Environment, Interactions Between Trypanosoma Cruzi and Its Host, and Health Meio-Ambiente, Interações Entre Trypanosoma Cruzi

S32 REVISÃO REVIEW

Environment, interactions between Trypanosoma cruzi and its host, and health

Meio-ambiente, interações entre Trypanosoma cruzi e seu hospedeiro e saúde humana

Antonio R. L. Teixeira 1 Clever Gomes 1 Silene P. Lozzi 1 Mariana M. Hecht 1 Ana de Cássia Rosa 1 Pedro S. Monteiro 1 Ana Carolina Bussacos 1 Nadjar Nitz 1 Concepta McManus 1

Abstract The ancient game

1 Faculdade de Medicina, An epidemiological chain involving Trypanoso- To understand the relationships amongst liv- Universidade de Brasília, Brasília, Brasil. ma cruzi is discussed at the environmental level, ing organisms with extensive epidemiological and in terms of fine molecular interactions in chains, affecting both health and species surviv- Correspondence invertebrate and vertebrate hosts dwelling in dif- al, requires that we begin when life began, in salt A. R. L. Teixeira 1,2 Laboratório Multidisciplinar ferent ecosystems. This protozoan has a complex, water . During the early Proterozoic era (the de Pesquisa em Doenças genetically controlled plasticity, which confers pre-Cambrian period) approximately 4.5 billion de Chagas, Faculdade de adaptation to approximately 40 blood-sucking years ago, primitive life forms such as eubacte- Medicina, Universidade de Brasília. triatomine species and to over 1,000 mammalian ria and archeabacteria emerged from the organic C. P. 04536, Brasília, DF species, fulfilling diverse metabolic requirements soup on the earth’s crust. These pro-karion mi- 70919-970, Brazil. in its complex life-cycle. The Tr. cruzi infections croorganisms incorporated organic constituents [email protected] are deeply embedded in countless ecotypes, where (proteins, nucleic acids, lipids and carbohy- they are difficult to defeat using the control meth- drates) which gave rise to life. In the presence of ods that are currently available. Many more field these ingredients, microorganisms initiated the and laboratory studies are required to obtain data synthesis of ATP (adenosine-triphosphate), uni- and information that may be used for the control versally used for energy storage 2,3. and prevention of Tr. cruzi infections and their Every miniscule organism on earth may come various disease manifestations. Emphasis should in to contact with every other, given sufficient be placed on those sensitive interactions at cellu- time 4. This “game” resulted in a revolution ap- lar and environmental levels that could become proximately 1.5 billion years ago. The approxi- selected targets for disease prevention. In the mation of archea and eubacteria led eventually short term, new technologies for social mobiliza- to their association. Subsequently, a biochemi- tion should be used by people and organizations cal event probably determined cooperation and working for justice and equality through health exchange of nutrients among these organisms. information and promotion. A mass media di- Extreme solidarity became symbiosis, resulting rected program could deliver education, informa- in the formation of undulipodia. This ancient tion and communication to protect the inhabit- protozoan retained its flagellum, which emerged ants at risk of contracting Tr. cruzi infections. from the mitochondrion of an archea bacteria spirochete. Other organelles from eubacteria Trypanosoma cruzi; Chagas Disease; Host-Para- completed the biochemical machinery of this eu- site Interactions; Environment karion, a unicellular living microorganism which

