Chagas Disease Diagnostic Applications: Present Knowledge and Future Steps.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is a lifelong and debilitating illness of major significance throughout Latin America and an emergent threat to global public health. Being a neglected disease, the vast majority of Chagasic patients have limited access to proper diagnosis and treatment, and there is only a marginal investment into R&D for drug and vaccine development. In this context, identification of novel biomarkers able to transcend the current limits of diagnostic methods surfaces as a main priority in Chagas disease applied research. The expectation is that these novel biomarkers will provide reliable, reproducible and accurate results irrespective of the genetic background, infecting parasite strain, stage of disease, and clinical-associated features of Chagasic populations. In addition, they should be able to address other still unmet diagnostic needs, including early detection of congenital T. cruzi transmission, rapid assessment of treatment efficiency or failure, indication/prediction of disease progression and direct parasite typification in clinical samples. The lack of access of poor and neglected populations to essential diagnostics also stresses the necessity of developing new methods operational in point-of-care settings. In summary, emergent diagnostic tests integrating these novel and tailored tools should provide a significant impact on the effectiveness of current intervention schemes and on the clinical management of Chagasic patients. In this chapter, we discuss the present knowledge and possible future steps in Chagas disease diagnostic applications, as well as the opportunity provided by recent advances in high-throughput methods for biomarker discovery.

Avian influenza surveillance in backyard poultry of Argentina.

Avian influenza (AI) is an exotic disease in Argentina. A surveillance program for AI was conducted in backyard poultry during 1998-2005 in two regions: 1) region A, which included the avian population in the provinces that border Brazil, Bolivia, and Paraguay, and 2) region B, which included the rest of the provinces of the country. More than 8000 serum samples were tested for antibodies by enzyme-linked immunosorbent assay and/or agar gel immunodiffusion tests, and more than 18,000 tracheal and cloacal swabs were tested for virus by isolation in embryonated specific-pathogen-free eggs. This study was part of the AI prevention program in Argentina, which includes other avian populations such as commercial poultry and all the controls for importation and exportation of live birds. The results from backyard poultry were negative for AI.

Classification of Marek's disease viruses according to pathotype: philosophy and methodology.

The concept of pathotype in Marek's disease (MD) probably dates from the recognition of a more virulent form of the disease in the late 1950s (Benton & Cover, 1957). Distinctions between MD virus strains were further expanded with the description of the vv pathotype in the early 1980s and of the vv+ pathotype in the 1990s. Pathotype designations reflect important biological properties that correlate with the break-through of vaccinal immunity in the field. However, pathotyping methods applied by various laboratories have not been uniform, preventing critical comparison of results. Better uniformity of pathotyping procedures is desirable.The Avian Disease and Oncology Laboratory (ADOL) method is based on induction of lymphoproliferative lesions in vaccinated chickens. This method has been used to pathotype more than 45 isolates and is the basis for the current pathotype classification of MD virus strains. Its limitations include requirements for a specific type of chickens (15x7 ab+), large numbers of animals, and a statistical method to compare lesion responses to those of JM/102W and Md5 control strains. Because of these limitations, it has not been and is not likely to be used in other laboratories. Comparability in pathotyping can be improved by the comparison of field isolates with standard prototype strains such as JM/102W, Md5 and 648A (American Type Culture Collection) or their equivalents. Data may be generated by different in vivo procedures that measure tumour induction, neurological disease (both neoplastic and non-neoplastic lesions), or solely non-neoplastic criteria (such as lymphoid organ weights or virus replication). Methods based on neoplastic criteria, especially when generated in MD-immunized chickens, will probably correlate most closely with that of the ADOL method and be most relevant to evolution of MD virus in the field. Based on data from several trials, a modification of the ADOL method that utilizes fewer chickens and can be conducted with commercial specific pathogen free strains is proposed. The modified method is based on "best fit" comparisons with prototype strains, and is expected to provide results generally comparable with the original method. A variety of other alternative criteria (see earlier) are also evaluated both for primary pathotyping and as adjuncts to other pathotyping methods. Advantages and disadvantages of alternative methods are presented.

Multiple overlapping epitopes in the repetitive unit of the shed acute-phase antigen from Trypanosoma cruzi enhance its immunogenic properties.

The repetitive shed acute-phase antigen (SAPA) from Trypanosoma cruzi was thoroughly mapped by SPOT peptides and phage display strategies, showing that a single SAPA repeat is composed of multiple overlapping B-cell epitopes. We propose that this intricate antigenic structure constitutes an alternative device to repetitiveness in order to improve its immunogenicity.

Comparative susceptibility of Marek's disease cell lines to chicken infectious anemia virus.

