Direct molecular identification of Trypanosoma cruzi discrete typing units in domestic and peridomestic Triatoma infestans and Triatoma sordida from the Argentine Chaco.

We assessed the distribution of Trypanosoma cruzi Discrete Typing Units (DTUs) in domestic and peridomestic Triatoma infestans and Triatoma sordida specimens collected in a well-defined rural area in Pampa del Indio, northeastern Argentina. Microscopically-positive bugs were randomly selected with a multi-level sampling design, and DTUs were identified using direct PCR strategies. TcVI predominated in 61% of 69 T. infestans and in 56% of 9 T. sordida. TcV was the secondary DTU in T. infestans (16%) and was found in 1 T. sordida specimen (11%). Three T. sordida (33%) were found infected with TcI, a DTU also identified in local Didelphis albiventris opossums. Mixed DTU infections occurred rarely (5%) and were detected both directly from the bugs' rectal ampoule and parasite cultures. The identified DTUs and bug collection sites of T. infestans were significantly associated. Bugs infected with TcV were almost exclusively captured in domiciles whereas those with TcVI were found similarly in domiciles and peridomiciles. All mixed infections occurred in domiciles. TcV-infected bugs fed more often on humans than on dogs, whereas TcVI-infected bugs showed the reverse pattern. T. sordida is a probable sylvatic vector of TcI linked to D. albiventris, and could represent a secondary vector of TcVI and TcV in the domestic/peridomestic cycle.

Impact of community-based vector control on house infestation and Trypanosoma cruzi infection in Triatoma infestans, dogs and cats in the Argentine Chaco.

The relative impact of two community-based vector control strategies on house infestation by Triatoma infestans and Trypanosoma cruzi infection in bugs, domestic dogs and cats was assessed in two neighboring rural areas comprising 40 small villages and 323 houses in one of the regions most endemic for Chagas disease in northern Argentina. The prevalence and abundance of domestic infestation were 1.5- and 6.5-fold higher, respectively, in the area under pulsed, non-supervised control actions operating under the guidelines of the National Vector Control Program (NCVP) than in the area under sustained, supervised surveillance carried out jointly by the UBA research team and NCVP. The prevalence of infestation and infection varied widely among village groups within each area. In the pulsed control area, the prevalence of infection in bugs, dogs and cats was two- to three-fold higher than in the area under sustained surveillance, most of the infected animals qualified as autochthonous cases, and evidence of recent transmission was observed. Infection was highly aggregated at the household level and fell close to the 80/20 rule. Using multiple logistic regression analysis clustered by household, infection in dogs was associated positively and significantly with variables reflecting local exposure to infected T. infestans, thus demonstrating weak performance of the vector surveillance system. For high-risk areas in the Gran Chaco region, interruption of vector-mediated domestic transmission of T. cruzi requires residual insecticide spraying that is more intense, of a higher quality and sustained in time, combined with community participation and environmental management measures.

Domestic dogs and cats as sources of Trypanosoma cruzi infection in rural northwestern Argentina.

The reservoir capacity of domestic cats and dogs for Trypanosoma cruzi infection and the host-feeding patterns of domestic Triatoma infestans were assessed longitudinally in 2 infested rural villages in north-western Argentina. A total of 86 dogs and 38 cats was repeatedly examined for T. cruzi infection by serology and/or xenodiagnosis. The composite prevalence of infection in dogs (60%), but not in cats, increased significantly with age and with the domiciliary density of infected T. infestans. Dogs and cats had similarly high forces of infection, prevalence of infectious hosts (41-42%), and infectiousness to bugs at a wide range of infected bug densities. The infectiousness to bugs of seropositive dogs declined significantly with increasing dog age and was highly aggregated. Individual dog infectiousness to bugs was significantly autocorrelated over time. Domestic T. infestans fed on dogs showed higher infection prevalence (49%) than those fed on cats (39%), humans (38%) or chickens (29%) among 1085 bugs examined. The basic reproduction number of T. cruzi in dogs was at least 8.2. Both cats and dogs are epidemiologically important sources of infection for bugs and householders, dogs nearly 3 times more than cats.