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contained membrane-wrapped DNA, in a pack- ment and a constant change in the temperature. age-type nucleus. In this environment macroscopic plants grew, At present, morphological evidence at the ul- with roots for uptake from soils and vascular sys- tra structural and molecular levels are consistent tems for distribution through the plant; in turn, with this pathway of primitive life 2. these plants became a major source of food for An occurrence 670 million years ago resulted animals. Those changes made grounds for co- in ancestral eu-karion undulipodia giving rise to evolution of Annelida-Molusca, which are close primitive trypanosomes, which were promptly relatives of Arthropods, and of vascular plants. recognized in fish. The flagellate protozoa Try- Paleontological data indicate that the invasion panosoma gray, which occurs in crocodiles, of the terrestrial environment by vascular plants, is related to mammal trypanosomes 5. It was, arthropods and higher vertebrates occurred rela- therefore, assumed that reptiles and batrachi- tively late, after the invertebrate phyla were well ans were primitive vehicles that brought these established in marine environments. Among the flagellates onto solid ground. The presence of vascular plants, the pteridophytes relied upon trypanosomatids in the blood of aquatic inverte- spores for dispersal. Among animals, complete brates and vertebrates favored secondary acqui- adaptation to plant-sucking resulted from the sition of whole microorganisms by a host during development of mouth-parts with a pump con- the Phanerozoic period, 570 million years ago 4. nected to the proboscis 5. Experiments using pte- These interactions among the trypanosomes and ridophytes and living arthropods indicate that cold-blooded animals require further study. The some spores remain viable after passing through construction and remodeling of the undulipodia the gut and hence it is believed that this feeding served to fabricate metazoans. These occurrenc- habit may have also been advantageous to some es explain multicellular organization, which de- early plants for propagule dispersion 6. It has pends on relationships at molecular and physi- been proposed that many occurrences provided ological levels. The concepts in this review paper the grounds for diversification into over a mil- show that life is a continuous process and con- lion species of arthropods in the Class Insecta: stantly changing. Hemiptera that presently inhabit the earth 7. This The protozoa that belong to the Class Zoo- reasoning can help to explain the happenings mastigophorea (Eukaryota, Excavata, Eugleno- during the Devonian epoch, 360 million years zoa) include the Kinetoplastid flagellates of the ago, where accounts of wide scale exchange of Trypanosomatidae family and have a major im- organelles, messengers and moieties have been pact on public health and veterinarian medicine. reported among early species of life 2,8. Numer- Phylogenetic analyses have placed the protozoan ous representatives of the Class Insecta may have Tr. gray, that can be found in the blood of croc- become second stage vehicles for the delivery of odiles and possibly date to around 480 million macromolecules which were widely exchanged years ago, at the root of the kinetoplastids, next between species. It appears that these insects to bodonids (Bodo saltans). These kinetoplastids became dependent on vascular plant carbohy- include Tr. cruzi, the agent of Chagas disease in drates that were sucked in by its mouth parts, the Americas, Tr. brucei, the agent responsible for leading to the accumulation of large energy sur- sleeping sickness in Africa, and the Leishmania pluses. These surpluses could be readily used for species that infect mammals and produce differ- subsequent transportation and delivery of whole ent forms of diseases worldwide 3. The life-cycles microorganisms or their spare-parts during fur- of these trypanosomatids account for their major ther interactions with newcomers. division into Salivarian and Stercorarian branches, completing the infective metacyclic stages, respectively, in the salivary gland and in the hind Efficient oxygen transportation system gut of invertebrate vectors. This feature intro- in warm-blooded animals duces important diversifications in the mode of infection transmission to mammals. During early life, the plant and animal kingdoms branched into two different energy-producing systems. Photosynthetic chloroplasts in green Vascular plants and the emergence of plants captured energy directly from the solar invertebrate-vectors system, and mitochondrion cytochrome in ex- tant microorganisms used anaerobic metabo- During the Paleozoic and Silurian periods, around lism which enabled them to survive during a time 434 million years ago, drastic changes in the mix- when oxygen on the earth’s surface was scarce ture of atmospheric gases led to an increase in 2,8. Following modifications in the gas composi- the oxygen diffusion coefficient in the environ- tion of the atmosphere, a respiratory chain de-

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veloped in vertebrate animals that carry ionized reptiles were ancient reservoirs of Tr. cruzi pop- Fe2+ in the hemoglobin molecule. A constant ulations, now infecting man and domesticated flux of hemoglobin-rich red blood cells made mammals. Some insects deciphered that hema- it possible for hemin-bound Fe2+ to become an tophagy helped their development and growth, important link in a metabolic pathway which hence small animals that populated the Ameri- is closely related to species fertility and repro- can Continent became providers of blood for duction 9,10. In vertebrate animals, an increase these predators. in oxygen led to complex biochemical pathways Triatomine and Tr. cruzi coevolution is gener- using a high consumption of glucose and energy ally considered to have been amazingly success- production, resulting from a reaction requiring ful. It was shown that following blood ingestion, ATPases, enzymes that hydrolyse ATP into Ad- the flagellate protozoa remains in the insect’s enosine Difosfato (ADP) with the release of in- fore-gut for a few days. There, the ingested blood organic phosphorus 11. These energy-producing trypomastigotes transform into epimastigotes, metabolic pathways generated efficient species which multiply by binary fission and colonize mobilization, growth and reproduction through- the gut. The insect’s hind-gut epimastigotes out the kingdom. This biochemical pathway was transform into non-dividing metacyclic tripo- promptly acquired by marsupials, which are the mastigotes that pass with excreta. Amastigotes most ancient mammals on earth, being present may usually be found in the mid-gut of insects since the Permian era, 245 million years ago 3. subjected to prolonged fasting 12. The develop- Thereafter, the circulation of blood increased ment of the parasitic forms in the triatomine’s considerably, with numerous species of small gut depends on interactions between the para- mammals appearing on earth during the Trias- site and the intestine lining mucosal cells as the sic and Mesozoic periods, 208 million years ago. epimastigotes forms adhere to a peri-microvillar It took a long time until an efficient hemoglobin membrane. This membrane protects the muco- system for oxygen capture and transportation sal cells against trauma by pathogens, and by tox- appeared in the triatomines, during the Creta- ins and chemical compounds 13,14,15. The partial ceous period, 100 million years ago. At present, permeability of the peri-microvillar membrane there are approximately 14,000 species of insects, to macromolecules regulates the flow of nutri- which depend on ionized iron [Fe2+] bound to ents to mucosal cells at different compartments a heme protein, in the core of the hemoglobin of the insect’s gut. Also, it has been considered molecule, to complete their complex life-cycles. that 10 days after T. infestans and Rhodnius pro- There follows a short account of these insects in lixus feeding, the peri-microvillar membrane be- the triatomine subfamily that suck blood from a comes a selective physiologic barrier for enzyme wide variety of vertebrate animals and initiate a regulation of absorption and digestion of blood broad chain of transmission of blood-born try- nutrients 16,17. The full development of peri-mi- panosomes. crovillar membrane lining mucosal cells of triatomines depends on abdominal distension after a full blood meal, provoking neurosecretion of a The coevolution of Trypanosomes and prothoracicotrophic hormone 18. This hormone Triatomines acts upon prothoracic glands producers of molt- inducing ecdisone 19. Also, hydrophobic proteins The adaptation of trypanosomes to the intestine and carbohydrate residues that bind the Tr. cruzi of triatomines created grounds for species sur- to peri-microvillar membrane lining mucosal vival, growth of Tr. cruzi forms and differentia- cells have been identified 19. The study showed tion of infective metacyclic trypomastigotes. The significant differences among trypomastigote coevolution of trypanosomes and triatomines and amastigote membrane bound molecules appeared to result from their gradual adapta- such as lipids, carbohydrates and proteins. Ac- tion to invertebrate and vertebrate hosts, pos- tually, the Tr. cruzi genome sequencing revealed sibly between 99.8 and 93.5 million years ago. an increasing plethora of genes expressing met- Although there are no secure recordings of such aloproteases, mucin and transialidase associ- putative adaptation steps, there are closely re- ated proteins related to the parasite evasion of lated accounts among lizards and insect-vectors the insect’s innate and acquired immunities 20. in Baja California. In the absence of hot-blooded Furthermore, it was shown that some insect’s gut animals in that ecosystem, infections of lizards enzymes are essential for the parasite develop- that ingested triatomines contaminated with Tr. ment. Serine-, cystein- and aspartic-proteases, cruzi, as well as the complete life-cycle of the carboxi-peptidases and aminopeptidases in the parasite, can be observed 3. These observations insect’s gut are important for blood digestion and are of practical importance, since it appears that for absorption of its by-products 21. However, the

