Lutzomyia whitmani (Diptera: Psychodidae) as vector of Leishmania (V.) braziliensis in Paraná state, southern Brazil

The phlebotomine sandflies in the northern areas of the state of Paraná, Brazil, particularly those in the '16a' health region, were investigated over a 3-year period. Using CDC light traps (with and without hamster bait) and Shannon traps (with lights and horse or human bait), 16 species were collected from seven municipal districts which were known foci for cutaneous leishmaniasis: Arapongas; Apucarana; Cambira; Marumbi; Faxinal; Florestópolis; and Sabáudia. Although the frequency at which each species was collected varied with the collection site, Lutzomyia whitmani predominated (62.0% of all the sandflies collected), followed by Lu. fischeri (13.3%), Lu. pessoai (10.8%), Lu. migonei (8.2%) and Lu. intermedia (2.8%). Lutzomyia monticola, Lu. shanonni, Lu. firmatoi, Lu. lanei, Lu. alphabetica, Lu. misionensis, Lu. correalimai, Lu. cortellezzii, Lu. longipenis, Brumptomyia brumpti and B. nitzulescui together represented the remaining 3.0% of the collected sandflies. Three of the 1961 female sandflies collected and dissected in the municipal district of Cambira, where a recent case of cutaneous leishmaniasis had been registered, were found to have flagellates in their guts. All three were Lu. whitmani. The parasites from each of these infections were successfully isolated in NNN and 'Tobie and Evans' media and/or by inoculation into a hind foot of a golden hamster. The results of isoenzyme electrophoresis indicated that all three isolates were of Leishmania (Viannia) braziliensis.     

Detection and identification of Leishmania species in field-captured phlebotomine sandflies based on mini-exon gene PCR

Leishmaniasis is one of the most diverse and complex of all vector-borne diseases. Because it involves several overlapping species and sandfly vectors, the disease has a complex ecology and epidemiology. Adequate therapy and follow-up depend on parasitological diagnosis, but classical methods present several constraints when identifying species. We describe a polymerase chain reaction (PCR) which uses primers designed from mini-exon repetitive sequences that are specific for subgenus LeishmaniaViannia (PV), as well as sequences with specificity for genus (PG) that can distinguish between Leishmania species from other insect flagellates with minor differences in PCR products. For standardization, these PCR were tested in experimentally infected sandflies, and Leishmania infection in these insects was successfully confirmed. This methodology identified a 3.9% infection rate in field-captured phlebotomine sandflies from an endemic region in Brazil. Natural infection by Leishmania species was identified in three samples of Lutzomyia longipalpis, of which two were Leishmania (L.) chagasi and one Leishmania (L.) amazonensis. Irrespective of specific epidemiological conclusions, the method used in this study was able to identify Leishmania infections both in experimentally infected and field-captured phlebotomine sandflies, and could be a useful tool in epidemiological studies and strategic planning for the control of human leishmaniasis.     

Cross-immunity experiments between different species or strains of Leishmania in rhesus macaques (Macaca mulatta)

This study evaluates cross-immunity in rhesus monkeys (Macaca mulatta) previously infected with one species of Leishmania and have had self-cured disease or were cured by antimony-based therapy upon development of full-blown disease. We found that a self-healing cutaneous leishmaniasis (CL) following experimental infection with Leishmania (Leishmania) major induces significant protection for L. (L.) amazonensis and L. (Viannia) guyanensis, and was dependent on time of re-challenge by L (L.) amazonensis after animals had recovered from primary lesions, but lacked protection against L. (V.) braziliensis. In contrast, monkeys that recovered from L. (V.) braziliensis CL or L. (L.) chagasi visceral leishmaniasis following chemotherapeutic intervention were protected by challenge with L. (V.) braziliensis and L (L.) amazonensis. These findings indicate the relative variability in protection after self-cure or drug-cured experimental leishmaniasis to challenge by heterologous leishmanial parasites. Further studying the immune response may provide information regarding relevant factors influencing cross-protective immunity.     

