Evaluation of TNF-alpha, IL-4, and IL-10 and parasite density in spleen and liver of L. (L.) chagasi naturally infected dogs

Dogs are the main domestic reservoirs of L. (L.) chagasi. Once in the vertebrate host, the parasite can cause visceral leishmaniasis, which can also be transmitted to humans. Cytokines are key elements of the host immune response against Leishmania spp. To investigate whether tumor necrosis factor (TNF)-alpha, interleukin (IL)-4 and IL-10 are associated with pattern infection in dogs, these cytokines were quantified in the spleen and liver of dogs naturally infected with L. (L.) chagasi, with or without clinical manifestations, and their levels were correlated with the parasite load verified in these organs. A total of 40 adult dogs naturally infected with L. (L.) chagasi were assessed, together with 12 uninfected control dogs. Samples from spleen and liver were used to determine the cytokine levels by capture ELISA and for quantifying parasite load by real-time PCR. Statistical analysis was performed using the minimum Chi square method and group means were compared using the Tukey test. TNF-alpha, IL-4 and IL-10 levels in infected dogs were higher than in control groups; the liver was the main cytokine-producing organ during infection. The level of splenic TNF-alpha showed correlation with parasite load and may represent an important marker for infection process evolution, with the participation of IL-10. These results may contribute to a clearer understanding of the immune response in dogs infected with L. (L.) chagasi, which may lead to the development of prophylactic or preventive measures for these animals.

Comparison between ELISA using total antigen and immunochromatography with antigen rK39 in the diagnosis of canine visceral leishmaniasis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecular characterization of Cryptosporidium spp. from fecal samples of birds kept in captivity in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparative evaluation of enzyme-linked immunosorbent assay based on crude and purified antigen in the diagnosis of canine visceral leishmaniasis in symptomatic and oligosymptomatic dogs

This study evaluated the performance of crude total antigen (CTA) and fucose-mannose ligand antigen (FML) in an enzyme-linked immunosorbent assay for diagnosis of canine visceral leishmaniasis (CVL). The assays used sera from known negative controls (n = 30), clinically symptomatic (n = 30) and oligosymptomatic (n = 30) parasitologically proven infection (by microscopy). Aspirates of popliteal lymph node from infected canines were colleted to score parasitism and compared with the ELISA results. The study indicated that FML used in ELISA provided high sensitivity for detecting oligosymptomatic dogs (90%) and CTA showed greater sensitivity than FML for symptomatic canines (90%). In oligosymptomatic dogs, specificity was 100% for CTA-ELISA, but in symptomatic dogs, FML specificity was higher (96.7%) than CTA-ELISA (93.3%). A significant correlation was observed between the degree of parasitism and the results obtained in CTA-ELISA. Since no available antigen offers 100% specificity and sensitivity for CVL diagnosis, the choice of antigen used must depend on the aim of the investigation. (C) 2008 Elsevier B.V. All rights reserved.

Histopathology of the reproductive system of male sheep experimentally infected with Toxoplasma gondii

The aim of this study was to investigate the histopathological changes in reproductive system (testicles, epididymis, seminal vesicles, and prostate) of small male ruminants after Toxoplasma gondii infection. Eight sheep were inoculated with T. gondii: group I, four sheep (2.0 x 10(5) P-strain oocysts); group II, four sheep (1.0 x 10(6) RH-strain tachyzoites); and group III, two uninfected sheep maintained as control. Infection with T. gondii was confirmed by seroconversion (indirect fluorescent antibody test-IgG) in all the infected animals beginning on post-inoculation day (PID) 7. on PID 70, all the animals were euthanized and tissue samples (testicles, epididymis, seminal vesicles, and prostate) were collected and processed for histological analysis. The main changes detected were a focal mononuclear interstitial inflammatory infiltrate in the prostate and seminal vesicles; diffuse testicular degeneration associated with calcification foci and a multifocal mononuclear interstitial inflammatory infiltrate; and a mononuclear interstitial infiltrate and focal necrotic areas of the muscle fibers surrounding the seminal vesicles. The histopathological findings of this work, along with the detection of T. gondii in the examined parenchyma tissues (immunohistochemistry) and the results obtained by other authors examining different tissues, suggest that histological changes diagnosed in the reproductive system of rams infected with T. gondii are strongly suggestive of toxoplasmatic infection.

