TESA-blot for the diagnosis of Chagas disease in dogs from co-endemic regions for Trypanosoma cruzi, Trypanosoma evansi and Leishmania chagasi.
ABSTRACT We standardized serodiagnosis of dogs infected with Trypanosoma cruzi using TESA (trypomastigote excreted-secreted antigen)-blot developed for human Chagas disease. TESA-blot showed 100% sensitivity and specificity. In contrast, ELISA using TESA (TESA-ELISA) or epimastigotes (epi-ELISA) as antigen yielded 100% sensitivity but specificity of 94.1% and 49.4%, respectively. When used in field studies in an endemic region for Chagas disease, visceral leishmaniasis and Trypanosoma evansi (Mato Grosso do Sul state, Central Brazil), positivities were 9.3% for TESA-blot, 10.7% for TESA-ELISA and 32% for epi-ELISA. Dogs from a non-endemic region for these infections (Rondonia state, western Amazonia) where T. cruzi is enzootic showed positivity of 4.5% for TESA-blot and epi-ELISA and 6.8% for TESA-ELISA. Sera from urban dogs from Santos, São Paulo, where these diseases are absent, yielded negative results. TESA-blot was the only method that distinguished dogs infected with T. cruzi from those infected with Leishmania chagasi and/or Trypanosoma evansi.
- SourceAvailable from: Shoaib Zafar[Show abstract] [Hide abstract]
ABSTRACT: The present report describes a case of Trypanosoma evansi infection (surra) in a 5 months old male bulldog pup weighing 13 kg. Clinical signs included a moderate fever (39.6 o C), pulse (140 beats/min) and an accelerated respiration rate (45/min; normal 10–30/min), anemia, watery eyes and dehydration. Microscopic examination of wet blood films revealed an extremely large number of trypo-mastigotes and thin blood smears stained with Dip Quick™ stain revealed a large numbers of trypanosomes with a characteristic flagellum, kinetoplast and undulating membrane. The pup was treated with 4 mg Cymelarsan® (Rhone Meriux, France) powder dissolved in 3 ml distilled water and administered by deep intramuscular injection once and oral administration of 100 mg manganese chloride dissolved in 10 ml water daily for 5 days. Wet blood films and Dip Quick ™ stained blood smears were negative for trypanosomes on day 3, 7, and 14 of post treatment and all vital parameters returned to normal on day–4 post–medication. MS (2014). Use of Cymelarsan ® and Manganese Chloride for Treatment of the Canine Trypanosomiasis (Surra): A Research report. Adv. Anim. Vet. Sci. 2 (2): 104 – 105. Surra is a protozoan disease caused by Trypanosoma evansi that affects a wide variety of mammalian hosts. Several reports have described the occurrence of surra in adult dogs (Rashid et al., 2008; Eloy and Lucheis, 2009; Defontis et al., 2012). However, as far as could be ascertained, documentation of treatment of this disease in pups doesn't exist. The present report describes surra in a 5–month old bulldog male pup and subsequent treatment.Advances in Animal and Veterinary Sciences 07/2014; 2(2):104-105.