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triatomines’ digestion appears to be trypsin-free ily blocked by an actin inhibitor 24. This finding and catepsin-dependent, which favor adaptation is in keeping with electron microscopy images, to its acidic intestine environment. Also, lectins, where there is a lack of pseudopodia at the site hemolytic factors and α-D-globin peptides in- of the Tr. cruzi invasion in the plasma membrane fluence the development of the parasite in the of the host cell 24. insect’s gut 13,21. In addition, high levels of su- It appears that the signaling pathways ini- peroxide and the presence of a cationic peptide tiated upon parasite contact with the host cell named defensin prevent the parasite invasion are critical checkpoints in the invasion process in the insect’s heme lymph. Lately, it has been 25,26. The invasion occurs after recruitment of shown that the inoculation of Tr. cruzi in the R. vacuoles beneath the plasma membrane level, prolixus heme lymph led to production of im- which invaginates to increase the ratio of fusion mune-related molecules such as nitrophorin, with lysosomes. It has been said that lysosomal iron-related transferins and a prophenoloxidase fusion is essential for the retention of Tr. cruzi activating protease 22. inside host cells 24. Biochemical events related to parasite invasion include increasing concentra- Interactions of Trypanosoma cruzi with tion of Ca2+ intracellular levels prior to lysosome vertebrate hosts fusion, which is triggered by parasite-induced stress at the plasma membrane level 25,26,27. Al- The Stercorarian protozoan Tr. cruzi has a com- so, increasing levels of cAMP (cyclic adenosine plex life-cycle. It undergoes extracellular multi- monophosphate) play an important role in the plication in the invertebrate host, but grows by internalization of the parasite into the host cell obligate intracellular multiplication cycles in and adenyl cyclase inhibitors reduce the rate of vertebrate hosts. The metacyclic trypomastigotes invasion 24. In contrast, increasing intracellular enter the human body through triatomine-made levels of cAMP and Ca2+ have been associated skin abrasions at the point of the insect bite. It with exocytosis in many cell types 24. appears that insectivorous mammals used to get In summary, the parasite invasion of a host the Tr. cruzi infections per os. Regardless of the cell evokes plasma membrane vesicle transporta- route of entry, the parasite interacts with skin his- tion, requiring blockage of the cytoskeleton actin tiocytes or with intestinal mucosal mononuclear barrier prior to fusion with the lysosomes. After cells, and macrophages occur immediately. After internalization, the length of permanence of the cycles of multiplication into the phagocytes, the parasitic form in the parasitophorous vacuole Tr. cruzi trypomastigotes are released into blood appears to be short; the infections may be con- circulation and the infection spreads to the body trolled by innate and acquired immune response tissues. The parasitic forms can enter any cell mechanisms which are efficient in eliminating type in the body, except the neurons 3. the parasites. On the other hand, the virulent try- The entry of Tr. cruzi forms into host cells pomastigotes readily disrupt the lysosome-like causes a wide variety of interactions with sur- vacuole, and the parasitic forms are set free for face membrane glycoproteins, hydrolytic en- replication in the cytosol of the phagocytes 24. zymes and diverse signaling metabolic path- Some proteins that have been associated with ways of cell growth and differentiation 10,23. An- iron transport 10 may well be essential for parasite cient biochemical signaling pathways associat- replication within macrophages and non-phago- ing cell growth and differentiation were found to cytic cells. For Tr. cruzi molecular interplays of be similar in the protozoa and in its vertebrate the host cell associated with parasite location, host cell. It was also shown that some strategic invasion and retention, the reader should consult features of parasite-host cell invasion appear a recent review 24. to be unique to this infectious process. Earlier The escape of parasites into the host cell cy- studies had shown that invasion of the host cell toplasm may occur during S phase, in which a by Tr. cruzi had a feature similar to that involving stress-induced burst of oxygen leads to an in- bacterium and a phagocyte, in which actin cyto- crease in glucose consumption and energy pro- skeleton mobilization and emission of pseudo- duction, thus triggering signaling pathways for podia led to engulfment of the microorganism parasite and host cell growth and differentiation. by the host cell 24. Other experiments showed Tr. cruzi infections of monocytes, macrophages that the Tr. cruzi invasion augments signifi- and tissue histiocytes appear to be self-limiting, cantly when host cells are treated with a specific possibly because the parasite may not tolerate actin inhibitor 25,26. Furthermore, it was shown the acidic environment of parasitophorous vacu- that the treatment of cells with cytochalasin did oles or because parasite overload kills the host not prevent the T. cruzi invasion, whereas the cell. However, under a moderate parasitic load, entry of the intracellular Salmonella was read- over a short period of time, the dividing amas-

