyaviridae family. As other bunyaviruses, the genome of TOSV consists of 3 segments(S for small, M for Medium, and L for Large) respectively encoding non structural and capsid proteins, envelope structural proteins, and the viral RNA-dependant RNA-polymerase. It is transmitted by sand flies. Therefore its distribution is dictated by that of the arthropod vectors, and virus circulation peaks during summertime when sandfly populations are active. Here, we reviewed the epidemiology of TOSV in the old world. First evidence of its pathogenicity for humans, specifically its propensity to cause central nervous system(CNS) infections such as meningitis and encephalitis, was reported in central Italy. After 2000, it was recognized that TOSV had a much larger geographic distribution than initially believed, and was present in most of the Western European countries located on the northern border of the Mediterranean Sea(Portugal, Spain, France, Greece, Croatia) as well as eastern countries such as Cyprus and Turkey. In the countries where TOSV is present, it is among the three most prevalent viruses in meningitis during the warm seasons, together with enteroviruses and herpesviruses. Up to now, epidemiological data concerning Northern Africa and other countries located south of the Mediterranean are scarce. TOSV must be considered an emerging pathogen. Despite the important role played by TOSV in CNS infections, it remains a neglected agent and is rarely considered by physicians in diagnostic algorithms of CNS infections and febrile illness during the warm season, probably because of the lack of information.

Insecticide resistance in phlebotomine sandflies in Southeast Asia with emphasis on the Indian subcontinent

Background:Visceral leishmaniasis,commonly known as kala-azar in India,is a global public health problem.In Southeast Asia,Bangladesh,Bhutan,India,Nepal,Sri Lanka and Thailand are endemic for visceral leishmaniasis.The role of sandflies as the vector of kala-azar was first confirmed in 1942 in India.Insecticide resistance in Phlebotomus argentipes Annandale and Brunetti,the vector of kala-azar in the Indian subcontinent,was first reported in 1987 in Bihar,India.This article provides a scoping review of the studies undertaken from 1959 to 2015 on insecticide resistance in P.argentipes and P.papatasi(Scopoli),the vectors of visceral and cutaneous leishmaniasis respectively,in Southeast Asia,mainly in Bangladesh,India,Nepal and Sri Lanka.Results:Studies undertaken in areas of Bihar and West Bengal in India where kala-azar is endemic have reported resistance of P.argentipes to DDT,while in non-endemic areas it has been reported to be susceptible.In areas of Nepal bordering India,there are indications of resistance to DDT;biochemical resistance has been reported in Sri Lanka.No laboratory studies have been undertaken in Bangladesh;however,the sandfly vector is reported to be still susceptible to pyrethroids in all kala-azar endemic areas in the aforementioned countries.Conclusions:Studies are needed to determine the resistance of sandfly vectors to all available classes of potential insecticides in kala-azar endemic areas.There is a need to assess the impact of indoor residual spraying with DDT and pyrethroids on the incidence of kala-azar in India where 54 districts remain endemic for the disease,strengthen entomological surveillance capacity,and develop and implement an insecticide management plan.Alpha-cypermethrin indoor residual spraying has been introduced in 33 kala-azar endemic districts in Bihar State of India in a pilot trial;the outcomes should be used to inform decisions on expanding coverage with alpha-cypermethrin in all remaining endemic districts to achieve the revised goal of elimination of visceral leishmaniasis by 2020.

Phlebotomine sand flies(Diptera:Psychodidae)transmitting visceral leishmaniasis and their geographical distribution in China:a review

After the existence of phlebotomine sand flies was first reported in China in 1910,the distribution of different species and their role in the transmission of visceral leishmaniasis(VL)have been extensively studied.Up until 2008,four species have been verified as vectors of VL,namely,Phlebotomus chinensis(Ph.sichuanensis),Ph.longiductus(Ph.chinensis longiductus),Ph.wui(Ph.major wui),and Ph.alexandri.The sand fly species vary greatly depending on the natural environments in the different geographic areas where they are endemic.Ph.chinensis is euryecious and adaptable to different ecologies,and is thus distributed widely in the plain,mountainous,and Loess Plateau regions north of the Yangtze River.Ph.longiductus is mainly distributed in ancient oasis areas south of Mt.Tianshan in the Xinjiang Uygur autonomous region.Ph.wui is the predominant species in deserts with Populus diversifolia and Tamarix vegetation in Xinjiang and the western part of the Inner Mongolia autonomous region.Finally,Ph.alexandri is steroecious and found only in stony desert areas,such as at the foot of the mountains in Xinjiang and the western Hexi Corridor,in Gansu province.This review summarized the relationship between the geographic distribution pattern of the four sand fly species and their geographical landscape in order to foster research on disease distribution and sand fly control planning.Furthermore,some problems that remained to be solved about vectors of VL in China were discussed.

Ecological niches and blood sources of sand fly in an endemic focus of visceral leishmaniasis in Jiuzhaigou,Sichuan,China

Background:Sand fly Phlebotomus chinensis is a principle vector for the visceral leishmaniasis(VL)in China with a wide geographic distribution.Jiuzhaigou,Sichuan is a mountain type endemic area of VL in China.Long term effective control efforts in the region have successfully reduced VL transmission.To assess the current status of the sand flies and their ecological aspects in the region,a survey was conducted in the summer of 2014 and 2015.Methods:Sand fly specimens were collected by light traps in a village and blood sources were identified by PCR and sequencing of the mitochondrial cytochrome b gene.Results:In a rock cave,65.2%–79.8%of collected sand flies were male.On a rabbit farm,92.9%–98.8%of specimens were female.In pig pens,61.1%of specimens were female.Some females had visible blood residues.The feeding rate was 49.4%from the pig pens,12.3%from the cave,and only 1.7%from the rabbit farm.Pig,rabbit,chicken,dog,and human blood were detected in the fed specimens.Swine blood,present in all tested samples,was a preferred blood source,while chicken and dog blood were present in a third of the samples.Conclusions:In Jiuzhaigou County,Sichuan Province of China,the considerable sandfly density and the peridomestic feeding behavior all increases the risk of VL transmission,and insecticide spraying in animal sheds could be exploited to reduce sand fly populations in human surroundings.