Chicken infectious anemia virus (CIAV) is known to infect and replicate in various Marek's disease chicken cell lines (MDCCs) derived from Marek's disease (MD) tumors. One line, MDCC-MSB1, has been the substrate used in most studies. We compared a total of 26 MDCCs, including two sublines of MDCC-MSB1, MSB1 (L) and MSB1 (S), four other MD tumor-derived lines, and 20 lines derived from MD virus-induced local lesions, for susceptibility to the Cux-1 and CIA-1 strains of CIAV. The cell lines represented six phenotypic groups of T cells based on the expression of CD4, CD8, and TCR-2 and -3 surface markers. Susceptibility was measured by the number of cells positive for viral antigen in immunofluorescence (IF) tests at 3-10 days postinfection. No clear-cut differences were found in susceptibility related to phenotype, although CD4-/8+ lines and CD4-/8- lines might be more susceptible than CD4+/8- lines. However, several individual lines were more susceptible to Cux-1 than the two MSB1 sublines tested. Contrary to an earlier report, cells of MDCC-CU147, a CD8+, TCR3+, local-lesion derived line, were found to be susceptible to CIA-1. In fact, CU147 was distinguished by very high susceptibility to both CIAV strains. In direct comparisons with MSB1, CU147 detected approximately 10-fold lower doses of virus. Also, virus spread was faster (P < 0.05) in CU147 than in MSB1 and other lines. Results from polymerase chain reaction (PCR) tests to detect infection in titrations were in general agreement with IF test results although PCR detected infection in a few terminal dilution cultures that were negative by IF.

Tandem amino acid repeats from Trypanosoma cruzi shed antigens increase the half-life of proteins in blood.

Proteins containing amino acid repeats are widespread among protozoan parasites. It has been suggested that these repetitive structures act as immunomodulators, but other functional aspects may be of primary importance. We have recently suggested that tandem repeats present in Trypanosoma cruzi trans-sialidase stabilize the catalytic activity in blood. Because the parasite releases trans-sialidase, this delayed clearance of the enzyme might have implications in vivo. In the present work, the ability of repetitive units from different T. cruzi molecules in stabilizing trans-sialidase activity in blood was evaluated. It is shown that repeats present on T. cruzi shed proteins (antigens 13 and Shed-Acute-Phase-Antigen [SAPA]) increase trans-sialidase half-life in blood from 7 to almost 35 hours. Conversely, those repeats present in intracellular T. cruzi proteins only increase the enzyme half-life in blood up to 15 hours. Despite these results, comparative analysis of structural and catalytic properties of both groups of chimeric enzymes show no substantial differences. Interestingly, antigens 13 and SAPA also increase the persistence in blood of chimeric glutathione S-transferases, thus suggesting that this effect is inherent to these repeats and independent of the carrier protein. Although the molecular basis of this phenomenon is still uncertain, its biotechnological potential can be envisaged.

Relationship between the immunosuppressive potential and the pathotype of Marek's disease virus isolates.

Isolates of Marek's disease virus (MDV) representing three pathotypes of differing virulence were compared for relative immunosuppressive properties in genetically susceptible P2a-strain and genetically resistant N2a-strain chickens. Criteria of immunosuppression were 1) persistence of early cytolytic infection (i.e., a delay or failure to enter latency) in lymphoid organs, 2) atrophy of the bursa of Fabricius and thymus as measured by organ weight proportional to body weight at 8 and 14 days postinfection (DPI), and 3) histopathologic evidence of necrosis and atrophy in lymphoid organs. No significant differences in infection level were observed among the pathotypes during the early (4-5 DPI) period of infection. However, the extent of persistent cytolytic infection at 7-8 DPI, based on numbers of tissues positive and mean scores in immunofluorescence tests, was greater (P < 0.05) for three isolates (RK1, 584A, 648A) in the highest virulence pathotype (very virulent-plus MDV [vv + MDV]) than for two isolates (JM16, GA5) in a lower virulence (virulent MDV [vMDV]) pathotype. Results from two isolates (RB1B, Md5) classified in the intermediate very virulent pathotype (very virulent MDV [vvMDV]) fell between those from the other two pathotypes. Similarly, there was a stepwise effect of viral pathotype in which the vv + MDV isolates caused the most severe damage to lymphoid organs in terms of atrophy (relative organ weights) and histopathologic changes. Organs from chickens infected with vv + MDVs showed little recovery between 8 and 14 DPI. The vMDV isolates caused the least severe damage, and lymphoid organs showed a significant return toward normal by 14 DPI; vvMDV isolates induced intermediate degrees of atrophy and recovery. The same pattern of relationship between virulence pathotype and degree of bursal and thymic atrophy was also observed in genetically resistant N2a chickens. These results suggest that the degree of immunosuppression is linked to virulence and that a simple measure of atrophic changes (relative organ weights) in the bursa of Fabricius and thymus might be useful in determining the pathotype classification of new MDV isolates. The basis for differences in immunosuppressive potential of MDV isolates needs further clarification.