Differential detection of Blastocrithidia triatomae and Trypanosoma cruzi by amplification of 24salpha ribosomal RNA genes in faeces of sylvatic triatomine species from rural northwestern Argentina.

Flagellates indistinguishable from Trypanosoma cruzi were detected by microscopy in faecal samples of 2/110 Triatoma guasayana and 2/283 Triatoma garciabesi captured in a rural area of northwestern Argentina. Inoculation of faecal homogenates to mice followed by xenodiagnosis, haemoculture, histopathology and culture from cardiac homogenates, and PCR based on T. cruzi minicircle and nuclear sequences failed to detect T. cruzi infection, pointing to another trypanosomatidean. A PCR strategy targeted to the D7 domain of 24salpha ribosomal DNA genes amplified a 250 bp sequence from one T. guasayana and one T. garciabesi faecal lysate. Sequence analysis revealed 100% identity with 24salpha rDNA amplicons from Blastocrithidia triatomae obtained from faeces of reared Triatoma infestans bugs. Phylogenetic analysis clustered this sequence with C. fasciculata and L. major, separated from the Trypanosoma branch (bootstrap: 968/1000), in concordance with a Neighbour-joining dendrogram based on 18s rDNA sequences. This PCR procedure provides a rapid sensitive tool for differential diagnosis of morphologically similar trypanosomatids in field surveys of Chagas disease vectors and laboratory-reared triatomines used for xenodiagnosis.

Long-term reduction of Trypanosoma cruzi infection in sylvatic mammals following deforestation and sustained vector surveillance in northwestern Argentina.

Long-term variations in the dynamics and intensity of sylvatic transmission of Trypanosoma cruzi were investigated around eight rural villages in the semiarid Argentine Chaco in 2002-2004 and compared to data collected locally in 1984-1991. Of 501 wild mammals from 13 identified species examined by xenodiagnosis, only 3 (7.9%) of 38 Didelphis albiventris opossums and 1 (1.1%) of 91 Conepatus chinga skunks were infected by T. cruzi. The period prevalence in opossums was four-fold lower in 2002-2004 than in 1984-1991 (32-36%). The infection prevalence of skunks also decreased five-fold from 4.1-5.6% in 1984-1991 to 1.1% in 2002-2004. Infection in opossums increased with age and from summer to spring in both study periods. The force of infection per 100 opossum-months after weaning declined more than six-fold from 8.2 in 1988-1991 to 1.2 in 2002-2004. Opossums were mainly infected by T. cruzi lineage I and secondarily by lineage IId in 1984-1991, and only by T. cruzi I in 2002-2004; skunks were infected by T. cruzi IId in 1984-1991 and by IIc in 2002-2004. The striking decline of T. cruzi infection in opossums and skunks occurred in parallel to community-wide insecticide spraying followed by selective sprays leading to very low densities of infected Triatoma infestans in domestic and peridomestic habitats since 1992; to massive deforestation around one of the villages or selective extraction of older trees, and apparent reductions in opossum abundance jointly with increases in foxes and skunks. These factors may underlie the dramatic decrease of T. cruzi infection in wild reservoir hosts.

PCR-based screening and lineage identification of Trypanosoma cruzi directly from faecal samples of triatomine bugs from northwestern Argentina.

This study applied improved DNA extraction and polymerase chain reaction strategies for screening and identification of Trypanosoma cruzi lineages directly from faeces of triatomines collected in a well-defined rural area in northwestern Argentina. Amplification of the variable regions of the kinetoplastid minicircle genome (kDNA-PCR) was performed in faecal lysates from 33 microscope (MO)-positive and 93 MO-negative Triatoma infestans, 2 MO-positive and 38 MO-negative Triatoma guasayana and 2 MO-positive and 73 MO-negative Triatoma garciabesi. kDNA-PCR detected T. cruzi in 91% MO-positive and 7.5% MO-negative T. infestans, which were confirmed by amplification of the minicircle conserved region. In contrast, kDNA-PCR was negative in all faecal samples from the other triatomine species. A panel of PCR-based genomic markers (intergenic region of spliced-leader DNA, 24Salpha and 18S rRNA genes and A10 sequence) was implemented to identify the parasite lineages directly in DNA lysates from faeces and culture isolates from 28 infected specimens. Two were found to be infected with TCI, 24 with TCIIe, 1 with TCIId and 1 revealed a mixed TCI+TCII infection in the faecal sample whose corresponding culture only showed TCII, providing evidence of the advantages of direct typing of biological samples. This study provides an upgrade in the current diagnosis and lineage identification of T. cruzi in field-collected triatomines and shows T. cruziII strains as predominant in the region.

