Sandfly surveillance and investigation of Leishmania spp. DNA in sandflies in Kosovo

In the past decade, leishmaniasis seems to be re-emerging in Balkan countries. There are serious implications that Kosovo is a visceral leishmaniasis endemic region with autochthonous transmission; nevertheless, surveillance of vectors, reservoirs or the disease is not yet established. Gaining knowledge about sandfly vector species is a prerequisite for the development of a monitoring and control plan in the future. After a long gap in research of over 70 years, sandfly studies in Kosovo were resumed in 2014. During this presence/absence study, nine sandfly species were detected: Phlebotomus papatasi, Ph. perfiliewi, Ph. tobbi, Ph. neglectus, Ph. simici, Ph. balcanicus, Ph. alexandri, Ph. mascittii and Sergentomyia minuta. Three species are new with regard to the fauna of Kosovo – Ph. alexandri, Ph. balcanicus and Ph. mascittii. Besides increased diversity, changes in the number of collected specimens and distribution range of species were recorded, with Ph. neglectus being the most dominant species with the widest distribution. Testing of randomly chosen females for Leishmania spp. DNA resulted the in detection of L. tropica in a specimen of Ph. neglectus. The presence of numerous vector species in the sandfly fauna of Kosovo pose a threat for the re-emergence of vector-borne diseases. Therefore, continuous surveillance is recommended with regular updates on vector distribution and abundance.     

First report on isolation of Leishmania tropica from sandflies of a classical urban Cutaneous leishmaniasis focus in southern Iran

Shiraz district in south of Iran is a classical focus of Cutaneous leishmaniasis (CL) and previous research has consistently documented the etiologic agent to be Leishmania tropica and Leishmania major in urban and rural areas, respectively. However, none of the Phlebotomus sergenti, a known vector for L. tropica, of the region has been found infected. We report the first isolation of L. tropica from sandflies in urban community of southern part of Shiraz city. Parasite polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) and gene sequencing analyses indicate CL cases in this community were caused by either L. major or L. tropica. Sandflies of P. sergenti were infrequent, however, three out of 10 (30.0%) females captured in urban area were found infected with L. tropica. But, no human cases were found to be infected with L. tropica. Phlebotomus papatasi were found the most dominant and infected species where 41 out of 207 (20%) tested individuals harboring L. major in suburb area of the city. Patients have been lived in the suburb area of the city where people keep normally domestic animals in their houses which provide appropriate environment for completion of sandfly life cycle and expansion of CL disease in the region.     

Molecular identification of bloodmeals and species composition in Culicoides biting midges

Investigations of host preferences in haematophagous insects, including Culicoides biting midges (Diptera: Ceratopogonidae), are critical in order to assess transmission routes of vector‐borne diseases. In this study, we collected and morphologically identified 164 blood‐engorged Culicoides females caught in both light traps and permanent 12‐m high suction traps during 2008–2010 in Sweden. Molecular analysis of the mitochondrial cytochrome c oxidase subunit I (COI) gene in the biting midges was performed to verify species classification, discern phylogenetic relationships and uncover possible cryptic species. Bloodmeal analysis using universal vertebrate cytochrome b primers revealed a clear distinction in host selection between mammalophilic and ornithophilic Culicoides species. Host sequences found matches in horse (n = 59), sheep (n = 39), cattle (n = 26), Eurasian elk (n = 1) and 10 different bird species (n = 18). We identified 15 Culicoides species previously recorded in Scandinavia and four additional species haplotypes that were distinctly different from the described species. All ornithophilic individuals (n = 23) were caught exclusively in the suction traps, as were, interestingly, almost all mammalophilic species (n = 41), indicating that many biting midge species may be able to cover long distances after completing a bloodmeal. These results add new information on the composition of Culicoides species and their host preferences and their potential long‐distance dispersal while blood‐engorged.     