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tigotes in the cytoplasm of muscle cells appear of the translocation, the p15 locus was altered, to undergo a full cell cycle, whereby free-swim- resulting in elimination of p15 mRNA 33. This ming trypomastigotes are released and reach the phenomenon produced gene knock-out, which blood stream to infect other cells in the body. is a molecular pathology stemming from mobi- For instance, after reaching muscle cells, divid- lization of a kDNA-LINE-1 mutation. It appears ing amastigotes appear to be out of the reach that the mutation-made genomic modification of immune system factors. Natural infections in and subsequent transcript variation is consistent humans and experimental chronic infections with the hypothesis that genotype induced phe- in laboratory animals show small, non-dividing notype alterations might be a causal component Tr. cruzi amastigote forms to be dormant, due of parasite-independent, autoimmune-driven to the lack of tissue reactions in their surround- lesions in Chagas disease. ings. This parasite form in healthy muscle tissue The above description detailing fine inter- is herein called “hypnomastigont”. The finding of actions among parasitic forms and host cells is muscle cell “hypnomastigont” nests (http://www. elaborated in this review article, as we aim to ecb.epm.br/~renato/nest.JPG) may explain the show the importance of environmental factors long lasting chronic infections in patients show- at the molecular level, which can cause severe ing fully mature specific immune responses. effects on human health. “The Trypanosoma cruzi perpetuation would hardly be possible without the renewing forces of sexual reproduction” 28 (p. 64). In spite of Cha- The triatomines and mammal reservoirs gas’ 28 opinion, Tr. cruzi sexual reproduction was not considered an option for explaining the ge- The triatomines of the Reduviidae family include netic diversity of Tr. cruzi populations that circu- those strictly hematophagous insects belonging late in invertebrate and vertebrate hosts. More to the subfamily Triatominae, that became read- recently, some studies have shown that trypano- ily adapted to different types of ecosystems 35. somes isolated from a single Chagas patient are The broad diversity among triatomine vectors of made up of diversifying populations 3. It was Tr. cruzi infections results from sexual reproduc- expected that each Tr. cruzi was a clone, giv- tion 35. The eggs hatch nymphs of first instars, ing rise to a homogeneous parasite population. which reach fifth instars after four molts and then However, it has been shown that such Tr. cruzi become adult. Each triatomine stage is strictly populations show enormous plasticity, and are hematophagous. A full blood meal is required potentially able to transpose their own lineages. for molting; however, when a nymph reaches the A direct explanation to the above observations adult stage it requires multiple blood meals for was given by Gaunt and co-workers 29, showing copulation and oviposition. This feature of the that haploid and aneuploid Tr. cruzi forms origi- insect life-cycle, requiring constant feeding on nate from sexual reproduction. In this regard, different prey, appears to have broad epidemio- Devera et al. 30 propose the designation “cruzi logical importance, as it is a multiplication factor complex”, which encloses the entire potential for transmission of infections. of parasite population diversity. It appears that One aspect deserving specific attention is sexual reproduction generates the enormous the divergence that took place during the Cre- genetic diversity that has been observed in Tr. taceous period in the early Mesozoic era, in- cruzi isolates from humans, as well as from wild troducing modifications in existing triatomine invertebrate and vertebrate animal reservoirs. tribes of some major ecosystems, which meant In recent years, a highly intimate interaction that the fulfillment of feeding patterns required between intracellular Tr. cruzi forms and the host for completion of the insects’ life-cycles became cell has been brought onto the scientific stage by possible. The Rhodniini tribe dwells in the hu- the work performed at our laboratory 31,32,33,34. mid tropical broad-leaf forest, and is mainly These authors have described the horizontal adapted to life in palm trees. On the other hand, transfer of Tr. cruzi mitochondrial minicircle the Triatomini tribe became adapted to rock DNA to the genomes of mammals and birds. The crevices, tree burrows, under tree barks, and in minicircle integrations within LINE-1 retrotrans- burrows in the ground, which serve as dwell- poson appeared to create the potential for for- ings for small mammals in the dry ecosystems eign DNA mobility within the host genome via of the cerrado (or savannah) and caatinga. the machinery associated with that element. On Table 1 shows the main species of triatomines one occasion, the minicircle sequence integrated inhabiting major ecosystems. The diversity of into a LINE-1 retrotransposon, and subsequently triatomines adapted to dry climate ecosystems relocated to another genomic location in asso- is observed alongside the large range of mam- ciation with the parasitic DNA. As a consequence mals that serve as blood sources for the insect