The repetitive domain of Trypanosoma cruzi trans-sialidase enhances the immune response against the catalytic domain.

Trypanosoma cruzi trans-sialidase consists of a C-terminal domain composed essentially of immunodominant amino acid repeat units (SAPA-repeats) and an amino region responsible for the enzymatic activity (catalytic domain). To investigate the possible function(s) of SAPA-repeats, recombinant trans-sialidases either containing or lacking the C-terminal region were tested in mice. The presence of SAPA-repeats in the intravenously injected protein has two consequences. First, they enhance the persistence of the trans-sialidase activity in blood. Second, SAPA-repeats promoted the production of antibodies directed to the catalytic domain that inhibit trans-sialidase activity. These results suggest that SAPA-repeats modulate the trans-sialidase activity in blood.

Regulation of ovarian follicle differentiation in gonadotrophin-stimulated rats.

The aim of the present study was to investigate the regulation of the in vitro DNA synthesis of ovarian cells recovered from prepubertal rats 48 h after administration of pregnant mare's serum gonadotrophin alone (granulosa cells) or followed by human chorionic gonadotrophin (luteal cells). Isolated granulosa cells were cultured in serum-free medium, different stimuli added for periods of 48 h, and 3H-thymidine incorporation was measured. Both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) inhibited 3H-thymidine incorporation by cultured granulosa cells in a dose-dependent manner (FSH: 10, 100, 200 ng/mL = 26, 41, 49% inhibition, respectively; LH: 0.1, 1, 10 ng/mL = 11, 37, 75% inhibition, respectively). On the other hand, estradiol was found to stimulate 3H-thymidine incorporation in granulosa cells (Estradiol: 5, 50, 500 ng/mL = 17, 37, 76% stimulation, respectively). In luteal cells, the rate of basal 3H-thymidine incorporation was very low (granulosa cells: 2560 +/- 310; luteal cells: 661 +/- 92 cpm/100,000 cells) and not modified by any stimulus. To determine the possible production of an inhibitory growth factor by the early corpus luteum, 3H-thymidine incorporation by granulosa cells was assessed in the presence of 10% conditioned media (CM) recovered from luteal cell cultures. A marked inhibition both in basal and estradiol-stimulated 3H-thymidine incorporation was observed (74 and 76% of inhibition, respectively). Results suggest that an inhibitory growth factor produced by luteal cells after luteinizing gonadotrophin stimulus could be involved in the differentiation of growing follicles to corpus luteum.

A putative pyruvate dehydrogenase alpha subunit gene from Trypanosoma cruzi.

A full-length DNA clone encoding a putative pyruvate dehydrogenase alpha subunit (E1 alpha) gene was isolated from a Trypanosoma cruzi (RA strain) DNA library. Sequencing of this clone revealed it to encode a 378 amino acid protein (M(r) 42774) with high sequence similarity to E1 alpha obtained from different sources. The highest score is obtained with human E1 alpha: 43,3% similarity. Southern blot analysis is consistent with the existence of a single copy of this putative T. cruzi E1 alpha gene per haploid genome in different parasite strains. Expression of this gene was demonstrated by Northern blot analysis and its trans-splicing acceptor site was identified by Polymerase Chain Reaction-mediated amplification of its cDNA.

Identification of chicken infectious anaemia, isolation of the virus and reproduction of the disease in Argentina.

Three chicken infectious anaemia virus (CIAV) isolates were obtained from broiler flocks with anaemia and poor performance, and were designated AIP-1, 2 and 3. All isolates were resistant to treatment with chloroform and were able to pass through 50-nm pore-size filters. The CIAV isolates induced signs and lesions of chicken infectious anaemia (CIA): thymus atrophy, bone marrow aplasia, low haematocrit values, and body weight reduction when inoculated into 1-day-old susceptible chicks. Microscopic lesions were a reflection of macroscopic observations. CIAV-specific antigens have been demonstrated in tissues of experimentally-infected chicks using monoclonal antibodies specific for CIAV. The presence of antibodies against CIAV in breeders was determined by indirect immunofluorescence tests. Although the chicks derived from breeder flocks that possessed antibodies against CIAV, they were not refractory to infection. These findings, the characteristics of the virus and the virus-induced lesions, demonstrate that CIAV is present in Argentina.

Effect of reticuloendotheliosis virus and infectious bursal disease virus on Marek's disease herpesvirus latency.