Bionomics of phlebotomine sandflies in the Galilee focus of cutaneous leishmaniasis in northern Israel

The bionomics of phlebotomine sandflies (Diptera: Psychodidae) were studied for three years (2001-2003) in the Galilee focus of cutaneous leishmaniasis in northern Israel, where the causative Leishmania tropica (Kinetoplastida: Trypanosomatidae) is transmitted by Phlebotomus (Adlerius) arabicus Theodor and Phlebotomus (Paraphlebotomus) sergenti Parrot, comprising 22% and 8%, respectively, of the local sandfly fauna sampled by light traps. The predominant species overall was Phlebotomus (Larroussius) tobbi Adler & Theodor (51%) with lesser numbers of Phlebotomus (Adlerius) simici Theodor (11%), Phlebotomus (Larroussius) syriacus Adler & Theodor (5%), Phlebotomus (Larroussius) perfiliewi Perfil'ev (3%) and Phlebotomus (Phlebotomus) papatasi Scopoli (0.05%). Sandfly adult populations were prevalent from April to November and peaked between June and August, being more abundant through the summer in irrigated habitats, such as gardens and orchards, than in open grassland. Of the two cutaneous leishmaniasis vectors, P. sergenti preferred boulder mounds located at the outskirts of settlements, whereas P. arabicus was more abundant overall and near houses in particular. Females of all these sandfly species displayed a peak of activity after sunset (20.00-22.00 hours), whereas activity of males persisted longer through the night. Another slight increase in activity was noted before dawn (02.00-04.00 hours). Phlebotomus arabicus appears to be the main vector of L. tropica in the Galilee focus, due to its denser populations, more endophily and preference for peridomestic habitats than shown by P. sergenti in northern Israel.     

Cutaneous leishmaniasis infection in Balb/c mice using a Leishmania tropica strain isolated from Turkey.

Leishmania tropica, which is endemic in Turkey, is the causative agent of cutaneous leishmaniasis. Leishmania tropica promastigotes (2 x 10(7)) isolated from a patient with dermal leishmaniasis and reproduced in NNN medium were inoculated subcutaneously into the footpads of 10 Balb/c mice. Cutaneous leishmaniasis developed on the footpads of 4 mice approximately 45 days later. Leishmania tropica amastigotes were observed in smear slides and then cultivated in NNN medium. Balb/c mice are a suitable laboratory model for this isolate of L. tropica and thus a source of amastigotes for studies on the immunology, chemotherapy, and pathogenicity of cutaneous leishmaniasis.     

Phlebotomine sandflies and leishmaniasis risks in Colombian coffee plantations under two systems of cultivation

The phlebotomine sandfly fauna of traditional (shaded) and intensified (unshaded) coffee plantations in Colombia was sampled by a variety of methods and the species composition and density under the two systems compared. Twenty species of Lutzomyia sandflies (Diptera: Psychodidae: Phlebotominae) were collected, of which eight were found only in the 'Coffee Axis' ('Eje Cafetero') of the departments of Caldas, Risaralda and Quindio, six were exclusive to the department of Norte de Santander and six occurred in both regions. Four species were collected only in traditional plantations and two exclusively in intensified ones. At least 13 species occurred in both plantation types. Fifteen species are opportunistic man-biters and eight are suspected vectors of leishmaniasis caused by Le. braziliensis, Le. panamensis or Le. mexicana. Seven species were collected inside houses and may be involved in intradomiciliary transmission of Leishmania. The dominant species in Norte de Santander was Lu. spinicrassa, which made up 93.8% of all the sandflies collected in this department. This species was absent from the Eje Cafetero and a number of others among the 15 recorded there might be responsible for Leishmania transmission in this region, including Lu. trapidoi, Lu. yuilli, Lu. gomezi, L. hartmanni and Lu. ovallesi. Sandfly population densities were significantly higher in traditional plantations than in intensified ones. Residents of traditional plantations were able to describe sandflies in significantly more detail than those of intensified plantations, based on seven basic characteristics related to the appearance and biting behaviour of the insects.     

Seasonal variation of Leishmania major infection rates in sandflies from rodent burrows in Isfahan province, Iran

Abstract. In preparation for field trials of killed Leishmania major vaccine, natural infections with Leishmania promastigotes were monitored in Phlebotomine sandfly vectors from villages of Borkhar rural district, northeast of Isfahan in central Iran, where Lmajor zymodeme MON-26 (=LON-l) has been identified as causing zoonotic cutaneous leishmaniasis (ZCL). Sandflies were collected and dissected weekly, from burrows of rodent colonies, during the 'sandfly season', June-October 1991. Leptomonad infection rates were 12% of 26 Phlebotomus ansarii , 8% of 280 P.caucasicus , 11 % of 1042 P.papatasi and none of 126 Sergentomyia sintoni , being greatest during late August through September, coinciding with peak activity of the sandflies, 2–3 months before the highest incidence of ZCL human cases in November-December.     

