Paraorientatractis semiannulata n. g., n. sp. (Cosmocercoidea: Atractidae) from the large intestine of the side-necked turtle, Podocnemis unifilis Troschel, 1848 (Testudines: Pelomedusidae) in Brazil

Specimens collected from the large intestine of the side-necked turtle Podocnemis unifilis Troschel, 1848, in the region of Cumina and Trombetas rivers near Para, Brazil are assigned to a new genus and Paraorientatracti semiannulata. The new genus is separated from the nearest genus Orientatractis by the funnel-shaped mouth opening, the presence of 4 distinct lips, 4 papillae in the internal cycle, one on each lip margin, 2 lateral amphids with large amphidial pores and absence of submedian papillae. It is also separated from Orientatractis and Proatractis by the presence of striated lateral alae which curve dorsally extending from mid oesophagus to mid tail, the difference in size of the vulvar opening and the presence of large transverse ridges or semiannules on the dorsal surface. The new species can be separated from the species of the genera Orientatractis and Proatractis by the characters that distinguish the genera and the arrangement of the caudal papillae on the male. A host/parasite list for Podocnemis spp. is included.

Paraorientatractis semiannulata n. g., n. sp. (Cosmocercoidea: Atractidae) from the large intestine of the side-necked turtle, Podocnemis unifilis Troschel, 1848 (Testudines: Pelomedusidae) in Brazil.

Specimens collected from the large intestine of the side-necked turtle Podocnemis unifilis Troschel, 1848 in the region of Cuminá and Trombetas rivers near Pará, Brazil are assigned to a new genus and new species of the nematode superfamily Cosmocercoidea and family Atractidae and named Paraorientatractis semiannulata. The new genus is separated from the nearest genus Orientatractis by the funnel-shaped mouth opening, the presence of 4 distinct lips, 4 papillae in the internal cycle, one on each lip margin, 2 lateral amphids with large amphidial pores and absence of submedian papillae. It is also separated from Orientatractis and Proatractis by the presence of striated lateral alae which curve dorsally extending from mid oesophagus to mid tail, the difference in size of the vulvar opening and the presence of large transverse ridges or semiannules on the dorsal surface. The new species can be separated from the species of the genera Orientatractis and Proatractis by the characters that distinguish the genera and the arrangement of the caudal papillae on the male. A host/parasite list for Podocnemis spp. is included.

[New sources of lectins to differentiate between Trypanosoma cruzi y T. rangeli]. / Nuevas fuentes de lectinas para la diferenciación entre Trypanosoma cruzi y T. rangeli.

Extracts of 176 species of Colombian plant seeds, corresponding to 49 families and 147 genera, were tested for detecting agglutinins against human red blood cells from A+, B+ and O+ groups, dog, horse and rabbit. Extracts with haemagglutination activity were used for agglutination of Trypanosoma cruzi and T. rangeli. In addition the hemolymph of 16 native species of invertebrates were tested in the same conditions. Serial dilution of extracts were used for agglutination reactions. Both T. cruzi and T. rangeli epimastigotes showed agglutination with the extract of seven different species of plant seeds and with two types of haemolymph of invertebrates. The seeds of five plants exclusively agglutinated the epimastigotes of T. cruzi and thus can be used for the differentiation between culture forms of the trypanosomes. The secretion of the lung of a snail (Bulimus sp.) lysed entirely the epimastigotes of T. cruzi but did not affect T. rangeli forms. No extracts were found which agglutinated or lysed exclusively the epimastigotes of T. rangeli.

Nuevas fuentes de lectinas para la diferenciación entre Trypanosoma cruzi y T. rangeli / Further sources of lectins for differentiation between Trypanosoma cruzi y T. rangeli

