Detection of Leishmania infantum DNA in the non-parasitized lung of dogs with visceral leishmaniasis

Background

Despite the very low or absent parasitism in the lungs, the interstitial pneumonitis is a common lesion found in humans and dogs with visceral leishmaniasis. The lung is a neglected organ in the study of dogs and humans with visceral leishmaniasis, but interstitial pneumonitis represents an important lesion characterized by thickening of the alveolar septum due to fibrosis and inflammatory exudate, and its pathogenesis is still uncertain. In this study, the polymerase chain reaction (PCR) was used to detect Leishmania infantum in paraffin-embedded lung biopsies from naturally infected dogs from an endemic area in Minas Gerais State, Brazil; PCR was compared to histological and immunohistochemical techniques for detecting Leishmania.

Results

Eighteen dogs in which leishmaniasis had been diagnosed by serological tests - indirect immunofluorescence assay (IFAT), enzyme-linked immunosorbent assay (ELISA) and complement fixation tests (CFT) - were classified as asymptomatic, oligosymptomatic or symptomatic. Nine of the 18 dogs studied had a positive PCR (50%) but parasites were not detected by histopathological and immunocytochemistry methods.

Conclusions

These data indicate that PCR on DNA extracted from paraffin-embedded tissue is a valuable method for detecting Leishmania infantum parasites in lungs of naturally infected dogs, despite the apparent absence of parasites from standard HE (hematoxylin and eosin) stained slides and of labeled parasites from immunocytochemical preparations.

     

Leishmania chagasi/infantum: further investigations on Leishmania tropisms in atypical cutaneous and visceral leishmaniasis foci in Central America

In Central America, apparently genetically identical Leishmania chagasi/infantum parasites cause cutaneous (CL) and visceral leishmaniasis (VL), the latter being more frequent in young children. The present study investigated if there were pathology-related differences in virulence between Honduran CL and VL strains using Mediterranean L. infantum strains as a reference. Macrophage infectivity and serum sensitivity, properties thought to be associated with virulence, were similar between CL and VL strains from both regions. Attention focused on the genome organisation of genes for two candidate virulence factors: Leishmania mitogen activated protein kinase (LMPK) and cysteine proteinase b (Cpb). Interestingly, the Mediterranean strains exhibited restriction enzyme polymorphisms associated with tropism for both LMPK and Cpb genes whereas no differences were observed for the Honduran strains. We also report relative genetic homogeneity of the Honduran strains as compared to the Mediterranean strains and discuss it in terms of the probable origin for the Central American L. chagasi/infantum.     

Courtship behaviour in the sandfly Lutzomyia longipalpis, the New World vector of visceral leishmaniasis

Courtship behaviour in Lutzomyia longipalpis Lutz and Neiva (Diptera: Psychodidae) was examined to determine the sequence of behaviours that occur prior to copulation. Courtship consisted of a series of male and female touching and wing-flapping behaviours, with males performing a greater variety of wing-flapping behaviours than previously described. Occurrence of male approach-flapping, semi-circling and female stationary-flapping were all predictors of eventual copulation, and may coincide with the dispersal of pheromones or the production of auditory signals important to courtship. Touching occurred in the majority of observations, with contact most often made with the tips of the legs and antennae. This behaviour, not previously described in sandflies, was initiated by males and females, and may indicate the use of contact pheromones, a form of communication previously overlooked in L. longipalpis. Future studies are required to separate the auditory and chemical signals associated with wing-flapping, and to confirm whether L. longipalpis possesses cuticular hydrocarbons capable of inducing behavioural responses. The identification of signals that inhibit sexual behaviour during courtship may be particularly relevant to developing mating disruption techniques against L. longipalpis.     

Xenodiagnosis using Lutzomyia youngi in Venezuelan cases of cutaneous leishmaniasis due to Leishmania braziliensis

Eight patients infected with Leishmania braziliensis were used for xenodiagnosis with Lutzomyia youngi, before and after specific antileishmanial treatment with "glucantime" and "gabbromycin". All of them infected sandflies fed on the borders of the skin lesions before the treatment, suggesting that infected persons might act as reservoirs of infection for an indoor-biting sandfly species. The negative results obtained by xenodiagnosis carried out after specific treatment of the same individuals indicated cure of the patients, and a reduction of risk for further intradomiciliary transmission.     

