Leishmaniasis in Brazil. XXI. Visceral leishmaniasis in the Amazon Region and further observations on the role of Lutzomyia longipalpis (Lutz & Neiva, 1912) as the vector

Further evidence is presented incriminating the sandfly Lutzomyia longipalpis as the vector of Leishmania chagasi, the causative agent of American visceral leishmaniasis, in the Amazon Region of Brazil. During an outbreak of the disease in Santarém, Pará State, this insect was shown to be the only species of sandfly consistently present in and around the patient's homes, where it often occurred in very large numbers. Of 491 specimens dissected, 35 (7.14%) proved to be infected, and isolates of L. chagasi were made from 16 of 27 of these sandflies following the inoculation of the promastigotes into hamsters. Finally, the parasite was transmitted to four other hamsters which had been subjected to the bites of large numbers of wild-caught Lu. longipalpis. Isolates of Leishmania from Lu. longipalpis captures in Santarém, and in another focus of visceral leishmaniasis on the Island of Marajó, Pará, have been shown to be biologically and biochemically indistinguishable from the parasite infecting man, dogs and foxes in Pará, and from stocks obtained from man elsewhere in Brazil (Bahia and Ceará States).     

Leishmaniasis in Brazil. XIX: visceral leishmaniasis in the Amazon Region, and the presence of Lutzomyia longipalpis on the Island of Marajó, Pará State

Sporadic cases of visceral leishmaniasis in Amazonian Brazil appear limited to Pará State, in the lower Amazon valley and principally near the Atlantic coast. The fox Cerdocyon thous (L.) has been incriminated as a natural host of the causative parasite, Leishmania donovani chagasi, but past doubts have existed over the identification of the most likely vector as Lutzomyia (Lutzomyia) longipalpis (Lutz & Neiva, 1912). Investigations on two of five recent cases of visceral leishmaniasis of man in the Districts of Cachoeira do Arari and Salvaterra, on the eastern part of the Island of Marajó, Pará showed undoubted Lu. longipalpis to be abundant in one house and in numerous chicken-houses. This is the first record of Lu. longipalpis on Marajó Island, and the finding supports previous implication of this sandfly in the epidemiology of visceral leishmaniasis in other parts of Pará. Morphological differences have been noted between this insect from Marajó and other specimens from more highly endemic regions in the States of Ceará and Minas Gerais, Brazil.     

Lutzomyia longipalpis naturally infected by Leishmania (L.) chagasi in Várzea Grande, Mato Grosso State, Brazil, an area of intense transmission of visceral leishmaniasis

The American visceral leishmaniasis (AVL) is caused by parasites belonging to the genus Leishmania (Trypanosomatidae) and is transmitted to humans through the bite of certain species of infected phlebotomine sand flies. In this study, we investigated the natural infection ratio of Lutzomyia longipalpis, the main vector species of AVL in Brazil, in Várzea Grande, Mato Grosso State. Between July 2004 and June 2006, phlebotomine sand flies were captured in peridomestic areas using CDC light-traps. Four hundred and twenty (420) specimens of Lu. longipalpis were captured. 42 pools, containing 10 specimens of Lu. longipalpis each, were used for genomic DNA extraction and PCR (polymerase chain reaction) amplification. Leishmania spp. DNA was detected in three out of the 42 pools tested, resulting in a minimal infection ratio of 0.71%. Restriction fragment length polymorphism (RFLP) analysis indicated that Leishmania (L.) chagasi was the infective agent in the positive pools.     

Leishmaniasis in Brazil. XXII: Characterization of Leishmania from man, dogs and the sandfly Lutzomyia longipalpis (Lutz & Neiva, 1912) isolated during an outbreak of visceral leishmaniasis in Santarém, Pará State

During epidemiological studies on an outbreak of visceral leishmaniasis in Santarém, Pará State, north Brazil, isolates of Leishmania from two children, three dogs and six naturally infected specimens of the sandfly Lutzomyia longipalpis were compared, biochemically, by starch-gel enzyme electrophoresis. They have proved to be indistinguishable from each other, and from a reference strain of Leishmania chagasi Cunha & Chagas, 1937 from a case of human visceral leishmaniasis from Bahia State, north-east Brazil, on their enzyme profiles for ASAT, ALAT, PGM, GPI, MDH and MPI. Lu. longipalpis is the principal, and possibly the only vector to man in the Amazon Region of Brazil.     