Os extratos de 176 espécies de sementes de plantas colombianas, correspondentes a 49 famílias e 147 gêneros foram testados para detectar a presença de aglutininas frente a hemácias humanas dos grupos A+, B+, O+ e de cachorro, cavalo e coelho. Os extratos que apresentaram alguma atividade hemaglutinante, foram usados para testar a aglutinaçao de Trypanosoma cruzi e T. rangeli. Além disso, a hemolinfa de 16 espécies nativas de invertebrados foram testadas nas mesmas condiçoes. Diluiçoes seriadas dos extratos foram usadas para as aglutinaçoes. Ambas epimastigotas de T. cruzi e T. rangeli foram aglutinadas com os extratos de sete sementes de plantas diferentes e com dois tipos de hemolinfa de invertebrados. As sementes de cinco plantas aglutinaram exclusivamente as epimastigotas de T. cruzi podendo assim ser usadas para a diferenciaçao entre as formas de cultura desses tripanossomos. A secreçao do pulmao do caramujo (Bulimus sp.) lisou completamente as epimastigotas de T. cruzi mas nao afetou as formas de T. rangeli. Nao foram encontrados extratos que aglutinaram ou lisaram exclusivamente as epimastigotas de T. rangeli.

Babesia sp. in Colombian bats (Microchiroptera).

Two leaf-chinned bats (Mormoops megalophylla) collected in 1963 in central Colombia were heavily infected with Babesia sp., probably Babesia vesperuginis. Both bats had pronounced splenomegaly. This is the first report of a Babesia sp. infection of a bat in the Americas.

[Presence of neuraminidase in Rhodnius prolixus infected with Trypanosoma rangeli]. / Estudio sobre la presencia de neuraminidasa en Rhodnius prolixus infectado con Trypanosoma rangeli.

Using three different methods, the activity of neuraminidase was studied in the promesenteron, postmesenteron, rectal ampulla, haemolymph and salivary glands in 600 Rhodnius prolixus experimentally infected with Trypanosoma rangeli stock San Agustín. The haemagglutination method with peanut lectin, and the fluorescence test with peanut lectin conjugated with fluorescein isothiocyanate, and the fluorescence emitted by 4-methylumbelliferone showed in all cases the presence of neuraminidase in the supernatant culture of T. rangeli in Tobie's medium between 8 to 15 days growth. None of the three methods was able to detect the presence of neuraminidase in R. prolixus infected with T. Rangeli, thus suggesting that this enzyme is not produced in vivo, and consecutively is not implicated in the pathogenicity that this trypanosome has to its vector.

A modification of Diamond's medium for the axenic culture of Entamoeba histolytica.

The use of casein hydrolysate in Diamond's axenic culture medium TPS-1 in replacement of trypticase allowed good growth of the trophozoites of Entamoeba histolytica. This modified medium also supported growth of trophozoites preserved for 16 months in liquid nitrogen. Considerable labour and cost of serum can be saved by using 5% instead of 10% bovine serum in combination with this modified medium.

[Antigenic differentiation among 3 life-cycle stages of Toxoplasma gondii using fluorescent antibodies]. / Diferenciación antigénica entre tres estadios del ciclo de vida de Toxoplasma gondii mediante anticuerpos fluorescentes.

Antigenic differentiation between three stages of the life cycle of Toxoplasma gondii by fluorescent antibodies. Antisera were prepared in three groups of twenty mice each with three different antigens of Toxoplasma gondii: the first group was inoculated with tachyzoites of RH strain and received sulfadiazine treatment; the second with tissue cysts of T-100-cat-6751 strain and the third with oocysts of the same strain, both without treatment. In the indirect immunofluorescent antibody technique each antigen was tested with its homologous and heterologous antisera, determining qualitative and quantitative antigenic differences according to the fluorescence patterns. Some stages of Toxoplasma when reacting with their heterologous antibodies showed a central, partial posterior or total fluorescence during a certain period of development. The difference in fluorescence was sufficient to distinguish whether the origin of the infection was via cyst, oocysts and/or via tachyzoite when observation was made before day 65 post-infection.

Use of monoclonal antibodies for the differential detection of Trypanosoma cruzi and T. rangeli in epidemiological studies and xenodiagnosis.

T. cruzi and T. rangeli have the same insect and mammalian hosts, including man, and in addition share approximately half the antigenic determinants recognised by the humoral response. Thus serodiagnosis of T. cruzi infection in areas where T. rangeli is endemic may include an unknown rate of false positives due to this antigenic cross-reactivity. Similarly, the results of xenodiagnostic procedures and epidemiological surveys of insect vectors are prone to distortion because of the close morphological resemblance of the epimastigote stages. The description of a T. cruzi epimastigote specific monoclonal antibody, 2A2, which reacts with both culture and insect derived epimastigotes provides a more reliable basis for differential diagnosis of these two parasites.

