The status of the Lutzomyia longipalpis species complex and possible implications for Leishmania transmission

The sand fly Lutzomyia longipalpis sensu latu has been identified as the principal vector of American visceral leishmaniasis, a potentially fatal disease that primarily affects children in several countries of South and Central America. Over the past several years increases have occurred both in the number of reported cases and the population at risk: approximately 1.6 million people reside in highly endemic areas with 16,000 cases reported annually. Several studies have attempted to relate the epidemiology of this disease to variability in Lu. longipalpis that is now recognized to be a complex of at least three sibling species. Morphological variation in this species was first noted by Mangabeira (1969). Since then physiological and biochemical differences have been reported by several investigators. Recent reports in Costa Rica of the presence of Lu. longipalpis in a focus of cutaneous leishmaniasis caused by Leishmania chagasi may be an additional indication of variability in this species. While existing evidence indicates that the morphospecies Lu. longipalpis may represent a complex of sibling species, genetic, epidemiological and ecological distinctions have not been fully resolved. Thus, delimitation of systematic boundaries within the complex and corresponding to geographic distributions and roles in transmission remain unresolved. The purpose of this review is to summarize from the literature observations of polymorphism in this morphospecies and consider what significance this reported variability may have to the epidemiology of visceral leishmaniasis.

Palaearctic origin of Leishmania

The hypothesis of a Palaearctic origin of Leishmania in the early Cenozoic, dispersal to the Nearctic in the late Eocene and to the Neotropical in the Pliocene is presented. It is further hypothesized that murid rodents and their immediate ancestors have been important mammalian reservoirs since the origination of Leishmania. Biochemical, molecular, biogeographical, entomological, mammalalogical and ecological support for these hypotheses are reviewed.

Establishment and characterization of a new continuous cell line from Lutzomyia longipalpis (Diptera: Psychodidae) and its susceptibility to infections with arboviruses and Leishmania chagasi

Embryonic tissue explants of the sand fly Lutzomyia longipalpis (Lutz & Neiva 1912) the main vector of Leishmania chagasi (Cunha and Chagas), were used to obtain a continuous cell line (Lulo). The tissues were seeded in MM/VP12 medium and these were incubated at 28ºC. The first subculture was obtained 45 days after explanting and 96 passages have been made to date. Lulo is composed of epithelioid cells, showed a 0.04 generations/hour exponential growth rate and population doubling time at 24.7 h. The cell line isoenzymatic profiles were determined by using PGI, PGM, MPI and 6-PGDH systems, coinciding with patterns obtained from the same species and colony's pupae and adults. The species karyotype characteristics were recognized (2n = 8), in which pair 1 is subtelocentric and pairs 2, 3 and 4 are metacentric. Lulo was free from bacterial, fungal, mycoplasmic and viral infection. Susceptibility to five arbovirus was determined, the same as Lulo interaction with Leishmania promastigotes.

Canine experimental infection: intradermal inoculation of Leishmania infantum promastigotes

Five mixed breed dogs were inoculated intradermally (ID) with cultured virulent stationary phase promastigotes of Leishmania infantum Nicole, 1908 stocks recently isolated. Parasite transformations in the skin of ID infected dogs were monitored from the moment of inoculation and for 48 h, by skin biopsies. Anti-Leishmania antibody levels were measured by indirect immunofluorescence assay, counterimmunoelectrophoresis and direct agglutination test, and clinical conditions were examined. Thirty minutes after ID inoculation the first amastigotes were visualised and 3 to 4 h after inoculation the promastigotes were phagocyted by neutrophils and by a few macrophages. These cells parasitised by amastigotes progressively disappeared from the skin and 24 h after inoculation parasites were no longer observed. Local granulomes were not observed, however, serological conversion for antibodies anti-Leishmania was achieved in all dogs. Direct agglutination test was the only technique positive in all inoculated dogs. Amastigotes were found in the popliteal lymph node in one dog three months after inoculation. This work demonstrates that, with this inoculum, the promastigotes were transformed into amastigotes and were up taken by neutrophils and macrophages. The surviving parasites may have been disseminated in the canine organism, eliciting a humoral response in all cases.

