Experimental infection of Phlebotomus perniciosus and Phlebotomus tobbi with different Leishmania tropica strains

Cutaneous leishmaniasis due to Leishmania tropica is increasingly documented in Europe and the Middle East. Besides its specific vector, Phlebotomus sergenti, permissive Phlebotomus sand flies are suspected as potential vectors of L. tropica. We investigated the susceptibility of two widely distributed species, Phlebotomus perniciosus and Phlebotomus tobbi. Laboratory-reared sand flies were infected experimentally with L. tropica strains differing in lipophosphoglycan epitopes, geographical distribution and epidemiology. High infection rates, heavy parasite loads and fully developed late-stage infections including colonization of the stomodeal valve were observed in all parasite-vector combinations. Our findings demonstrate that P. perniciosus and P. tobbi are susceptible to different L. tropica strains and may play a role in their circulation in endemic foci of Europe, the Middle East and North Africa.     

Comparative real-time kinetic analysis of human complement killing of Leishmania infantum promastigotes derived from axenic culture or from Phlebotomus perniciosus

Although Leishmania metacyclic promastigotes are generally considered resistant to human complement, studies of in vitro-cultured axenic stationary promastigotes using serum concentrations that approximate physiological plasma conditions indicate complement sensitivity. Natural Leishmania infection is caused by sand fly-inoculated promastigotes, whose complement resistance has not been analyzed systematically. We compared Leishmania susceptibility to human complement in L. infantum promastigotes derived from in vitro cultures and from sand flies. Phlebotomus perniciosus sand flies were fed with axenic promastigotes, L. infantum-infected U-937 cells, or spleen cells from L. infantum-infected hamsters. On selected days post-feeding, flies were dissected and promastigotes isolated; in addition, axenic promastigotes were obtained from culture at equivalent days of growth. In near-physiological serum concentration and temperature conditions, measurement of real-time kinetics of propidium iodide uptake showed that approximately 90% of axenic- and sand fly-derived promastigotes were rapidly killed by complement. We found no substantial differences between promastigotes from axenic culture, those isolated from flies on different post-feeding days, or those generated in flies fed with distinct inocula. The results indicate that Leishmania susceptibility to human complement is independent of promastigote developmental stage in the sand fly mid-gut and in axenic culture.     

Infection parameters in the sand fly vector that predict transmission of Leishmania major

To identify parameters of Leishmania infection within a population of infected sand flies that reliably predict subsequent transmission to the mammalian host, we sampled groups of infected flies and compared infection intensity and degree of metacyclogenesis with the frequency of transmission. The percentage of parasites within the midgut that were metacyclic promastigotes had the highest correlation with the frequency of transmission. Meta-analysis of multiple transmission experiments allowed us to establish a percent-metacyclic "cutoff" value that predicted transmission competence. Sand fly infections initiated with variable doses of parasites resulted in correspondingly altered percentages of metacyclic promastigotes, resulting in altered transmission frequency and disease severity. Lastly, alteration of sand fly oviposition status and environmental conditions at the time of transmission also influenced transmission frequency. These observations have implications for transmission of Leishmania by the sand fly vector in both the laboratory and in nature, including how the number of organisms acquired by the sand fly from an infection reservoir may influence the clinical outcome of infection following transmission by bite.     

Leishmania Metacyclogenesis Is Promoted in the Absence of Purines

Leishmania parasites, the causative agent of leishmaniasis, are transmitted through the bite of an infected sand fly. Leishmania parasites present two basic forms known as promastigote and amastigote which, respectively, parasitizes the vector and the mammalian hosts. Infection of the vertebrate host is dependent on the development, in the vector, of metacyclic promastigotes, however, little is known about the factors that trigger metacyclogenesis in Leishmania parasites. It has been generally stated that “stressful conditions” will lead to development of metacyclic forms, and with the exception of a few studies no detailed analysis of the molecular nature of the stress factor has been performed. Here we show that presence/absence of nucleosides, especially adenosine, controls metacyclogenesis both in vitro and in vivo. We found that addition of an adenosine-receptor antagonist to in vitro cultures of Leishmania amazonensis significantly increases metacyclogenesis, an effect that can be reversed by the presence of specific purine nucleosides or nucleobases. Furthermore, our results show that proliferation and metacyclogenesis are independently regulated and that addition of adenosine to culture medium is sufficient to recover proliferative characteristics for purified metacyclic promastigotes. More importantly, we show that metacyclogenesis was inhibited in sand flies infected with Leishmania infantum chagasi that were fed a mixture of sucrose and adenosine. Our results fill a gap in the life cycle of Leishmania parasites by demonstrating how metacyclogenesis, a key point in the propagation of the parasite to the mammalian host, can be controlled by the presence of specific purines.     

