Plasmodium carmelinoi n. sp. (Haemosporida: Plasmodiidae) of the lizard Ameiva ameiva (Squamata: Teiidae) in Amazonian Brazil.

Plasmodium carmelinoi n. sp. is described in the teiid lizard Ameiva ameiva from north Brazil. Following entry of the merozoites into the erythrocyte, the young, uninucleated trophozoites are at first tear-shaped and already possess a large vacuole: with growth, they may assume an irregular shape, but eventually become spherical or broadly ovoid. The vacuole reduces the cytoplasm of the parasite to a narrow peripheral band in which nuclear division produces a schizont with 8-12 nuclei. At first the dark, brownish-black pigment granules are restricted to this rim of cytoplasm but latterly become conspicuously concentrated within the vacuole. The mature schizonts are spherical to ovoid and predominantly polar in their position in the erythrocyte. They average 5.4 x 4,9 microm (4.4 x 4.4 - 6.6 x 5,9 microm), shape index 1.1, n = 50: 8-12 merozoites are produced and measure approximately 2.0 x 1,0 microm. Mature gametocytes are also polar in position, and spherical to subspherical. The macrogametocytes measure 5.7 x 5,2 microm (4.4 x 4.0- 5.9 x 5,1 microm), shape index 1.1, n = 50 and, following staining by Giemsa's method, possess a compact, pink-staining nucleus and a clear blue, faintly stained cytoplasm. Microgametocytes are slightly larger, 6.0 x 5,0 microm (5.2 x 4.4 - 6.2 x 5,2 microm), shape index 1.2, n = 45. They stain an over-all pink colour due to the dispersed nuclear chromatin. The vacuoles in both the macro- and microgametocytes are considerably smaller than those of the schizonts and of ovoid or spindle shape: they contain most of the pigment granules. The sex ratio, as seen in an inicial intense infection, was 1 male to 2.2 females. Prevalence of infection was low (5%) but, due to the very low parasitaemia which may result in a failure to detect parasites, it is probably higher than this.

Immunopathogenic competences of Leishmania (V.) braziliensis and L. (L.) amazonensis in American cutaneous leishmaniasis.

The immunopathogenic competences of Leishmania (V.) braziliensis and L. (L.) amazonensis were reviewed in the light of more recent features found in the clinical and immunopathological spectrum of American cutaneous leishmaniasis. It was shown a dichotomy in the interaction between these Leishmania species and human T-cell immune response; while L. (V.) braziliensis shows a clear tendency to lead infection from the localized cutaneous leishmaniasis (LCL), a moderate T-cell hypersensitivity form at the centre of the spectrum, toward to the mucocutaneous leishmaniasis (MCL) at the T-cell hypersensitivity pole and with a prominent Th1-type immune response, L. (L.) amazonensis shows an opposite tendency, leading infection to the anergic diffuse cutaneous leishmaniasis (ADCL) at the T-cell hyposensitivity pole and with a marked Th2-type immune response. Between the central LCL and the two polar MCL and ADCL, the infection can present an intermediary form known as borderline disseminated cutaneous leishmaniasis, characterized by an incomplete inhibition of T-cell hypersensitivity but with a evident supremacy of Th1 over Th2 immune response (Th1 > or = Th2). These are probably the main immunopathogenic competences of L. (V.) braziliensis and L. (L.) amazonensis regarding the immune response dichotomy that modulates human infection outcome by these Leishmania parasites.

Trypanosoma (Megatrypanum) saloboense n. sp. (Kinetoplastida: Trypanosomatidae) parasite of Monodelphis emiliae (Marsupiala: Didelphidae) from Amazonian Brazil.

