Recombinant Cysteine Proteinase B from Leishmania braziliensis and Its Domains: Promising Antigens for Serodiagnosis of Cutaneous and Visceral Leishmaniasis in Dogs.

Leishmaniasis represents a group of parasitic diseases caused by a protozoan of the genus Leishmania and is widely distributed in tropical and subtropical regions. Leishmaniasis is one of the major tropical neglected diseases, with 1.5 to 2 million new cases occurring annually. Diagnosis remains a challenge despite advances in parasitological, serological, and molecular methods. Dogs are an important host for the parasite and develop both visceral and cutaneous lesions. Our goal was to contribute to the diagnosis of canine cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) using the recombinant cysteine proteinase B (F-CPB) from Leishmania braziliensis and its N- and C-terminal domains (N-CPB and C-CPB) as antigens in an enzyme-linked immunosorbent assay (ELISA). Sera from dogs from Northwest Argentina diagnosed with CL were tested by ELISA against a supernatant of L. braziliensis lysate, the F-CPB protein, and its domains. We found values of sensitivity (Se) of 90.7%, 94.4%, and 94.3% and specificity (Sp) of 95.5%, 90.9%, and 91.3% for F-CPB and its N- and C-terminal domains, respectively. In sera from dogs diagnosed with VL from Northeast Argentina, we found Se of 93.3%, 73.3%, and 66.7% and Sp of 92.3%, 76.9%, and 88.5% for F-CPB and its N- and C-terminal domains, respectively. These results support CPB as a relevant antigen for canine leishmaniasis diagnosis in its different clinical presentations. More interestingly, the amino acid sequence of CPB showed high percentages of identity in several Leishmania species, suggesting that the CPB from L. braziliensis qualifies as a good antigen for the diagnosis of leishmaniasis caused by different species.

Benznidazole treatment in chronic children infected with Trypanosoma cruzi: serological and molecular follow-up of patients and identification of Discrete Typing Units.

A total of 221 children from two rural settlements in Northeast Argentina were examined for T. cruzi infection. Blood samples were taken for serology tests and PCR assays. In addition, T. cruzi Discrete Typing Units (DTUs) were determined by hybridization with specific DNA probes of the minicircle hypervariable regions (mHVR). Serological results indicated that 26% (57/215) were reactive against T. cruzi antigens. PCR analyses were performed on seropositive samples showing presence of parasite DNA in 31 out of 53 samples (58.5%). All seropositive children underwent specific chemotherapy with Benznidazole (5mg/kg/day) for a period of two months and were monitored two and five years after treatment. Overall the treatment was well tolerated and low side effects were observed. Serological conversion was observed at two years post -treatment in one child form Pampa Ávila and at five years in two children from Tres Estacas. However, at the end of the follow-up period, T. cruzi DNA could not be detected by PCR in samples from treated children, except in two cases. In addition, the results of hybridizations with specific DNA probes showed that DTU TcV was detected in 68% (21/31), TcVI in 7% (2/31) and TcV/VI in 3% (1/31) of the samples. Altogether, results of the follow-up of treated children showed a low rate of seroconversion; however trend toward seroconversion was evident at five years post-treatment. On the other hand, detection of T. cruzi DNA by PCR significantly decreased after Benznidazole treatment. The existence of data regarding serological and molecular follow-ups from controlled studies in the Chaco Region will be important for future treatment efforts against T. cruzi infection in this region. The results obtained in the present study represent a contribution in this regard.

Inheritance of resistance to pyrethroids in Triatoma infestans, the main Chagas disease vector in South America.

