Transmission of Leishmania infantum MON-98 to hamsters by the bite of Phlebotomus langeroni Nitzulescu (Diptera: Psychodidae)

The ability of Phlebotomus langeroni to successfully acquire and transmit Leishmania infantum MON-98 to hamsters was demonstrated. Sand flies and Leishmania both originated from an infantile visceral leishmaniasis focus in El Agamy Egypt. P. langeroni females were infected by feeding on lesions of needle-inoculated hamster and on infected blood suspension using a chick-skin membrane apparatus. Infection rate of sand flies fed on membrane was 88% compared to 7.8% for flies fed on leishmanial lesion. The transmission to hamster took place by the bites of infective flies taking a second blood meal, on the 8th to 10th day post-feeding. Furthermore, successful transmission was by the bites of flies that took no blood or that took full blood meal. Whereas flies that took full blood meal were not infective as indicated by dissection. In three hamsters, lesions developed after three months. Leishmania amastigotes were demonstrated from the lesion as well as from the liver and spleen of infected hamsters.     

Refractory barriers in the sand fly Phlebotomus papatasi (Diptera: Psychodidae) to infection with Leishmania panamensis.

The life cycle of Leishmania panamensis in Phlebotomus papatasi was studied to characterize barriers limiting parasite colonization, differentiation, migration, and attachment in an unnatural sand fly host. The insects were fed a suspension of L. panamensis-infected macrophages and human erythrocytes, and were examined up to 16 days post-infection by light and electron microscopy. Histologic examination of 401 flies showed the peritrophic membrane to be the first important barrier to parasite establishment in the gut lumen. In most flies, parasites were unable to escape from the closed peritrophic sac, which was either excreted or retained intact in the midgut. After five days, only 31% of the flies were infected; attached parasites colonized the pylorus-ileum and/or colon regions of the hindgut. Anterior migration into the cardia region of the midgut occurred in less than 1% of infected flies; no parasites colonized the foregut. In the bloodmeal and residual bloodmeal, five morphologic forms developed from ingested amastigotes: stumpy, spatulate, elongate, short nectomonad promastigotes, and paramastigotes. Abnormal retention of amastigotes in macrophages and delayed development of promastigote stages was observed. The primary form attached in the hindgut was a pear-shaped haptomonad promastigote. Differentiation of L. panamensis in Ph. papatasi appeared to be similar to that described in natural hosts, except that metacyclic infective forms were not observed, and some forms developed in unusual locations. Phlebotomus papatasi was a partly refractory biological host for L. panamensis. The peritrophic membrane adversely affected the infection rate; rare anterior migration and a lack of metacyclic promastigotes may preclude transmission by bite.     

Experimental infection of Phlebotomus perniciosus and determination of the natural infection rates of Leishmania infantum in dogs

Direct xenodiagnosis has been used to determine the infection rates in sand flies of 12 dogs parasitized by Leishmania infantum and classified in function of clinical signs. The dogs were divided into three clinical groups and no statistically significant differences in the infection rates of female sand flies were observed among them. This result indicates the epidemiological importance of asymptomatic and oligosymptomatic dogs. Indirect xenodiagnosis uses sand flies fed on the blood of dogs ex vivo. This technique was not found to be useful as an alternative to the direct method for the detection of dogs parasitized by L. infantum. The clinical worsening of the disease was not reflected in the increasing number of parasites detected in the animals' blood by the indirect technique, thereby rejecting its utility as an indicator of the prognosis of the disease.     

Detection and identification of Leishmania species within naturally infected sand flies in the andean areas of ecuador by a polymerase chain reaction

The surveillance of prevalent Leishmania and sand fly species in endemic areas is important for prediction of the risk and expansion of leishmaniasis. In this study, we developed a polymerase chain reaction (PCR)-based method for detection of Leishmania minicircle DNA within individual sand flies. Using this method, we detected minicircle DNA in 6 (3.3%) of 183 sand flies, while 5 (3.5%) of 143 were positive for Leishmania promastigotes in the same areas by microscopic examination. The species were identified as Leishmania (Leishmania) mexicana by nucleotide sequencing of the cytochrome b gene. Additionally, all the Leishmania-positive sand flies were identified as Lutzomyia ayacuchensis by the restriction enzyme digestion of the PCR-amplified 18S ribosomal RNA gene fragments. Since this combined method is relatively easy and can process a large number of samples, it will be a powerful tool for the rapid identification of prevalent sand fly and Leishmania species as well as monitoring the infection rate in sand fly populations in endemic areas.     