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vectors co-inhabiting animal dwellings. These broad diversity of insect-vectors spread the Tr. reservoir hosts, upon whom triatomines prey, cruzi infections to those mammals dwelling in participate in the transmission cycle of Tr. cruzi 19 defined ecosystems (Figure 1) 36. Although and belong to the Classes, Marsupialia, Edenta- field studies, aiming at the discovery of wild life ta, Chiroptera, Carnivora, Arthiodactyla, Roden- triatomines, have been designed on the basis of tia and Primata. Over 1,150 wildlife mamma- the political division of the Brazilian Federation, lian species belonging to these seven classes are it is expected that future studies will focus on potential reservoirs of Tr. cruzi 3. Additionally, a specific features of the ecosystems, determining

Figure 1

Main ecosystems in Brazil.

18 17 14

15 19 16

9 10 11

8 12

7 4

3 4 6

5 4

(1) Savannah; (2) Araucaria forest; (3) Mato Grosso Pantanal swamp; (4) Cerrado (savannah); (5) Inland Atlantic forest; (6) Coast- al Atlantic forest; (7) Flooded grasslands; (8) Southeast Amazon forest; (9) Rondonia and Mato Grosso rain forest; (10) Choco- Darien humid Forests; (11) Tapajós/Xingu humid rain-forest; (12) Caatinga (Scrub Forest); (13) Tocantins humid rain-forest; (14) Guiana humid rain-forest; (15) Amapá humid rain-forest; (16) Uarama humid forest; (17) Guyana savannah; (18) Guiana humid forest; (19) Juruá/Negro humid forest. Source: Dinerstein et al. 36.

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species selection pressure and disease transmis- maintain the Tr. cruzi infections among mammal sion. Pressures imposed by climate, vegetation reservoirs, was readily introduced into Amerindi- and fauna are main actors in the huge Tr. cruzi ans. By that time, it appears that recrudescence epidemiological chain, which is independent of of epidemics took place after small mammals political divisions of the national territory. The were domesticated and the insect-vectors ini- field study approach by ecosystems appears to tiated colonization of human dwellings, about serve better future strategies for control and 9,000 years ago 38,39,40. The readiness with which prevention of disease transmission. In this re- pathways made by insect-vectors carrying the spect, many more field studies are required to Tr. cruzi infections were reconstructed when the elucidate triatomine distribution in major eco- new settlers arrived on the American continent systems. Studies aiming at prevention of insect- and acquired Chagas disease should be noted. borne disease should consider as top priority These reconstructions appear to be essential for environmental public health problems. Policy- understanding this major zoonanthroponosis, makers should build up County, State and Cen- which is now considered an important public tral Federation consortia to deal with disease health problem. At present, new studies about Tr. prevention activities. cruzi infections in different ecosystems are fun- A wide range of factors in the major ecosys- damental for obtaining sound information about tems pose a real, every day threat for the spread their many camouflage features, hidden within of the epidemic and these should to be dealt with wild and human populations 41, 42 on a permanent basis. Meanwhile, various wild In this review, we consider insecticide spray- species of animals interact with elements par- ing, leading to subsequent dislodgment of Tri- ticipating in the Tr. cruzi epidemiologic chain and atoma infestans from human huts in the Brazil- require original solutions to create novel strate- ian dry ecosystems of the cerrado and caatinga, gies for control of infections transmitted to the to be a very important activity, as it resulted in human population. It is impossible to eradicate a spectacular decrease in the ratios of vector- many actors belonging to diverse phylum in the transmitted Tr. cruzi infections in these human kingdom from nature, which have been interact- populations. ing in the various ecosystems for several million However, data showing the distribution of tri- years, therefore making the prevention of Chagas atomines in these dry ecosystems reveal at least disease a very difficult task. This concern leads us six other main species carry Tr. cruzi infections to suppose that interactions within such a com- and, therefore they are potentially infective to plex epidemiological chain require proper use human populations 35. On the other hand, each of the land and preservation of ecosystems for of the 19 main ecosystems, composing the Bra- securing human health. The anthrop predation zilian landscape, harbor triatomine species con- that causes outbreaks of acute Tr. cruzi infections taminated with Tr. cruzi. These species include in human populations is described in the follow- Panstrongylus megistus adapted to humid eco- ing section. systems, which is the main transmitter of the infections in the moist Atlantic forest and where this branches into many different ecosystems Emerging Chagas disease along streams and swamps. The importance of P. megistus, a major vector transmitting the Tr. The American trypanosomiasis is an ancient cruzi infections, has been recognized since the zoonosis that emerged approximately 95 mil- year 1909 when Chagas discovered the flagellate lion years ago 3. Since that time, the triatomine in the insect’s gut in 43. The ubiquitous habits of vectors that had developed hematophagy could P. megistus, characterized by its adaptability to sustain the symbiotic Tr. cruzi in their gut. It is peridomicile and sylvatic life, where it is capable important to note that the insect and protozoan of obtaining blood from different animal hosts, were brought together due to a metabolic ne- as well as from humans, make this triatomine an cessity, the association with Fe2+ needed for fer- important target for future field studies aimed at tility, reproduction and completion of their life the prevention of transmission of Tr. cruzi infec- cycles. Nowadays, possibly as a consequence of tions in the humid ecosystems where this species these interactions, insect vectors and mammal dwells (Table 1). hosts sympatrically occupy vast areas of South Other species thriving in the humid Atlantic America. tropical forest are equally important: T. tibiamac- The introduction of Homo sapiens in the en- ulata, T. vitticeps, P. geniculatus and P. lignarius. zootic areas may have occurred upon arrival of Frequently, these triatomines have been captured Polynesians on the Continent about 50,000 years in a variety of palm tree species of various ecosys- ago 37. An early sylvatic cycle, which used to tems 35. Each of these species has been associ-

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Table 1

Feeding habits and ecotopes of main triatomine species transmitting Trypanosoma cruzi infections in ecosystems located in Brazil.