Natural infection of Lutzomyia longipalpis by Leishmania sp. in Teresina, Piauí State, Brazil

In Brazil, control of the vector Lutzomyia longipalpis is one of the main strategies used to limit the expansion of American visceral leishmaniasis. However, studies on the ecology of this sand fly are rare, especially regarding its natural infection with species of Leishmania. A study of the natural infection of Lu. longipalpis by Leishmania sp. was carried out in the Bela Vista neighborhood in the city of Teresina, Piauí State, Brazil, an important area of American visceral leishmaniasis transmission. From February 2004 to January 2005, sand flies were captured with CDC light traps. Approximately 10 female sand flies in each capture were dissected and examined for the presence of evolutionary forms of Leishmania sp. Two sand fly species were identified: 1,832 were Lu. longipalpis and six Lu. whitmani. Twenty female sand flies (1.1%), all Lu. longipalpis, were infected with procyclic and nectomonad forms of Leishmania sp., found mostly in the hindgut. Higher proportions of infected sand flies were found four months after the rainy season, suggesting that environmental factors may predict not only vector abundance (as already known) but also their level of infection.     

Molecular analysis of natural infection of Lutzomyia longipalpis in an endemic area for visceral leishmaniasis in Brazil

The main purpose of this study was to investigate natural infection by Leishmania chagasi in female sand flies in a visceral leishmaniasis (VL) focus on S?o Luís Island, Maranh?o State, Brazil. Molecular analysis by polymerase chain reaction (PCR) was applied to determine the rate of natural infection of Lutzomyia longipalpis by L. chagasi in areas of old and recent human settlement on S?o Luís Island. Based on a sample of 800 female specimens captured from March to August 2005, the natural infection rate was 1.25% in an area of old settlement and 0.25% in two recently settled areas. Infection of L. longipalpis was detected in both areas, regardless of the number of reported human VL cases, indicating that other factors modulating infection in the wild need to be investigated. The results confirm PCR as a specific technique and an important tool for epidemiological surveillance.     

Mixed infection with Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi in a naturally infected dog from Rio de Janeiro, Brazil

We report here the first case of co-infection with Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi in a naturally infected dog from Rio de Janeiro, Brazil. Isoenzyme characterisation identified the parasites isolated in culture from the cutaneous lesion as L. (V.) braziliensis and the isolates from blood and lymph node as L. (L.) chagasi. PCR analysis using specific primers followed by molecular hybridisation for direct Leishmania species identification in tissue fragments confirmed the presence of L. (V.) braziliensis DNA in the cutaneous lesion and of L. (L.) chagasi DNA in spleen and popliteal lymph node fragments. This report emphasises the importance of identification of Leishmania species infecting seropositive dogs in endemic areas, and the consequent re-assessment of control and epidemiological surveillance measures for the control of leishmaniasis, as is the case in Brazil.     

The FML-vaccine (Leishmune) against canine visceral leishmaniasis: a transmission blocking vaccine

Transmission blocking vaccines are one of the control strategies for vector-transmitted protozoan diseases. Antibodies raised in the vaccinated host prevent the development of the parasite in the insect vector, interrupting the epidemiological cycle. The FML antigen of Leishmania donovani in combination with saponin (FML-vaccine and Leishmune) induced 92-97% of protections against zoonotic visceral leishmaniasis. We assayed the ability of FML to inhibit Leishmania donovani and Leishmania chagasi procyclic promastigote-binding to dissected Lutzomyia longipalpis midguts. We found a dose-dependent inhibition, more pronounced on L. donovani (80%) than on L. chagasi promastigotes (p<0.001). On the other hand, the Fab-IgG serum fraction of Leishmune vaccinated dogs (IgG2 predominant), also inhibited parasite binding in a dose-response (p<0.0001) with an equally potent effect against L. donovani or L. chagasi (p = 0.061). The transmission blocking properties of the Leishmune vaccine was also assessed by an in vivo membrane assay, with sand flies fed with 1.5 x 10(7) amastigotes, human blood and, vaccinated or normal control dog sera. Significantly higher values were found in rate of infection (p<0.025) and intensity of infection (number of parasites/insect) (p<0.05) of control sand flies, making a very reduced infection index (20.7%) in the vaccine group. Our results disclosed that the Leishmune vaccine is a TBV, and that the dog antibodies present in sera, even 12 months after vaccination, lead to a significant effective protection of 79.3%.     