Trypanosoma (Megatrypanum) soloboense n. sp., is described in the Brazilian opossum Monodelphis emiliae (Thomas, 1912) from primary forest in the Salobo area of the Serra dos Carajás (6 degrees S, 50 degrees 18' W) Pará State, North Brazil. Two morphologically different trypomastigotes were noted. Slender forms, regarded as immature parasites, have a poorly developed undulating membrane adhering closely to the body: large, broad forms with a well developed membrane are considered to be the mature trypomastigotes and have a mean total length of 71.2 microm (62.4-76.2) and a width of 6.1 (5.0-8.0). Infections studied in two opossums were of very low parasitaemia. The large size of T. (M.) saloboense readily distinguishes it from the two previously described members of the subgenus Megatrypanum of neotropical marsupials, T. (M.) freitasi Régo et al., 1957 of Didelphis ozarae and D. marsupialis, and T. (M.) samueli Mello, 1977 of Monodelphis domesticus, which measure only 49.0-51.5 microm and 42.4 microm respectively. No infections were obtained in hamsters inoculated with triturated liver and spleen from one infected M. emiliae, or in laboratory mice inoculated with epimastigotes from a blood-agar culture. No division stages could be detected in the internal organs or the peripheral blood.

New species of Eimeria and Isospora (Protozoa: Eimeriidae) in Geochelone spp. (Chelonia: Testudinidae) from Amazonian Brazil.

Tetrasporocystic, dizoic oocysts of reptiles have been separated by some authors into the genera Eimeria, Choleoeimeria and Acroeimeria (Protozoa: Eimeriidae), based on the site and mode of development of their endogenous stages. The majority of Eimeria species have been, and still are, however, described on oocyst morphology alone. Four different oocysts with this basic morphology were encountered in the faeces of Brazilian tortoises, Geochelone carbonaria Spix, 1824 and are assigned to the genus Eimeria, with the view that they can readily be transferred to the genus Choleoeimeria or Acroeimeria if this is indicated by a future examination of their endogenous development. A morphological comparison distinguishes the oocysts from those of Eimeria spp., previously described in chelonids of the family Testudinidae, and the names E. amazonensis, E. carbonaria, E. carajasensis and E. wellcomei n. spp. are proposed. Coccidial infection appears to be common in G. carbonaria, with three of seven animals examined passing oocysts. Oocysts of Isospora rodriguesae n. sp. (Protozoa: Eimeriidae) are described in the faeces of Geochelone denticulata Linnaeus, 1766. They are morphologically very different from those of Isospora testudae, Davronov, 1985 in Testudo horsfieldi. Eimeria motelo Hurková et al., 2000, previously described in Geochelone denticulata from Peru, is here recorded in the some chelonid from Amazonian Brazil.

Natural and experimental infection of the lizard Ameiva ameiva with Hemolivia stellata (Adeleina: Haemogregarinidae) of the toad Bufo marinus.

Developmental stages of a haemogregarine in erythrocytes of the lizard Ameiva ameiva (Teiidae), from Pará State, north Brazil, were shown to be those of Hemolivia by the nature of the parasite's sporogonic cycle in the tick Amblyomma rotondatum. The type species, Hemolivia stellata Petit et al., 1990 was described in the giant toad Bufo marinus and the tick Amblyomma rotondatum, also from Pará State, and in view of the fact that A. ameiva and Bufo marinus share the same habitat and are both commonly infested by A. rotondatum, the possibility that the parasite of A. ameiva is H. stellata had to be considered. Uninfected lizards fed with material from infected ticks taken from B. marinus, and others fed with liver of toads containing tissue-cysts of H. stellata, were shown to subsequently develop typical Hemolivia infections, with all stages of the development similar to those seen in the naturally infected lizards. Conversely, a juvenile, uninfected toad became infected when fed with sporocysts of Hemolivia in a macerated tick that had fed on an infected A. ameiva and pieces of liver containing tissuecysts from the same lizard. The remarkable lack of host specificity shown by H. stellata, in hosts so widely separated as an amphibian and a reptile, is discussed.

Parasites of the squirrel Sciurus spadiceus (Rodentia: Sciuridae) from Amazonian Brasil, with particular reference to Eimeria damnosa n. sp. (Apicompiexa: Eimeriidae).