Comparative real-time kinetic analysis of human complement killing of Leishmania infantum promastigotes derived from axenic culture or from Phlebotomus perniciosus

Although Leishmania metacyclic promastigotes are generally considered resistant to human complement, studies of in vitro-cultured axenic stationary promastigotes using serum concentrations that approximate physiological plasma conditions indicate complement sensitivity. Natural Leishmania infection is caused by sand fly-inoculated promastigotes, whose complement resistance has not been analyzed systematically. We compared Leishmania susceptibility to human complement in L. infantum promastigotes derived from in vitro cultures and from sand flies. Phlebotomus perniciosus sand flies were fed with axenic promastigotes, L. infantum-infected U-937 cells, or spleen cells from L. infantum-infected hamsters. On selected days post-feeding, flies were dissected and promastigotes isolated; in addition, axenic promastigotes were obtained from culture at equivalent days of growth. In near-physiological serum concentration and temperature conditions, measurement of real-time kinetics of propidium iodide uptake showed that 90% of axenic- and sand fly-derived promastigotes were rapidly killed by complement. We found no substantial differences between promastigotes from axenic culture, those isolated from flies on different post-feeding days, or those generated in flies fed with distinct inocula. The results indicate that Leishmania susceptibility to human complement is independent of promastigote developmental stage in the sand fly mid-gut and in axenic culture.     

Leishmania infantum proteophosphoglycans regurgitated by the bite of its natural sand fly vector,Lutzomyia longipalpis,promote parasite establishment in mouse skin and skin-distant tissues

We demonstrate that a proteophosphoglycan-rich gel secreted by Leishmania infantum inside the midgut of Lutzomyia longipalpis sand flies (promastigote secretory gel) is regurgitated along with an average dose of 500 L. infantum metacyclic promastigotes per infected bite. Using both low (10³) and high (10⁵) doses of parasites in the ears of BALB/c mice we show that the infections benefit from the presence of vector saliva and parasite gel in the skin. However, chronic infection of the spleen was only enhanced in high dose co-infections with gel. These results provide the framework for a more natural experimental model of visceral leishmaniasis.     

Stage-Specific Adhesion of Leishmania Promastigotes to Sand Fly Midguts Assessed Using an Improved Comparative Binding Assay

Background

The binding of Leishmania promastigotes to the midgut epithelium is regarded as an essential part of the life-cycle in the sand fly vector, enabling the parasites to persist beyond the initial blood meal phase and establish the infection. However, the precise nature of the promastigote stage(s) that mediate binding is not fully understood.

Methodology/Principal Findings

To address this issue we have developed an in vitro gut binding assay in which two promastigote populations are labelled with different fluorescent dyes and compete for binding to dissected sand fly midguts. Binding of procyclic, nectomonad, leptomonad and metacyclic promastigotes of Leishmania infantum and L. mexicana to the midguts of blood-fed, female Lutzomyia longipalpis was investigated. The results show that procyclic and metacyclic promastigotes do not bind to the midgut epithelium in significant numbers, whereas nectomonad and leptomonad promastigotes both bind strongly and in similar numbers. The assay was then used to compare the binding of a range of different parasite species (L. infantum, L. mexicana, L. braziliensis, L. major, L. tropica) to guts dissected from various sand flies (Lu. longipalpis, Phlebotomus papatasi, P. sergenti). The results of these comparisons were in many cases in line with expectations, the natural parasite binding most effectively to its natural vector, and no examples were found where a parasite was unable to bind to its natural vector. However, there were interesting exceptions: L. major and L. tropica being able to bind to Lu. longipalpis better than L. infantum; L. braziliensis was able to bind to P. papatasi as well as L. major; and significant binding of L. major to P. sergenti and L. tropica to P. papatasi was observed.