An outbreak of pyrethroid resistance was recently detected in Triatoma infestans from northern Argentina. To analyze the inheritance of the resistant phenotype, we carried out experimental crosses between resistant (R) and susceptible (S) strains captured in Argentina during 2005. The R strain was collected from sprayed houses in the north of the province of Salta while the S strain was collected in the province of Chaco. Both strains were bred in the laboratory for reciprocal crosses (F1), intercrosses (F2) and backcrosses (BC). The descendents were tested by a standard insecticide resistance bioassay. Resistance ratios were 1 for S strain, 103.36 for R strain and 18.34 for F1. The regression lines of F1 generations (R×S and S×R) showed no significant differences and were closer to that of the R parents, indicating that inheritance of deltamethrin resistance in T. infestans is autosomal and incompletely dominant (D=0.20). Chi-square analysis from responses of intercross and backcross progenies rejected the hypothesis of a single gene being responsible for resistance. The minimum number of independent segregation genes was three, as calculated with Lande's method. The genetic basis here described for the resistant phenotype indicate that, under pyrethroid selective pressure, the resistant genotypes could be easily spread to susceptible insects from resistant individuals, posing a major threat to vectorial control of Chagas disease.

Enzymes of the antioxidant network as novel determiners of Trypanosoma cruzi virulence.

Virulence of Trypanosoma cruzi depends on a variety of genetic and biochemical factors. It has been proposed that components of the parasites' antioxidant system may play a key part in this process by pre-adapting the pathogen to the oxidative environment encountered during host cell invasion. Using several isolates (10 strains) belonging to the two major phylogenetic lineages (T. cruzi-I and T. cruzi-II), we investigated whether there was an association between virulence (ranging from highly aggressive to attenuated isolates at the parasitemia and histopathological level) and the antioxidant enzyme content. Antibodies raised against trypanothione synthetase (TcTS), ascorbate peroxidase (TcAPX), mitochondrial and cytosolic tryparedoxin peroxidases (TcMPX and TcCPX) and trypanothione reductase (TcTR) were used to evaluate the antioxidant enzyme levels in epimastigote and metacyclic trypomastigote forms in the T. cruzi strains. Levels of TcCPX, TcMPX and TcTS were shown to increase during differentiation from the non-infective epimastigote to the infective metacyclic trypomastigote stage in all parasite strains examined. Peroxiredoxins were found to be present at higher levels in the metacyclic infective forms of the virulent isolates compared with the attenuated strains. Additionally, an increased resistance of epimastigotes from virulent T. cruzi populations to hydrogen peroxide and peroxynitrite challenge was observed. In mouse infection models, a direct correlation was found between protein levels of TcCPX, TcMPX and TcTS, and the parasitemia elicited by the different isolates studied (Pearson's coefficient: 0.617, 0.771, 0.499; respectively, P<0.01). No correlation with parasitemia was found for TcAPX and TcTR proteins in any of the strains analyzed. Our data support that enzymes of the parasite antioxidant armamentarium at the onset of infection represent new virulence factors involved in the establishment of disease.

The identification of sandfly species, from an area of Argentina with endemic leishmaniasis, by the PCR-based analysis of the gene coding for 18S ribosomal RNA.

The area around Río Blanco, in the Orán department in the north of the Argentinian province of Salta, is endemic for American tegumentary leishmaniasis. In an attempt to facilitate the identification of the Lutzomyia species in this area, sequences of the gene coding for the 18S ribosomal RNA (rRNA) of sandflies caught in a Shannon trap were explored, by a combination of PCR and analysis of restriction-fragment-length polymorphism (RFLP). The products from the PCR, which employed two primers developed specifically for this study (Lu.18S 1S and Lu.18S AR), were cloned into a commercial vector (pGEM-T Easy) so that their nucleotide sequences could be investigated. In the RFLP analysis, the products of single and double digestion with the AfaI and HapII restriction enzymes were separated by electrophoresis in 3% or 4% agarose. Taken together with the results of a morphological investigation of the flies, the resultant DNA fragment patterns were sufficient to identify most of the sandflies caught as Lu. neivai. Although two other species, Lu. cortelezzii and Lu. sallesi, were collected, they were relatively rare and only identified morphologically. A single digestion of the 18S-rRNA gene sequences with AfaI or HapII appeared sufficient and useful for the identification of Lu. neivai from the north of Salta province, and for several other Lutzomyia species.