A description is given of the mature oocysts and endogenous stages of Eimeria damnosa n. sp. from the small intestine of the red squirrel, Sciurus spadiceus, from the State of Acre, north Brazil. Ten of 12 animals examined were infected. Oocysts ovoid to ellipsoidal, occasionally cylindrical but not with parallel sides, 30.2 x 20.0 microm (18.0 x 15.0-40.2 x 30.0), shape-index (ratio length/width) 1.5 (1.3-1.8), n = 40. Oocyst wall smooth, colourless, with no micropyle, apparently of a single layer measuring approximately 1.0-1.5 microm thick. No oocyst residuum, but approximately 50 % of the oocysts with a single spherical, ovoid or dumbbell-shaped polar body. Sporocysts pear-shaped, 15.0 x 8.0 microm (11.0 x 6.0-16.0 x 8.0), shape index 1.9 (1.8-2.0), n = 33. Stieda body, if it merits this name, appears only as a slight thickening of the sporocyst wall at the more pointed extremity. Endogenous stages intracytoplasmic in the epithelial cells of the duodenum and throughout the ileum, above the host cell nucleus. Sporulation frequently completed in the lumen of the intestine, but most oocysts mature outside the host at some time within 24 hours. Massive infections may result in extensive desquamation of the gut epithelium, and sometimes in the death of the animal. In addition to this coccidian, one squirrel showed abundant trophozoites of a Giardia sp., in the ileum. The liver of two others contained developing and mature meronts, producing large numbers of slender merozoites, and other cyst-like bodies containing a small number of large zoites (sporozoites?). No parasites were detected in the blood of any of the squirrels that could be associated with this unidentified protozoan. Histological sections of the ileum of one squirrel revealed a globidium-like parasite in the lamina propria: it contained a very large number of slender, curved zoites. Three animals were with a sheathed microfilaria in the peripheral blood and liver smears. Finally, a Trypanosoma cruzi-like trypanosome was isolated from the blood of one squirrel and a T. lewisi-like trypanosome from two others.

Haemoproteus (Apicomplexa: Haemoproteidae) of tortoises and turtles.

It is the general opinion that the haemoproteid blood parasites of chelonians belong to the genus Haemoproteus. Different specific names have long been assigned to this parasite in birds, but some past authorities have accepted only a single species, H. metchnikovi, for all those haemoproteids recorded in a wide range of chelonian genera throughout the world. In the present study, a comparison of one such organism in the tortoise Geochelone denticulata with another in the river turtle Peltocephalus dumerilianus, from Amazonian Brazil, has revealed clear morphological differences. These distinguish the parasites from each other, H. metchnikovi and the other named species of chelonian Haemoproteus for which adequate descriptions are available. We have assigned to them the names Haemoproteus geochelonis n.sp. and Haemoproteus peltocephali n.sp.

Production of a specific monoclonal antibody for the identification of Leishmania (Leishmania) venezuelensis.

We describe a monoclonal antibody (Mab), V1, specific for Leishmania (Leishmania) venezuelensis. Previous Mabs and DNA probes were not specific for this parasite, and so it was not directly possible to distinguish L. (L.) venezuelensis from other Leishmania species. Immunofluorescent staining using Mabs may be performed on very few parasites, whereas other methods of identification usually require far greater numbers of organisms. L. (L.) venezuelensis frequently dies on subculture. Mab V1 can be used to identify this parasite by indirect immunofluorescence and radioimmunoassay.

The American leishmaniases: some observations on their ecology and epidemiology.