Leishmania and HIV co-infection: dermatological manifestations

Leishmania species can cause a wide spectrum of cutaneous disease in HIV-positive patients: asymptomatic, localized cutaneous, mucosal, muco-cutaneous, diffuse cutaneous or post-kala-azar leishmaniasis. In such cases, which are usually severely immunocompromised, the leishmanial parasites reach the skin of the human host by dissemination after either a new infection (resulting from the bite of infected sandfly or, probably, the sharing of contaminated syringes by intravenous-drug users) or the re-activation of a latent infection. Recent experience and past observations on the dermatology of leishmaniasis in those with Leishmania/HIV co-infection are reviewed here.     

Immunopathogenic competences of Leishmania (V.) braziliensis and L. (L.) amazonensis in American cutaneous leishmaniasis

The immunopathogenic competences of Leishmania (V.) braziliensis and L. (L.) amazonensis were reviewed in the light of more recent features found in the clinical and immunopathological spectrum of American cutaneous leishmaniasis. It was shown a dichotomy in the interaction between these Leishmania species and human T-cell immune response; while L. (V.) braziliensis shows a clear tendency to lead infection from the localized cutaneous leishmaniasis (LCL), a moderate T-cell hypersensitivity form at the centre of the spectrum, toward to the mucocutaneous leishmaniasis (MCL) at the T-cell hypersensitivity pole and with a prominent Th1-type immune response, L. (L.) amazonensis shows an opposite tendency, leading infection to the anergic diffuse cutaneous leishmaniasis (ADCL) at the T-cell hyposensitivity pole and with a marked Th2-type immune response. Between the central LCL and the two polar MCL and ADCL, the infection can present an intermediary form known as borderline disseminated cutaneous leishmaniasis, characterized by an incomplete inhibition of T-cell hypersensitivity but with a evident supremacy of Th1 over Th2 immune response (Th1 ≥ Th2). These are probably the main immunopathogenic competences of L. (V.) braziliensis and L. (L.) amazonensis regarding the immune response dichotomy that modulates human infection outcome by these Leishmania parasites.     

Assessment of PCR in the detection of Leishmania spp in experimentally infected individual phlebotomine sandflies (Diptera: Psychodidae: Phlebotominae)

DNA amplification by the polymerase chain reaction (PCR) was applied in the investigation of the presence of Leishmania (Kinetoplastida: Trypanosomatidae) parasites in single phlebotomine sandflies. Three phlebotomine/parasite pairs were used: Lutzomyia longipalpis/Leishmania chagasi, Lutzomyia migonei/Leishmania amazonensis and Lutzomyia migonei/Leishmania braziliensis, all of them incriminated in the transmission of visceral or cutaneous leishmaniasis. DNA extraction was performed with whole insects, with no need of previous digestive tract dissection or pooling specimens. The presence of either mouse blood in the digestive tract of the sandflies or the digestive tract itself did not interfere in the PCR. Infection by as few as 10 Leishmania sp. per individual were sufficient for DNA amplification with genus-specific primers. Using primers for L. braziliensis and L. mexicana complexes, respectively, it was possible to discriminate between L. braziliensis and L. amazonensis in experimentally infected vectors (L. migonei).     

Endemically exposed asymptomatic individuals show no increase in the specific Leishmania (Viannia) panamensis-Th1 immune response in comparison to patients with localized cutaneous leishmaniasis

In Colombia, most cases of human cutaneous leishmaniasis are caused by Leishmania (Viannia) panamensis. Interestingly, up to 30% of the exposed population do not suffer from clinical leishmaniasis although it is likely that they are continuously infected with Leishmania parasites. Since it is believed that the induction of efficient Th1 immune responses protects against Leishmania infections both in humans and in animal models, we determined if endemically exposed asymptomatics showed stronger Leishmania-specific Th1 immune responses than patients with active localized cutaneous leishmaniasis (LCL). We found that Montenegro skin test responses were slightly higher among asymptomatic individuals compared to patients suffering from LCL. However, PBMC from patients with LCL showed similar Leishmania-specific proliferative responses compared to PBMC from asymptomatic individuals. Furthermore, PBMC from both groups also secreted similar amounts of IFN-gamma, IL-12p40 and IL-10 after in vitro exposure to L. panamensis. No IL-4 was detected in the supernatants. Taken together our results suggest that lack of LCL development in endemically exposed asymptomatics cannot be explained by stronger systemic anti-Leishmania Th1 immune responses or decreased Th2 responses in these individuals in comparison to individuals who develop LCL. It may be possible that other mechanisms are responsible for resistance to cutaneous leishmaniasis in Colombia in endemically exposed asymptomatics.     