A suspected new species of Leishmania, the causative agent of visceral leishmaniasis in a Thai patient

A suspected new species of Leishmania is described as the causative agent of the third reported case of autochthonous visceral leishmaniasis in a Thai man living in Southern Thailand. The results of PCR-restriction fragment length polymorphism and sequence analysis of the internal transcribed spacer 1 of ssrRNA and the mini-exon genes were different from those of previously reported Leishmania species. A direct agglutination test (DAT) revealed that antibody against Leishmania infection was detected in nine domestic cats. No potential vectors could be identified. A large-scale epidemiological survey of leishmaniasis should be urgently conducted since visceral leishmaniasis is considered an emerging disease of public health concern in Thailand.     

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.     

Dispersal pattern of the sand fly Lutzomyia neivai (Diptera: Psychodidae) in a cutaneous leishmaniasis endemic rural area in Southeastern Brazil

The dispersal pattern of the sand fly Lutzomyia neivai was studied through mark-release-recapture experiments in an American cutaneous leishmaniasis endemic rural area in Southeastern Brazil. Over 6500 specimens were marked with fluorescent powder and released in forest edge and peridomicile habitats from August to November 1999, February and April 2000. Recapture attempts were made using Shannon and CDC traps up to eight successive nights after releases. A total of 493 (7.58%) specimens were recaptured. The number of recaptured males and females of L. neivai in CDC traps was not affected by the distance between the trap and the release points. Approximately 90% of males and females recaptured in CDC traps were caught up to 70 m from the release points. The maximum female flight range recorded was 128 m. The average flight range per day was less than 60 m for males and females. Of the flies released in forest edge, approximately 16% of the recaptured females were caught in Shannon traps in the peridomicile habitat. The results indicate that the movements of L. neivai are spatially focal and the possibility of dispersion from forest to peridomicile habitat may be an important way of contracting leishmaniasis in dwellings.     

Development and evaluation of Leishmania infantum rK26 ELISA for serodiagnosis of visceral leishmaniasis in Iran

The purpose of this study was to prepare recombinant K26 antigen from Leishmania infantum and evaluate its performance by enzyme-linked immunosorbent assay (ELISA) test for serodiagnosis of visceral leishmaniasis (VL) in endemic regions of Iran. The results were compared with those obtained by direct agglutination test (DAT) and whole cell ELISA using crude parasite antigen. Of 93 sera from patients with confirmed VL, 90 sera were positive with rK26 ELISA (sensitivity=96.8%), whereas 85 sera were positive with DAT (sensitivity=91.4%) and 89 sera were positive with whole cell ELISA (sensitivity=95.7%). Of 130 subjects who either had other infectious diseases (n=30) or were healthy (n=100), rK26 ELISA were negative in all cases (specificity=100%), whereas DAT were negative in 116 cases (specificity=89.2%) and whole cell ELISA was negative in 114 cases (specificity=87.7%). The results of this study indicate that the rK26 ELISA is more sensitive and specific than conventional methods and could be used for reliable diagnosis of VL caused by Leishmania infantum.     

Potential distributions of phlebotomine sandfly vectors of human visceral leishmaniasis caused by Leishmania infantum in Morocco

Zoonotic visceral leishmaniasis is a common vector-borne systemic disease caused by Leishmania infantum (Kinetoplastida: Trypanosomatidae). In Morocco the situation is complex: many sandfly species have been collected in areas in which the disease is endemic, but only Phlebotomus ariasi, Phlebotomus perniciosus and Phlebotomus longicuspis (Diptera: Psychodidae) have been confirmed to have vectorial roles. The objective of the present study was to ascertain the potential distribution of L. infantum and its vectors in Morocco, using ecological niche modelling. Vector records were obtained from field collections of the Laboratory team and from previously published entomological observations. Epidemiological data for L. infantum modelling were obtained from Moroccan Ministry of Health reports. The jackknife test indicated that the bioclimatic variables with the greatest influence on model development for all species were annual precipitation and precipitation in the driest quarter of the year. MaxEnt model representations for sandfly species that act as vectors of L. infantum showed the widespread geographic distribution of these species in Morocco, specifically in northern and central Morocco, where foci of visceral leishmaniasis are found. The ecological niche modelling points out areas in which the probability of occurrence of these species is higher. This information should be considered as a starting point for further research to fully elucidate the ecology and epidemiology of these species, as well as of the pathogens they transmit.     