Triatomine Climate/Ecosystem Ecotopes Feeding habits

Triatoma T. infestans Dry/Cerrado, Caatinga Artificial: intra- and peri-domicile; sylvatic: Synantrophic but highly (scrub forest) and savannah mammal dwellings on rocks and in the ground. antropophylic

T. tibiamaculata Moist/Atlantic coastal forest Sylvatic: palm trees in proximity to Synantrophic mammal dwellings; artificial: peri-domicile.

T. viticipes Moist/Atlantic coastal forest Same as T. tibiamaculata Synantrophic

T. sordida Dry/Cerrado Sylvatic: burrows, and under tree bark; Synantrophic artificial: peri- and intra-domicile

T. braziliensis Dry/Caatinga Sylvatic: on rocks close to rodents’ Synantrophic dwellings; artificial: peri- and intra-domicile.

T. pseudomaculata Dry/Caatinga Sylvatic: birds’ nests; Natural: intra- Ornitophylic; occasionally and peri-domicile anthropohylic

T. rubrovaria Dry/Savannah flat land Natural: intradomicile Anthropophylic

Panstrongylus P. megistus Moist/Atlantic coast and in broad Sylvatic: on rocks, wood bark and on Synantrophic and highly leaf areas within any ecosystem. the ground. Artificial: peri- and intra-domicile antropophylic

P. lutzi Dry/Cerrado and caatinga Sylvatic: dwellings of armadillos; Synantrophic artificial: peri- and intra-domicile

P. geniculatus Humid/Ubiquitous Sylvatic: palm trees Ornitophylic, occasionally antropophylic

P. lignarius Humid/Broad leaf forest Sylvatic: palm trees Ornitophylic

Rhodnius R. neglectus Ubiquitous Sylvatic: palm trees; Artificial: peri-domicile Ornitophylic, synantrophic

R. nasutus Ubiquitous Sylvatic: palm trees; Artificial: peri-domicile Ornitophylic, antropophylic

R. pictipes Humid/Broad leaf forest Sylvatic: palm trees; Artificial: Synantrophic, antropophylic peri- and intra-domicile

R. robustus Humid/Broad leaf forest Sylvatic: palm trees Synantrophic

R. brethesi Humid/Broad leaf forest Sylvatic: palm trees Synantrophic

Note: the data compiled from previous publications (Coura et al. 42; Teixeira et al. 41; Diotaiuti 35) show that triatomines are considered to be important in the transmission of Tr. cruzi infections in Brazil.

ated with transmission of the Tr. cruzi infections if they knew that for each acute case there are to the human population. An outbreak of acute around 100 infections that go unperceived. The Chagas disease in a resource area of the Atlantic epidemiologic study in the area where these cas- coast 44, comprising the county of Navegantes, es were identified showed a direct correlation be- in the State of Santa Catarina, Brazil, was read- tween the ingestion of sugar-cane juice and the ily identified and immediately broadcasted, but infections. Palm trees were found at a particular generated great public concern. This epidemic spot where sick people had drank this juice. The was apparently limited to less than 30 people and search for triatomines in the palm clefts revealed direct sampling of their blood confirmed the di- T. tibiamaculata that harbored the Tr. cruzi forms agnosis of acute Tr. cruzi infections. Two patients in their guts 45. It was assumed that the triato- died. However, the public would be shocked mines with Tr. cruzi in their intestinal contents