Association between nutritional indicators and infectivity of dogs seroreactive for Trypanosoma cruzi in a rural area of northwestern Argentina.

The association between the nutritional state of mongrel dogs naturally infected with Trypanosoma cruzi and their infectivity to Triatoma infestans bugs and immune response to Trypanosoma cruzi were studied in the rural village of Amamá, northwestern Argentina. All of the 97 evaluated dogs were classified into one of three categories of external clinical aspect (ECA) based on the degree of muscle development, external evidence of bone structures, state of the hair of the coat, existence of fatty deposits, and facial expression. ECA was significantly associated with two nutritional indicators, hematocrit and skin-fold thickness, but not with total serum proteins. For all dogs, hematocrit was significantly correlated with skin-fold thickness. The 2-year survival probability decreased significantly from 60.7% for dogs with good ECA to 45.9% and 31.2% for those with regular and bad ECA, respectively. The age-adjusted relative odds of infection for Triatoma infestans xeno-diagnosis nymphs that fed once on a dog seroreactive for Trypanosoma cruzi decreased significantly as ECA improved, when tested by multiple logistic regression analysis. A delayed hypersensitivity reaction was observed in all of the seroreactive dogs with good ECA but only in 45-50% of those with regular or bad ECA. Dogs with bad ECA had a 2.6 and 6.3 times greater probability of infecting triatomines after a single full blood meal than dogs with regular or good ECA, respectively. Our study shows that the reservoir competence of dogs for Trypanosoma cruzi was associated with ECA, which is a surrogate and valid index of nutritional state.

Immunodiagnosis of Trypanosoma cruzi (Chagas' disease) infection in naturally infected dogs.

This study reports on the standardization of an enzyme-linked immunosorbent assay (ELISA) for detecting specific antibodies anti-Trypanosoma cruzi in naturally infected dogs. Sera from 182 mongrel dogs of all ages residing in four rural villages in Santiago del Estero, Argentina, were collected in November 1994 and preserved in buffered neutral glycerin. All sera were tested by indirect hemagglutination test (IHAT), indirect immunofluorescence test (IFAT), and ELISA using the flagellar fraction of T. cruzi as antigen. Dog sera from an area without vectorial transmission were used to calculate ELISA specificity and cut-off value. Eighty-six percent of sera had concordant results for all tests. All sera reactive for IHAT and IFAT were also reactive for ELISA, except in one case. Sera tested by ELISA when diluted 1:200 allowed a clearer division between non-reactive and reactive sera than when 1:100 with greater agreement among serologic techniques. The specificity of ELISA was 96.2%. Among 34 adult dogs with a positive xenodiagnosis, sensitivity was 94% both for ELISA and IFAT. ELISA is the first choice for screening purposes and one of the pair of techniques recommended for diagnostic studies in dog populations.

Immunodiagnosis of Trypanosoma cruzi (Chagas' disease) infection in naturally infected dogs

This study reports on the standardization of an enzyme-linked immunosorbent assay (ELISA) for detecting specific antibodies anti-Trypanosoma cruzi in naturally infected dogs. Sera from 182 mongrel dogs of all ages residing in four rural villages in Santiago del Estero, Argentina, were collected in November 1994 and preserved in buffered neutral glycerin. All sera were tested by indirect hemagglutination test (IHAT), indirect immunofluorescence test (IFAT), and ELISA using the flagellar fraction of T. cruzi as antigen. Dog sera from an area without vectorial transmission were used to calculate ELISA specificity and cut-off-value. Eighty-six percent of sera had concordant results for all tests. All sera reactive for IHAT and IFAT were also rective for ELISA, except in one case. Sera tested by ELISA when diluted 1:200 allowed a clearer division between non-reative and reactive sera than when 1:100 with greater agreement among serologic techniques. The specificity of ELISA was 96,2 per cent. Among 34 adult dogs with a positive xenodiagnosis, sensitivity was 94 per cent both for ELISA and IFAT. ELISA is the first choice for screening purposes and one of the pair of techniques recommended for diagnostic studies in dog populations.