Molecular and Seroepidemiological Survey of Visceral Leishmaniasis among Humans and Domestic Dogs in Mazandaran Province,North of Iran

Background

New cases of visceral leishmaniasis (VL) have been reported recently in some parts of Mazandaran Province, north of Iran where the first human case of VL was reported in 1949. This study aimed to determine the present status of Leishmania infantum infection among humans and domestic dogs using serological and molecular methods in central parts of Mazandaran Province.

Methods

In this cross-sectional study, blood samples were randomly collected from 402 humans and forty-nine domestic dogs throughout 2009 and 2010 in the central part of Mazandaran Province including Semeskadeh and Kiakola districts where recent cases of human visceral leishmaniasis had been reported there. All the collected samples were tested by direct agglutination test (DAT) for the detection of anti-Leishmania infantum antibodies as well as convenience PCR assay on whole blood samples for detection of leishmanial infection and identification of Leishmania species.

Results

None of 402 collected human (402) and dog (49) blood samples showed anti Leishmania infantum antibodies at titers 1:3200 and 1:320 as cut-off values of DAT, respectively but only 2 of domestic dogs (4.1%) were found PCR-positive corresponding to L.infantum.

Conclusion

This study confirms the circulation of L. infantum at least among domestic dogs and highlights the sporadic pattern of VL in the studied areas. Further investigations regarding to sand flies fauna and wild canines as reservoir hosts of the disease, are recommended.     

Standardization of a rapid immunochromatographic test with the recombinant antigens K39 and K26 for the diagnosis of canine visceral leishmaniasis

The serological diagnosis of canine visceral leishmaniasis (CVL) remains problematic because there areno reliable commercially available tests. Most laboratories use domestically prepared tests such as the enzyme-linked immunosorbent assay (ELISA) or the indirect immunofluorescent antibody test (IFAT). We evaluated rapid immunochromatographic (RICH) test kits for the diagnosis of CVL. The tests were assembled with either Leishmania chagasi recombinant antigens K39 or K26 and with either gold-labelled Staphylococcus aureus protein A or Streptococcus pyogenes protein G. Fifty sera from dogs with CVL, 14 sera from dogs with Chagas disease, and 50 sera from normal healthy dogs were tested. The results show that the RICH test using recombinant antigen K39 has a sensitivity of 96% and 100% specificity for the diagnosis of CVL. No significant differences were observed in the tests assembled with either protein A or protein G. The RICH tests using recombinant antigen K26 were equally specific but less sensitive than those using K39. However, the 2 antigens complemented each other and increased the overall sensitivity of the test. Because of its simplicity and performance the RICH test is a quick and reliable alternative for the diagnosis of CVL either in conventional laboratories or for remote areas where laboratories are not readily accessible for conventional assays.     

Atypical American visceral leishmaniasis caused by disseminated Leishmania amazonensis infection presenting with hepatitis and adenopathy

Leishmania amazonensis is widely recognised as a cause of cutaneous leishmaniasis in Latin America, but it can also disseminate to produce atypical visceral leishmaniasis with hepatitis and lymphadenopathy. The patient, an 8-year-old Brazilian boy, presented with a febrile illness and hepatosplenomegaly, elevated liver enzymes and generalised adenopathy. Serological tests using antigens of L. chagasi, the typical cause of visceral leishmaniasis in Latin America, were inconclusive. Leishmania amazonensis was eventually isolated in a culture of a lymph node. The patient recovered fully after treatment with meglumine antimoniate. As this case illustrates, L. amazonensis produces a spectrum of disease that includes atypical American visceral leishmaniasis with evidence of hepatocellular injury and generalised lymphadenopathy.     