Cutaneous leishmaniasis

Cutaneous leishmaniasis is endemic in the tropics and neotropics. It is often referred to as a group of diseases because of the varied spectrum of clinical manifestations, which range from small cutaneous nodules to gross mucosal tissue destruction. Cutaneous leishmaniasis can be caused by several Leishmania spp and is transmitted to human beings and animals by sandflies. Despite its increasing worldwide incidence, but because it is rarely fatal, cutaneous leishmaniasis has become one of the so-called neglected diseases, with little interest by financial donors, public-health authorities, and professionals to implement activities to research, prevent, or control the disease. In endemic countries, diagnosis is often made clinically and, if possible, by microscopic examination of lesion biopsy smears to visually confirm leishmania parasites as the cause. The use of more sophisticated diagnostic techniques that allow for species identification is usually restricted to research or clinical settings in non-endemic countries. The mainstays of cutaneous leishmaniasis treatment are pentavalent antimonials, with new oral and topical treatment alternatives only becoming available within the past few years; a vaccine currently does not exist. Disease prevention and control are difficult because of the complexity of cutaneous leishmaniasis epizoology, and the few options available for effective vector control.     

新疆克拉玛依地区几株利什曼原虫分离物的同源性分析

目的：确定新疆克拉玛依皮肤利什曼病患者和硕大白蛉吴氏亚种体内利什曼原虫的种。方法：通过酶切电泳及ＤＮＡ杂交的方法对当地病人体内和蛉体内的利什曼原虫以及参考虫株的ｎＤＮＡ及ｋＤＮＡ的同源性分析，研究克拉玛依病人和蛉体内利什曼原虫的基因型。结果：经ｎＤＮＡ基因型分析，表明病人与蛉体内原虫与婴儿利什曼原虫同源性大。结论：当地皮肤利什曼病的病原体为婴儿利什曼原虫，硕大白蛉吴氏亚种为该病的媒介。     

Changes in the antigenic profile of Leishmania parasites following shifts in temperature.

We have examined by immunoblotting the antigen profiles of Leishmania parasites which have undergone upward shifts in ambient temperature during culture. Parasites in the promastigote insect vector stage were grown to stationary growth phase at 25 degrees C, and then further cultured at the 37 degrees C temperature experienced in the mammalian host. Changes in the immunoblot profiles of the parasites occurred within one day of culture at mammalian ambient temperature. Serum antibodies from patients with active Leishmania infections showed reactivity with antigenic determinants of greater than Mr 38,000 that were expressed by parasites at 37 degrees C, and which were not comparably observed on immunoblots of 25 degrees C cultured organisms. The promastigotes of Leishmania species which cause either cutaneous or visceral leishmaniasis express differing forms of the 37 degrees C induced high molecular weight determinants, however, these molecules express cross-reactive epitopes. Previous studies have suggested that temperature may play a role in the differentiation process between the insect and host life cycle stages of Leishmania. Our results suggest that the antigenic profile of Leishmania parasites may also be affected by the expression of products from temperature sensitive biosynthetic pathways.     

新疆克拉玛依皮肤利什曼病传播媒介的研究

１９９４年的研究表明，在克拉玛依从皮肤利什曼病患者皮肤损害部位和从硕大白岭吴氏亚种消化道内分离出来的利什曼原虫，经ＤＮＡ基因型的分析，证实与婴儿利什曼原虫同源。本文报道，在皮肤利什曼病流行区内硕大白蛉吴氏亚种的数最颇大，亲人性强，在野外和居民点内该蛉的前鞭毛体自然感染率分别为５．９％（５８／９８５）和２．９％（１３／４４９），前鞭毛体在该蛉的消化道内能大量繁殖并可移行至咽及喙部；而在非流行区，该蛉的数量很少或无，也未查见前鞭毛体的感染。综合以往和本文的研究结果，作者确认硕大白蛉吴氏亚种为克拉玛依山婴儿利什曼原虫所致的皮肤利什曼病的传播媒介。     