Conclusions/Significance

The results demonstrate that Leishmania gut binding is strictly stage-dependent, is a property of those forms found in the middle phase of development (nectomonad and leptomonad forms), but is absent in the early blood meal and final stages (procyclic and metacyclic forms). Further they show that although gut binding may be necessary for parasite establishment, in several vector-parasite pairs the specificity of such in vitro binding alone is insufficient to explain overall vector specificity. Other significant barriers to development must exist in certain refractory Leishmania parasite-sand fly vector combinations. A re-appraisal of the specificity of the Leishmania-sand fly relationship is required.     

Leishmania parasites (Kinetoplastida: Trypanosomatidae) reversibly inhibit visceral muscle contractions in hemimetabolous and holometabolous insects

Female sand flies can acquire protozoan parasites in the genus Leishmania when feeding on an infected vertebrate host. The parasites complete a complex growth cycle in the sand fly gut until they are transmitted by bite to another host. Recently, a myoinhibitory peptide was isolated from Leishmania major promastigotes. This peptide caused significant gut distension and reversible, dose-dependent inhibition of spontaneous hindgut contractions in the enzootic sand fly vector, Phlebotomus papatasi. The current study further characterizes myoinhibitory activity in L. major and other kinetoplastid parasites, using the P. papatasi hindgut and other insect organ preparations. Myoinhibitory activity was greatest in cultured promastigotes and in culture medium in late log-phase and early stationary-phase, coinciding with development of infective Leishmania morphotypes in the sand fly midgut. L. major promastigote lysates inhibited spontaneous contractions of visceral muscle preparations from hemimetabolous (Blattaria and Hemiptera) and holometabolous (Diptera) insects. Inhibition of visceral muscle contractions in three insect orders indicates a conserved mode of action. Myoinhibitory activity was detected also in Leishmania braziliensis braziliensis, a Sudanese strain of Leishmania donovani, and the kinetoplastid parasite Leptomonas seymouri. Protozoan-induced myoinhibition mimics the effect of insect myotropins. Inhibiting host gut contractions protects Leishmania parasites from being excreted after blood meal and peritrophic matrix digestion, allowing development and transmission of infective forms.     

Dissemination of Leishmanias to the organs of Syrian hamsters following intrasplenic inoculation of promastigotes

Four strains of leishmanial promastigotes were inoculated intrasplenically into male Syrian hamsters (Mesocricetus auratus). The dissemination of the parasites as amastigotes to various organs was followed at closely spaced intervals for 3.5 mo.An Israeli strain of Leishmania tropica and a strain isolated in Israel from a gerbil (Meriones shawi) displayed practically identical distribution patterns. Migration was outward from the spleen to the body surface, where intense multiplication of the amastigotes occurred, primarily in the ear pinnae and in the extremities of the limbs. A cryptic visceral infection persisted in the spleen, and most of the internal organs studied also developed cryptic infections. The bone marrow became rather heavily infected, and the epididymis, exceptionally heavily infected.An Indian strain of L. donovani led to a severe visceral infection in the spleen, liver, and bone marrow; and to mild to cryptic infections in most of the other visceral organs studied. However, no invasion of the genitalia occurred, nor did the body surface become infected.An Ethiopian strain of diffuse cutaneous leishmaniasis (DCL) was noninfective to Syrian hamsters, following either the intrasplenic or the intradermal inoculation of promastigotes.     