Birds infected with reticuloendotheliosis virus (REV) were exposed to Marek's disease virus (MDV) to determine if the establishment of MDV latency was affected by REV-induced immunosuppression, while other chickens, already latently infected with MDV, were challenged with REV or infectious bursal disease virus (IBDV) to determine if the consequent immunosuppression caused a return to cytolytic infection. Immunosuppression was assessed by in vitro mitogen stimulation assays with spleen cells. Latently MDV-infected cells were free of viral internal antigen(s) (VIA) but could be identified by their ability to produce VIA after in vitro cultivation. The results were unexpected: chickens infected with either of these viruses had very low, and often undetectable, levels of MDV infection when compared with appropriate controls. REV infection interfered with early cytolytic MDV infection, and IBDV and REV both failed to activate latent MDV infection in the face of inferred (IBDV) or demonstrated (REV) immunosuppression by these viruses. Apparently, both viruses reduced the number of MDV infected cells since neither cytolytic nor latent infection could be demonstrated. This was based on an absence of cells with VIA either before or after cultivation and, in the case of REV infection, on failure to detect MDV-DNA using a dot-blot hybridisation technique.

Maintenance of Marek's disease herpesvirus latency in vitro by a factor found in conditioned medium.

Chicken spleen cells latently infected with Marek's disease virus were cultured with and without conditioned medium (CM) obtained from concanavalin A-stimulated chicken spleen cell cultures. The expression of viral internal antigen(s) (VIA), which is usually associated with cultivation, was prevented or markedly reduced by the CM. This effect required the continued presence of CM, since its removal after 48 h resulted in the subsequent appearance of VIA. Although CM contains both gamma interferon (IFN-gamma) and interleukin 2, our studies suggest that the 'latency-maintaining activity' (LMF) may not be associated with either of these products of stimulated lymphocytes. However, IFN-gamma may also have had some suppressive effect. LMF appears to have an Mr greater than 10,000 and to be inactivated by heating to 90 degrees C for 5 min.

Effect of immunocompetence on the establishment and maintenance of latency with Marek's disease herpesvirus.

Marek's disease virus (MDV) infections normally have an early cytolytic phase in lymphoid organs at 3 to 6 days post-infection followed by a period of latent infection. Little is known about the mechanisms that govern latency with herpesvirus infections, including Marek's disease (MD). To investigate the importance of immunocompetence for either the establishment or the maintenance of latency in MD, immunosuppressive treatments were applied prior to infection with MDV or after latency was established. These included cyclosporin (Cs) or betamethasone (BM) treatments, neonatal thymectomy plus cyclophosphamide treatment (Tx/Cy), and infection at a young age before full competence. The effect of all the treatments was determined by examining tissues and spleen cells for evidence of virus replication before and after cultivation in vitro. Induced (Cs or Tx/Cy treatments) or natural (infection at a young age) incompetence resulted in prolonged and more widespread early cytolytic infection. Immunosuppression by Cs after latency had developed resulted in a reappearance of cytolytic infection in the spleen and it enhanced the cytolytic infection in the thymus and the bursa of Fabricius. After immunosuppression with Cs, cytolytic infection was found mostly in T cells, although many of the virus-positive cells did not have markers for either T cells or B cells. Immunosuppression by BM after latency had developed also resulted in the reappearance of cytolytic infection in the spleen but only at a very low level. These results suggest that immunocompetence is required for the establishment and maintenance of latency.

Cell-culture virus-neutralization test and enzyme-linked immunosorbent assay for evaluation of immunity in chickens against fowlpox.

Two serological tests--the virus-neutralization (VN) test in chicken embryo fibroblasts (CEF) using a cell-culture-adapted virus, and the enzyme-linked immunosorbent assay (ELISA)--were used for evaluating the immune response in chickens against fowlpox virus. The VN test was conducted in 96-well tissue-culture plates using a fowlpox virus that was adapted to induce cytopathic effects (CPE) in CEF in 48 hr. The ELISA was carried out with an antigen prepared by precipitation of a cell-culture-propagated virus suspension with ammonium sulfate and concentration by centrifugation. A 0.1 M acetate buffer, pH 5, was used as the sensitizing solution for maximum specific binding of the antigen to the microplate plastic well. No antibodies were detected by the VN test in 228 serum samples taken from chickens at irregular intervals between 1 and 39 weeks of age, even though the birds were vaccinated against fowlpox at 13 weeks of age. However, in sera collected 4 weeks after a sample of laying hens was challenged with fowlpox virus, VN titers of 1/10 to 1/40 were detectable. On the other hand, significant antibody reactions were detected by the ELISA on sera from chickens during the growing period, following vaccination and challenge. Although no maternal antibodies were found at 1 week of age, a continuous increase in the mean ELISA titers to fowlpox was demonstrated during the entire experimental period. This study showed that the ELISA was considerably more sensitive and practical than the VN test.