Preliminary description of a new entomoparasitic nematode infecting Lutzomyia longipalpis sand fly,the vector of visceral leishmaniasis in the New World

Phlebotomine sandflies are vectors of important pathogens world-wide, including Leishmania spp. in the Neotropics. Entomoparasites have been described from phlebotomines, including virus, bacteria, protozoa, fungi, nematodes, and mites, some of which are capable of killing the host. In the present study, interference, fluorescence, and scanning electron microscopies were used for the first time to detect and morphologically characterize a new entomoparasite infecting Lutzomyia longipalpis. Several filiform larvae and eggs in different stages were encountered in the abdomen of female and male insects. Pairs of large egg-bearing nematodes found within cyst-like structures or free in the hemocel accompanied by larvae could be the adult sexual stages. This entomoparasite infects sand flies naturally in the field. We believe that stress caused by the colonization procedure produced an increase in the infection rate among sand flies affecting their development. These findings could be applied to future biological control studies of sand fly vectors.     

Leishmania (L.) infantum chagasi isolated from skin lesions of patients affected by non-ulcerated cutaneous leishmaniasis lead to visceral lesion in hamsters

In Central America, Leishmania (L.) infantum chagasi infection causes visceral leishmaniasis (VL) and non-ulcerated cutaneous leishmaniasis (NUCL). The aim of the present study was to evaluate the course of an experimental infection in hamsters caused by L. (L.) infantum chagasi isolated from patients affected by NUCL compared with a strain isolated from a patient with VL. Stationary phase parasites in culture were inoculated through subcutaneous and intraperitoneal routes in hamsters. Following the post-infection times, a histopathological study, parasite load and cytokine determination in skin from the cutaneous inoculation site and viscera were performed. Animals subcutaneously infected with the different strains did not develop macroscopic lesions at the inoculation site, and the histopathological changes in the dermis were very slight. Regarding the histopathological study of the viscera, we observed the portal mononuclear inflammatory infiltrate, the presence of nodules in the hepatic parenchyma and the proliferation of macrophages in the spleen, which increased over the infection course. Overall, the parasite load in the liver and spleen and in the total IgG titres in the sera of infected hamster showed an increase with the time of infection, regardless of the route of inoculation. Regarding cellular immunity, we did not observe an increase or decrease in pro- and anti-inflammatory cytokines compared to the healthy control, except for IL-10, which was evident in the infected animals. The data showed that strains isolated from NUCL cause visceral lesions in the hamsters regardless of the route of inoculation, and they were similar to parasites isolated from VL humans.     

Enhanced attraction of sand fly vectors of Leishmania infantum to dogs infected with zoonotic visceral leishmaniasis

BackgroundThe sand fly Phlebotomus perniciosus is the main vector of Leishmania infantum, etiological agent of zoonotic visceral leishmaniasis in the Western Mediterranean basin. Dogs are the main reservoir host of this disease. The main objective of this study was to determine, under both laboratory and field conditions, if dogs infected with L. infantum, were more attractive to female P. perniciosus than uninfected dogs.Methodology/Principal findingsWe carried out a series of host choice experiments and found that infected dogs were significantly more attractive to P. perniciosus than uninfected dogs in the laboratory as well as in the field. Significantly more P. perniciosus fed on infected dogs than on uninfected dogs. However, the fecundity of P. perniciosus fed on infected dogs was adversely impacted compared to uninfected dogs by lowering the number of laid eggs. Phlebotomus perfiliewi, the second most abundant sand fly species in the field site and a competent vector of L. infantum had similar trends of attractivity as P. perniciosus toward infected dogs under field conditions.ConclusionsThe results strongly suggest that L. infantum causes physiological changes in the reservoir host which lead to the host becoming more attractive to both male and female P. perniciosus. These changes are likely to improve the chance of successful transmission because of increased contact with infected hosts and therefore, infected dogs should be particularly targeted in the control of zoonotic visceral leishmaniasis in North Africa.     