Distinct mitochondrial HSP70 homologues conserved in various Leishmania species suggest novel biological functions

We report the identification of two distinct homologues of the 70-kDa mitochondrial heat shock protein (mtHSP70) from Leishmania chagasi/Leishmania infantum (Lc2.1 and Lc2.2). in Leishmania species, multiple genes encoding Lc2.2 are present whilst single genes encode Lc2.1. Strikingly, genes encoding Lc2.1-like proteins are absent from Trypanosoma species. Lc2.2 is characterized by a poly-glutamine rich C-terminus, absent from Lc2.1 or mtHSP70 homologues outside the trypanosomatids. Lc2.1 displays unique substitutions within its peptide-binding domain which modify amino acids strictly conserved in cytoplasmic and mitochondrial HSP70 proteins alike. Affinity purified antibodies recognize mainly a single protein in extracts from promastigotes/epimastigotes of various Leishmania/Trypanosoma species. Upon differentiation of Leishmania amazonensis into amastigotes a second protein (presumably Lc2.1) is induced and becomes the predominant mtHSP70 homologue expressed. Subcellular localization of these proteins was investigated and ratified a distribution throughout the mitochondrial matrix. Our results imply novel mtHSP70 functions which evolved within the genus Leishmania. (C) 2008 Elsevier B.V. All rights reserved.

Immune response and performance of growing Santa Ines lambs to artificial Trichostrongylus colubriformis infections

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Relationship of abomasal histology and parasite-specific immunoglobulin A with the resistance to Haemonchus contortus infection in three breeds of sheep

This study was carried out to evaluate the relationship of abomasal inflammatory cells and parasite-specific immunoglobulin A (IgA) in mucus, with the resistance to Haemonchus contortus infection in three breeds of sheep naturally infected with gastrointestinal nematodes. The breeds were the native Santa Ines sheep, and the European Suffolk and Ile de France breeds. Mast cells, eosinophils and globule leucocytes were enumerated in abomasal mucosa. Eosinophils within the sub-mucosa also were counted separately. Histamine concentration was estimated in abomasal tissue samples. Enzyme-linked immunosorbent assay was carried out in mucus samples to determine the level of IgA anti-H. contortus third and fifth instar. There were no significant differences among group means of these variables (P > 0.05). The correlation coefficients between fecal egg counts (FEC) x mast cells (r = -0.490; P < 0.05) and FEC x eosinophils in sub-mucosa (r = -0.714; P < 0.01) was significant in the Santa Ines sheep. In the Ile de France group, the correlation coefficients between globule leucocytes x FEC (r = -0.879; P < 0.001) and histamine x worm burden (r = -0.833; P < 0.01) were also significant. In the Santa Ines and Ile de France sheep, correlation coefficients between IgA anti-L3 x worm burden and IgA anti-L3 x FEC were negative. In general, inflammatory cells and IgA-parasite-specific in abomasum were inversely associated with H. contortus worm burden and FEC indicating that they may impair parasite development or fecundity in the three breeds of sheep. However, similar mean values of inflammatory cells and IgA were found in the resistant (Santa Ines) and in the susceptible (Suffolk and Ile de France) breeds of sheep. The enumeration of cells by histological assessment does not provide information on their functional activity, which may be different among breeds. Thus, the effect of breed on the functional activity of these and other inflammatory cells is an important area for further study. (c) 2004 Elsevier B.V. All rights reserved.

Telomere biology of trypanosomatids: beginning to answer some questions

Studies of telomere structure and maintenance in trypanosomatids have provided insights into the evolutionary origin and conservation of some telomeric components shared by trypanosomes and vertebrates. For example, trypanosomatid telomeres are maintained by telomerase and consist of the canonical TTAGGG repeats, which in Trypanosoma brucei can form telomeric loops (t-loops). However, the telomeric chromatin of trypanosomatids is composed of organ ism-specific proteins and other proteins that share little sequence similarity with their vertebrate counterparts. Because telomere maintenance mechanisms are essential for genome stability, we propose that the particular features shown by the trypanosome telomeric chromatin hold the key for the design of antiparasitic drugs.

