Frequency of IFNγ-producing T cells correlates with seroreactivity and activated T cells during canine Trypanosoma cruzi infection.

Vaccines to prevent Trypanosoma cruzi infection in humans or animals are not available, and in many settings, dogs are an important source of domestic infection for the insect vector. Identification of infected canines is crucial for evaluating peridomestic transmission dynamics and parasite control strategies. As immune control of T. cruzi infection is dependent on humoral and cell-mediated immune responses, we aimed to define a serodiagnostic assay and T cell phenotypic markers for identifying infected dogs and studying the canine T. cruzi-specific immune response. Plasma samples and peripheral blood mononuclear cells (PBMCs) were obtained from forty-two dogs living in a T. cruzi-endemic region. Twenty dogs were known to be seropositive and nine seronegative by conventional serologic tests two years prior to our study. To determine canine seroreactivity, we tested sera or plasma samples in a multiplex bead array against eleven recombinant T. cruzi proteins. Ninety-four percent (17/18) of dogs positive by multiplex serology were initially positive by conventional serology. The frequency of IFNγ-producing cells in PBMCs responding to T. cruzi correlated to serological status, identifying 95% of multiplex seropositive dogs. Intracellular staining identified CD4+ and CD8+ T cell populations as the sources of T. cruzi lysate-induced IFNγ. Low expression of CCR7 and CD62L on CD4+ and CD8+ T cells suggested a predominance of effector/effector memory T cells in seropositive canines. These results are the first, to our knowledge, to correlate T. cruzi-specific antibody responses with T cell responses in naturally infected dogs and validate these methods for identifying dogs exposed to T. cruzi.

Trans-sialidase inhibition assay detects Trypanosoma cruzi infection in different wild mammal species.

The detection of Trypanosoma cruzi infection in mammals is crucial for understanding the eco-epidemiological role of the different species involved in parasite transmission cycles. Xenodiagnosis (XD) and hemoculture (HC) are routinely used to detect T. cruzi in wild mammals. Serological methods are much more limited because they require the use of specific antibodies to immunoglobulins of each mammalian species susceptible to T. cruzi. In this study we detected T. cruzi infection by trans-sialidase (TS) inhibition assay (TIA). TIA is based on the antibody neutralization of a recombinant TS that avoids the use of anti-immunoglobulins. TS activity is not detected in the co-endemic protozoan parasites Leishmania spp and T. rangeli. In the current study, serum samples from 158 individuals of nine wild mammalian species, previously tested by XD, were evaluated by TIA. They were collected from two endemic areas in northern Argentina. The overall TIA versus XD co-reactivity was 98.7% (156/158). All 18 samples from XD-positive mammals were TIA-positive (co-positivity, 100%) and co-negativity was 98.5% (138/140). Two XD-negative samples from a marsupial (Didelphis albiventris) and an edentate (Dasypus novemcinctus) were detected by TIA. TIA could be used as a novel tool for serological detection of Trypanosoma cruzi in a wide variety of sylvatic reservoir hosts.

trans-Sialidase neutralizing antibody detection in Trypanosoma cruzi-infected domestic reservoirs.

The detection of Trypanosoma cruzi infection in domestic dogs and cats is relevant to evaluating human transmission risks and the effectiveness of insecticide spraying campaigns. However, the serological assays routinely used are associated with cross-reactivity in sera from mammals infected with Leishmania spp. We used a trans-sialidase inhibition assay (TIA) for T. cruzi diagnosis in serum samples from 199 dogs and 57 cats from areas where these types of infections are endemic. TIA is based on the antibody neutralization of recombinant trans-sialidase, an enzyme that is not detected in the coendemic Leishmania species or Trypanosoma rangeli parasites. T. cruzi infection was also evaluated by conventional serology (CS) (indirect immunofluorescence, indirect hemagglutination, enzyme-linked immunosorbent assay, and immunochromatographic dipstick test) and xenodiagnosis. Sera from 30 dogs and 15 cats from areas where these organisms are not endemic and 5 dogs with visceral leishmaniasis were found to be nonreactive by TIA and CS. Samples from dogs and cats demonstrated 91 and 95% copositivities between TIA and CS, whereas the conegativities were 98 and 97%, respectively. Sera from xenodiagnosis-positive dogs and cats also reacted by TIA (copositivities of 97 and 83%, respectively). TIA was reactive in three CS-negative samples and was able to resolve results in two cat serum samples that were CS inconclusive. Our study is the first to describe the development of trans-sialidase neutralizing antibodies in naturally infected dogs and cats. High CS conegativity and the absence of trans-sialidase neutralization in dog sera from areas where leishmaniasis is not endemic and from dogs with visceral leishmaniasis support TIA specificity. The TIA may be a useful tool for T. cruzi detection in the main domestic reservoirs.