Influence of humans and domestic animals on the household prevalence of Trypanosoma cruzi in Triatoma infestans populations in northwest Argentina.

In three rural villages of northwest Argentina, the overall proportion of domiciliary Triatoma infestans infected with Trypanosoma cruzi was 49% among 1,316 bugs individually examined for infection in March and October 1992). Most of the variation among individual households in the proportion of infected triatomines was explained by variations among houses in the proportion of bugs that fed on dogs or cats, the prevalence of infected dogs or cats, and the proportion of bugs that fed on humans, according to a logistic multiple regression analysis. The effects of human infection rates on bug infection rates were not statistically significant. After adjusting for the effects of other predictors, the presence of chickens in bedroom areas had negative and significant effects on the proportion of infected Triatoma infestans, and positive and significant effects on the number of T. cruzi-infected triatomines collected per person-hr per house. Dog or cat infection rates and the proportion of bugs that fed on dogs or cats and on chickens explained 80% of the total variance of infected-bug numbers in a linear multiple regression model. This is the first study to use detailed field data to show that variations in triatomine infection rates depend on bug host feeding patterns and dog or cat infection rates, while the presence of chickens in bedroom areas exerts opposite effects on the proportion and number of infected triatomines. Domestic animals play a crucial role in the domiciliary transmission of T. cruzi.

Evaluation of dogs as sentinels of the transmission of Trypanosoma cruzi in a rural area of north-western Argentina.

Dogs, the main domestic reservoirs of Trypanosoma cruzi in the Argentine chaco, may be useful as sentinels of vector-mediated transmission of Trypanosoma cruzi in control programmes if canine infections acquired by all other routes could be excluded. In the present study, in the province of Santiago del Estero, Argentina, the dog populations in the neighbouring villages of Amamá, Trinidad, Mercedes, Villa Matilde and Pampa Pozo were studied immediately before spraying of residual insecticides (in 1992 in Amamá, Trinidad and Mercedes and 1993-1994 in the other villages) and twice during a subsequent programme of triatomine surveillance (in 1994 and 1996). Overall seropositivity for Trypanosoma cruzi infection (i.e. the proportion of dogs found positive by at least two different serological tests in any one survey) steadily declined from 65% (54/83) in 1992 to 39% (70/182) in 1994 and 15% (36/237) in 1996. No seroconversion was detected between the 1994 and 1996 surveys in any dog aged > 2 years. However, among the dogs aged < 2 years (which were born after virtual elimination of Triatoma infestans from the villages), seropositivity fell from 15% (12/81) in 1994 to 5% (6/131) in 1996. Thirteen demographic, behavioural and entomological variables were analysed by logistic multiple-regression analysis for their association with seropositivity in dogs born locally during the surveillance programme. The total number of Triatoma guasayana collected in the bedroom areas of the owner's house, having a mother which was seropositive for Trypanosoma cruzi, and the number of seropositive dogs with which it cohabited were all found to be significant predictors of seropositivity in any dog. The results are the first indication that Triatoma guasayana is the vector responsible for the new cases of Trypanosoma cruzi infection seen in dogs in domestic or peridomestic sites during the surveillance programme.

Probability of infection with Trypanosoma cruzi of the vector Triatoma infestans fed on infected humans and dogs in northwest Argentina.

The probability that an uninfected Triatoma infestans would become infected with Trypanosoma cruzi after a single feed on people or dogs seropositive for T. cruzi was estimated in Amama, a rural village in northwest Argentina where transmission had resurged four years earlier. The prevalence of seropositivity for T. cruzi was 34.2% among 225 people tested, and 65.1% among 83 dogs tested. Parasitemia was detected by xenodiagnosis in 29.3% of 41 seropositive persons and in 85.3% of 34 seropositive dogs. Parasitemia decreased with age more sharply in seropositive people than in seropositive dogs. Seropositive humans infected 2.6% (95% confidence interval = 1.6%-3.6%) of 963 third or fourth instar nymphs fed once on them, whereas dogs infected 48.7% (44.7%-52.7%) of 610 nymphs. The probability of bug infection increased significantly with instar and was positively related to molting success. The infectivity to bugs of seropositive dogs was 12 times higher than that of seropositive children, and 100 times higher than that of seropositive adults. The weighted probability of infection of an uninfected bug fed randomly on any dog (0.3082) was about 50 times higher than that of bugs fed on any human (0.0062). Such differences in relative infectivity, combined with the relative host-feeding preference of domiciliary Triatoma infestans for dogs, reinforces the important role of domestic dogs as a risk factor for the domestic transmission of T. cruzi.