Clinical Trypanosoma rangeli infection as a complication of Chagas' disease.

Laboratory studies on a group of 20 patients from the Rio Negro Valley, Colombia selected for detailed study showed that 14 gave antibody reactions on immunoassay consistent with Trypanosoma cruzi or T. rangeli infections. Four were diagnosed as having T. rangeli infection, 4 had mixed infections and 6 were infected with T. cruzi alone. Immunoprecipitation analysis showed that sera from T. cruzi-infected patients recognized a similar range of trypomastigote-derived polypeptides as sera from patients in Brazil, and all of the Colombian sera reacted with the 160 kiloDalton (kDa) polypeptide associated with active infection. Although sera from patients with T. rangeli infection alone gave a positive immunofluorescence or ELISA reaction with T. rangeli, they failed to bind to parasite polypeptides by either immunoprecipitation or Western blotting. Intriguingly, sera from patients with mixed infections consistently gave a stronger, but qualitatively similar, binding reaction in immunoprecipitation and Western blotting compared to sera from patients infected with T. cruzi alone.

Rastreo epidemiológico de donantes de sangre chagásicos en una zona endémica (Norte de Santander, Colombia) / Seroepidemiological survery of chagasic blood-donors from an endemic zone (North Santander, Colombia)

La enfermedad de Chagas prevalece en áreas rurales en donde generalmente no existen facilidades para realizar pruebas diagnósticas. En el presente estudio se utilizó el método de Imunofluorescencia Indirecta (IFI) colectando las muestras por absorción en papel de filtro (IFI-PF) para ser analizadas en un laboratorio central. El uso de este método práctico de diagnóstico demostró alta confiabilidad. El índice de positividad obtenido fue del 7.5% sobre un total de 491 muestras analizadas

The affinity of the lectins Ricinus communis and Glycine maxima to carbohydrates on the cell surface of various forms of Trypanosoma cruzi and Trypanosoma rangeli, and the application of these lectins for the identification of T. cruzi in the feces of Rhodnius prolixus.

Flagellates of Trypanosoma cruzi (stock Molino 1), obtained from the intestine of experimentally infected Rhodnius prolixus, grown in cellular or acellular culture, as well as from the blood of infected mice, were examined by a direct fluorescence test using the lectins RCA (Ricinus communis-120) and SBA (soy bean agglutinin; Glycine maxima), conjugated with fluorescein isothiocyanate, for the detection of beta-D-galactose and alpha,beta-N-acetyl-D-galactosamine on the membranes of the flagellates. The same reactions were carried out using Trypanosoma rangeli (stock San Agustin), obtained from the intestine, hemo-lymph or salivary glands of experimentally infected R. prolixus, as well as from cultures and from the blood of experimentally infected CFW mice. The results indicate that the membrane of T. rangeli in the salivary glands of the vector contains beta-D-galactose, but that this sugar is absent from all other developmental stages of this trypanosome. All stages of intestinal and cultured. T. cruzi presented positive reactions with RCA-FITC and SBA-FITC. The high specificity of this technique makes it useful for the examination of R. prolixus, previously used in xenodiagnosis of Chagas' disease and for the examination of intradomiciliary or sylvatic vectors in epidemiological surveys in areas where T. cruzi and T. rangeli coexist. Formaldehyde fixed samples can be examined months later and false reports due to T. rangeli can be avoided.

[Laboratory maintenance of Trypanosoma (Herpetosoma) rangeli Tejera, 1920]. / Mantenimiento en el laboratorio de Trypanosoma (Herpetosoma) rangeli Tejera, 1920.

Two laboratory maintenance systems of Trypanosoma rangeli were compared. The maintenance by weekly subinoculations in Tobie's culture medium and the intrafemoral inoculation of Rhodnius prolixus with cultured flagellates, resulted in loss of infectivity of the metacyclic salivarian trypomastigotes for mice, ten months after maintenance in culture. With the system of cyclical passes through culture-Rhodnius-mouse-culture-Rhodnius, the infectivity of the metacyclic trypomastigotes for mice, was maintained during the three years of the experiment. The number and percentage of metacyclic trypomastigotes formed in the salivary glands of R. prolixus, previously inoculated intrafemorally or intracoelomically with culture forms of T. rangeli, did not show correlation with the inoculated dose, however the inoculated quantity demonstrated a direct relation with the mortality rate of the insects. The results indicate that T. rangeli requires an adequate maintenance system, so that under experimental condition the biological characteristics, normally expressed under natural conditions, are conserved.

