TESA-blot for the diagnosis of Chagas disease in dogs from co-endemic regions for Trypanosoma cruzi, Trypanosoma evansi and Leishmania chagasi
Introduction
Dogs are important reservoirs of Trypanosoma cruzi and Leishmania chagasi and play an important role in the transmission of Chagas disease and visceral leishmaniasis to humans in rural areas of Central and South America. T. cruzi infection, which is primarily a zoonosis, is widespread and has a high prevalence in the region extending from the southern half of the USA to the southern countries of South America (Miles et al., 2003). In regions where human Chagas disease is endemic, T. cruzi circulates between humans and domestic animals and is transmitted by home-dwelling blood-sucking triatomine bugs. Several mammals and triatomine species sustain domestic and sylvatic transmission cycles, while domestic mammals (dogs and cats) and peridomestic mammals (rodents and marsupials) are responsible for the interaction between these two cycles, playing an important role in the transmission of T. cruzi from sylvatic to domestic cycles. Dogs from rural areas frequently enter wild environments where T. cruzi enzootic populations circulate between wild mammals and triatomines (Gürtler et al., 1998, Gürtler et al., 2007, Crisante et al., 2006, Estrada-Franco et al., 2006, Cardinal et al., 2007, Cardinal et al., 2008).
Domestic dogs are a risk factor for Chagas disease because they can be a source of T. cruzi infection in humans, and close contact between humans and dogs, particularly when the latter are kept inside a house overnight, can significantly enhance transmission of this disease to humans. Because of their persistent parasitemia, dogs have a greater capacity for infecting triatomine bugs than do humans and have been used as efficient natural sentinels to assess T. cruzi reinfections in vector surveillances (Gürtler et al., 1993, Castanera et al., 1998, Estrada-Franco et al., 2006, Cardinal et al., 2006a).
The importance of domestic dogs as reservoirs and as a risk factor for the transmission of T. cruzi to humans has been extensively studied in Argentina. The prevalence of infected T. infestans in households was found to increase about 3–4 times with the number of infected dogs, and the seroprevalence of infected humans doubled in households with 1–2 infected dogs (Gürtler et al., 1993, Gürtler et al., 1996, Gürtler et al., 2007, Diosque et al., 2004, Cardinal et al., 2007). In Mexico, dogs are known to provide frequent blood meals for Triatomabarberi and T. pallidipennis, and T. cruzi infected triatomines and have been associated with human infections (Ramsey et al., 2005, Estrada-Franco et al., 2006). Finding of a direct correlation between seropositivity in humans and dogs suggest that the diagnosis of T. cruzi infected domestic dogs can be of help in assessing the risk of transmission of human Chagas disease.
In Brazil, studies conducted in rural communities in Northeast Brazil found high prevalence of T. cruzi infected domestic animals, with high importance of dogs and cats in the domestic transmission cycles (Barrett et al., 1979). The human seropositivity was five times greater in houses inhabited by infected dogs and cats than in houses with non-infected domestic animals (Mott et al., 1978).
Surveys of canine Chagas disease have also been carried out in rural areas of regions that are endemic for the human disease in Chile (Burchard et al., 1996), Venezuela (Crisante et al., 2006); Paraguay (Fujita et al., 1994), and Mexico (Ramsey et al., 2005, Estrada-Franco et al., 2006, Jimenez-Coello et al., 2008). Prevalence rates of T. cruzi in dogs from these countries varied widely. The highest rates (65–83%) were reported in Northwest Argentina before the spraying of residual insecticides (Castanera et al., 1998) and were followed by a rate of 67% infection for dogs in Venezuela (Crisante et al., 2006), 38% in Paraguay (Chapman et al., 1984), 21% in Mexico (Estrada-Franco et al., 2006), 20.3% in Texas, USA (Kjos et al., 2008), and 19% in Brazil (Barrett et al., 1979). There have been several reports of T. cruzi-infected dogs in the southern USA, where human Chagas disease is not endemic but dogs infected with T. cruzi have been associated with domestic transmission of human infection (Barr et al., 1991, Shadomy et al., 2004, Duprey et al., 2006, Kjos et al., 2008).
Diagnosis of canine Chagas disease is usually made by serological methods developed for humans (Barr et al., 1991, Lauricella et al., 1998, Shadomy et al., 2004, Cardinal et al., 2006b, Estrada-Franco et al., 2006). Although several conventional serological tests based on crude antigens from epimastigotes, such as ELISA (enzyme-linked immunosorbent assay), IFA (indirect immunofluorescence assay) and IHA (indirect hemagglutination assay), showed good sensitivity, their specificity decreased when sera from hosts with visceral leishmaniasis were tested (Frank et al., 2003, Caballero et al., 2007, Ferreira et al., 2007). Nevertheless, most serological surveys of dogs infected with T. cruzi have used methods that have not been evaluated in terms of cross-reaction with Leishmania spp. In addition, these methods also ignored cross-reaction with other important canine pathogens, including Trypanosoma evansi that is endemic in overlapping areas of T. cruzi in Brazil, Venezuela, Colombia and Bolivia (Ventura et al., 2002, Herrera et al., 2004), and that shares antigens with T. cruzi (Desquesnes et al., 2007).