Furoxan derivatives as cytotoxic agents: preliminary in vivo antitumoral activity studies.

Furoxan derivatives with in vitro cytotoxic activity were investigated as antitumoral agents in vivo. The compounds were tested in murine models of both CCRFS-180 II sarcoma and mammary adenocarcinoma. Two of the furoxan derivatives considered here, 3-formyl-4-phenyl-1,2,5-oxadiazole N2-oxide and 3-carbonitrile-4-phenyl-1,2,5-oxadiazole N2-oxide, present in vivo antitumoral activity. They were able to produce more than 90% of tumoral necrosis under the experimental protocol of administration and posology employed. NO-releasing capacity of furoxans may explain the anti-neoplastic activity of these compounds.

Canine infection and the possible role of dogs in the transmission of American tegumentary leishmaniosis in Salta, Argentina.

Some Leishmania species affect humans in two principal forms: visceral and cutaneous leishmaniosis (CL). Several studies have identified dogs as the main reservoirs of the visceral leishmaniosis (VL) caused by Leishmania infantum. The purpose of this work was to carry out a survey of the canine population associated with human cases of American tegumentary leishmaniosis (ATL), in order to establish the clinical, parasitological, serological and immunological characteristics of the canine disease, in an endemic region for both ATL and Chagas' disease in the province of Salta, in northwestern Argentina. Two hundred and eight dogs from the endemic area were examined and 41 (19.7%) of them presented lesions compatible with leishmaniosis. In order to investigate the presence of antibodies against Leishmania spp. and Trypanosoma cruzi, sera were screened by ELISA using two complex antigens from these parasites and, because of cross-reactions between them, a specific antigen for diagnosis of T. cruzi infection. Sixty-two (29.8%) of 208 dogs were positive for the complex antigen F45 from Leishmania and 50 (24%) were positive for the complex antigen F105 from T. cruzi. Nine dogs (4.3%) were positive for the specific Ag163B6-cruzipain suggesting that these dogs were truly infected with T. cruzi. Furthermore, three of these nine dogs presented Leishmania sp. in their skin lesions and therefore were considered as infected by both, T. cruzi and Leishmania parasites. The prevalence of Leishmania infection detected by lesions and/or positive serology was 27.4% (57/208). On the basis of previous observations regarding the clustered appearance of human ATL, the dog population was divided into two groups: zone A, dogs living within a 100 m radius from houses with human cases, and zone B, dogs living beyond this limit. The prevalence of ATL in dogs was significantly higher in zone A (34.6%) than in zone B (7.3%), suggesting a strong correlation between canine and human cases. The average time required for a parasitological diagnosis by microscopy was six times longer for dog samples than human ones, and the average number of parasites per 100 microscopic fields was 14-fold lower in canine samples. The high prevalence of Leishmania infection and the close association with human cases, demonstrated that dogs are a very susceptible host for Leishmania infection, but the scarcity of parasites in their lesions suggests that they may not be the main reservoir of the parasite in this endemic area.

Enzymatic activity, protein expression, and gene sequence of cruzipain in virulent and attenuated Trypanosoma cruzi strains.

Protein expression, characterized in Western blots and gelatinolytic activity, of cruzipain (Cr), the major Trypanosoma cruzi cysteine proteinase, was compared among 3 attenuated T. cruzi strains (TUL 0, TCC, and Y null) and their virulent counterparts (TUL 2, Tulahuen, and Y). All attenuated strains displayed a weaker gelatinolytic activity as compared with their virulent counterparts. The electrophoretic mobility and immunological reactivity revealed quantitative and qualitative differences, with the attenuated parasites showing bands of less density in all strains and lower mobility in 2 of them, as compared with the virulent strains. Sequence analysis of 1 Cr gene in the Tulahuen and TCC strains indicated 37/1404 base pair substitutions, corresponding to 20 amino acid changes in the attenuated strain. A similar comparative analysis of 1 Cr gene between Y and Y null strains showed 13/1404 base pair substitutions, corresponding to 8 amino acid changes in the attenuated strain. Although enough variability exists in the Cr gene to allow for less- or nonfunctional isoforms of the protein, further clones should be analyzed to establish whether attenuation is regularly associated with specific sequence changes of this enzyme.