Host specificity of sheep and cattle nematodes in São Paulo state, Brazil

The trial was carried out to investigate parasite host specificity and to analyse the dynamics of infection with nematodes parasitizing sheep and catt:le raised together or separately in São Paulo state, Brazil, and, also to clarify doubts about the systematics of species of the genus Haemonchus on the basis of cytological and morphological studies. Ten steers and 32 ewes were randomly assigned to three paddocks (P), as follows: P1, 5 steers; P2, 5 steers and 16 ewes; and P3, 16 ewes. The animals remained on these paddocks in continuous grazing throughout the trial (1-yr period). Faecal exams and larvae counting on pasture were performed fortnightly. Once a month two tracer lambs were placed in each paddock, while two tracer calves were also placed, but only in the eighth month of the trial. All these animals were slaughtered for worm identification and counting. At the end of the trial, one steer and one ewe from P2, which showed high faecal egg counts, were also slaughtered for the same purpose. Nematodes identified cytogenetically as H. placei presented spicule hooks longer than those identified as H. contortus. The following distribution of parasites in cattle and sheep was observed: Bunostomum phlebotomum, H. similis, Mammomonogamus laryngeus strongly adapted to cattle, H. placei and Cooperia punctata more adapted to cattle than to sheep, Trichostrongylus axel and C. spatulata apparently more adapted to cattle, T. colubriformis strongly adapted to sheep, H. contortus more adapted to sheep than to cattle and C. curticei apparently more adapted to sheep. Cross-infection was shown to occur involving some species, however, with time the animals apparently eliminate the species that are not well adapted to them. Therefore, grazing management systems using cattle and sheep appear to be promising for worm control in southeastern Brazil. (C) 1997 Elsevier B.V. B.V.

Schistosoma mansoni: Evaluation of an RNAi-based treatment targeting HGPRTase gene

Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) is an essential gene of the parasite Schistosoma mansoni and it is well conserved in its hosts (mouse and human) at the protein but not at the RNA level. This feature prompted us to assess RNA interference (RNAi) to combat schistosomiasis. Small interfering RNAs (siRNAs) were Produced against HGPRTase, injected in infected mice and the number of worms was counted six days after injection. The total number of parasites was reduced by approximately 27% after treatment. RT-PCR analyzes showed a significant reduction in parasite target mRNA but not in host's homologue. The use of low doses of molecules did not oversaturate si- or miRNA pathways as mice survival rates were not affected by siRNAs. This is the first successful in vivo demonstration of a RNAi-based treatment against schistosomiasis. We believe that improvements in molecule delivery and an increase on siRNA dose could rapidly eliminate parasite. (c) 2007 Elsevier B.V. All rights reserved.

Leishmania amazonensis: Partial purification and study of the biochemical properties of the telomerase reverse transcriptase activity from promastigote-stage

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Sir2-Related Protein 1 from Leishmania amazonensis is a glycosylated NAD(+)-dependent deacetylase

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Study of Toxoplasma infection in Brazilian wild mammals: Serological evidence in Dasypus novemcinctus Linnaeus, 1758 and Euphractus sexcinctus Wagler, 1830

Toxoplasmosis is a worldwide distributed zoonosis that affects man and most warm-blooded animals, with a great economic impact in animal and public health. Serum samples from nine 9-banded armadillos, three 6-banded armadillos, three coatimundis, two opossums and one nutria were submitted for anti-Toxoplasma gondii antibody detection by means of a modified direct agglutination method. Encephalic tissue of three 6-banded armadillos, one 9-banded armadillo, one coatimundi and one nutria were digested in acid pepsin solution and inoculated into Swiss mice for parasite isolation. Only one serum sample from a nine-banded armadillo and two from six-banded armadillos reacted producing titers equal to 256, 512 and 512, respectively. T gondii was isolated in two 6-banded armadillos, one of which was not positive in the serological test. (c) 2005 Elsevier B.V. All rights reserved.

Toxoplasma gondii and Leptospira spp. infection in free-ranging armadillos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)