An appraisal of the taxonomy and nomenclature of trypanosomatids presently classified as Leishmania and Endotrypanum.

We propose a taxonomic revision of the dixenous trypanosomatids currently classified as Endotrypanum and Leishmania, including parasites that do not fall within the subgenera L. (Leishmania) and L. (Viannia) related to human leishmaniasis or L. (Sauroleishmania) formed by leishmanias of lizards: L. colombiensis, L. equatorensis, L. herreri, L. hertigi, L. deanei, L. enriettii and L. martiniquensis. The comparison of these species with newly characterized isolates from sloths, porcupines and phlebotomines from central and South America unveiled new genera and subgenera supported by past (RNA PolII gene) and present (V7V8 SSU rRNA, Hsp70 and gGAPDH) phylogenetic analyses of the organisms. The genus Endotrypanum is restricted to Central and South America, comprising isolates from sloths and transmitted by phlebotomines that sporadically infect humans. This genus is the closest to the new genus Porcisia proposed to accommodate the Neotropical porcupine parasites originally described as L. hertigi and L. deanei. A new subgenus Leishmania (Mundinia) is created for the L. enriettii complex that includes L. martiniquensis. The new genus Zelonia harbours trypanosomatids from Neotropical hemipterans placed at the edge of the Leishmania-Endotrypanum-Porcisia clade. Finally, attention is drawn to the status of L. siamensis and L. australiensis as nomem nudums.

Partial transformation of human thyroid epithelial cells by mutant Ha-ras oncogene.

We have previously shown that activation of ras oncogenes by mutation is a frequent early event in human thyroid neoplasia. Using amphotropic retroviral vectors to achieve gene transfer, we demonstrate here that human primary thyroid epithelial cells can be partially transformed by an activated cellular or viral Ha-ras oncogene, in the absence of a cooperating oncogene. The transformation event induced by ras involves temporary rescue from senescence for up to 20 rounds of cell division together with morphological alteration, growth factor independence and anchorage independence. It has therefore been possible to reconstruct in vitro a key early event in the genesis of human epithelial neoplasia.

Evidence against involvement of APC mutation in papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is one of several tumours associated with familial adenomatous polyposis (FAP), an inherited tumour syndrome which appears to result from germ-line mutation of the APC tumour suppressor gene. Here we investigate the possibility that somatic mutation of APC might play a role in sporadic PTC. 16 cases of PTC together with matched normal tissue were examined by single-strand conformation polymorphism (SSCP) analysis, concentrating on the mutation cluster region (MCR) of the APC gene (codons 1286-1513). No evidence of mutation was observed in any sample. We conclude that APC mutation, at least in the MCR, is not a significant causal mechanism in sporadic PTC.

High frequency deletion of the tumour suppressor gene P16INK4a (MTS1) in human thyroid cancer cell lines.

p16INK4a (MTS1) is an important negative regulator of mammalian cell proliferation, acting via inhibition of CDK4/cyclin D-dependent phosphorylation of pRb to prevent progression through the G1 phase of the cell cycle. Loss of p16 activity by either gene deletion, mutation or transcriptional inactivation has now been found in a wide range of human cancers of both epithelial and mesenchymal origin, at a frequency rivalling that of p53 mutation. As a first step towards investigating its possible role as a tumour suppressor gene in thyroid tumorigenesis, we have carried out a Southern blot analysis of the p16 gene locus in a series of cell lines derived from differentiated human thyroid cancers. Homozygous deletion of the entire p16 coding sequence was observed in two of three follicular and two of four papillary cancer cell lines, but not in normal tissue or normal cells immortalised by SV40 T antigen. Given the co-existence of p16 abnormalities in primary tumours and cell lines observed in other tumour types, this high frequency of deletion suggests that p16 is a key tumour suppressor gene in the genesis of differentiated thyroid cancer.

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.

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: 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.

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. 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.

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.

Chagas's Disease in the Amazon Basin: 1. Trypanosoma cruzi infections in silvatic mammals, triatomine bugs and man in the State of Pará, north Brazil.