Long-lasting protection against canine visceral leishmaniasis using the LiESAp-MDP vaccine in endemic areas of France: double-blind randomised efficacy field trial

Vaccination against visceral leishmaniasis has received limited attention compared with cutaneous leishmaniasis, although the need for an effective vaccine against visceral leishmaniasis is pressing. Dogs constitute the major reservoir of Leishmania infantum/chagasi responsible for human visceral leishmaniasis. We have recently demonstrated that the combination of naturally excreted/secreted antigens, easily purified from culture supernatant of Leishmania infantum promastigotes (LiESAp) as vaccine antigen in formulation with muramyl dipeptide (MDP) as adjuvant, conferred 100% protection to dogs experimentally infected with L. infantum by inducing in vaccinees a significant, stable and long-lasting Th1-type cell response [Lemesre JL, Holzmuller P, Cavaleyra M, Bras Gon?alves R, Hottin G, Papierok G. Protection against experimental visceral leishmaniasis infection in dogs immunised with purified excreted secreted antigens of L. infantum promastigotes. Vaccine 2005; 23:2825-2840; Holzmuller P, Cavaleyra M, Moreaux J, Kovacic R, Vincendeau P, Papierok G, Lemesre JL. Lymphocytes of dogs immunised with purified excreted secreted antigens of L. infantum co-incubated with Leishmania-infected macrophages produce IFN-gamma resulting in nitric oxide-mediated amastigote apoptosis. Vet. Immunol. Immunopathol. 2005, 106:247-257]. In this report, protection against visceral leishmaniasis is investigated in naturally exposed dogs of endemic areas of the South of France vaccinated with LiESAp/MDP vaccine. A double-blind randomised efficacy field trial was developed on a large-scale dog population composed of vaccinees (n=205) and placebo-treated animals (n=209), which were prospectively studied for a 2-year period. 0f the initial 414 enrolled dogs, 340 (175 controls and 165 vaccinees) were analysed for clinical, serological and parasitological studies at 24 months post-vaccination, after two sand fly seasons. Strong seroconversion disclosed by an L. infantum indirect immunofluorescence antibody test (IFAT) associated with suspicious clinical symptoms, considered an indication that the animals had an established progressive infection, was only observed in the placebo group. The seropositive and/or symptomatic dogs were selected for further examination for possible Leishmania infection by culturing parasites from bone-marrow aspirate. The presence of leishmanial infection was also evaluated by means of the PCR analysis of bone marrow samples in all enrolled dogs prior to vaccination and in all evaluated animals (175 controls and 165 vaccinees) at 24 months post-vaccination. After two transmission cycles completed, the Leishmania infection rate was 0.61% (1/165) in vaccinated dogs and 6.86% (12/175) in the placebo group. The efficacy of the vaccine was calculated to be 92% (P=0.002). A clear difference between the dogs that received vaccine and those that received placebo was also established by the results of their immune status. Increased anti-LiESAp IgG2 reactivity and significant enhanced NO-mediated anti-leishmanial activity of canine macrophages in response to higher IFN-gamma production by T cells were almost exclusively revealed in vaccinees. The LiESAp-MDP vaccine induced a significant, long-lasting and strong protective effect against canine visceral leishmaniasis in the field.     

Current epidemiological status of visceral leishmaniasis in Northeastern Brazil

The objective was to describe the current epidemiological status of visceral leishmaniasis in the State of Pernambuco, Northeastern Brazil. For that, a search for scientific production on epidemiology of visceral leishmaniasis in Pernambuco was carried out in electronic databases MEDLINE, SciELO and LILACS. Additionally, relevant papers that have not been retrieved from the searches were analyzed. The 18 studies selected for this review indicate that: visceral leishmaniasis is widely spread in Pernambuco; human cases are often associated to anthropic pressure on the environment; and children have been more frequently affected by the disease. These results showed the need for further studies on risk factors for visceral leishmaniasis in humans, the role of Leishmania chagasi hosts in the zoonotic transmission cycle, and the vector behavior in different geographical regions.     