As the first species of Leishmania encountered were the agents of human visceral and cutaneous leishmaniasis, it is understandable that studies on these parasites for a long time concentrated on those organisms commonly causing disease in man. Epidemiological studies over the past 20 years or so, however, have led to the inescapable conclusion that the genus Leishmania is comprised of numerous species of well adapted parasites, in a wide range of mammals, throughout most of those tropical and subtropical regions of the world where phlebotomine sandflies exist (Diptera: Psychodidae). Many of the leishmanias probably never gain entrance into man: due either to an incapacity to survive in his tissues, or (more likely) because the natural sandfly vectors do not feed on him. The leishmanias that do infect man are, nevertheless, among the greatest protozoological scourges of mankind, and a better understanding of their life-cycles may well help in future prevention or control of the diseases they cause. With few exceptions the leishmaniases are zoonoses, with a major source of infection in wild or domestic animals. In the Americas, the disease is essentially a rural one, and most commonly acquired by those penetrating forested or wooded regions. The following paper deals with the better known human leishmaniases of the New World, and some new ones, and discusses the major historical events in the laborious task of elucidating their ecology and epidemiology.

Leishmaniasis in Brazil. XIV. Leishmanial and trypanosomal IgA antibody in patients with leishmaniasis and Chagas's disease.

Preserved sera from patients with various forms of leishmaniasis, acute and chronic Chagas's disease and from normal individuals were examined for the presence of IgG and IgA antibodies by the IFA test. The leishmanial antigen used in the IFA test was prepared from a hamster infected with the PH8 strain of Leishmania mexicana amazonensis; amastigotes were isolated from a skin lesion and fixed in formal phosphate-buffered saline-glucose for slide antigen. There were significantly more positive reactions with IgA conjugate in patients with mucocutaneous leishmaniasis than in those with other forms. There was no association between IgA and IgG titres. The only two sera with trypanosomal IgA antibody were from two patients with acute Chagas's disease but neither sera produced cytoplasmic or surface fluorescence of leishmanial amastigotes. The implications of the presence of leishmanial and trypanosomal IgA antibody is discussed in detail.

Leishmaniasis in Brazil: X. Some observations of intradermal reactions to different trypanosomatid antigens of patients suffering from cutaneous and mucocutaneous leishmaniasis.

In spite of the difficulty of antigen standardization, paired intradermal skin tests demonstrated qualitative differences between leishmanin and in vitro exo-antigen. Some of these differences may be due to the destruction of certain antigens by phenol, while others seem to reflect basic antigenic differences. Leishmania mexicana amazonensis in vitro exo-antigen produced immediate anaphylactic reponses in 74.4% of the patients with parasitologically proven cutaneous leishmaniasis. Although more individuals responded to leishmanin, 82.4% as compared to 73.9%, the leishmanial in vitro exo-antigen gave significantly larger delayed responses, Trypanosoma cruzi trypamosomin elicited delayed reactions, while trypanosomal in vitro exo-antigen only produced immediate anaphylactic reactions in persons with active cutaneous leishmaniasis.

Pneumocystis and Histoplasma infections in wild animals from the Amazon region of Brazil.

Routine examination of tissues from wild forest rodents from Amapá, north Brazil, revealed Pneumocystis carinii in lung smears from a newly captured Oryzomys capito (Cricetidae). Acute, fatal infections with this parasite are also recorded in a number of captive "coatimundis", Nasua narica (Carnivora: Procyonidae) and a sloth, Bradypus tridactylus (Edentata). Pneumocystis was also encountered in lung smears from a newly captured and apparently healthy sloth, Choloepus didactylus. The presence of infection in newly captured animals leads us to believe that the fatal, fulminating pneumocystosis seen in the captive Nasua and Bradypus was due to exacerbation of pre-existant infections acquired in their natural forest environment. Pneumocystis carinii is a well known cause of fatal, interstitial plasma-cell pneumonia in human infants and sometimes the weakened adult: the keeping of exotic pets such as the coatimundi is, therefore, not without some hazard in this respect. Histoplasma, another well known pathogen for man, was isolated from 4 rodents, Proechimys guyanensis (Echimyidae), all from virgin forest along the newly opened Trans Amazon Highway, Pará State, and from a single sloth, Choloepus didactylus, from near Belém, Pará. All these animals showed no symptoms of infection: isolation of the parasite was made by the inoculation of laboratory hamsters with saline suspensions of triturated liver and spleen.

