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The little villains of leishmaniosis

Wild rats are the hosts for the microbes of this tropical disease

A study developed over the last five years under the coordination of the parasitologist Jeffrey Jon Shaw, of the University of São Paulo (USP), has proven for the first time that two little rodents – the South American Field [rato-do-mato] (Bolomys lasiurus) and the Black Rat (Rattus Rattus) – are the wild animals that naturally carry the protozoan Leishmania (Viannia) braziliensis, which causes American Tegumentary Leishmaniosis (ATL). This is the most common form of leishmaniosis in human beings in this country.

The discovery allows for a better understanding of the parasite’s life cycle and its transmission from its natural host to man. It also opens up the way for new proposals of combating leishmaniosis, since one of the factors which inhibited control measures was the lack of information on the wild hosts of L. (V.) braziliensis, a species of protozoan found throughout the country. The identification of the hosts, also called reservations, is important as it is in them that the parasite finds the conditions to reproduce.

Toxic medicines
The ATL is an disease of lengthy and difficult treatment, since it requires very toxic medicines: pentavalent Antimony compounds and amphotericin B, which can cause acute renal inefficiency and pentamidine, associated with the beginning of mellitus diabetes. The ATL can show itself in two forms: cutaneous and mucocutaneous. The first provokes injuries to the skin, in varied sizes and number – from a lesion the size of a pimple to large ulcerations. The mucocutaneous form is more aggressive: it destroys mucous membranes and cartilage of the mouth, nose and throat, causing deformities, though only in rare cases is the disease fatal.

For the World Health Organization (WHO), leishmaniosis is one of the six most important parasite-infection illnesses. It is calculated that there are1.5 million new cases per year and that there exist 12 million contaminated people throughout the world. In Brazil, it is a public health problem that extends through all regions: between 1985 and 2000, it reached 422,500 victims and over the last two years 66,800 new cases of the disease were detected as the disease remains out of control.

Hunting the parasite
A professor at the Parasitology Department of the Biomedical Sciences Institute (ICB) of USP, Jeffrey Shaw, has been studying the protozoa of the genre Leishmania since he came to Brazil in 1965. It has been over thirty years of work, which began through a fatality until the recent identification of the natural hosts of the parasite, in field research carried out in the states of São Paulo and Pernambuco.

Though since the decade of the 70s, there have been studies that pointed towards rodents and marsupials as the possible reservations of L. (V.) braziliensis, not one of them could prove that the protozoan found in the animals was of this species. Shaw considers that the identifying of the two rodents as the natural hosts of the parasite was one of the most memorable moments in his lifetime.

Between 1965 and 1994, Dr. Shaw investigated the species of protozoa that exist in the Amazon forest, studying the life cycle of the parasites, their possible reservations and their possible carriers – the insects that transmit the parasites. Finally, in order to study the ATL caused by the L. (V.) braziliensis, he had to leave the Amazon and come back to the regions of São Paulo and Pernambuco, where the infection for this species was endemic and there was no register of illnesses caused by other species of Leishmania.

The work involved a search in the areas close to the cities of three São Paulo regions and a municipality in the Jungle Zone of the state of Pernambuco. In search of the protozoan, Shaw’s team captured in these areas 770 examples of wild animals and close to 20 species and 79,446 insects of the group of the phlebotomies – called vectors, since they transmit the parasite from the host to another. The most important result came from part of the project developed in Pernambuco between 1996 and 2000, in collaboration with Sinval Brandão Filho, of the Aggeu Magalhães Research Center – a unit of the Oswaldo Cruz Foundation in Recife.

Jungle Zone
Shaw and Brandão studied leishmaniosis in the municipality of Amaraji, an area dating back to the colonial days about a hundred kilometers from Recife, in the southern portion of the Jungle Zone. The local vegetation is formed from vestiges of the Atlantic Rain Forest – hence the name, the original habitat of the vector insects. Over the last fifteen years the largest number of new cases of ATL in Pernambuco have been registered in and around Amaraji. The researchers analyzed the material collected in the lesions of people and domestic animals – dogs, horses and donkeys – as being suspected ATL cases.

During the period, 309 new cases in humans were registered: of these, 205 cases were analyzed and in 30 of them it was proven that the agent that caused the illness was the L. (V.) braziliensis. In relation to the domestic animals included in the study, the test with the technique of Polymerase Chain Reaction (PCR) showed that around 20% of the 61 dogs and 14% of the 58 equines tested showed infection by Leishmania. Nonetheless, the exams did not allow for the proof of the protozoa species.

Also blood sucking insects were collected, as well as wild animals and mammals that live close to the houses. The capturing was done in three physical areas: in the interior of houses, in their vicinity (outhouses and stables) and in the external environment (plantations and the remains of the forest). 588 examples of rodents and marsupials were captured, within a total of 11 species. The vast majority were formed from three species of wild rodents: Water Rat (Nectomys squamipes), South American Rat (Bolomys lasiurus) and the Black Rat (Rattus rattus).

The researchers extracted material from 460 of these wild animals for submission to PCR testing, done in collaboration with Lucile Floeter-Winter, of the Department of Parasitology of ICB-USP, and of Edna Ishikawa, of the Evandro Chagas Institute of Belém. The results showed that close to 18% of them (81 rodents) were positive for leishmaniosis. In spite of this, the examination did not allow for the identification of the species of parasite – only of the sub genre Viannia. “Although there was no definite guarantee, the chance of the agent causing leishmaniosis to be the species L. (V.) Braziliensis is of 99% in these regions”, reveals Shaw.