- [Show abstract] [Hide abstract]
ABSTRACT: We investigated whether ELISA using crude antigens from insect and plant trypanosomatids, which are non-pathogenic and easily cultivated in large scale, has the same positivity data as Leishmania (Leishmania) chagasi, the etiological agent of human visceral leishmaniasis (VL) or canine leishmaniasis (CanL), or as T. cruzi, the etiological agent of Chagas disease (CD). The antigens from Crithidia fasciculata, Crithidia luciliae, and Leptomonas seymouri showed 100% cross-reactivity with VL and CanL samples, with no statistically titers differences from L. (L.) chagasi, however, 34% (17/50) of VL samples revealed higher titers using the insect trypanosomatids than the homologous antigen. On the other hand, antigens from Strigomonas culicis, Angomonas deanei, and Phytomonas serpens showed low cross-reactivity with VL and CanL samples. The sera from patients with American tegumentary leishmaniasis showed low levels of cross-reactivity with all trypanosomatids investigated, even with L. (L) chagasi, without titers dissimilarity among them. These parasites were also worthless as antigen source for detection of CD cases, which required homologous antigens to reach 100% positivity. This study showed, by ELISA, that crude extract of Crithidia and Leptomonas have epitopes similar to L. (L.) chagasi, which supports the idea of using them as antigens source for the serodiagnosis of visceral leishmaniasis.Acta tropica 11/2013; · 2.79 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Introduction: The aim of this study was to evaluate the serological cross-reactivity between Leishmania sp. and other canine pathogens. Methods: Positive serum samples for Ehrlichia canis, Babesia canis, Toxoplasma gondii, Neospora caninum and Trypanosoma cruzi were tested using three serological methods enzyme linked immunosorbent assay (ELISA), indirect immunofluorescent antibody test (IFAT) and Kalazar Detect™, for canine visceral leishmaniasis. Results: Of the 57 dog samples tested, 24 (42.1%) tested positive using one of the three serological methods: 10/57 (17.5%) for ELISA, 11/57 (19.3%) for IFAT and 3/57 (5.3%) for Kalazar Detect™. Conclusions: Our results demonstrated that the presence of other infectious agents may lead to cross-reactivity on leishmaniasis serological tests.Revista da Sociedade Brasileira de Medicina Tropical 12/2013; 47(1):105-7. · 0.93 Impact Factor
Acta Tropica 111 (2009) 15–20
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/actatropica
TESA-blot for the diagnosis of Chagas disease in dogs from co-endemic regions
for Trypanosoma cruzi, Trypanosoma evansi and Leishmania chagasi
E.S. Umezawaa,b, A.I. Souzac, V. Pinedo-Cancinoa, M. Marcondesd, A. Marcilie, L.M.A. Camargoe,
A.A. Camachoc, A.M.S. Stolfa, M.M.G. Teixeirae,∗
aUniversidade de São Paulo - Instituto de Medicina Tropical de São Paulo - Av. Dr. Enéas de Carvalho Aguiar, 470, São Paulo, SP, Brazil
bUniversidade de São Paulo, Faculdade de Medicina, Departamento de Medicina Preventiva, SP, Brazil
cUniversidade do Estado de São Paulo (FCAV-UNESP), Jaboticabal, SP, Brazil
dUniversidade do Estado de São Paulo (UNESP), Arac ¸atuba, SP, Brazil
eUniversidade de São Paulo, Instituto de Ciências Biomédicas, Departamento de Parasitologia - Av. Lineu Prestes 1374, 05508-900, São Paulo, SP, Brazil
a r t i c l ei n f o
Received 12 September 2008
Received in revised form 9 December 2008
Accepted 7 January 2009
Available online 19 January 2009
Canine Chagas disease
a b s t r a c t
We standardized serodiagnosis of dogs infected with Trypanosoma cruzi using TESA (trypomastigote
excreted-secreted antigen)-blot developed for human Chagas disease. TESA-blot showed 100% sensi-
tivity and specificity. In contrast, ELISA using TESA (TESA-ELISA) or epimastigotes (epi-ELISA) as antigen
yielded 100% sensitivity but specificity of 94.1% and 49.4%, respectively. When used in field studies in an
endemic region for Chagas disease, visceral leishmaniasis and Trypanosoma evansi (Mato Grosso do Sul
state, Central Brazil), positivities were 9.3% for TESA-blot, 10.7% for TESA-ELISA and 32% for epi-ELISA.
Dogs from a non-endemic region for these infections (Rondonia state, western Amazonia) where T. cruzi
is enzootic showed positivity of 4.5% for TESA-blot and epi-ELISA and 6.8% for TESA-ELISA. Sera from
urban dogs from Santos, São Paulo, where these diseases are absent, yielded negative results. TESA-blot
was the only method that distinguished dogs infected with T. cruzi from those infected with Leishmania
chagasi and/or Trypanosoma evansi.
© 2009 Published by Elsevier B.V.
Dogs are important reservoirs of Trypanosoma cruzi and Leish-
mania 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 coun-
tries 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 domes-
tic 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 circu-
late between wild mammals and triatomines (Gürtler et al., 1998,
∗Corresponding author. Tel.: +55 11 3091 7268; fax: +55 11 30917417.
E-mail address: firstname.lastname@example.org (M.M.G. Teixeira).
2007; Crisante et al., 2006; Estrada-Franco et al., 2006; Cardinal et
al., 2007, 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.,
The importance of domestic dogs as reservoirs and as a risk
factor for the transmission of T. cruzi to humans has been exten-
households was found to increase about 3–4 times with the num-
ber of infected dogs, and the seroprevalence of infected humans
doubled in households with 1–2 infected dogs (Gürtler et al., 1993,
1996, 2007; Diosque et al., 2004; Cardinal et al., 2007). In Mex-
ico, dogs are known to provide frequent blood meals for Triatoma
barberi 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
0001-706X/$ – see front matter © 2009 Published by Elsevier B.V.