Leishmaniasis in Brazil. XXIV. Natural flagellate infections of sandflies (Diptera: Psychodidae) in Pará State, with particular reference to the rôle of Psychodopygus wellcomei as the vector of Leishmania braziliensis braziliensis in the Serra dos Carajás.

Between July 1983 and December 1984 natural flagellate infections were found in 114 (1%) of 11,586 female phlebotomine sandflies (Diptera: Psychodidae) of 21 species. A further 1084 females of 17 other species were not infected. Identification of the organisms on a number of occasions confirms the exclusive parasite/vector relationship of Leishmania mexicana amazonensis/Lutzomyia flaviscutellata and Le. braziliensis braziliensis/Psychodopygus "wellcomei". Undescribed or unidentified Leishmania spp. were isolated from Lu. shawi, Lu. ubiquitalis, Lu. whitmani, Ps. hirsutus, Ps. paraensis Ps. "wellcomei", and trypanosomes from Lu. nordestina and Lu. trinidadensis. Flagellate infections were recorded in 8 of 21 species examined for the first time, and some were isolated directly from insects into cultures. Le. b. braziliensis was transmitted to a hamster by the bite of a wild-caught, naturally infected Ps. "wellcomei". 7 of the 35 infected Ps. "wellcomei" were allowed to oviposit and the eggs were reared to adults. Four produced Ps. wellcomei males only, confirming the rôle of this species as the major vector of Le. b. braziliensis.

Leishmaniasis in Brazil: XX. Prevalence of "enzootic rodent leishmaniasis" (Leishmania mexicana amazonensis), and apparent absence of "pian bois" (Le. braziliensis guyanensis), in plantations of introduced tree species and in other non-climax forests in eastern Amazônia.

In Amazonian Brazil most human leishmaniasis is due to Leishmania braziliensis s.l. and is acquired during the clearing of primary climax forest. One of the largest deforestation projects has taken place on the JARI property where plantations of exotic tree species are grown for paper pulp. The ability of the regional leishmaniasis enzootics to invade plantations was investigated. CDC light-trap catches indicated the phletobomine vectors of Le. b. guyanensis (causing "pian bois" in man) to be very scarce in JARI plantations compared to native-forest controls. It is concluded (drawing on other observations) that the vectors of "pian bois" are unlikely to thrive in any secondary forest. In contrast, catches from mammal traps and rodent-baited (Disney) traps demonstrated the presence in JARI plantations of infected Proechimys guyannensis and large populations of Lutzomyia flaviscutellata, respectively the major rodent reservoir and sandfly vector of Le. mexicana amazonensis. Alone amongst the local vectors of human cutaneous leishmaniasis, Lu. flaviscutellata is adapted to non-climax forests (primary or secondary, natural or man-made; synopsis given). It is predicted that the public health importance of Le. m. amazonensis is unlikely to diminish following the development of Amazônia. This is worrying because ca. 30% of Le. m. amazonensis infections in man cause highly-disfiguring, incurable "diffuse cutaneous leishmaniasis".

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.

American visceral leishmaniasis: wild animal hosts.

In Colombia it has been suggested that the rodent Proechimys canicollis may be a reservoir-host of Leishmania (L.) chagasi, the cause of American visceral leishmaniasis, based on polymerase chain reaction/hybridization tests. We have detected no infection in laboratory-bred specimens of another species, P. guyannensis, after their inoculation with promastigotes or amastigotes of this parasite from Amazonian Brazil.

Leishmaniasis in Brazil: XVII. Enzymic characterization of a Leishmania from the armadillo, Dasypus novemcinctus (Edentata), from Pará State.