Metagenomic analysis of taxa associated with Lutzomyia longipalpis, vector of visceral leishmaniasis, using an unbiased high-throughput approach

Background

Leishmaniasis is one of the most diverse and complex of all vector-borne diseases worldwide. It is caused by parasites of the genus Leishmania, obligate intramacrophage protists characterised by diversity and complexity. Its most severe form is visceral leishmaniasis (VL), a systemic disease that is fatal if left untreated. In Latin America VL is caused by Leishmania infantum chagasi and transmitted by Lutzomyia longipalpis. This phlebotomine sandfly is only found in the New World, from Mexico to Argentina. In South America, migration and urbanisation have largely contributed to the increase of VL as a public health problem. Moreover, the first VL outbreak was recently reported in Argentina, which has already caused 7 deaths and 83 reported cases.

Methodology/Principal Findings

An inventory of the microbiota associated with insect vectors, especially of wild specimens, would aid in the development of novel strategies for controlling insect vectors. Given the recent VL outbreak in Argentina and the compelling need to develop appropriate control strategies, this study focused on wild male and female Lu. longipalpis from an Argentine endemic (Posadas, Misiones) and a Brazilian non-endemic (Lapinha Cave, Minas Gerais) VL location. Previous studies on wild and laboratory reared female Lu. longipalpis have described gut bacteria using standard bacteriological methods. In this study, total RNA was extracted from the insects and submitted to high-throughput pyrosequencing. The analysis revealed the presence of sequences from bacteria, fungi, protist parasites, plants and metazoans.

Conclusions/Significance

This is the first time an unbiased and comprehensive metagenomic approach has been used to survey taxa associated with an infectious disease vector. The identification of gregarines suggested they are a possible efficient control method under natural conditions. Ongoing studies are determining the significance of the associated taxa found in this study in a greater number of adult male and female Lu. longipalpis samples from endemic and non-endemic locations. A particular emphasis is being given to those species involved in the biological control of this vector and to the etiologic agents of animal and plant diseases.     

Isolation of Leishmania infantum, zymodeme MON-1 from canine and human visceral leishmaniasis on Margarita Island, Venezuela.

An increase in the incidence of human visceral leishmaniasis (HVL) has been detected in recent years on Margarita Island, located off the NE coast of Venezuela. Recent studies have revealed reactivity to rK39 antigen (Leishmania chagasi) in 20% of 541 sera from domestic dogs in endemic communities; PCR reactions were positive using primers for the L. donovani complex. Here we report that isolates from human and canine infection, identified by isoenzyme analysis, correspond to L. infantum, zymodeme MON-1. This appears to be the first isolation and identification of an isolate from HVL on Margarita Island and demonstrates the presence of this zymodeme in the canine population.     

Schizodeme and zymodeme characterization of Leishmania in the investigation of foci of visceral and cutaneous leishmaniasis

Leishmania parasites were isolated from humans and canines in foci of cutaneous and visceral leishmaniasis. After in vitro cultivation the parasites were examined by the following biochemical techniques: (i) restriction analysis of kinetoplast DNA (kDNA) also known as schizodeme analysis (Morel et al., 1980); (ii) zymodeme analysis (Barret et al., 1980); by agarose gel electrophoresis and (iii) isoelectricfocusing in polyacrylamide gels. The strains of cutaneous and visceralizing leishmanias studied could be differentiated by schizodeme analysis, using the endonuclease MspI, into three complexes agreeing with those accepted for human New World leishmaniasis. In the municipality of Rio de Janeiro, isolates from a focus of cutaneous leishmaniasis were identified as L. braziliensis braziliensis and from a focus of visceral leishmaniasis were identified as L. donovani by zymodeme characterization. Identical restriction enzyme profiles of kDNA from human and canine isolates indicated that in the cutaneous focus at Jacarepaguá, Rio de Janeiro, the same strain was probably circulating in both the canine and human populations. This suggests a possible role for dogs as a reservoir host for L. braziliensis braziliensis. In addition, our results confirm the importance of dogs as reservoirs in visceral leishmaniasis. The stability of the electrophoretic patterns of restriction digest ("fingerprints") of Leishmania kDNA as well as differences in the sensitivity of the techniques used were demonstrated. Strains from widely different geographical areas as well as strains maintained in vivo and in vitro showed identical kDNA restriction patterns, while strains showing similar banding patterns by enzyme electrophoresis could be differentiated by schizodeme analysis. These results demonstrate the usefulness of an integrated biochemical approach in the identification of Leishmania.