A total of 1,197 wild animals from Pará State north Brazil, were examined for haematozoa. Trypanosoma cruzi-like parasites were found in 13 different species, and were particularly common in a variety of marsupials (Didelphidae), porcupines (Coendou spp.), armadillos (Dasypus novemcinctus) and coatimundis(Nasua nasua). Three human infections are reported, making a total of seven autochthonous cases of Chagas's disease from Pará since the first were described in 1969. A serological survey of 5,319 inhabitants from the Belém suburbs revealed 14 positive reactions with immunofluorescent antibody titres greater than 1:64, and 15 equivocal reactions at 1:16. No evidence was found of silvatic species of triatomine bugs (Hemiptera: Reduviidae) colonizing houses, but occasional infected specimens of Panstrongylus geniculatus and Rhodnius pictipes were found in suburban houses, near the forest. Human infection is considered to be silvatic in origin. Chagas's disease could become endemic in the Amazon Basin if silvatic triatomine bugs were able to adapt to a domestic habitat: the real danger, however, is in the possible importation of already domiciliated vector-species along the newly opened highways, from other endemic regions of Brazil. Other Trypanosoma species were recorded in a variety of wild animals. Haemogregarines were found in some marsupials and rodents, and piroplasms in marsupials and a single armadillo.

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

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

Leishmaniasis in Brazil: 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.

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

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

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.

Leishmaniasis in Brazil: XV. Biochemical distinction of Leishmania mexicana amazonensis, L. braziliensis braziliensis and L. braziliensis guyanensis--aetiological agents of cutaneous leishmaniasis in the Amazon Basin of Brazil.

Enzymic profiles of the three known agents of human cutaneous leishmaniasis in the lower Amazon region are compared. Of 14 enzymes, 10 (ASAT, ALAT, GPI, G5PD, MDH, ACON, PEP, HK, MPI and ACP) differentiate Leishmania mexicana amazonensis from L. braziliensis braziliensis or L. braziliensis guyanensis: this supports their taxonomic status as distinct species. In contrast, only slight mobility differences of four enzymes (ASAT, ALAT, PGM, MPI) separate L. b. braziliensis and L. b. guyanensis, which are distinguished biochemically for the first time: this indicates that they are closely related. Four stocks of L. b. panamensis correspond with L. b. guyanensis on mobilities of 10 enzymes (ASAT, ALAT, PGM, GPI, G6PD, MDH, PK, HK, MPI, ACP), although these two subspecies are known to be separable by kinetoplast DNA buoyancies and the enzyme 6PGDH. The generation of practical, regional biochemical keys to the medically important leishmanias is discussed.

Leishmaniasis in Brazil: XVI. Isolation and identification of Leishmania species from sandflies, wild mammals and man in north Para State, with particular reference to L. braziliensis guyanensis causative agent of "pian-bois".

A total of 125 wild mammals (14 different species) were examined for evidence of infection with Leishmania in an area of primary forest highly endemic for "pian-bois", due to Leishmania braziliensis guyanensis, in north Pará State, Brazil. Parasites isolated were characterized biologically, and biochemically on enzymic profiles. L. b. guyanensis was isolated from the viscera of one lesser anteater (Tamandua tetradactyla) and one opossum (Didelphis marsupialis), and the skin of one rodent (Proechimys guyannensis). The isolates were indistinguishable from 10 others previously made from the sandfly vectors Lutzomyia umbratilis (five) and Lu. whitmani (five), and nine isolates from field-workers who became infected during these studies. Leishmania mexicana amazonensis was obtained from the skin of 21 animals, including three species of opossums (D. marsupialis, Philander opossum and Metachirus nudicaudatus) and two species of rodents (proechimys guyannensis and Dasyprocta sp.). A peripylarian Leishmania isolated from the viscera of two armadillos (Dasypus novemcinctus) was shown to be different, biologically and biochemically, from L. b. guyanensis and L. m. amazonensis. Four other isolates of Leishmania, from the rodents Rhipidomys leucodactylus (one) and P. guyannensis (three) have yet to be characterized owing to their very poor growth in both hamster skin and in vitro culture: they appear closest, however, to L. braziliensis braziliensis. The complexity of Amazonian leishmaniasis is discussed, and attention drawn to the importance of edentates as reservoir hosts of some leishmanias in the New World. Whereas L. mexicana subspecies appear largely restricted to the skin of their natural hosts, subspecies of L. braziliensis are commonly found in the viscera.