E.S. Umezawa et al. / Acta Tropica 111 (2009) 15–20
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 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
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 Mex-
ico (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 sev-
eral reports of T. cruzi-infected dogs in the southern USA, where
have been associated with domestic transmission of human infec-
tion (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 serolog-
ical 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 immunofluo-
good sensitivity, their specificity decreased when sera from hosts
with visceral leishmaniasis were tested (Frank et al., 2003;
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 (try-
pomastigote excreted–secreted antigen) as antigen. In this method
no cross-reactivity with sera from humans infected with Leishma-
nia spp., T. rangeli or other pathogens is observed (Umezawa et al.,
1996, 2001; Caballero et al., 2007). In the present study, whose
objective was to standardize a highly specific and sensitive sero-
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
visceral leishmaniasis and T. evansi infection.
2. Materials and methods
TESAs (trypomastigote excreted–secreted antigens) from the Y
strain of T. cruzi were obtained as previously described (Umezawa
(in serum-free medium or with 2% FCS) infected with T. cruzi were
10–20×106/mL. After being centrifuged at 1800×g for 15min at
4◦C, the supernatant containing TESA was then re-submitted to a
second centrifugation (7000×g for 5min at 4◦C) and used directly
without any further treatment or stored at −80◦C in small aliquots.
pared with fresh parasites cultivated in LIT medium as described
previously (Umezawa et al., 2001). Briefly, 500?g of epimastigote
forms were incubated with 0.3N NaOH (500?L) and, after 18h at
4◦C, neutralized with 0.3N HCl, centrifuged at 12,000g for 1min
at 4◦C and used after determining the protein content (Micro-BCA
protein reagent kit, Pierce Co.).
2.2. ELISA using TESA or epimastigote forms of T. cruzi as antigen
ELISAs using TESA (TESA-ELISA) and epimastigotes (epi-ELISA)
were performed as previously described (Umezawa et al., 2001).
For this study, the optimal antigen concentration and dilution
of dog sera were previously determined by checkerboard titra-
tion. The sera were diluted 1:200 for TESA-ELISA and 1:100 for
epi-ELISA. Horseradish peroxidase-labeled anti-dog immunoglob-
ulin G (Sigma Co.) was used to detect antigen-specific antibodies,
the mixture was incubated with hydrogen peroxide and O-
phenylenediamine dihydrochloride (OPD-tablets, Sigma Co.), and
the reaction stopped by adding 4N HCl. The absorbance (A492nm)
was measured in an ELISA reader (Labsystems Multiskan MS). All
the experiments were carried out in duplicate and repeated at least
twice on different days. The cutoff values for ELISAs were calcu-
lated as the mean plus three standard deviations of the absorbance
(A492 nm) values for 30 true negative dog sera.
Proteins from TESA were separated by SDS-PAGE, transferred to
nitrocellulose sheets, and blocked with PBS containing 5% fat-free
milk for 1h at room temperature. Membrane strips (5mm) were
incubated with canine sera (1:100) diluted in PBS with 1% milk
for 2h or overnight at room temperature, washed, and the bound
antibodies were detected with horseradish peroxidase-labeled
by addition of 0.05% hydrogen peroxide and 4-chloro-1-naphthol
when a large 150–160kDa band and/or five bands between 130 and
200kDa were observed (Umezawa et al., 1996).
2.4. Serum sampling and T. cruzi isolates
Domestic canine sera were obtained from blood samples col-
lected by femoral puncture from domestic dogs living in rural areas
and stored at −20◦C. TESA-blot was compared with TESA-ELISA
and epi-ELISA using 10 positive control sera from dogs chroni-
cally infected with T. cruzi, eight from dogs experimentally infected
Furnas de Dionísio, Mato Grosso do Sul State, and were diagnosed
by xenodiagnosis followed by isolation in culture of parasites from
the guts of the triatomines (xenocultures). Molecular diagnosis of
cultured isolates was performed using the PCR method described
by Fernandes et al. (2001). Lineage genotyping was carried out as
described previously (Martins et al., 2008; Marcili et al., 2009).