Short report: surveillance of Leishmania sp. among sand flies in Sicily (Italy) using a fluorogenic real-time polymerase chain reaction

Leishmaniasis caused by Leishmania infantum is a complex zoonotic disease, resulting in cutaneous and visceral manifestations in both dogs and humans. The present study involved a published Taqman fluorogenic real-time polymerase chain reaction (PCR) assay for surveillance of Leishmania sp. parasites among sand flies trapped in two provinces in Sicily, Catania and Agrigento, during the summer and fall of 2003. Only male specimens were identified to species level, while females were used to evaluate Leishmania sp. infection by PCR testing. The two most prevalent sand fly species found were Phlebotomus perfiliewi and P. perniciosus. Of the female sand flies tested, 2.9% were positive for Leishmania sp. DNA by the PCR.     

Natural infection of Lutzomyia neivai with Leishmania spp. in northwestern Argentina

The natural infection of Lutzomyia neivai with Leishmania in the endemic area of American cutaneous leishmaniasis (ACL) in northwestern Argentina was analyzed by the polymerase chain reaction (PCR)-hybridization technique. Phlebotominae sand flies were captured in the provinces of Tucumán and Salta between 1999 and 2003. From a sample of 440 Lu. neivai females analysed for the detection of the Leishmania (Viannia) and Leishmania (Leishmania) subgenera, 9.1% of the samples resulted infected with a parasite of the subgenus Viannia and none with the Leishmania. This is the first report of naturally infected sand flies in Argentina besides the first report of infected Lu. neivai sensu strictu. Our results contributed to further incrimination of this specie as vector of leishmaniasis in the area and the identification of the main circulating parasite as belonging to the Leishmania (Viannia) subgenera.     

Prevalence of sand flies and Leishmania donovani infection in a natural population of female Phlebotomus argentipes in Bihar State, India

Leishmaniasis is a vector-borne disease, and in the Indian subcontinent the female Phlebotomus argentipes is the vector for Leishmania donovani. However, data on the extent of sand fly infection rates in natural settings using molecular methods have not been extensively reported in India. In this study a PCR technique was applied targeting the 18S rRNA encoding region to determine the prevalence of Leishmania infection in female P. argentipes captured in the field. For this study, sand flies were collected from 897 houses selected from 50 villages endemic for visceral leishmaniasis (VL) in Muzaffarpur district, Bihar state, using CDC miniature light traps and mouth aspirators. A total of 14,585 sand flies were collected of which 449 were female P. argentipes divided into 132 pools. Molecular detection using PCR targeting the 18S rRNA gene was carried out for the identification of P. argentipes and Leishmania. The overall prevalence of infection was 4.90-17.37% for L. donovani in female P. argentipes in endemic regions of Bihar state. In this study no correlation was found between the presence of infected sand flies and the occurrence of clinical VL. This study provides the first report evaluating the prevalence of Leishmania infection in sand flies in a region endemic for VL in India. Sergentomyia species are the most common species of sand fly. Knowledge of the infection rate in female P. argentipes may help in predicting severity of disease and in vector elimination programs.     

Establishment of a mass screening method of sand fly vectors for Leishmania infection by molecular biological methods

Surveillance of the prevalence of Leishmania and its vector, sand fly species, in endemic and surrounding areas is important for prediction of the risk and expansion of leishmaniasis. In this study, a method for the mass screening of sand flies for Leishmania infection was established. This method was applied to 319 field-captured specimens, and 5 positive sand flies were detected. Sand fly species were identified by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) of the18S rRNA gene, and all the positive flies were Lu. hartmanni. Furthermore, cytochrome b (Cyt b) gene sequence analyses identified all the parasites as Endotrypanum species including a probable novel species. Because the method requires minimum effort and can process a large number of samples at once, it will be a powerful tool for studying the epidemiology of leishmaniasis.     

Natural infection of Lutzomyia tortura with Leishmania (Viannia) naiffi in an Amazonian area of Ecuador

Natural infection of sand flies with Leishmania parasites was surveyed in an Amazonian area in Ecuador where leishmaniasis is endemic. Seventy-one female sand flies were dissected and one was positive for Leishmania protozoa. The species of this sand fly was identified as Lutzomyia (Lu.) tortura on the basis of morphologic characteristics. Analysis of the cytochrome b gene sequence identified the parasite as L. (Viannia) naiffi. We report the distribution of L. (V.) naiffi in Ecuador and detection of a naturally infected sand fly in the Ecuadorian Amazon and natural infection of Lu. tortura with Leishmania parasites in the New World.     

Sand fly feeding on noxious plants: a potential method for the control of leishmaniasis.

