Expression of IL-4, IL-10 and IFN-γ in the liver tissue of cattle that are naturally infected with Fasciola hepatica.

The role of interleukin IL-4, IL-10 and interferon gamma cytokines on natural Fasciola hepatica infection was investigated by quantifying the mRNA levels in liver tissue from chronically infected cattle. IL-4 and IL-10 had higher expression relative to interferon gamma in the liver tissue of infected animals when compared with the control group. The higher levels of IL-10 and IL-4 observed in the present study suggest a synergism between these cytokines, as well as involvement in the suppression of TH1 cell responses and a consequent induction of decreased interferon gamma expression in chronic cattle fascioliasis. The cytokine ratios were positively correlated, indicating a predominance of IL-4 in the chronic phase of infection with respect to interferon gamma and IL-10. Interferon gamma was predominant expressed in the controls, suggesting the involvement of IL-10 in modulating the immune response in favor of IL-4 in infected animals. Our results suggest that the TH2 polarized host immune response previously observed in experimental infection may also be responsible for establishing chronic phase and the maintenance of the natural infection of cattle from endemic areas that are in continuous contact with parasite.

The MASP family of Trypanosoma cruzi: changes in gene expression and antigenic profile during the acute phase of experimental infection.

BACKGROUND: Trypanosoma cruzi is the etiological agent of Chagas disease, a debilitating illness that affects millions of people in the Americas. A major finding of the T. cruzi genome project was the discovery of a novel multigene family composed of approximately 1,300 genes that encode mucin-associated surface proteins (MASPs). The high level of polymorphism of the MASP family associated with its localization at the surface of infective forms of the parasite suggests that MASP participates in host-parasite interactions. We speculate that the large repertoire of MASP sequences may contribute to the ability of T. cruzi to infect several host cell types and/or participate in host immune evasion mechanisms. METHODS: By sequencing seven cDNA libraries, we analyzed the MASP expression profile in trypomastigotes derived from distinct host cells and after sequential passages in acutely infected mice. Additionally, to investigate the MASP antigenic profile, we performed B-cell epitope prediction on MASP proteins and designed a MASP-specific peptide array with 110 putative epitopes, which was screened with sera from acutely infected mice. FINDINGS AND CONCLUSIONS: We observed differential expression of a few MASP genes between trypomastigotes derived from epithelial and myoblast cell lines. The more pronounced MASP expression changes were observed between bloodstream and tissue-culture trypomastigotes and between bloodstream forms from sequential passages in acutely infected mice. Moreover, we demonstrated that different MASP members were expressed during the acute T. cruzi infection and constitute parasite antigens that are recognized by IgG and IgM antibodies. We also found that distinct MASP peptides could trigger different antibody responses and that the antibody level against a given peptide may vary after sequential passages in mice. We speculate that changes in the large repertoire of MASP antigenic peptides during an infection may contribute to the evasion of host immune responses during the acute phase of Chagas disease.

Genomic analyses, gene expression and antigenic profile of the trans-sialidase superfamily of Trypanosoma cruzi reveal an undetected level of complexity.

The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a highly debilitating human pathology that affects millions of people in the Americas. The sequencing of this parasite's genome reveals that trans-sialidase/trans-sialidase-like (TcS), a polymorphic protein family known to be involved in several aspects of T. cruzi biology, is the largest T. cruzi gene family, encoding more than 1,400 genes. Despite the fact that four TcS groups are well characterized and only one of the groups contains active trans-sialidases, all members of the family are annotated in the T. cruzi genome database as trans-sialidase. After performing sequence clustering analysis with all TcS complete genes, we identified four additional groups, demonstrating that the TcS family is even more heterogeneous than previously thought. Interestingly, members of distinct TcS groups show distinctive patterns of chromosome localization. Members of the TcSgroupII, which harbor proteins involved in host cell attachment/invasion, are preferentially located in subtelomeric regions, whereas members of the largest and new TcSgroupV have internal chromosomal locations. Real-time RT-PCR confirms the expression of genes derived from new groups and shows that the pattern of expression is not similar within and between groups. We also performed B-cell epitope prediction on the family and constructed a TcS specific peptide array, which was screened with sera from T. cruzi-infected mice. We demonstrated that all seven groups represented in the array are antigenic. A highly reactive peptide occurs in sixty TcS proteins including members of two new groups and may contribute to the known cross-reactivity of T. cruzi epitopes during infection. Taken together, our results contribute to a better understanding of the real complexity of the TcS family and open new avenues for investigating novel roles of this family during T. cruzi infection.

Infectivity of seropositive dogs, showing different clinical forms of leishmaniasis, to Lutzomyia longipalpis phlebotomine sand flies.

Visceral leishmaniasis (VL) is a growing zoonosis with an increasing number of new cases and a rapid geographical spreading of the disease. In the present study, a canine survey was carried out in the city of Montes Claros (320,000 inhabitants), an endemic area of American visceral leishmaniasis in the state of Minas Gerais, Brazil. A total number of 4795 dogs were examined by serology, which showed a rate of seropositivity of 5%. Isoenzymatic analysis confirmed Leishmania infantum chagasi as the local aetiological agent of CVL. Canine tissues were assayed for the presence of Leishmania parasite DNA using different techniques. The infectivity of asymptomatic, oligosymptomatic and symptomatic seropositive dogs was tested by xenodiagnosis using laboratory reared Lutzomyia longipalpis. Rates of infection of 5.4%, 5.1% and 28.4% were found for the phlebotomine sand flies that fed in asymptomatic, oligosymptomatic and symptomatic dogs, respectively. Our results indicate that, under experimental conditions, symptomatic dogs are about four times more infective to VL vectors than oligosymptomatic or asymptomatic animals. The lower infectivity rates of dogs displaying any of the last two clinical forms of leishmaniasis, however, must be taken into account in the epidemiology of CVL.

Use of PCR-RFLP to identify Leishmania species in naturally-infected dogs.

Tissue imprints on Giemsa stained slides from dogs were used to investigate the presence of Leishmania amastigotes by either optical microscopy (OM) or Polymerase chain reaction (PCR) detection of DNA. Samples from skin, spleen, lymph node, liver and bone marrow from a Leishmaniasis endemic area dogs where Leishmania (Leishmania) chagasi and Leishmania (Viannia) braziliensis are sympatric were studied. Dogs were initially diagnosed by Indirect Immunofluorescence (IIF), as which 39 were IIF positive (> or = 1:40) and 16 negative. The IIF positive dogs were clinically grouped as symptomatic (n = 15), oligosymptomatic (n = 12) and asymptomatic (n = 12). Although PCR positivity was higher in symptomatic dogs, specially their skin samples, there was no significant difference among clinical groups or organs examined. Ten (62.5%) out of 16 IIF and OM negative animals were positive for PCR in at least one organ. Forty-eight positive PCR amplicons were further submitted to RFLP for Leishmania identification. All dogs were infected with L. (L.) chagasi except one, infected with L. (V.) braziliensis. PCR was more efficient than IIF and OM to diagnose canine visceral Leishmaniasis (CVL), regardless of the organ examined and the clinical form present. The use of PCR together with serology helps determining the extension of sub clinical infection in CVL endemic areas and provides a better estimate of the number of dogs to be targeted for control measures. In conclusion, our data reinforce the need for a specific diagnosis of canine infection in areas where diverse Leishmania species are sympatric and demonstrate that PCR-RFLP can be used to identify Leishmania species in dog tissue imprint stained slides.