Evidence for a neotropical origin of Leishmania

Contradictory biogeographic hypotheses for either a Neotropical or a Palaearctic origin of the genus Leishmania have been proposed. Hypotheses constructed on the basis of biogeographic data must be tested against an independent dataset and cannot be supported by biogeographic data alone. In the absence of a fossil record for the Leishmania these two hypotheses were tested against a combined dataset of sequences from the DNA polymerase A catalytic subunit and the RNA polymerase II largest subunit. The phylogeny obtained provided considerable support for a Neotropical origin of the genus Leishmania and leads us to reject the hypothesis for a Palaearctic origin.

Further support for a palaearctic origin of Leishmania

The fossil record and systematics of murid rodents, reservoirs of zoonotic cutaneous leishmaniasis in the Palaearctic, Oriental, African, Nearctic and Neotropical, strongly support a Palaearctic origin of Leishmania. The fossil record and systematics of phlebotomine sand flies reinforce this idea. Interpretations of molecular data that place the origin of Leishmania in the Neotropical are inconsistent with the natural histories of reservoirs and vectors. The evolutionary pattern of New World rats (Sigmodontinae) indicates that they may be the most important reservoirs of zoonotic cutaneous leishmaniasis throughout their range.

Speculations on the origin and evolution of the genus Leishmania

Recently two hypotheses have been proposed for the evolution of Leishmania involving respectively a Neotropical or Paleartic origin for the species. Here an alternative proposal on the phylogeny of Leishmania based on the major divisions within the genus is presented. In this hypothesis a Neotropic origin is retained for L. (Viannia) and Paraleishmania, a recently desribed section within the genus Leishmania, while an African origin is proposed for L. (Leishmania) and possibly Sauroleishmania. The current distribution of Leishmania in the Neotropics is explained as the product of multiple introductions of Leishmania parasites into the New World. Problems with organismal identity in Sauroleishmania and the use of molecular sequence data in inferring phylogenies are also discussed.

Sand fly evolution and its relationship to Leishmania transmission

The evolutionary relationships of sand flies and Leishmania are discussed in this report, which draws distinctions between co-association, co-evolution and co-speciation (or co-cladogenesis). Examples focus on Phlebotomus vectors of Le. infantum and Le. major in the Mediterranean subregion.

Acidic ribosomal proteins and histone H3 from Leishmania present a high rate of divergence

Another additional peculiarity in Leishmania will be discussed about of the amino acid divergence rate of three structural proteins: acidic ribosomal P1 and P2b proteins, and histone H3 by using multiple sequence alignment and dendrograms. These structural proteins present a high rate of divergence regarding to their homologous protein in Trypanosoma cruzi. At this regard, L. (V.) peruviana P1 and T. cruzi P1 showed 57.4% of divergence rate. Likewise, L. (V.) braziliensis histone H3 and acidic ribosomal P2 protein exhibited 31.8% and 41.7% respectively of rate of divergence in comparison with their homologous in T. cruzi.

Retention of Leishmania (Leishmania) Mexicana in naturally infected rodents from the State of Campeche, Mexico

In the State of Campeche, Mexico, zoonotic cutaneous leishmaniasis is mainly due to Leishmania (L.) mexicana. The parasite population is maintained in a mammalian species, a reservoir in which the ideal course of infection should be long and relatively nonpathogenic. The objective of the present study was to document the retention of L. (L.) mexicana in 29 naturally infected rodents. These cricetids lived in captivity for up to two years and were tested monthly for the presence of the parasite, by cultures of needle aspirates from the base of the tail. Peromyscus yucatanicus and Ototylomys phyllotis were incriminated as the primary reservoir hosts. The finding that the multiplication of parasites in P. yucatanicus might be triggered by temperature, suggests that this animal would be a good choice for further research on L. (L.) mexicana.