Influence of the inoculation route in BALB/c mice infected by Leishmania infantum

Mice of the BALB/c strain are frequently used, due to their high susceptibility to Leishmania infection. Most of the studies in visceral leishmaniasis use the endovenous or the intraperitoneal routes to inoculate the parasites. In this study, the development of experimental visceral leishmaniasis infection was evaluated in BALB/c mice inoculated with Leishmania infantum parasites by endovenous (EV group) or intraperitoneal (IP group) routes. The results shows that both inoculation routes were able to produce progressive infection in mice. However, a higher dispersion of the values of parasite density was detected among the animals of the EV group than the IP group. Our results indicate that the intraperitoneal inoculation results in a higher homogeneity of infections, so we suggest that this route should be preferentially used in experimental infections in the mice model, particularly when pools of samples are required for immune cellular studies.     

Leishmaniasis in Greece I. Isolation and identification of the parasite causing human and canine visceral leishmaniasis

Three human and 19 canine leishmanial stocks were typed according to their excreted factor serotype and the electrophoretic mobility of their MDH, GPI, G6PDH and 6PGDH and shown to be identical with regard to these characters and, thus with Leishmania donovani infantum. This verifies the opinion of earlier researchers, who suggested that the parasites which cause human and canine visceral leishmaniasis in Greece are the same organism and that dogs are the reservoir for the human infection. The complexities raised by the co-existence of human cutaneous leishmaniasis in Greece caused by L. tropica (formerly L. t. minor) are stressed. A comparison was made of the clinical symptomatology, serological diagnosis by IFA and ELISA tests and parasitological diagnosis of the human cases and canine infections.     

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.     

The sandfly fauna in the visceral-leishmaniasis focus of Gedaref,in the Atbara-River area of eastern Sudan

Visceral leishmaniasis (VL) is an acute public-health problem in Sudan. Between 1997 and 2000, four, brief entomological surveys were carried out in Barbar El Fugarra, a village in the state of Gedaref, in the Atbara-River area of eastern Sudan. Between 1996 and 1999, 658 cases of VL occurred among the village's population of about 4000. CDC miniature light-traps set inside and outside human dwellings were used to collect a total of 12,745 sandflies, including five species of the genus Phlebotomus and 19 of Sergentomyia. Phlebotomus papatasi and P. orientalis made up 7% and 5% of the collected sandflies, respectively. Seasonal variation was observed in the numbers of P. orientalis, P. papatasi, S. schwetzi and S. magna caught. Almost all (88%) of the sandflies collected were caught inside houses or granaries and there appeared to be particularly large indoor populations of P. orientalis, P. papatasi, S. schwetzi, S. magna and S. clydei. Phlebotomus orientalis could be responsible for the indoor transmission of the parasites causing the local VL, between humans and between humans and local dogs (which have been found infected by some of the Leishmania zymodemes found in humans). The co-occurrence in this focus of P. papatasi and Arvicanthis niloticus, which are known vectors and reservoir hosts, respectively, of L. major, indicates the possibility that outbreaks of human cutaneous leishmaniasis might occur in the area.     

Natural infection of Nyssomyia neivai by Leishmania (Viannia) spp. in the state of Paraná, southern Brazil, detected by multiplex polymerase chain reaction

Natural sandfly infection by Leishmania spp. in an area endemic for American cutaneous leishmaniasis was analyzed using multiplex polymerase chain reaction (PCR). The sandflies were captured using Falc?o light traps in an endemic area of the municipality of Doutor Camargo during March, April, and June 2008. In total, 1803 females were analyzed; 1755 were Nyssomyia neivai (Pinto) and 48 were Nyssomyia whitmani (Antunes and Coutinho). Multiplex PCR analyses using MP3H-MP1L and 5Llcac-3Llcac primers showed the presence of Leishmania (Viannia) spp. in 4/181 pools of sandflies, all Ny. neivai, that is, a minimal infection rate of 0.22%. This study showed, for the first time, the presence of DNA of Leishmania (Viannia) spp. in Ny. neivai. This suggests the existence of natural infection by Leishmania (Viannia) spp. in Ny. neivai in the state of Paraná. Multiplex PCR is an important tool in the detection of Leishmania infection in sandflies.     