Detection of amastigote-like forms in the valve of Phlebotomus papatasi infected with Leishmania major

A massive and homogeneous amount of amastigote-like forms was detected in the stomodeal valve (SV) and the thoracic mid-gut (TMG) of Leishmania major-infected Phlebotomus papatasi, which received a second blood meal 13 to 21 days post-infection on healthy anaesthetized hamsters. After re-feeding, the infected sand flies were dissected out to examine the morphology of the parasite in SV, TMG and the abdominal mid-gut (AMG). Different promastigote forms were seen in the infected flies. Among these included typical promastigotes (nectomonads and haptomonads), paramastigotes, metacyclic promastigotes and, in some samples, the here-reported amastigote-like forms. The Leishmania amastigote-like forms were detected in the SV of sand flies with 14, 18 and 21 days of infection as well as in the TMG at 13 and 18 days post-infection. However, the amastigote-like forms were not detected in the AMG. Factors such as the acidic pH predominating the TMG and the SV, as well as the temperature of the ingested blood, among others, are suggested as contributing to the transformation of the typical promastigotes into the amastigote-like forms. The significance of this finding is discussed and the possible biological advantage for transmission of Leishmania is considered.     

Leishmania (Viannia) braziliensis metacyclic promastigotes purified using Bauhinia purpurea lectin are complement resistant and highly infective for macrophages in vitro and hamsters in vivo

In this study we characterised metacyclogenesis in axenic culture of Leishmania (Viannia) braziliensis, the causative agent of mucocutaneous leishmaniasis in the New World. Metacyclogenesis of other species of Leishmania has been shown by morphological changes as well as molecular modifications in the lipophosphoglycan, the major cell surface glycoconjugate of the promastigotes. In order to obtain metacyclic forms of L. braziliensis we tested a panel of different lectins. Our results showed that Bauhinia purpurea lectin facilitated the purification of metacyclic promastigotes from stationary-phase culture by negative selection. The B. purpurea non-agglutinated promastigotes had a slender short cell body and long flagella, typical of metacyclic morphology. The ultrastructural analysis showed that B. purpurea non-agglutinated promastigotes have a dense and thicker glycocalyx. They are resistant to complement lysis, and highly infective for macrophage in vitro and hamsters in vivo. Contrary to procyclic promastigotes, B. purpurea non-agglutinated forms were poorly recognised by sand fly gut epithelial cells. These results suggest that the B. purpurea non-agglutinated promastigotes are the metacyclic forms of L. braziliensis.     

Transmission of Leishmania metacyclic promastigotes by phlebotomine sand flies

A thorough understanding of the transmission mechanism of any infectious agent is crucial to implementing an effective intervention strategy. Here, our current understanding of the mechanisms that Leishmania parasites use to ensure their transmission from sand fly vectors by bite is reviewed. The most important mechanism is the creation of a "blocked fly" resulting from the secretion of promastigote secretory gel (PSG) by the parasites in the anterior midgut. This forces the sand fly to regurgitate PSG before it can bloodfeed, thereby depositing both PSG and infective metacyclic promastigotes in the skin of a mammalian host. Other possible factors in transmission are considered: damage to the stomodeal valve; occurrence of parasites in the salivary glands; and excretion of parasites from the anus of infected sand flies. Differences in the transmission mechanisms employed by parasites in the three subgenera, Leishmania, Viannia and Sauroleishmania are also addressed.     

Canine antibody response to Phlebotomus perniciosus bites negatively correlates with the risk of Leishmania infantum transmission

Background

Phlebotomine sand flies are blood-sucking insects that can transmit Leishmania parasites. Hosts bitten by sand flies develop an immune response against sand fly salivary antigens. Specific anti-saliva IgG indicate the exposure to the vector and may also help to estimate the risk of Leishmania spp. transmission. In this study, we examined the canine antibody response against the saliva of Phlebotomus perniciosus, the main vector of Leishmania infantum in the Mediterranean Basin, and characterized salivary antigens of this sand fly species.

Methodology/Principal Findings

Sera of dogs bitten by P. perniciosus under experimental conditions and dogs naturally exposed to sand flies in a L. infantum focus were tested by ELISA for the presence of anti-P. perniciosus antibodies. Antibody levels positively correlated with the number of blood-fed P. perniciosus females. In naturally exposed dogs the increase of specific IgG, IgG1 and IgG2 was observed during sand fly season. Importantly, Leishmania-positive dogs revealed significantly lower anti-P. perniciosus IgG2 compared to Leishmania-negative ones. Major P. perniciosus antigens were identified by western blot and mass spectrometry as yellow proteins, apyrases and antigen 5-related proteins.