Fifty negative control serum samples were obtained from
healthy domestic dogs (without any clinical symptoms) from
Santos, a coastal city in São Paulo State, Southern Brazil that is non-
endemic for Chagas disease, visceral leishmaniasis and T. evansi.
E.S. Umezawa et al. / Acta Tropica 111 (2009) 15–20
Results obtained using TESA-blot, TESA-ELISA and epi-ELISA for detection ofT. cruzi infection in dogs, cross-reactivity with other canine pathogens and evaluation of TESA-blot
for field studies of canine Chagas’ disease.
Status of dogs Number of cases% (Number) of positive canine sera by:
T. cruzi-infected dogs (% positivity)a
Dogs without T. cruzi infection
Leishmania chagasi – total
Arac ¸atuba, SP
Campo Grande, MS
% specificity (including Leishmania)b
% Specificity (excluding Leishmania)c
Furnas do Dionísio, Mato Grosso do Sul
Monte Negro, Rondônia
aSera (10 samples) from T. cruzi-infected dogs including two naturally infected from Furnas de Dionísio (MS), and 8 experimentally infected with Esmeraldo strain. Specificity
analysis of TESA-blot calculated including sera from dogs with (b) or without (c) visceral leishmaniasis.
Sera from 106 dogs naturally infected with visceral leishmaniasis
(n=50 from Arac ¸atuba, São Paulo, Brazil, and n=56 from Campo
Grande University Hospital, Mato Grosso do Sul State) were pos-
itive in parasitological tests (microscopic examination of lymph
node smears) and serological (IFA) were used as control of canine
visceral leishmaniasis. Sera from two dogs from Campo Grande,
MS, naturally infected with T. evansi diagnosed by examination of
Giemsa-stained blood smears and confirmed by species-specific
mentally infected dogs that were positive by parasitological and/or
serological methods for the following hemoprotozoan infections
Babesia canis (n=3), Ehrlichia canis (n=3), Neospora canis (n=3) and
Toxoplasma gondii (n=3).
2.5. Field surveys
Field surveys were carried out by testing 119 sera collected from
domestic dogs in houses from two rural areas of Brazil: (i) 75 sera
of dogs from Furnas de Dionísio (S20◦06?19??, W54◦25?60??), Mato
Grosso do Sul State, Central Brazil, which is an endemic region
for Chagas disease and visceral leishmaniasis in humans. While
this region is also endemic for visceral leishmaniasis and T. evansi
in dogs, the prevalence of T. cruzi infection in this animal has
never been evaluated (Cortada et al., 2004; Pompilio et al., 2005;
Herrera et al., 2004; Savani et al., 2005); (ii) 44 sera from Monte
Negro (S10◦15?06??, W63◦17?13?), Rondonia State in western Brazil-
ian Amazonia, where autochthonous Chagas disease and visceral
leishmaniasis are absent but cutaneous leishmaniasis is endemic
in humans (Shaw et al., 2007). Dogs from these regions were not
3.1. Standardization of TESA-blot for diagnosis of T. cruzi-infected
The TESA-blot developed for diagnosis of human Chagas dis-
ease 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 150–160kDa 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 resulted in brown
bands (not shown). Sensitivity of TESA-blot was not determined
due to limited number of parasitologically positive dogs.
The sera from dogs that were not infected with T. cruzi (n=170),
infections (n=120) showed negative results by TESA-blot and posi-
tivities of 5.9% by TESA-ELISA and 50.6% by epi-ELISA (Table 1). Sera
of healthy dogs (n=50) from urban areas of Santos, São Paulo, not
endemic for T. cruzi, L. chagasi and T. evansi, were not reactive by
either TESA-blot or epi-ELISA, but one of these samples (2%) was
positive by TESA-ELISA with a low titer (Table 1).
Sera from 106 dogs with visceral leishmaniasis (from Arac ¸atuba
and Campo Grande) confirmed by parasitological (lymph node
smears) and IFA methods were not reactive for TESA-blot while
showed overall positivities of 6.6% (7/106) by TESA-ELISA and 77.4%
(82/106) by epi-ELISA. Samples from Arac ¸atuba dogs showed pos-
itivity of 4.0% and 76% for TESA-ELISA and epi-ELISA, respectively.