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were attracted by light and, then, they may have total) below the age of 10. Seroprevalence of Tr. contaminated the sugar-cane mill. Other case cruzi infections in the Paço do Lumiar County studies have implicated different species of tri- human population was in the absence of hema- atomines, thriving in different ecosystems, with tophagous insects or their vestiges in the dwell- Tr. cruzi contamination of food ingested by peo- ings. A search for triatomines was carried out ple during barbecues 46,47. in the ecosystem surrounding the households Nowadays, Tr. cruzi infections occupy every where the population was infected or continues ecosystem where suitable insect-vectors have to be at risk of contracting the infections. This been captured and identified. Regardless of cur- approach counted on the local population for rent knowledge, early maps showing distribution the capture of triatomines in their houses, which of human Tr. cruzi infections in Brazilian territory were kept in plastic containers. These contain- did not show human Chagas disease in the major ers were delivered to the laboratory for micro- humid broad-leaf tropical forest. Nevertheless, scopic search of metacyclic trypomastigotes in many outbreaks of acute Chagas disease arising the insect’s hind gut. In addition, palm trees in in the Amazon Basin over the last three decades the backyards of households in five villages were have been reported 47,48. These observations cut down and dissected 41. meant that frequent outbreaks of the Tr. cruzi Using this householder-assisted surveillance infections in the Amazon have been brought to and capture method, 36 R. pictipes and 16 R. ne- the public’s attention. It has been hypothesized, glectus were obtained. On the basis of this infor- therefore, that severe anthropic modifications mation, it was concluded that triatomine excreta introduced in the Amazon basin, may correlate and molted skins in these houses were either not with outbreaks of acute Tr. cruzi infections in reported by the inhabitants or detected by field that major ecosystem, with expected dramatic workers. Also, different developmental stages of changes in ratios of disease prevalence in the re- 133 triatomines were captured in clefts of palm gion 41,42,47,48. This hypothesis was examined in a frond-sheets carefully dissected in backyards study area in the county of Paço do Lumiar, in the in five villages. However, the remains of animal State of Maranhão 41. This case study is presented species were found in their nests in palm clefts below. where triatomine bugs rest and prey. Marsupials and birds were easily detected on palm fronds and crowns. Furthermore, we identified molds Trophic network and the cycle of and captured and identified different species of transmission of Trypanosoma cruzi from various taxa of invertebrate and vertebrate an- palm trees in the Amazon imals upon dissection of 23 palm trees. Molds were found in stipes, fronds, and crowns, and in- The tropical moist broadleaf forests of the Ama- sects in roots, stipes, inflorescence, fruits, fronds, zon Basin are increasingly subjected to anthrop crowns and leaves. The clefts formed by frond modifications. Although blocks of original habi- sheets were rich in Amphibia, Arachnida, and tats are relatively intact, some ecoregions have Hemiptera. Triatomines were detected at the bot- been converted or degraded, and elements of tom of clefts where marsupials build their nests. their biodiversity have been eroded. Correspond- Bird nests were found in the fronds and crowns ingly, population numbers of wildlife species where abundant species of insects were available have decreased where human settlers have es- for predation. tablished land colonization and villages, which Also, ten marsupials were captured in their are now considered threats to native species and nests in palm trees throughout the neighbor- communities. We hypothesize that human pred- hood. These marsupials presented few blood atory economic activity in a defined ecoregion of flagellates, which could not be demonstrated by the major tropical broad-leaf moist forest poses a direct microscope examination. However, the risk for transmission of Tr. cruzi infections. A field metacyclic flagellates were recovered from nine study was conducted to assess seropositivity for out of ten marsupials subjected to xenodiagno- these infections in the human population of Paço ses and hemocultures. Furthermore, these flag- do Lumiar County and to search for triatomines ellates were expanded in liver infusion tryptose that harbor these infections in palm trees 41. medium (LIT) cultures aiming at further isolat- An indirect immunofluorescence test was ing characterization. Phenotype and genotype used to search for anti-Tr. cruzi antibodies in hu- molecular markers were used to demonstrate man blood, and positive results were found to whether these isolates are virulent Tr. cruzi. In increase in younger sections of the populations the first group, antibodies in serum of chronic studied, where recent transmission and acute in- Chagas disease patients reacted with antigens fections were found in 0.18% of children (46 in on culture forms of archetype Tr. cruzi Berenice

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stock and, also, with isolates DM1, DM2, and present time, the blood-borne Tr. cruzi infections DM3 from Didelphis marsupialis and with Rp1 are transmitted by triatomine vectors to around from R. prolixus. In the second, genotype kDNA 1,150 mammal species belonging to seven major and nuclear DNA markers were used to charac- classes. Therefore, the complexity of the Tr. cruzi terize these wild flagellates. PCR amplification of life-cycle involving at least 40 species of triato- DNA from each of these isolates showed patterns mines and over 1,000 mammal hosts, inhabit- that were similar in standard virulent Tr. cruzi and ing 19 major ecosystems, supports the assertion in test isolates 3. The molecular characterization that presently available strategies for preventing was further confirmed by in situ hybridization of the Tr. cruzi infections may not be sufficient to wild Tr. cruzi isolates with a probe derived from protect the human population. We believe that Tr. cruzi Berenice 3. the described gamut of actors, playing important It was shown that trophic networks compris- roles in a multi-factorial chain of events related ing six different levels sustain the cycle of trans- to transmission of Tr. cruzi infections in different missions of Tr. cruzi in Babassu trees located in ecosystems, requires further studies in laborato- the backyards of households in five villages in ries and in the field, to find novel strategies for its Paço do Lumiar County, Maranhão State, Brazil. prevention and control. Developmental stages of R. pictipes, R. neglec- The different levels of interactions of Tr. cru- tus and P. lignarius were captured in palm trees zi in invertebrate and vertebrate hosts require (68% Tr. cruzi infected) and in houses (28% Tr. much research at the parasitological, genetic, cruzi infected). These triatomines feed on birds, molecular biochemistry, immunology and pa- marsupials, rodents, dogs and horses. However, thology levels. Future research and development 6.8% of R. pictipes captured inside households should unravel intricate features of parasite-host had fed on human blood. Immunologic, genetic cell ‘cross-talking’ that leads to either a long-last- and molecular biology assays disclosed that the ing symbiotic relationship with no harm to the flagellates infecting reservoir hosts and humans host, or to an autoimmune type disease causing are indeed virulent Tr. cruzi that could be associ- several biochemical and molecular disturbances ated with a growing prevalence of the infections at physiological and pathological levels, leading in the young human population. The data show to clinical manifestations and Chagas disease. It the importance of an intact trophic network to should be emphasized that the hierarchy distinc- keep the Tr. cruzi transmitters in their sylvatic en- tion among laboratory and field research work is vironment. Anthropic predation of fauna, with solely defined by quality that yields a real con- resulting scarcity of wild animals to feed upon, tribution to knowledge. Such epistemological is considered a primary cause of the spread of Tr. definition is essential, because a broad variety cruzi infections to the human population. of questions have been brought into play by an enormously complex epidemiologic chain of events related to Tr. cruzi infections and Chagas Discussion and conclusions disease. This requires a multidisciplinary approach and hard work to unravel the problem Existing evidence suggests that about 670 mil- over several years. lion years ago an ancestral undulipodia gave Hopefully, many unresolved questions may rise to primitive trypanosomes, which were drive scientific research to generate answers promptly recognized in fishes inhabiting the and new tools for the prevention and control oceans ever since. These are considered to be of human Chagas disease. Meanwhile, the self- the closest relatives of the trypanosomes. It ish competition that tends to push laboratory therefore appears that amphibians, not mam- (academic) and field (pragmatic) workers apart mals, are ancient reservoirs of Trypanosomatids should be avoided at all costs. In this respect, the that gave rise to Tr. cruzi. We believe that life results stemming from a field study that led to flows and changes continuously and, therefore, the description of a trophic network connecting the trypanosome evolution feature, which links the risk of contamination of the human popula- Tr. cruzi relatives to early reptiles and amphib- tion with the Tr. cruzi infections emerging from ians, should be carefully considered by scholars, palm trees in the Amazon was described. This before propagating eradication of Tr. cruzi infec- required a multidisciplinary approach and close tions and Chagas disease. collaboration among laboratory workers and During the cretaceous period, around 100 field researchers. An alternative route could be to million years ago, insects in the subfamily triato- build up accessible knowledge on organization mine, sucking up blood from a broad variety of of space, promotion empowerment and develop- vertebrate animals, founded the basis for a broad ment, aiming at disease prevention and environ- chain of transmission of trypanosomes. At the mental preservation. This knowledge could lead