Force of infection and evolution of lesions of canine tegumentary leishmaniasis in northwestern Argentina.

A clinical-serological follow-up was carried out in a canine population in endemic foci of Leishmania braziliensis spread in northwestern Argentina. Each dog was studied in at least two visits, 309+/-15 days (X+/-SE) apart. Some initially healthy dogs (n=52) developed seroconversion or lesions. The clinical evolution of the disease in dogs resembles in many aspects the human disease. Similarities include the long duration of most ulcers with occasional healing or appearance of new ones and the late appearance of erosive snout lesions in some animals. Yearly incidence rates of 22.7% for seroconversion and of 13.5% for disease were calculated as indicators of the force of infection by this parasite upon the canine population.

Synthesis and antitrypanosomal evaluation of E-isomers of 5-nitro-2-furaldehyde and 5-nitrothiophene-2-carboxaldehyde semicarbazone derivatives. structure-activity relationships.

Several novel semicarbazone derivatives were prepared from 5-nitro-2-furaldehyde or 5-nitrothiophene-2-carboxaldehyde and semicarbazides bearing a spermidine-mimetic moiety. All derivatives presented the E-configuration, as determined by NMR-NOE experiments. These compounds were tested in vitro as potential antitrypanosomal agents, and some of them, together with the parent compounds, 5-nitro-2-furaldehyde and 5-nitrothiophene-2-carboxaldehyde semicarbazone derivatives, were also evaluated in vivo using infected mice. Structure-activity relationship studies were carried out using voltammetric response and lipophilic-hydrophilic balance as parameters. Two of the compounds (1 and 3) displayed the highest in vivo activity. A correlation was found between lipophilic-hydrophilic properties and trypanocidal activity, high R(M) values being associated with low in vivo effects.

Studies on the virulence and attenuation of Trypanosoma cruzi using immunodeficient animals.

Tissue invasion and pathology by Trypanosoma cruzi result from an interaction between parasite virulence and host immunity. Successive in vivo generations of the parasite select populations with increasing ability to invade the host. Conversely, prolonged in vitro selection of the parasite produces attenuated sublines with low infectivity for mammals. One such subline (TCC clone) has been extensively used in our laboratory as experimental vaccine and tested in comparative experiments with its virulent ancestor (TUL). The experiments here reviewed aimed at the use of immunodeficient mice for testing the infectivity of TCC parasites. It has not been possible to obtain virulent, revertant sublines by prolonged passaged in such mice.

Endemic Trypanosoma cruzi infection in Indian populations of the Gran Chaco territory of South America: performance of diagnostic assays and epidemiological features.

The relative specificities and sensitivities of several serological assays for the diagnosis of Trypanosoma cruzi infection were estimated in Indian populations of Argentina and Paraguay. The results obtained with the assays, which proved to be most reliable, were used to study the distribution of the parasite in these populations. Serological evidence of T. cruzi infection was demonstrated in 256 (37.7%) of 679 Indians living in relatively small and isolated communities in the Salta province of northern Argentina and in western Paraguay, regions that are part of the tropical territory called Gran Chaco. In contrast, none of the 94 Indians examined in south-western Argentina was positive. Infection in the Gran Chaco Indians increased with age and clustered in families. Marked differences in seroprevalence were observed between the 16 Indian communities examined in Gran Chaco. These differences seem to be associated both with the risk of transmission from the sylvatic reservoirs of the parasite and with the frequency with which vector-spraying campaigns have been implemented.

Levels of tumor necrosis factor alpha, gamma interferon, and interleukins 4,6, and 10 as determined in mice infected with virulent or attenuated strains of Trypanosoma cruzi.