Phlebotomine sandflies associated with a focus of cutaneous leishmaniasis in Valle del Cauca, Colombia

Abstract. A survey was made of the phlebotomine sandfly fauna of La Guaira, a village with coffee plantations near Cali, Colombia, from which cases of American cutaneous leishmaniasis had been reported due to Leishmania (Viannia) panamensis and Le. ( V. ) braziliensis. Among six species of sandfly collected on human bait, Lutzomyia youngi was most important in terms of biting nuisance. Lu. columbiana, Lu. lichyi and Lu. scorzai as well as Lu. youngi adults occurred throughout the year. Sandfly man-biting activity occurred throughout the night and was highest within 2 h of sunset. Despite its abundance in nocturnal samples, Lu. youngi was rarely taken in diurnal resting site collections. In contrast, Lu. lichyi was collected on tree-trunks during the day in large numbers and was the only species biting in daylight. The implications of these and other findings for leishmaniasis control measures in La Guaira are considered.     

Leishmania (Leishmania) amazonensis-induced cutaneous leishmaniasis in the primate Cebus apella: a model for vaccine trials

A primate model of leishmaniasis was developed with the objective of future vaccine testing. Lesion development and immunological parameters were studied upon primary and secondary infections. Seven Cebus apella were injected subcutaneously with 2×10⁶ Leishmania (Leishmania) amazonensis promastigotes. Erythematous nodules appeared 19–29 days p.i., which disappeared 100 days p.i. Four months later, six of the monkeys were challenged with the same inoculum; three of them developed erythematous nodules after 7 days p.i., with ulcer formation in two of these subjects. The lesions were short-lived and all were cured 40 days post challenge. Anti-Leishmania IgG antibodies were detected and they increased after the challenge infection. Leishmania antigen-induced lymphoproliferation was found 1 month post-primary infection, which coincided with IFN-γ production and lesion development. It decreased to control levels afterwards, but at the time of the challenge dose, it was significantly above the initial level. After the challenge infection, it first increased then decreased sharply at 40 days post-challenge, coinciding with the healing of the lesion. It increased again to a higher level at 60 days post-challenge. Leishmania (Leishmania) amazonensis-infection in C. apella did not induce complete protection against a secondary infection with a homologous parasite although specific antibody production and lymphoproliferation with IFN-γ production were observed. This fact indicates that vaccine has to be better than infection in the induction of protective immunity, and raises a question on in vitro parameters that should be considered as a counterpart of expected protection induced by vaccine candidate. In addition, we conclude that this is a useful primate model for the evaluation of candidate vaccines.     

Characterization and Pathogenicity of Rhizoctonia spp. from Onion in Amasya, Turkey

Forty‐two isolates of Rhizoctonia spp. were obtained from onion in Amasya, Turkey. Of these, 29% were Rhizoctonia solani (AG‐4), 69% were Waitea circinata var. zeae (Rhizoctonia zeae) and 2% were binucleate Rhizoctonia (AG‐B). Most of the isolates were recovered from rhizosphere soil. In pathogenicity tests on onion, R. solani AG‐4 caused the greatest disease severity, those of W. circinata var. zeae were moderately virulent but binucleate Rhizoctonia isolates were of low virulence. This is the first report of binucleate Rhizoctonia AG‐B and W. circinata var. zeae occurring on onion in Turkey.     

The roles of Phlebotomus martini and P. celiae (Diptera: Phlebotominae) as vectors of visceral leishmaniasis in the Aba Roba focus, southern Ethiopia