Standardization of serological tests for detecting anti-Trypanosoma cruzi antibodies in dogs

his paper reports on the standardization of four serological reactions currently used in human serodiagnosis for the detection of anti-Trypanosoma cruzi antibodies in naturally and experimentally infected dogs. Indirect immunofluorescence test (IFAT) and hemagglutination test (IHAT) were standardized, and complement fixation test (CFT) and direct agglutination test (DAT) were used for diagnostic confirmation. Four hundred and eighty one mongrel dogs that were studied by xenodiagnosis were used: (1) parasitemic dogs of two localities of endemic area (EA) of Santiago del Estero province in Argentina (n = 134); (2) non-parasitemic dogs of the same area (n = 285); (3) dogs experimentally infected with T. cruzi in the patent period (n = 6); (4) non-infected dogs (n = 56) which were born in the city of Buenos Aires (BA), one non-EA for Chagas' disease. For IFAT, parasitemic dogs EA showed 95 per cent of reactive sera. Non parasitemic dogs EA showed 77 per cent of non reactive sera. None sera from BA were reactive for dilutions higher than four. For IHAT, 84 per cent of sera of parasitemic dogs EA showed serological reactivity and among non parasitemic dogs BA, 61 per cent were non reactive, while the remainder showed at most titres of 1/16. The cut-off titres for IFAT and IHAT were 1/16 and 1/32 respectively, and for CFT and DAT 1/1 and 1/128 respectively. Sensitivity for IFAT, IHAT, CF and DAT were 95 per cent , 84 per cent , 97 per cent and 95 per cent respectively

Standardization of serological tests for detecting anti-Trypanosoma cruzi antibodies in dogs.

This paper reports on the standardization of four serological reactions currently used in human serodiagnosis for the detection of anti-Trypanosoma cruzi antibodies in naturally and experimentally infected dogs. Indirect immunofluorescence test (IFAT) and hemagglutination test (IHAT) were standardized, and complement fixation test (CFT) and direct agglutination test (DAT) were used for diagnostic confirmation. Four hundred and eighty one mongrel dogs that were studied by xenodiagnosis were used: (1) parasitemic dogs of two localities of endemic area (EA) of Santiago del Estero province in Argentina (n = 134); (2) non-parasitemic dogs of the same area (n = 285); (3) dogs experimentally infected with T. cruzi in the patent period (n = 6); (4) non-infected dogs (n = 56) which were born in the city of Buenos Aires (BA), one non-EA for Chagas' disease. For IFAT, parasitemic dogs EA showed 95% of reactive sera. Non parasitemic dogs EA showed 77% of non reactive sera. None sera from BA were reactive for dilutions higher than four. For IHAT, 84% of sera of parasitemic dogs EA showed serological reactivity and among non parasitemic dogs BA, 61% were non reactive, while the remainder showed at most titres of 1/16. The cut-off titres for IFAT and IHAT were 1/16 and 1/32 respectively, and for CFT and DAT 1/1 and 1/128 respectively. Sensitivity for IFAT, IHAT, CF and DAT were 95%, 84%, 97% and 95% respectively.

Infectivity to the vector Triatoma infestans of dogs infected with Trypanosoma cruzi in north-west Argentina.