Sera from Campo Grande (MS) dogs had positivities of 8.9% and
78.6% for TESA-ELISA and epi-ELISA, respectively. Sera from dogs
infected with T. evansi (n=2) were negative for TESA-blot although
canis, Neospora canis or Toxoplasma gondii, two from dogs infected
with N. canis cross-reacted in epi-ELISA at low titers (Table 1).
Specificity calculated by testing sera from dogs non-infected
with T. cruzi (n=170) was higher for TESA-ELISA (94%) than for epi-
ELISA (49.4%). However when calculated by excluding sera from
dogs with visceral leishmaniasis, specificities were similar for both
specificity of epi-ELISA is essentially due to cross-reactivity with
Leishmania (Table 1). All the 170 sera from dogs that were either
tozoans were negative by TESA-blot (did not react with either the
150–160 or the 130–200kDa bands).
3.2. Occurrence of T. cruzi-infected dogs in endemic or
non-endemic areas for Chagas disease, visceral leishmaniasis and
T. evansi infection
Comparison of the diagnostic methods tested for field surveys
was performed by comparing the performance of TESA-blot, TESA-
E.S. Umezawa et al. / Acta Tropica 111 (2009) 15–20
ELISA and epi-ELISA in detecting T. cruzi infection in dogs living in
regions with different endemicities for Chagas disease and other
infectious diseases. Different seroprevalences were obtained for
the 119 dogs surveyed depending on their geographic origin and
the test used. In Amazonia (Rondonia state), an endemic region for
cutaneous but not for visceral leishmaniasis, the percentage of pos-
itive dogs was 6.8% (3/44) by TESA-ELISA, 4.5% (2/44) by epi-ELISA,
and 4.5% (2/44) by TESA-blot. On the other hand, in the State of
Mato Grosso do Sul that is endemic for T. cruzi, Leishmania spp.
and T. evansi, the prevalences of dog positive for T. cruzi were 9.3%
(7/75) by TESA-blot, 10.7% (8/75) by TESA-ELISA, and 32% (24/75)
by epi-ELISA (Table 1). Only 7 (9.3%) dogs from this region were
confirmed by TESA-blot to have T. cruzi infection, with a large con-
cordance between this method and TESA-ELISA. Conflicting results
between methods using TESA antigen and epi-ELISA were due to
high cross-reactivity of sera from dogs infected with L. chagasi,
as demonstrated in field survey of canine Chagas disease in Mato
Grosso do Sul.
3.3. Genotyping of T. cruzi isolates from dogs
dogs from the State of Mato Grosso do Sul. Two isolates obtained in
cultures were genotyped as TCIIc using the methods employed by
Martins et al. (2008) and Marcili et al. (2009).
tion in dogs from North to South America (Barr et al., 1991; Gürtler
et al., 1993, 1998, 2007; Crisante et al., 2006; Estrada-Franco et
al., 2006; Jimenez-Coello et al., 2008; Cardinal et al., 2007, 2008)
indicates a high prevalence of the infection in this animal in these
Chagas disease in rural communities in endemic Northeast Brazil.
The household rate of seroreactivity to T. cruzi was five times grater
than in houses with non-infected dogs and cats (Mott et al., 1978;
Barrett et al., 1979).
Serological surveys describing dogs infected with T. cruzi have
used methods that have not been evaluated regarding cross-
reaction with Leishmania spp. and/or T. evansi. However, validation
tion in regions co-endemic for L. chagasi and/or T. evansi infection
requires testing sera from dogs infected with these parasites to rule
out the possibility of cross-reactivity. Dogs are the most important
reservoirs of L. chagasi, agent of serious disease in dogs that causes
visceral leishmaniasis in humans. Domestic dogs infected with L.
chagasi have been reported from North (Gaskin et al., 2002; Duprey
et al., 2006) to South America including Brazil, Venezuela, Colom-
bia and a recent focus in Argentina (Ferreira et al., 2007; Baneth et
al., 2008; Salomon et al., 2008). Dogs are also found infected by L.