Conclusions

Results suggest that monitoring canine antibody response to sand fly saliva in endemic foci could estimate the risk of L. infantum transmission. It may also help to control canine leishmaniasis by evaluating the effectiveness of anti-vector campaigns. Data from the field study where dogs from the Italian focus of L. infantum were naturally exposed to P. perniciosus bites indicates that the levels of anti-P. perniciosus saliva IgG2 negatively correlate with the risk of Leishmania transmission. Thus, specific IgG2 response is suggested as a risk marker of L. infantum transmission for dogs.     

Introductory Remarks: Symposium on Differentiation of Trypanosomatids

ABSTRACT. Leishmania differentiation in the gut of phlebotomine sand flies was evaluated based on five light and electron microscopic studies of natural (Leishmania panamensis/Lutzomyia gomezi, Leishmania chagasi/Lutzomyia longipalpis) and unnatural (Leishmania mexicana/Lutzomyia abonnenci, Leishmania panamensis/Phlebotomus papatasi, Leishmania major/Lutzomyia longipalpis) life cycles. In the bloodmeal, transformation of amastigotes into stumpy promastigotes occurred before or during division. Further division in pairs or rosettes resulted in the development of spatulate and/or elongate nectomonad (free-swimming) promastigotes. Elongate, short, and metacyclic nectomonad promastigotes, and nectomonad paramastigotes were present in the midgut lumen. Dividing short promastigotes predominated in the cardia, and appeared to generate metacyclic forms which were observed in three life cycles. Haptomonad (attached) forms of Leishmania panamensis in the hindgut were primarily spatulate promastigotes (natural host) or pear-shaped promastigotes (unnatural host); paramastigotes and dividing forms were rare. At the stomodeal valve, short haptomonad promastigotes predominated in unnatural hosts, while both short and pear-shaped haptomonads were abundant, along with paramastigotes in natural hosts. Haptomonad paramastigotes and pear-shaped promastigotes colonized the esophagus, while paramastigotes predominated in the pharynx. Metacyclics were free-swimming in the lumen of the foregut.     

Cooperation between Apoptotic and Viable Metacyclics Enhances the Pathogenesis of Leishmaniasis

Mimicking mammalian apoptotic cells by exposing phosphatidylserine (PS) is a strategy used by virus and parasitic protozoa to escape host protective inflammatory responses. With Leishmania amazonensis (La), apoptotic mimicry is a prerogative of the intramacrophagic amastigote form of the parasite and is modulated by the host. Now we show that differently from what happens with amastigotes, promastigotes exposing PS are non-viable, non-infective cells, undergoing apoptotic death. As part of the normal metacyclogenic process occurring in axenic cultures and in the gut of sand fly vectors, a sub-population of metacyclic promastigotes exposes PS. Apoptotic death of the purified PS-positive (PS^POS) sub-population was confirmed by TUNEL staining and DNA laddering. Transmission electron microscopy revealed morphological alterations in PS^POS metacyclics such as DNA condensation, cytoplasm degradation and mitochondrion and kinetoplast destruction, both in in vitro cultures and in sand fly guts. TUNEL^POS promastigotes were detected only in the anterior midgut to foregut boundary of infected sand flies. Interestingly, caspase inhibitors modulated parasite death and PS exposure, when added to parasite cultures in a specific time window. Efficient in vitro macrophage infections and in vivo lesions only occur when PS^POS and PS-negative (PS^NEG) parasites were simultaneously added to the cell culture or inoculated in the mammalian host. The viable PS^NEG promastigote was the infective form, as shown by following the fate of fluorescently labeled parasites, while the PS^POS apoptotic sub-population inhibited host macrophage inflammatory response. PS exposure and macrophage inhibition by a subpopulation of promastigotes is a different mechanism than the one previously described with amastigotes, where the entire population exposes PS. Both mechanisms co-exist and play a role in the transmission and development of the disease in case of infection by La. Since both processes confer selective advantages to the infective microorganism they justify the occurrence of apoptotic features in a unicellular pathogen.     