Abstract. During field studies (December 1988 to April 1990) to determine the vectors of visceral leishmaniasis (kala-azar) in the Aba Roba (Segen Valley) focus of southern Ethiopia, a total of 40,770 sandflies (Diptera: Psychodidae: Phlebotominae) were collected and identified as six species of Phlebotomus and seventeen Sergentomyia spp. Nine of these species were anthropophilic (four Phlebotomus and five Sergentomyia spp.), the dominant being Phlebotomus (Synphlebotomus) martini, P.(S.)celiae and Sergentomyia (Sergentomyia) schwetzi. P. celiae adult populations increased during the rainy season. Of 2326 P. martini and 1044 P. celiae dissected, the parous rate was 29.9% and 24.4%, respectively. Leishmania promastigotes were detected in sixteen P. martini (eleven identified as L. donovani by isoenzyme analysis and/or a DNA probe) and in three P. celiae (two identified by DNA probe), giving overall infection rates of 0.7% (1.9% parous infection rate) and 0.3% (1.2% parous infection rate), respectively. Four isolates from P. martini were typed by their isoenzyme profile as L. donovani zymodeme MON-37 = LON-44, identical to one of the zymodemes isolated from patients in this focus. This is the first evidence that P. celiae is a vector and the first time that P. martini has been implicated in Ethiopia. Based on observations of abundance, seasonality and prevalence of infection of the two Synphlebotomus spp., it is concluded that P. martini is the major vector and P. celiae a secondary vector in the Aba Roba focus. The risk of infection with L. donovani is greatest during the wet season.     

Heterogeneity among zymodemes of Leishmania infantum from HIV-positive patients with visceral leishmaniasis in south Italy

Abstract Eleven zymodemes of Leishmania infantum were identified among 38 parasite stocks isolated from Italian HIV-positive patients with visceral leishmaniasis (VL). Only one zymodeme is a common agent of Mediterranean VL in HIV-negative individuals, five zymodemes usually cause simple, self-resolving cutaneous leishmaniasis (CL), and five belong to unique genotypes which have not been previously reported from either VL or CL cases in immunocompetent individuals. This last group of parasites showed reassortaient patterns within electromorphs frequently observed in dermotropic L. infantum zymodemes. The highest zymodeme heterogeneity was found in south Italy (Sicily), with six zymodemes identified among 12 HIV-positive patients surveyed.     

Leishmania infection and host-blood feeding preferences of phlebotomine sandflies and canine leishmaniasis in an endemic European area,the Algarve Region in Portugal

The Algarve Region (AR) in southern Portugal, which is an international tourist destination, has been considered an endemic region of zoonotic leishmaniasis caused by Leishmania infantum since the 1980s. In the present study, phlebotomine and canine surveys were conducted to identify sandfly blood meal sources and to update the occurrence of Leishmania infection in vectors and dogs. Four sandfly species were captured: Phlebotomus perniciosus, Phlebotomus ariasi, Phlebotomus sergenti and Sergentomyia minuta. In one P. perniciosus female, L. infantum DNA was detected. Blood meal tests showed that this species had no host preferences and was an opportunistic feeder. An overall canine leishmaniasis (CanL) seroprevalence of 16.06% was found; the seroprevalence was 3.88% in dogs housed in kennels and 40.63% in dogs that attended veterinary clinics. The simultaneous occurrence of dogs and P. perniciosus infected with L. infantum in the AR indicates that the region continues to be an endemic area for CanL. Our results reinforce the need for the systematic spatial distribution of phlebotomine populations and their Leishmania infection rates and the need to simultaneously perform pathogen monitoring in both invertebrate and vertebrate hosts to investigate the transmission, distribution and spreading of Leishmania infection.     

Molecular identification of Leishmania spp. isolates causes cutaneous leishmaniasis (CL) in Sanliurfa Province,Turkey, where CL is highly endemic

Cutaneous leishmaniasis (CL) is an important public health problem in Turkey. CL has been most frequently seen in Sanliurfa. There is an expectation of increase in the population of leishmaniasis cases with the influence of Syrian refugees arriving in Turkey. In this study we aimed to diagnosis of CL and identifying of parasite from Leishmania isolates by using ITS 1 PCR RFLP. Samples were collected from 135 CL patients in Sanliurfa. After the specimens were inoculated in medium NNN, the ones which were cultures positive were cultivated in RPMI 1640 followed by PCR-RFLP. Genomic DNA was extracted phenol-chloroform procedure. Samples were examined by using ITS 1 PCR followed by RFLP analysis. Our results indicated that two species, L. tropica (132 samples) and L. major (3 samples), are responsible for cutaneous leishmaniasis in Sanl?urfa. Our study is the first scientific study in which it is reported molecular analyses of cutaneous leishmaniasis cases caused by L. major in Sanliurfa in Southestern Anatolia Region. Because CL cases caused by L.major are detected in our study, it is considered that genotyping is important for diagnosis of Leishmania and following change of epidemiology.     