The influence of reinfections on the infectivity to Triatoma infestans of dogs seropositive to Trypanosoma cruzi was investigated in Amamá, north-west Argentina, before (1984) and after (1986-1987) elimination of domestic bugs with residual insecticides in 1985. Infectivity was quantified by the percentage of infected bugs in each xenodiagnosis of a seropositive dog, i.e. the infective force (IF). Exposure to reinfection was represented by the domiciliary density of infected bugs and by an index of the potentially-infective contacts (IPIC) experienced by each dog (calculated as the product of the relative density of bugs per dog, the proportion of infected bugs, and the proportion of bugs taking blood meals from the dog). IF was unrelated to the dog's age or sex, or the level of specific antibodies to T. cruzi. Before vector elimination, the IF of seropositive dogs was significantly associated with both the density of infected bugs and the IPIC. After vector elimination, seropositive dogs resident at houses which had 10-70 infected bugs before spraying showed a higher median IF (67-75%) than those living in houses with zero to eight infected bugs before spraying (47-55%). The age-specific IF of seropositive dogs aged one year or older, recorded before (45-64%) and after (52-56%) the elimination of domestic bugs, did not differ significantly. Our data suggest that the infectivity of seropositive dogs to bugs is modified by previous or current exposure to bug-mediated reinfections, and that this relationship might be irreversible in the short term.

Chagas disease in north-west Argentina: infected dogs as a risk factor for the domestic transmission of Trypanosoma cruzi.

The association between household seroreactivity to Trypanosoma cruzi in dogs and children and T. cruzi infection rates in domestic Triatoma infestans was investigated in 1988-1989 in the rural community of Amamá, north-west Argentina, where house spraying with residual pyrethroids was carried out in 1985. Three years after spraying, a greater reduction of the average T. cruzi prevalence rate in dogs (from 83% to 40%) than in children (from 48% to 30%) was accompanied by a substantial decrease in vector infection rates from 51%-63% to 21%. At a household level, in homes with or without seroreactive children, the percentage of infected T. infestans was 4.5-4.7 times higher when seroreactive dogs were present (27.1%-34.8%) than when they were not (5.8%-7.7%; stratified relative risk [RR] = 4.58). The contribution of seroreactive children to bug infection rates was not significant (RR = 1.29). The combined effect of both seroreactive dogs and seroreactive children fitted equally well with additive or multiplicative transmission models. Bug infection rates showed an increasing trend with the number of seroreactive dogs and an inverse association with the age of the youngest seroreactive dog. Our study supports the hypothesis of a causal association between the presence and number of infected dogs and increased levels of T. cruzi transmission to domestic T. infestans.

The prevalence of Trypanosoma cruzi and the demography of dog populations after insecticidal spraying of houses: a predictive model.

A three-year demographic and seroparasitological follow-up of the canine population of a rural area of Argentina endemic for Chagas' disease was carried out in order to (a) describe the population dynamics of domestic dogs, and (b) predict the decrease in the prevalence of Trypanosoma cruzi among these reservoirs after indoor spraying of houses with residual insecticides. For the latter project we designed an age-structured model with a discrete time scale. We assumed a time-dependent exponential decay in the frequency of infected hosts proportional to the host mortality rate, and also assumed and that no differential mortality existed between infected and non-infected dogs. Validation of the model was carried out, and yielded an extremely close fit between observed and theoretical values. The relevance of the model as an aid to designing strategies for the control of animal domestic reservoirs of T. cruzi, and the role of dogs as efficient sentinels of re-infestation of an area, are discussed.

Natural Trypanosoma cruzi infection in dogs of endemic areas of the Argentine Republic.

The population dynamics and the prevalence of chagasic infection of 352 dogs living in 108 rural houses infested by triatomines were studied. The region was divided into three sections according to increasing distances to an urban area. Each animal was identified by means of its particular characteristics and built, and its owners gave information about its habits. By means of xenodiagnosis, serology and ECG studies, prevalences of infection, parasitological-serological correlation, percentage of altered electrocardiographic outlines and percentage of houses with parasitemic dogs, were determined. The rural area showed a characteristic T. cruzi infection pattern and differences in the canine population parameters with respect to the other areas were observed: a higher proportion of puppies than adult dogs, a more sedentary population, higher prevalences of infection, as measured by xenodiagnosis, in dogs, and the highest proportion of bedroom insects infected with T. cruzi. It is assumed that the sedentary characteristics of the human population in that rural area impinge in the blood offer to the triatomine population, and the high percentage of parasitemic dogs of the area, contribute to the rise of "kissing bugs" infected with T. cruzi found in bedrooms.