TESA-blot, previously described for the diagnosis of human Chagas disease, is a Western blotting technique that uses TESA (trypomastigote excreted–secreted antigen) as antigen. In this method no cross-reactivity with sera from humans infected with Leishmania spp., T. rangeli or other pathogens is observed (Umezawa et al., 1996, Umezawa et al., 2001, Caballero et al., 2007). In the present study, whose objective was to standardize a highly specific and sensitive serological assay for canine Chagas disease, we evaluated the diagnostic performance of TESA-blot and compared this method with TESA-ELISA and the conventional epi-ELISA. These methods were further evaluated for use in field epidemiology of canine Chagas disease in areas of Brazil that are endemic or non-endemic for Chagas disease, visceral leishmaniasis and T. evansi infection.
Section snippets
Antigens
TESAs (trypomastigote excreted–secreted antigens) from the Y strain of T. cruzi were obtained as previously described (Umezawa et al., 1996, Umezawa et al., 2001). Briefly, the supernatants of LLC-MK2 cell cultures (in serum-free medium or with 2% FCS) infected with T. cruzi were collected when the concentration of trypomastigotes reached about 10–20 × 106/mL. After being centrifuged at 1800 × g for 15 min at 4 °C, the supernatant containing TESA was then re-submitted to a second centrifugation (7000 ×
Standardization of TESA-blot for diagnosis of T. cruzi-infected dogs
The TESA-blot developed for diagnosis of human Chagas disease was standardized for detection of T. cruzi-infected dogs using sera from 10 domestic dogs infected with T. cruzi. These sera were 100% reactive by all methods TESA-blot, TESA-ELISA and epi-ELISA (Table 1). All sera reacted with a 150–160 kDa polypeptide in TESA-blot, resulting in an intense blue band when 4-chloro-1-naphthol was used as the substrate. This procedure was more accurate in detecting bands than using DAB substrate that
Discussion
The existence of a number of publications reporting T. cruzi infection in dogs from North to South America (Barr et al., 1991, Gürtler et al., 1993, Gürtler et al., 1998, Gürtler et al., 2007, Crisante et al., 2006, Estrada-Franco et al., 2006, Jimenez-Coello et al., 2008, Cardinal et al., 2007, Cardinal et al., 2008) indicates a high prevalence of the infection in this animal in these regions. In Brazil, dogs showed to be important risk factor for human Chagas disease in rural communities in
Acknowledgements
This study was supported by grants from LIM49-FMUSP and CNPq. The authors would like to thank G. Cavalcanti and S.M. Genari for providing sera from dogs from Rondonia, D. Yamada for her technical help, and Professor E. Camargo for his valuable comments on the manuscript. V. Pinedo-Cancino and A. Marcili are recipients of fellowships from CNPq.
References (49)
- et al.
Canine leishmaniosis – new concepts and insights on an expanding zoonosis: part one
Trends Parasitol.
(2008) - et al.
An outbreak of acute Chagas's disease in the São Francisco Valley region of Bahia Brazil: triatomine vectors and animal reservoirs of Trypanosoma cruzi
Trans. R. Soc. Trop. Med. Hyg.
(1979) - et al.
Impact of community-based vector control on house infestation and Trypanosoma cruzi infection in Triatoma infestans, dogs and cats in the Argentine Chaco
Acta Trop.
(2007) - et al.
Molecular epidemiology of domestic and sylvatic Trypanosoma cruzi infection in rural northwestern Argentina
Int J Parasitol.
(2008) - et al.
Leishmania (Viannia) braziliensis is the prevalent species infecting patients with tegumentary leishmaniasis from Mato Grosso State, Braz.
Acta Trop.
(2006) - et al.
Infected dogs as a risk factor in the transmission of human Trypanosoma cruzi infection in western Venezuela
Acta Trop.
(2006) - et al.
Detection of Chagas infections using Trypanosoma evansi crude antigen demonstrates high cross-reactions with Trypanosoma cruzi
Infect. Genet. Evol.
(2007) - et al.
Comparison of serological assays for the diagnosis of canine visceral leishmaniasis in animals presenting different clinical manifestations
Vet. Parasitol.
(2007) - et al.
Chagas disease in north-west Argentina: association between Trypanosoma cruzi parasitaemia in dogs and cats and infection rates in domestic Triatoma infestans
Trans. R. Soc. Trop. Med. Hyg.
(1993) - et al.
Enzootiology of Trypanosoma evansi in Pantanal, Brazil
Vet. Parasitol.
(2004)