Lovesongs and period gene polymorphisms indicate Lutzomyia cruzi (Mangabeira, 1938) as a sibling species of the Lutzomyia longipalpis (Lutz and Neiva, 1912) complex

The sand fly Lutzomyia cruzi (Mangabeira, 1938) is implicated as a vector of American visceral leishmaniasis (AVL) in some areas of Brazil. Lutzomyia cruzi is closely related to Lutzomyia longipalpis s.l. (Lutz and Neiva, 1912) the main Latin American vector of AVL and a species complex. Although females of the two species are identical, the males can be distinguished by differences in the genitalia. Nevertheless, pheromone analysis shows that Lu. cruzi males produce 9-methyl-germacrene-B, which has also been found in a number of Latin American populations of Lu. longipalpis s.l. In addition, analysis of microsatellite loci shows that the level of divergence between Lu. cruzi and Lu. longipalpis s.l. is similar to that observed among the Lu. longipalpis s.l. sibling species. Here we present the lovesongs of Lu. cruzi males which are similar to the Burst-type songs produced by one of the Lu. longipalpis s.l. sibling species. We also present data on the molecular polymorphisms of the period gene of Lu. cruzi that indicate this species as another sibling within the Lu. longipalpis complex. The results highlight the importance of an integrative approach to understand the patterns of genetic and phenotypic divergence among very closely related vector species.     

Heterologous prime-boost vaccination with a non-replicative vaccinia recombinant vector expressing LACK confers protection against canine visceral leishmaniasis with a predominant Th1-specific immune response

Leishmaniasis caused by Leishmania infantum is a severe endemic disease in the Mediterranean basin, being domestic dogs the main reservoir of the disease that plays a key role in the transmission to humans. Studies on vaccines against canine leishmaniasis, aimed to modify the T cell repertoire, have advanced in recent years. LACK vaccination assays, using protein or DNA vectors, show protection against cutaneous L. major infections by redirecting the early IL-4 responses to a protective Th1 response. The aim of this study was to define the effectiveness and type of immune response in a canine visceral leishmaniasis model of two poxvirus vectors (Western reserve strain, WR and modified vaccinia virus Ankara, MVA) expressing the LACK protein of L. infantum in prime/boost vaccination protocols. The results obtained showed that dog vaccination priming with DNA-LACK followed by a booster with MVA-LACK or rVV-LACK triggered a Th1 type of immune response, leading to protection against canine visceral leishmaniasis. This protection correlated with absence of visceral leishmaniasis symptoms, lower Leishmania-specific antibodies, higher degree of T cell activation in Leishmania-target organs and higher synthesis of Th1 cytokines. In addition, we found that dogs boosted with the non-replicative virus show less VL symptoms and higher degree of T cell activation, providing evidences for a clear advantage of MVA-LACK as a vaccination vector against canine visceral leishmaniasis.     

Leishmaniasis in domestic dogs: epidemiological aspects

In Barra de Guaratiba, an endemic area for American visceral leishmaniasis (AVL) in Rio de Janeiro, Brazil, control campaigns were unable to reduce canine infection rates. This difficulty prompted an in-depth study of dogs as a reservoir for Leishmania chagasi in the peri-urban environment through clinical and serological follow-up using the immunofluorescence and Western blot techniques. Recognition of 29 kDa and 32 kDa peptides by sera from dogs with proven L. chagasi infection was observed. Furthermore, only sera from symptomatic dogs recognized the 68.5 kDa antigen, so the latter should be considered a parameter for culling dogs from endemic areas. The WB technique proved to be more sensitive than IFA, since the 29 and 32 kDa peptide fractions were even recognized by sera from AVL seronegative dogs up to 8 months before IFA seroconversion. Proximity to wooded areas was an important risk factor for L. chagasi infection in dogs, possibly due to the presence of wild reservoirs.     