The guilty and the suspect
For there to be no doubt, the parasites isolated from the animals with a positive result for PCR were tested with monoclonal antibodies. Furthermore, at the Oswaldo Cruz Institute in Rio de Janeiro, the researcher Elisa Cupolillo attempted to identify the same protozoan samples with a technique that uses specific enzymes for each species. The tests demonstrated that in six rodents – five field rats and one Black rat – the parasite found was of the species L. (V.) braziliensis. Although the protozoan has not been isolated in the water rat, it remained under suspicion due to the positive results of the PCR test.

As well as the identification of the field rat and the black rat as reservations, the work brought to light data that suggests the possible pathway taken by the parasite to reach man. All of the water rats, for example, were captured in a wild environment. A small proportion (3%) of the field rats were found in the proximity of houses. However, with the black rat the reserve occurred: 10% came from a wild environment, 64% in the areas surrounding houses and the remainder within the houses themselves.

This data lead to the following hypothesis on the epidemiological evolution of the illness: little by little the vector insects for Leishmania transmit from the wild reservations to other rodents that live in the vicinity of dwellings. The transmission continues until it reaches the domestic animals and human beings.

In the case of Pernambuco, although they have not managed to isolate the protozoan from the 5,626 bloodsucking insects dissected, the probable vector for the parasite is the Lutzomyia whitmani, the most abundant specie in the region, with 98% of the 64,806 insects collected. There is evidence that the contamination of man occurs principally in the proximity of houses.

Change of habitat
The São Paulo part of the study was developed in the municipalities of Ilhabela, São Sebastião, Iguape and Eldorado (on the coast), Itupeva (on the Atlantic plateau) and Araçatuba and Guararapes (on the Western plateau). The results were not as conclusive as those of Pernambuco. For example, it was not possible to isolate the parasite in wild or domesticated animals nor even in man. However, the PCR test registered positive cases for Leishmania of the sub-genreViannia.

The work, which resulted in the doctorate thesis defended last year by José Eduardo Tolezano of ICB-USP, also pointed towards some changes in the situation of leishmaniosis in relation to the first half of the twentieth century. Up until the 50’s, ATL was considered to be an occupational disease in the state. It mainly affected men in their productive age who worked in activities related to economic expansion and the occupation of the interior of the state in areas being deforested. Beginning with human action and the destruction of the original vegetation, however, this profile began to change.

In the second half of the century, the progressive elimination of the original vegetation altered the habitat of the vector of the protozoan and the distribution of the insect. Currently, outbreaks of leishmaniosis happen in open areas, away from the forest. Based on the data collected in the Paraíba Valley, Tolezano suggests that, in the modified environments, the probable vector could be the insect Lutzomyia intermedia, while in the forest, there are two other species – Lutzomyia fischeri and Lutzomyia ayrozai – that take on this role.

The next steps
The research was part of a thematic project of three-year duration, financed by FAPESP, with the support of the Ministry of Health, the Science and Technology Support Foundation of the State of Pernambuco (Facepe) and of the People Training Coordination at Tertiary Level Foundation (Capes), of the Ministry of Education. It has already resulted in two doctorate theses and in articles published in 1999 in the magazine Transactions of the Royal Society of Tropical Medicine and Hygiene. The details of the discovery will be published in a scientific paper to be prepared by Shaw and Brandão in collaboration with six other researchers, which should be submitted for publication in the British magazine Nature.

From now on, the researchers will be attempting to isolate the L. (V.) braziliensis in other species in which the result of the PCR testing was positive and to evaluate the distribution of the animals in the regions in which they were captured. As well, they are going to study the process of the adaptation of the protozoan to ecological changes in the analyzed locations. “It would be naive to believe that in five years we could throw light on the complex relationship parasite-host of one of the most common forms of leishmaniosis in Latin America”, adds Shaw, already satisfied with what he has obtained.

Infected in London, he went to research in Belém
Jeffrey Jon Shaw began his study of leishmaniosis by chance. At the end of the 50’s, he was a doctorate student at the School of Hygiene and Tropical Medicine in London, England. As he didn’t like the London cold, his advisor Percy Garnham suggested to him that he could study protozoa common in Central America. He caught onto the idea. After some time in the region, there appeared on his neck some wounds which he couldn’t cure.

In search of a diagnosis, Shaw, at that time 22 years of age, returned to London where he spent three weeks interned in the university hospital in which he was a student, but the doctors didn’t manage to identify the cause of the lesions. Ralph Lainson, a scientific friend, brought up the suspicion of leishmaniosis. The hospital exams were negative, but tests done by the researcher himself confirmed the suspicion.

“I’m in a school of tropical medicine, and nobody but nobody recognizes the disease. I’m going to study it.” decided young Shaw who confesses to having a passion for the question. In 1965, through the indication of a friend, he arrived at the Evandro Chagas Institute of Belém, where he remained until his transfer to USP in 1994.

The Project
Ecoepidemiology of American Tegumentary Leishmaniasis in Brazil. States of São Paulo and of Pernambuco. Endemic areas of old colonization, corresponding to the Atlantic rainforest zone (nº 97/13015-1); Modality Thematic project; Coordinator Dr. Jeffrey Jon Shaw – Biomedical Sciences Institute of USP; Investment R$ 97,710.00