Inoculation of BALB/c mice with the virulent Tulahuen (TUL) strain of T. cruzi was shown to lead to progressive and eventually lethal infections, whereas infection with an attenuated strain (TCC) resulted in a hardly noticeable experimental disease producing no tissue damage. To determine whether differences in such infection outcome are associated with a particular pattern of cytokine response, a study was undertaken to investigate the serum levels of TNF-alpha, IFN-gamma, IL-4, IL-6, and IL-10 by using an enzyme-linked immunosorbent assay. Mice from both infected groups were bled at 5, 9, 15, 22, 30 and 48 days post-infection (p.i.), with the same interval being applied for obtention of serum samples in age-matched uninfected mice, a group that yielded negative results in all cases. Infection with the TUL strain of T. cruzi was accompanied by a significant increase of TNF-alpha serum concentrations at day 5 p.i., and detectable amounts of IFN-gamma by day 15 p.i., which were exclusively recorded in this group. Serum IL-4 was mostly present in TCC mice with values at day 15 pi being statistically significant in relation to TUL-infected mice. IL-10 was firstly detected at 3 weeks after infection, and showed higher levels in the TCC group, although comparisons with TUL-infected group were not significant. At our limit of detection, no samples were found to contain IL-6 serum concentrations. Infection with virulent parasites seems to be associated with presence of Th1-type cytokines, whereas challenge with the attenuated TCC strain appears as being related to a Th2-type profile.

[The transmission de Chagas disease in Salta and the detection of congenital cases]. / La transmisión de la enfermedad de Chagas en Salta y la detección de casos congénitos.

Data on the prevalence of Trypanosoma cruzi infection is presented for the province of Salta, Argentina. Special emphasis is given to the detection of congenital transmission and to the economic benefits of preventing Chagas' disease. Seroepidemiological data obtained from 20 year old army draftees revealed a reduction, from 22.7 to 11.11% between 1964 and 1985. In university students, a rate of 0.96% was found in 1998. Surveys carried out during 1996 showed that more than 15% of the pregnant women analyzed carried T. cruzi infection, particularly in the north of the province. This situation brings about a high risk of appearance of congenital cases and represents an opportunity to test the most adequate strategies for detection. By applying systematically microhematocrit, hemoculture and PCR methods, to umbilical chord blood, an increase in the early detection of congenitally infected babies is being achieved. In 1992-94, very high seroprevalence rates of infection were found among indians of the Chaco region of Salta. The overall rate was 37%, but there were 5 localities where more than 54% of the population was infected. These numbers indicate that, in vast areas of the provincial territory, fight against vector bugs must not merely consist of surveilance activities, but rather of renewed spraying attacks. The fight must include control of pregnant women and blood banks. An economic analysis of the economic return, calculated only for spraying activities and for the Department of Anta (Salta), indicated a net present value of over 7 million dollars and an internal rate of return exceeding 60%.

A cost-benefit analysis of Chagas disease control in north-western Argentina.

Chagas disease has been controlled in the Department of Anta, Province of Salta, Argentina, through a series of vector control interventions beginning in 1983. Based on data from this programme, together with estimates of the value of benefits accruing to the programme due to avoidance of new cases of Chagas disease, we present an analysis of costs and benefits of the vector control interventions. Under all assumptions, the interventions have been highly profitable from a societal point of view, with an internal rate of return in excess of 60%. The net present value of benefits accruing to the programme is estimated to be above US$7 million.

High prevalence of hantavirus infection in Indian communities of the Paraguayan and Argentinean Gran Chaco.

Serologic evidence of past infection with a Sin Nombre-like hantavirus(es) was demonstrated in 78 (40.4%) of 193 Indians living in western Paraguay and in 38 (17.1%) of 222 Indians inhabiting the Salta province of northern Argentina. In both populations seroprevalence increased with age, with the most striking increase occurring at 18 years of age in the Paraguayan population and at 35 years of age in the Salta population. The peak prevalences in both populations (66.6% and 44.0%, respectively) were seen in Indians > 53 years old. Although no sex difference was observed in the Paraguayan Indians, in the Salta population seroprevalence was greater in males than in females. Familiar clustering of the infection was observed. The data indicate that the Indian populations of the Gran Chaco are frequently exposed to and survive infection with a Sin Nombre-like virus(es). Possible explanations of this novel epidemiology are discussed.