克拉玛依地区的利什曼病 Ⅸ.白蛉自然感染前鞭毛体的鉴定

本文报道新疆克拉玛依地区的大沙鼠洞、旷野及人房等不同场所内白蛉自然感染利什曼原虫的情况、蛉体内原虫对实验动物的致病特征以及用利什曼原虫McAb的dot-ELISA法对白蛉自然感染的利什曼前鞭毛体的检测结果;并结合4年来对当地白蛉的生态和大沙鼠体内的利什曼原虫对白蛉的感染性等一系列的调查研究,证实蒙古白蛉和安氏白蛉为克拉玛依大沙鼠体内利什曼原虫的传播媒介。     

Monthly variation in natural infection of the sandfly Lutzomyia ayacuchensis with Leishmania mexicana in an endemic focus in the Ecuadorian Andes.

In order to collect information on the role of Lutzomyia ayacuchensis in the transmission of leishmaniasis in a newly discovered Andean endemic focus in Ecuador, a longitudinal field study was carried out over 13 months. Monthly dissections were made of a minimum of 200 anthropophilic sandflies, collected at night during the month. A total of 2600 flies was separated from a small number of Lu. osornoi, another anthropophilic species in the area, and dissected; 95 (3.65%) were naturally infected with Leishmania mexicana promastigotes. The parasites were always located in the sandfly midgut. The current study revealed a marked monthly variation both in natural infections with Leishmania and in biting activity of sandflies in the endemic area, demonstrating a high transmission rate during the period from the early rainy season to the early or mid dry season (February to July).     

Importance of cats in zoonotic leishmaniasis in Portugal

Leishmaniasis, caused by Leishmania infantum, is an endemic zoonosis in the Mediterranean basin. Dogs are considered the major host for these parasites, as well as the main reservoir for human visceral infection. In recent years, asymptomatic infection or clinical disease caused by L. infantum in cats has been reported in several countries where zoonotic leishmaniasis is present. The aim of the present study was to perform a leishmaniasis survey in cats from an endemic focus. Twenty-three adult stray cats were surveyed by clinical examination, and peripheral blood samples for serological and molecular analysis were collected. In 7 of the 23 cats (30.4%) Leishmania DNA was detected in blood. A low level of fluorescent antibodies was detected in four serum samples. All the animals were asymptomatic. Taking into account the high rate of asymptomatic feline leishmaniasis in this survey, it can be suggested that cats may act as a habitual reservoir host of L. infantum infection in endemic areas. Furthermore, it will be important in the future to add this parasitosis to the differential diagnosis of feline infections from leishmaniasis foci in cats. Feline leishmaniasis diagnosis should be accessed by molecular tools.     

Disseminated cutaneous leishmaniasis resembling post-kala-azar dermal leishmaniasis caused by Leishmania donovani in three patients co-infected with visceral leishmaniasis and human immunodeficiency virus/acquired immunodeficiency syndrome in Ethiopia

We report paired strains of Leishmania parasites, one from the viscera and the other from skin lesions that were isolated from three patients with visceral leishmaniasis and disseminated cutaneous leishmaniasis that were co-infected with human immunodeficiency virus. The causative parasites were characterized by polymerase chain reaction-restriction length polymorphism of the ribosomal DNA internal transcribed spacer 1 and by a panel of multilocus microsatellite markers. We demonstrated that the causative agent was Leishmania donovani in all cases, irrespective of the phenotype of the disease. The paired strains from viscera and skin lesions of the same patients showed genetic identity across the 14 microsatellite markers investigated. These findings demonstrate that the skin lesions in these human immunodeficiency virus-positive patients with visceral leishmaniasis were caused by dissemination of viscerotropic L. donovani parasites as a consequence of severe immunosuppression. However, in all three patients, rapid clearance of the skin lesions was observed after antimonial therapy.