Molecular identification of bloodmeal sources and trypanosomes in Glossina spp., Tabanus spp. and Stomoxys spp. trapped on cattle farm settlements in southwest Nigeria

The interactions of host, vector and parasite in bovine trypanosomiasis transmission cycles in southwest Nigeria are not yet well understood. Trypanosoma (Trypanosomatida: Trypanosomatidae) species infection prevalences and bloodmeal sources were determined in transmitting vectors of the genera Glossina (Diptera: Glossinidae), Tabanus (Diptera: Tabanidae) and Stomoxys (Diptera: Muscidae) collected using Nzi traps in cattle settlements in southwest Nigeria. Sequenced cytochrome B mitochondrial DNA segments obtained from vector digestive tracts identified bloodmeal sources from eight host species, namely human, cattle, hippopotamus, giraffe, gazelle, spotted hyena, long‐tailed rat and one unidentified species. Overall, 71.1% [95% confidence interval (CI) 63.0–78.1], 33.3% (95% CI 21.9–47.0) and 22.2% (95% CI 16.2–29.9), respectively, of Glossina, Tabanus and Stomoxys flies were positive for trypanosomes. The observed trypanosome species were Trypanosoma vivax, Trypanosoma congolense, Trypanosoma brucei, Trypanosoma evansi, Trypanosoma simiae and Trypanosoma godfreyi. Trypanosome DNA was more prevalent in tsetse (34.8% Tr. vivax, 51.1% Tr. b. brucei, 5.2% Tr. congolense, 4.4% Tr. simiae and 24.4% mixed infections) than in other flies and the main determinants in all flies were seasonal factors and host availability. To the best of the present group's knowledge, this is the first report of Trypanosoma species in Tabanus and Stomoxys flies in Nigeria. It indicates that vector control programmes should always consider biting flies along with tsetse flies in the control of human and animal trypanosomiasis.     

不同种株利什曼原虫前鞭毛体体外向无鞭毛体转化的研究

目的 观察不同种株利什曼原虫前鞭毛体在不同体外培养条件下向无鞭毛体的转化.方法 将6个对数生长期的不同种株利什曼原虫前鞭毛体接种于含M199和RPMI 1640复合培养液的24孔板内,在不同温度、pH值、血清、CO2下培养,第3天和第6天分类计数不同形态的原虫.结果 当培养温度为26℃,而其他培养条件发生变化时,利什曼原虫的增殖速度发生变化,大多数原虫保持前鞭毛体典型性形态,运动活跃.当培养温度上升至37℃,不论其他培养条件如何变化,原虫均沉积于培养孔底,停止增殖,运动减缓,显著地向中间体和无鞭毛体转化,酸性pH值和5%CO2可促进前鞭毛体转化为无鞭毛体,新生小牛血清可延长无鞭毛体的成活.但相同的培养条件下,杜氏利什曼原虫转化效率低于其他种株利什曼原虫.结论 温度是影响利什曼原虫前鞭毛体转化为无鞭毛体的基本因素,酸性pH值与CO2可协同温度促进转化,不同种株利什曼原虫的遗传异质性可影响转化效率.     

Amplification and analysis of human DNA present in mosquito bloodmeals

Abstract. DNA fingerprinting should permit the identification of individual human hosts of haematophagous arthropods, providing epidemi-ologically useful information, for example, the biting rates on different people and the impact of insecticide-impregnated bednets.
Investigations reported here demonstrate that it is possible to extract, amplify and fingerprint human DNA from the bloodmeals of individual female Anopheles gambiae mosquitoes kept at 24^oC for up to 10–15 h post-ingestion.     

Detection of Leishmania (Viannia) DNA in blood from patients with American cutaneous leishmaniasis

The aim of the present study was to investigate the presence of Leishmania (Viannia) subgenus DNA in peripheral blood from patients with cutaneous lesions due to American cutaneous leishmaniasis. The buffy coats from 68 blood samples were analyzed by polymerase chain reaction using the MP1L/MP3H primers. The parasite DNA was detected in 2 (3.4%) out of 59 patients who had amastigotes present in samples taken from lesions. The presence of Leishmania (Viannia) DNA in the blood of these patients indicates hematogeneous parasite dissemination.