Trypanosoma cruzi: effect of immunization on the risk of vector-delivered infection in guinea pigs.

The protective effect of experimental immunization was studied in guinea pigs exposed to vectorial infection by Trypanosoma cruzi. Immunized animals received an inoculum of live-attenuated T. cruzi epimastigotes into a granuloma previously induced by Freund's complete adjuvant in the hind footpad. Seven days later, a delayed-type hypersensitivity reaction was triggered by reinjection of the parasites in the front footpad. The animals were then placed in Triatoma infestans-colonized corrals and exposed to vectorial T. cruzi transmission of the parasite for up to 200 days. The effectiveness of this immunizing protocol was controlled in terms of the number of bites necessary for infection (NBNI) in immunized as compared with control animals. Periodic entomological census allowed for the determination of vector biting and infection rates and the calculation of NBNI. Although this measurement was quite variable between yards, an overall average of 4,973 bites was enough to infect a control guinea pig in 4 separate experiments. The corresponding figure for the experimental group was 21,307 bites, implying that immunized animals could resist a 4.28-fold increase (range: 1.99-8.32) in the number of vector bites before becoming infected.

Immunogenicity of the recombinant SAPA protein of Trypanosoma cruzi for mice.

The humoral and cellular immune responses induced by the recombinant SAPA (shed acute phase antigen) of Trypanosoma cruzi were studied in mice and correlated with the immunologic control of parasitemia. The immunizing schedule used consisted of 2 weekly injections of 50 micrograms glutathione-S-transferase (GST)-SAPA in Freund's adjuvant. Specific alpha GST-SAPA antibodies were detected by enzyme-linked immunosorbent assay 1 wk after each antigen dose, the concentration of antibodies after the second injection being 30-fold higher than after the first. Immediate- (ITH) and delayed-type hypersensitivity (DTH) reactions were observed as footpad swelling after injecting 50 micrograms GST-SAPA in preimmunized mice as compared to naive controls. Adoptive transfer experiments indicated that these cutaneous reactions were mediated by lymphoid cells and not by serum. Both humoral and cellular responses were specific for the GST-SAPA antigen and did not cross-react with either the GST or the recombinant GST-1 T. cruzi antigen. Immunized mice that had developed high levels of antibody and DTH reaction to GST-SAPA were able to control the level of parasitemia after challenge with 10(3) blood trypomastigotes. The levels of parasitemia obtained were lowered to about 1/3 (P < 0.05) and mortality at day 60 was reduced from 67 to 25% (P = 0.085). Comparison of this immunizing method with other schedules involving more injections or higher antigen doses indicates that control of parasitemia can be obtained with low amounts of antigen and seems to be associated with the development of DTH.

Endemic infection with human T cell leukemia/lymphoma virus type IIB in Argentinean and Paraguayan Indians: epidemiology and molecular characterization.

Human T cell leukemia/lymphoma virus (HTLV-II) type II infection was detected by polymerase chain reaction or serologic analyses (or both) in 22% of 697 Indians of six different ethnic back-grounds inhabiting the Argentinean and Paraguayan Gran Chaco. None was infected with HTLV-I. The prevalence of HTLV-II increased with age (14% in those < 13 years and 23% in those > or = 13 years). HTLV-II infection was found in all 20 Gran Chaco communities studied, but marked differences (44%-4%) in the rate of infection were observed even in communities separated by only a few miles. These variations correlated closely with ethnicity. In the high-incidence communities, infection clustered within families, with evidence for both sexual and perinatal transmission, primarily via breast-feeding. By contrast, only 2% of 94 Mapuche Indians from southern Argentina were positive for HTLV-II. Analyses of pol and long terminal repeat sequences from 15 Gran Chaco HTLV-II strains indicated that they constitute a highly conserved branch of the HTLV-IIB substrain.