A real-time PCR assay to estimate Leishmania chagasi load in its natural sand fly vector Lutzomyia longipalpis

Leishmania chagasi, transmitted mainly by Lutzomyia longipalpis sand flies, causes visceral leishmaniasis and atypical cutaneous leishmaniasis in Latin America. Successful vector control depends upon determining vectorial capacity and understanding Leishmania transmission by sand flies. As microscopic detection of Leishmania in dissected sand fly guts is laborious and time-consuming, highly specific, sensitive, rapid and robust Leishmania PCR assays have attracted epidemiologists' attention. Real-time PCR is faster than qualitative PCR and yields quantitative data amenable to statistical analyses. A highly reproducible Leishmania DNA polymerase gene-based TaqMan real-time PCR assay was adapted to quantify Leishmania in sand flies, showing intra-assay and inter-assay coefficient variations lower than 1 and 1.7%, respectively, and sensitivity to 10 pg Leishmania DNA ( approximately 120 parasites) in as much as 100 ng sand fly DNA. Data obtained for experimentally infected sand flies yielded parasite loads within the range of counts obtained by microscopy for the same sand fly cohort or that were around five times higher than microscopy counts, depending on the method used for data analysis. These results highlight the potential of quantitative PCR for Leishmania transmission studies, and the need to understand factors affecting its sensitivity and specificity.     

Seroprevalence of Leishmania infantum in a rural area of Senegal: analysis of risk factors involved in transmission to humans

Whereas Leishmania infantum, the agent of visceral leishmaniasis (VL), is well known in North Africa, very limited data exist on its spread in West Africa, where mainly cutaneous leishmaniasis has been widely reported. Nevertheless, dogs infected with L. infantum were recently found in the Mont Rolland District in Senegal. To provide a better understanding of L. infantum epidemiology in this area, clinical and serological surveys were carried out to determine the seroprevalence of L. infantum-specific antibodies in the human population. In parallel, an analysis of environmental and individual factors associated with Leishmania antigen seropositivity was conducted to identify potential risk factors for exposure. Although no cases of VL were detected within this study, a large part of the population (73/315; 23%) was exposed to infection, with a strong age effect (being >40 years old increased the risk of being seropositive). Moreover, the presence of Nebedaye trees (Moringa oleifera) and infected dogs in the household were factors increasing the risk of exposure in household members. These results may provide important information to identify the still unknown sandfly species involved in transmission.     

Isoenzymatic polymorphism of Leishmania infantum in southern Spain

Over a period of more than 10 years we have isolated and classified 161 Leishmania strains from cases of human visceral, cutaneous and mucosal leishmaniasis in immunocompetent subjects, from cases of visceral leishmaniasis in immunocompromised individuals with HIV, from dogs with leishmaniasis (visceral and cutaneous), from Rattus rattus and from sandflies. The strains were all L. infantum, the only species endemic in Spain, and corresponded to 20 different zymodemes. We describe the life cycle of these zymodemes for which, in most cases, we only partially know the hosts involved. We also discuss possible reasons for the greater polymorphism of L. infantum in southern Spain.     

Leishmanization revisited: Immunization with a naturally attenuated cutaneous Leishmania donovani isolate from Sri Lanka protects against visceral leishmaniasis

Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa and associated with three main clinical presentations: cutaneous, mucocutaneous and visceral leishmaniasis. Visceral leishmaniasis is the second most lethal parasitic disease after malaria and there is so far no human vaccine. Leishmania donovani is a causative agent of visceral leishmaniasis in South East Asia and Eastern Africa. However, in Sri Lanka, L. donovani causes mainly cutaneous leishmaniasis, while visceral leishmaniasis is rare. We investigate here the possibility that the cutaneous form of L. donovani can provide immunological protection against the visceral form of the disease, as a potential explanation for why visceral leishmaniasis is rare in Sri Lanka. Subcutaneous immunization with a cutaneous clinical isolate from Sri Lanka was significantly protective against visceral leishmaniasis in BALB/c mice. Protection was associated with a mixed Th1/Th2 response. These results provide a possible rationale for the scarcity of visceral leishmaniasis in Sri Lanka and could guide leishmaniasis vaccine development efforts.