The sand fly Phlebotomus papatasi transmits Leishmania major, which causes cutaneous leishmaniasis, in vast regions of the Old World. In addition to blood, the sand flies feed on plants. In a study of this diet, we observed that one night of feeding on branches of Solanum jasminoides, Ricinus communis, or Bougainvillea glabra drastically shortened the life span of the sand flies. Flowering B. glabra attracted P. papatasi in the field. Nevertheless, in the region endemic for L. major in yards abounding with vector sand flies, the number of P. papatasi trapped near hedges of B. glabra was eight times less (62 versus 502 flies trapped) than in the control sites. The results imply that B. glabra affords local protection against sand fly bites and decreases the risk of leishmaniasis. We suggest that this and other ornamental plants that are harmful to sand flies can be used as a tool for this purpose.     

An epidemic outbreak of canine cutaneous leishmaniasis in Colombia caused by Leishmania braziliensis and Leishmania panamensis

The largest recorded outbreak of cutaneous leishmaniasis in Colombia's history occurred during 2005-2009 in soldiers of the Colombian Army, with ~40,000 cases. This outbreak was caused by the influx of military personnel into the jungle with the mission of combat illicit crops and the guerrilla. The soldiers remain for long periods within the rainforest and are exposed to the bite of infected sand flies. During the military activities, soldiers work with dogs specially trained to detect landmines, and therefore, dogs are also exposed to the infected sand flies and show high incidence of cutaneous leishmaniasis (CL). This work describes an epidemic outbreak of canine CL caused by Leishmania braziliensis and Leishmania panamensis in Colombia, South America. The clinical features of the disease and the response to treatment with pentavalent antimonials observed in 72 guard dogs from the Colombian Army are described. A program for prevention and control of canine CL is also discussed.     

Detection of Leishmania within sand flies by kinetoplast DNA hybridization

A kinetoplast DNA hybridization probe method was used to detect Leishmania within sand flies and to distinguish it from the non-pathogenic flagellate, Endotrypanum. Eighty-one sand flies (74 Lutzomyia umbratilis, 1 Lu. anduzei, and 6 Lu. shannoni) collected outside Manaus, Brazil were dissected. Forty-four of these were found to be infected with flagellates, and 2 hybridized with a Leishmania braziliensis probe. Thirty-three of sixty-one flies reprobed with an Endotrypanum probe were positive.     

利什曼原虫胞内寄生相关基因的研究

利什曼病是由利什曼原虫无鞭毛体寄生在包括人在内的哺乳动物巨噬细胞而引起的疾病,由杜氏利什曼原虫引起的内脏利什曼病若不治疗则会致命.研究者对寄生在白蛉属消化道内的前鞭毛体和寄生在巨噬细胞内的无鞭毛体胞内高表达基因或蛋白进行研究,筛选出一些特异基因,为利什曼原虫疫苗候选抗原的确定和靶作用药物的确定提供了科学依据.     

利什曼原虫胞内寄生相关基因的研究

利什曼病是由利什曼原虫无鞭毛体寄生在包括人在内的哺乳动物巨噬细胞而引起的疾病,由杜氏利什曼原虫引起的内脏利什曼病若不治疗则会致命.研究者对寄生在白蛉属消化道内的前鞭毛体和寄生在巨噬细胞内的无鞭毛体胞内高表达基因或蛋白进行研究,筛选出一些特异基因,为利什曼原虫疫苗候选抗原的确定和靶作用药物的确定提供了科学依据.     

Molecular mass screening to incriminate sand fly vectors of Andean-type cutaneous leishmaniasis in Ecuador and Peru

Sand flies from the Andean areas of Ecuador and Peru were examined for Leishmania infections by using our recently established molecular mass screening method. Leishmanial minicircle DNA-positive sand flies were detected in 3 of 192 and 1 of 462 samples from Ecuador and Peru, respectively. Sand fly species were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the 18S ribosomal RNA (rRNA) gene, and the positive flies were Lutzomyia (Lu.) ayacuchensis and Lu. peruensis, respectively. Furthermore, cytochrome b and mannose-phosphate isomerase gene sequence analyses identified the parasites from Ecuador and Peru as Leishmania (Leishmania) mexicana and L. (Viannia) peruviana, respectively. Thus, the mass screening method was confirmed to be a powerful tool for sand fly research.     

利什曼原虫胞内寄生相关基因的研究

利什曼病是由利什曼原虫无鞭毛体寄生在包括人在内的哺乳动物巨噬细胞而引起的疾病,由杜氏利什曼原虫引起的内脏利什曼病若不治疗则会致命.研究者对寄生在白蛉属消化道内的前鞭毛体和寄生在巨噬细胞内的无鞭毛体胞内高表达基因或蛋白进行研究,筛选出一些特异基因,为利什曼原虫疫苗候选抗原的确定和靶作用药物的确定提供了科学依据.     