A comparison of enzyme profiles, by starch-gel electrophoresis, has distinguished a Leishmania of armadillos (Dasypus novemcinctus), from Pará State, north Brazil, from Leishmania braziliensis braziliensis, L. braziliensis guyanensis, L. mexicana amazonensis, L. donovani sensu lato (from Bahia, Brazil), and L. hertigi deanei. The parasite was separated from L. b. braziliensis and L. b. guyanensis by 8 of the 14 enzymes used (ASAT, ALAT, PGM, GPI, G6PD, PEP, MPI and GD), although differences in the mobility of some of the enzymes were small. At least 9 of the enzymes separated the organism from L. m. amazonensis, L. donovani s.l., and L. h. deanei.

Leishmaniasis in Brazil: XVIII. Further evidence incriminating the fox Cerdocyon thous (L) as a reservoir of Amazonian visceral leishmaniasis.

Major endemic areas of visceral leishmaniasis in Brazil are located in the drier, poorly forested regions, principally in the northeastern States such as Ceará and Bahia. Cases of the human disease in the Amazon Region are rare, very sporadic, and seldom present opportunities for epidemiological study. Following the report of a fatal case near Salvaterra, the Island of Marajó, Pará State, a preliminary investigation has resulted in the isolation of a parasite regarded as Leishmania donovani chagasi from the viscera and skin of an apparently healthy fox, Cerdocyon thous, captured in the same locality. This represents the third recorded isolation of the parasite from this species of fox in the Amazon Region. The inapparent nature of the infections supports the suggestion that this canid may represent the primitive natural host of L. d. chagasi. C. thous is commonly associated with forested or wooded areas, and enzymic profiles for the enzymes ASAT, ALAT, PGM, GPI, MDH, MPI, G6PD, PEP and ACP failed to distinguish an isolate of L. d. chagasi from this animal in Pará from others obtained from cases of human visceral leishmaniasis in the neighbouring States of Maranhão, Ceará and Bahia. This suggests that the major, present-day endemics may have originated from a primary silvatic enzootic.

Leishmaniasis in Brazil: XXIII. The identification of Leishmania braziliensis braziliensis in wild-caught neotropical sandflies using monoclonal antibodies.

Using the indirect immunofluorescence test natural flagellate infections of wild-caught sandflies, from the Serra dos Carajás region of Pará State, Brazil, were identified by sequentially staining smears made from the infected flies with monoclonal antibodies. With normal methods of isolation 30% of the infections were identifiable, but when monoclonal antibodies specific to Leishmania braziliensis braziliensis were used a further 26% were identified. The staining of organisms in smears of natural infections was different from that seen with culture forms and with forms from experimentally infected wild flies. A monoclonal antibody previously thought to be specific for Leishmania did not react with culture forms of Endotrypanum, but did react with those of monoxenous insect parasites.

Some methods for the enzymic characterization of Latin-American Leishmania with particular reference to Leishmania mexicana amazonensis and subspecies of Leishmania hertigi.

30 Brazilian stocks of Leishmania mexicana amazonensis and 13 stocks of subspecies of Leishmania hertigi were characterized by starch-gel electrophoresis, using 18 enzymes selected from a total of 36 investigated. L. m. amazonensis was separable from subspecies of L. hertigi by enzymic profiles of 11 enzymes. The L. m. amazonensis stocks, which were from a wide range of hosts in a large geographical area, were enzymically extremely homogeneous, and could only be subdivided on two enzymes; sub-groups did not relate to each other or to any differences in epidemiological characters, including the clinical form of the human disease. 12 stocks regarded as L. hertigi deanei, that were isolated from Coendou prehensilis prehensilis and Coendou sp. in Pará State, Brazil, were separable into two sub-groups by three enzymes. A single stock of L. hertigi hertigi from Panama was separable from both enzymic sub-groups of L. h. deanei, in each case by three enzymes. The significance of these and other characters of diversity is discussed, together with the use of enzymes for the identification of the leishmaniae.