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to the formation of a critical conscience united In this paper, we emphasize that control of with political participation, required to achieve vector-transmitted Tr. cruzi infections should rely social transformation that secures equality and initially on an information, education and com- social justice 49. munication program, which encourages control measures by the householder. For example, the identification of triatomine in the proximity of Concluding remarks the household and its elimination by cleaning and spraying with insecticide, the use of screens, New control strategies need to be devised before bed nets, and vegetation management with success can be obtained in eliminating the spe- conservation of local fauna, should be encour- cies close to human populations in rural areas, aged. Also, a program for preventing the human as well as the peripheries of towns and cities. We population from close contact with triatomines consider the risk factors associated with the pos- should be conducted directly in communities, sibility of emergence of Chagas disease in a major elementary schools, churches and social clubs, ecosystem as follows: (a) invasion of an ecosys- reinforced by social marketing and mass media tem by triatomine species which could be easily communications. Finally, further studies are also adapted to feeding upon human blood; (b) defor- needed. These may not necessarily be similar to estation and new population settlements, shift- those already shown to be partially effective in ing cultivation, and rapid human colonization of controlling the vectors of Tr. cruzi infections in the vectors’ natural ecotopes as well as predation various ecosystems. It appears that the inhab- of wild fauna, with a subsequent lack of multiple itants in each of 19 ecosystems in the Brazilian blood sources for the vectors. Therefore, the con- territory should be promptly informed about the trol of emerging Chagas disease in major ecosys- modes of transmission of the infections, with tems appears to be an enormous task for the rea- emphasis on measures which avoid contact with sons pointed out above, especially those related a broad variety of insect vectors and animal res- to its complex, multi-factorial trends associated ervoirs, excreta and fresh tissues that may pose with vector transmitted Tr. cruzi infections. daily threats to human health.

Resumo Contributors

Uma rede epidemiológica envolvendo o Trypanosoma This article is a team-work production. cruzi foi discutida nos níveis ambientais e de inte- rações moleculares nos hospedeiros que habitam em 19 diferentes ecossistemas. O protozoário tem uma enorme plasticidade controlada geneticamente que confere sua adaptação a cerca de quarenta espécies de triatomíneos e mais de mil espécies de mamíferos. Essas infecções estão profundamente embutidas em inúmeros ecótopos, onde elas estão inacessíveis aos métodos de controle utilizados. Muito mais estudos de campo e de laboratório são necessários à obtenção de dados e informação pertinentes ao controle e preven- ção das infecções pelo Tr. cruzi e as várias manifesta- ções da doença. Ênfase deve ser dada àquelas intera- ções que ocorrem nos níveis celulares e ambientais que se poderiam tomar como alvos seletivos para preven- ção da doença. Novas tecnologias para mobilização social devem ser disponibilizadas para os que trabal- ham pela justiça e pela igualdade, mediante informa- ção para a promoção da saúde. Um programa direcio- nado de educação de massa pode prover informação e comunicação necessárias para proteger os habitantes atualmente expostos ao risco de contrair as infecções pelo Tr. cruzi.

Trypanosoma cruzi; Doença de Chagas; Interações Hos- pedeiro-Parasita; Meio Ambiente

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