Quantification of the infectious dose of Leishmania major transmitted to the skin by single sand flies

Leishmaniasis is transmitted between mammalian hosts by the bites of bloodsucking vector sand flies. The dose of parasites transmitted to the mammalian host has never been directly determined. We developed a real-time PCR-based method to determine the number of Leishmania major parasites inoculated into the ears of living mice during feeding by individual infected flies (Phlebotomus duboscqi). The number of parasites transmitted varied over a wide range in the 58 ears in which Leishmania were detected and demonstrated a clear bimodal distribution. Most of the infected mice were inoculated with a low dose of <600 parasites. One in four received a higher dose of >1,000 and up to 100,000 cells. High-dose transmission was associated with a heavy midgut infection of >30,000 parasites, incomplete blood feeding, and transmission of a high percentage of the parasite load in the fly. To test the impact of inoculum size on infection outcome, we compared representative high- (5,000) and low- (100) dose intradermal needle infections in the ears of C57BL/6 mice. To mimic natural transmission, we used sand fly-derived metacyclic forms of L. major and preexposed the injection site to the bites of uninfected flies. Large lesions developed rapidly in the ears of mice receiving the high-dose inoculum. The low dose resulted in only minor pathology but a higher parasite titer in the chronic phase, and it established the host as an efficient long-term reservoir of infection back to vector sand flies.     

Naturally infected Lutzomyia sand flies in a Leishmania-endemic area of Brazil

In Brazil, Leishmania transmission involves several species of phlebotomine sand flies that are closely associated with different parasites and reservoirs, giving rise to different transmission cycles. The present study focused on naturally infected phlebotomines originating from Santa Luzia, a municipality near Belo Horizonte, capital of the Brazilian state of Minas Gerais, in which leishmaniasis are endemic. Systematic and non systematic approaches,involving the use of light traps and direct aspiration from resting sites, respectively, were used to collect females and flies. Identification of the captured insects and determination of natural infection by Leishmania spp. were performed using both conventional dissection methods and polymerase chain reaction (PCR). The dissection of 102 sand flies allowed five species of Lutzomyia to be identified, although no flagellate parasite forms were observed.In addition, 211 sand flies were identified, were separated according to species, and were combined into 11 pools of up to 20 individuals each. PCR analyses showed that two of these pools were infected with Leishmania:one pool of Lu. whitmani was infected with Le. (Viannia) spp. and another of Lu. cortelezzii was infected with Le. chagasi. This suggests that Lu. whitmani may be a possible vector of Leishmania in the study area, and more work needs to be performed to assess the role of Lu. cortelezzii as a vector.     

First report on natural Leishmania infection of Phlebotomus sergenti due Leishmania tropica by high resolution melting curve method in Southeastern Iran

Objective:To identify the Leishmania species in infected sand flies by Real-time PCR coupled with HRM analysis.Methods:Real-time PCR coupled with HRM analysis targeting the first internal transcribed spacer(ITS1)of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish Lelthmania species in sand flies specimens.Results:Three out of 115females of Phlebotomus sergenti(P.sergenti)(2.6%)were positive to Leishmania tropica(L.tropica).Conclusions:This is the first report on P.sergenti as the main and proven vector of anthroponitic cutaneous leishmaniasis in Dehbakri County using Real-time PCR coupled with HUM analysis.This method is rapid,sensitive and specific for diagnosing of parasites in infected Sand flies and ideal for large scale genotyping projects.     

Evidence for leishmania (viannia) parasites in the skin and blood of patients before and after treatment

BACKGROUND: American cutaneous leishmaniasis is considered to be a zoonotic disease transmitted by sand flies that feed on infected sylvatic mammals. However, the "domestication" of transmission and the increase in treatment failure with antimonial drugs have raised the suspicion of anthroponotic transmission of American cutaneous leishmaniasis. METHODS: The objective of the present study was to explore the potential of humans as a source of infection for sand flies. Biological (xenodiagnosis and culture) and molecular (polymerase chain reaction/Southern blot) detection methods were used to evaluate peripheral-blood monocytes and tissue fluids from sites accessible to sand flies from 59 adult patients with parasitologically confirmed American cutaneous leishmaniasis. RESULTS: Overall, 44.1% of patients (26/59) presented biological and/or molecular evidence of Leishmania parasites in normal skin, peripheral-blood monocytes, lesion scars, or lesion border (by xenodiagnosis) before (18/59 [30.5%]) or after (10/27 [37.0%]) treatment. Leishmania parasites were cultured from the unaffected skin of 2 (3.6%) of 55 patients, and xenodiagnosis gave positive results for 5 (8.8%) of 57 patients before treatment. CONCLUSIONS: The presence of Leishmania parasites in the unaffected skin and peripheral-blood monocytes of a high proportion of patients even after treatment and the acquisition of infection by sand flies support the plausibility of anthroponotic transmission of American cutaneous leishmaniasis.