Two separate growth phases during the development of Leishmania in sand flies: implications for understanding the life cycle

The life cycle of Leishmania alternates between two main morphological forms: intracellular amastigotes in the mammalian host and motile promastigotes in the sand fly vector. Several different forms of promastigote have been described in sandfly infections, the best known of these being metacyclic promastigotes, the mammal-infective stages. Here we provide evidence that for Leishmania (Leishmania) mexicana and Leishmania (Leishmania) infantum (syn. chagasi) there are two separate, consecutive growth cycles during development in Lutzomyia longipalpis sand flies involving four distinct life cycle stages. The first growth cycle is initiated by procyclic promastigotes, which divide in the bloodmeal in the abdominal midgut and subsequently give rise to non-dividing nectomonad promastigotes. Nectomonad forms are responsible for anterior migration of the infection and in turn transform into leptomonad promastigotes that initiate a second growth cycle in the anterior midgut. Subsequently, leptomonad promastigotes differentiate into non-dividing metacyclic promastigotes in preparation for transmission to a mammalian host. Differences in timing, prevalence and persistence of the four promastigote stages were observed between L. mexicana and L. infantum in vivo, which were reproduced in cultures initiated with lesion amastigotes, indicating that development is to some extent governed by a programmed series of events. A new scheme for the life cycle in the subgenus Leishmania (Leishmania) is proposed that incorporates these findings.     

Stage-Specific Differential Gene Expression of Glutathione Peroxidase in Leishmania Major and Leishmania Tropica

Background:Leishmania (L) major and L. tropica are the etiological agents of cutaneous leishmaniosis. Leishmania species cause a board spectrum of phenotypes. A small number of genes are differentially expressed between them that have likely an important role in the disease phenotype. Procyclic and metacyclic are two morphological promastigote forms of Leishmania that express different genes. The glutathione peroxidase is an important antioxidant enzyme that essential in parasite protection against oxidative stress and parasite survival. This study aimed to compare glutathione peroxidase (TDPX) gene expression in procyclic and metacyclic and also interspecies in Iranian isolates of L. major and L. tropica. Methods:The samples were cultured in Novy-Nicolle-Mc Neal medium to obtain the promastigotes and identified using PCR-RFLP technique. They were then grown in RPMI1640 media for mass cultivation. The expression level of TDPX gene was compared by Real-time PCR.Results:By comparison of expression level, up-regulation of TDPX gene was observed (5.37 and 2.29 folds) in L. major and L. tropica metacyclic compared to their procyclic, respectively. Moreover, there was no significant difference between procyclic forms of isolates, while 3.05 folds up-regulation in metacyclic was detected in L. major compared L. tropica.Conclusion:Our data provide a foundation for identifying infectivity and high survival related factors in the Leishmania spp. In addition, the results improve our understanding of the molecular basis of metacyclogenesis and development of new potential targets to control or treatment and also, to the identification of species-specific factors contributing to virulence and pathogenicity in the host cells.Key Words: Glutathione peroxidase, Leishmania, L. major, L. tropica, Quantitative Real-time PCR     

Infective stages of Leishmania in the sandfly vector and some observations on the mechanism of transmission

Infective stages of Leishmania (Leishmania) amazonensis, capable of producing amastigote infections in hamster skin, were shown to be present in the experimentally infected sandfly vector Lutzomyia flaviscutellata 15, 25, 40, 49, 70, 96 and 120 hours after the flies had received their infective blood-meal. Similarly, infective stages of Leishmania (L.) chagasi were demonstrated in the experimentally infected vector Lu. longipalpis examined 38, 50, 63, 87, 110, 135, 171 and 221 hours following the infective blood-meal, by the intraperitoneal inoculation of the flagellates into hamsters. The question of whether or not transmission by the bite of the sandfly is dependent on the presence of "metacyclic" promastigotes in the mouthparts of the vector is discussed.