Mantenimiento en el laboratorio de Trypanosoma (Herpetosoma) rangeli Tejera, 1920 / Maintenance in the laboratory of Trypanosoma (Herpetosoma) rangeli Tejera, 1920

Se compararon dos sistemas de mantenimiento de Trypanosoma rangeli en el laboratorio. El mantenimiento por repiques semanales en medio de Tobie e inoculación intrafemoral de Rhodnius prolixus con flagelados de cultivo dio por resultado que los tripomastigotes metacíclicos formados en las glándulas salivares, perdieron su infectividad para ratones después de diez meses de mantenimiento en cultivo. En contraste, un sistema de pasajes cíclicos seriados a través de cultivo-Rhodnius-ratón cultivo-Rhodnius, mostró que los tripomastigotes metacíclicos, conservaron su infectividad para ratones, durante los tres años del experimento. El número y proporción de los tripomastigotes metacíclicos formados en las glándulas salivares de los R. prolixus, previamente inoculados intrafemoral o intracelómicamente con formas de cultivo de T. rangeli, no presentó correlación con la dosis inoculada; no obstante, la cantidad aplicada mostró relación directa con la tasa de mortalidad de los insectos. Los resultados indican que T. rangeli requiere un sistema de mantenimiento adecuado, para que en condiciones experimentales, conserve las características biológicas que normalmente expresa en condiciones naturales

Comparative investigations of Latin American trypanosomes with lectins and complement lysis test.

Four-days old epimastigote culture forms of different stocks of Trypanosoma cruzi from Colombia, Costa Rica and Mexico as well of Colombian stocks of T. rangeli were tested with 27 lectins and the complement lysis test. While the stocks of T. cruzi and T. rangeli showed common agglutination reactions with the lectins of Canavalia ensiformis and Pisum sativum, only the stocks of T. cruzi were additionally agglutinated by Ricinus communis-120, Glycine maxima, Helix pomatia, Axinella polypoides, Bandeiraea simplicifolia, Bauhinia purpurea, Wistaria floribunda, Abrus precatorius, Aaptos papillata II, Limax flavus and Arachis hypogaea. On the basis of lectin typing, the strains of T. cruzi belong to the PNA-type. While the epimastigote culture forms of T. cruzi stocks were lysed by normal fresh human, rat, chicken, rabbit and guinea pig serum, the culture forms of T. rangeli were only lysed by chicken serum. Mouse serum had no lytic effect on either trypanosome species. Incubation of T. cruzi with neuraminidase did not alter the lytic effect of the sera. The membrane-associated N-acetylneuraminic acid on T. cruzi does not protect the cell surface against the activation of the alternative complement pathway. After treatment of T. cruzi with neuraminidase and subsequent incubation in culture medium, the PNA-type was restored. The cells reacted again with the lectins of Arachis hypogaea, Limax flavus, Aaptos papillata II but not with Triticum vulgaris.

Antibody response to experimental Trypanosoma rangeli infection and its implications for immunodiagnosis of South American trypanosomiasis.

Differential immunodiagnosis of T. rangeli and T. cruzi infections in man poses a particular problem, not only because these parasites share antigenic determinants, as detected by immunofluorescence, but also because they have a similar geographical distribution, the same host range and often identical insect vectors. We show here that whereas mouse anti-T. rangeli sera have significant cross reactivity with T. cruzi by immunofluorescence, they are entirely specific when tested by ELISA, using apparently similar antigen preparations. Immunoprecipitation analysis detected relatively little cross-reactivity between heterologous antisera and parasite combinations. Intriguingly, immunization with T. rangeli epimastigotes was much more powerful than similar immunization with trypomastigotes, and the majority of the antibody was directed against a single polypeptide of apparent Mr 73kDa.