Visceral Leishmaniosis caused by Leishmania (L.) mexicana in a Mexican patient with human immunodeficiency virus infection

A 36 year old male was admitted in December 1997 to hospital with afternoon fever, malaise and hepatosplenomegaly. He also had a dry cough, dyspnoea and anaemia. Pneumonia caused by Pneumocystis carinii and human immunodeficiency virus (HIV) infection were documented. The HIV infection was confirmed in 1997 with 290,000 virus copies. The patient had been in the Mexican State of Chiapas which is known to be endemic for visceral leishmaniosis (VL) and localized cutaneous leishmaniosis (LCL). The visceral symptoms were diagnosed as VL and the causal agent was identified as Leishmania (L.) mexicana. Identification of Leishmania was carried out by the analysis of amplified DNA with specific primers belonging to the Leishmania subgenus and by dot blot positive hybridisation of these polymerase chain reaction derived products with kDNA from the L. (L.) mexicana MC strain used as probe. This is the first case in Mexico of VL caused by a species of Leishmania that typically produces a cutaneous disease form.

Cutaneous leishmaniasis caused by members of Leishmania braziliensis complex in Nayarit, State of Mexico

An epidemiological study was carried out in the northern Mexican state, Nayarit. Fourteen patients with possible cutaneous leishmaniasis skin lesions gave positive Montenegro skin tests. Biopsies were taken from the skin ulcer and analyzed by polymerase chain reaction (PCR) with specific primers for the Leishmania mexicana complex; however all biopsies were not amplified. PCR carried out with specific primers for the L. braziliensis complex resulted in the amplification of all patient DNA. DNA from 12 out of 14 biopsies gave positive amplification with primers species specific for L. (Viannia) braziliensis and hybridized with a species specific L. (V.) braziliensis probe. These results demonstrate the presence in Nayarit of at least two members of the L. braziliensis complex. Most of the cutaneous lesions were caused by L. (V.) braziliensis and two by another species belonging to the L. braziliensis complex. As far as we are aware, this is the first report of L. (V.) braziliensis in Nayarit. The main risk factor associated with the contraction of this disease in Nayarit is attributed to working on coffee plantations.

Clinical picture of cutaneous leishmaniases due to Leishmania (Leishmania) mexicana in the Yucatan peninsula, Mexico

Localized cutaneous leishmaniasis (LCL), known as "chiclero's ulcer" in southeast Mexico, was described by Seidelin in 1912. Since then, the sylvatic region of the Yucatan peninsula has been identified as an endemic focus of LCL. The purpose of the present work was to describe the clinical picture of LCL caused by Leishmania (Leishmania) mexicana in the Yucatan peninsula. A total of 136 cases of LCL, based on isolation and characterization of L. (L.) mexicana by isoenzymes and/or monoclonal antibodies, were selected. Some variability of clinical features regarding number, type, size, form, location and time of evolution of the lesions was observed. The most frequently observed presentation was a single, ulcerated, rounded small lesion, located on the ear, with an evolution time of less than three months, with neither cutaneous metastases nor lymphatic nor mucosal involvement. This picture corresponds to previous studies carried out in the same endemic area where an organism of the L. mexicana complex has been incriminated as a major aetiological agent of classical "chiclero's ulcer", confirming that in the Yucatan peninsula LCL due to L. (L.) mexicana when located on the pinna of the ear is a remarkable characteristic.

Effect of a second bloodmeal on the oesophagus colonization by Leishmania mexicana complex in Lutzomyia evansi (Diptera: Psychodidae)

Migration and colonization of the oesophagus by Leishmania mexicana parasites were enhanced after digestion of a second bloodmeal intake in Lutzomyia evansi. This event has epidemiological significance since it affects the infection susceptibility of this sand fly species, which is a proven vector of L. chagasi in Colombian and Venezuelan visceral leishmaniasis foci. Also, it may explain the host seeking behaviour displayed by some partially bloodfed flies found inside houses.