braziliensis and L. amazonensis, agents of human cutaneous leish-
maniasis in Argentina and Brazil (Padilla et al., 2002; Tolezano et
In addition to T. cruzi and Leishmania spp., dogs can be infected
with T. rangeli and T. evansi, including mixed infection with more
than one of these trypanosomatids (Crisante et al., 2006; Savani
et al., 2005; Kjos et al., 2008). In South America, T. evansi is found
in wetland regions from Brazil, Colombia, Venezuela, Bolivia and
disease (Herrera et al., 2004; Savani et al., 2005). T. evansi shares
before this study cross-reactivity with T. cruzi of sera from T. evansi-
infected dogs had been not confirmed. T. rangeli shares vertebrate
hosts and vectors with T. cruzi. This species has been investigated
using sensitive and specific molecular markers. Dogs and several
species of wild mammals have been reported infected by T. rangeli
from Central (Panama) to South (Colombia, Venezuela, Peru and
has been also reported in Northeast, Central and Southern Brazilian
regions (Maia da Silva et al., 2007, 2009). T. cruzi and T. rangeli share
several antigens, and are known to cause mixed infection in dogs
(Crisante et al., 2006). Sera from dogs infected with T. rangeli were
not available for the present study. However, sera from humans
infected with T. rangeli did not cross-react with T. cruzi in TESA-blot
(Caballero et al., 2007).
conventional serological tests used to diagnose T. cruzi infection is
the main obstacle in the accurate diagnosis of canine Chagas dis-
ease. Although some studies refer to this problem, cross-reaction
In recent years, new methods have been developed specifically
for the diagnosis of canine Chagas disease. An immunochromato-
graphic dipstick to detect T. cruzi from endemic and non-endemic
areas in Argentina showed a specificity of 94% and a sensitivity of
96%, although authors mentioned that for dogs in Chaco Province
cross-reactivity with Leishmania spp. could be possible (Cardinal
et al., 2006b). A combination of conventional ELISA for screen-
ing followed by confirmation using a complex technique of Flow
Cytometry with live trypomastigotes was employed for detection
antigen in IFA and confirmation through Western blot assays was
al., 2008). Unfortunately, methods employed in these and in other
mania or T. evansi (Gaskin et al., 2002; Frank et al., 2003; Ramsey
et al., 2005; Duprey et al., 2006; Kjos et al., 2008). Thus, methods
to distinguish infections caused by T. cruzi from those caused by
other trypanosomatids that combine sensitivity and specificity are
required to assess the prevalence of T. cruzi infection in dogs.
In this study, we adapted TESA-blot, which was originally
designed for diagnosis of human Chagas disease, so that it could be
used to detect canine T. cruzi infection. This method is highly sen-
sitive and specific, is not subject to cross-reactivity with sera from
humans infected with Leishmania spp. or T. rangeli, and has been
employed as a reference test for human Chagas disease (Umezawa
et al., 1996, 2001; Caballero et al., 2007). In contrast, conventional
serological methods, such as IFA and ELISA using T. cruzi epimastig-
from Leishmania-infected patients (Frank et al., 2003; Caballero et
is 100% specific when tested with dogs infected with leishmania,
in contrast with the high cross-reactivity observed using epi-ELISA.
leishmaniasis (6.6%) compared with that of TESA-blot in the diag-
nosis of dogs (0%). This is in agreement with the results obtained
for these two tests when chagasic human serum is used (Umezawa
et al., 2001). High cross-reactivity was observed between T. cruzi
epimastigote antigens and sera from dogs with visceral leishma-
niasis as well as with T. evansi (100%) and N. canis (66.7%). These
results confirm that control sera from dogs infected with L. cha-
gasi, T. evansi and other pathogens are crucial in the validation of
diagnostic tests of canine Chagas disease suitable for field stud-
ies. Although TESA-blot requires trypomastigotes from cell culture,
antigen preparation does not demand purification and could be
stocked for years at −80◦C or in liquid nitrogen. Alternatively, and
E.S. Umezawa et al. / Acta Tropica 111 (2009) 15–20
taining TESA can be maintained at 4◦C for several months allowing
rapid and easy-to-perform diagnosis (Umezawa et al., 1996, 2001).
TESA-blot was employed for field epidemiological surveys of T.
cruzi infection in dogs in two areas of Brazil: one in Mato Grosso do
Sul (central region) and the other in Rondonia (western Amazonia).
These regions are endemic and non-endemic for Chagas disease,
visceral leishmaniasis and T. evansi infection, respectively.
be infected with T. cruzi and with visceral and cutaneous leishma-
niasis (Borges-Pereira et al., 2001; Pompilio et al., 2005; Carvalho
et al., 2006). Furnas de Dionísio is a small, poor, rural village in
sidered free of domestic triatomines, which used to inhabit palms
for T. evansi, exhibiting the highest prevalence in Brazil for this
canine infection (Ventura et al., 2002; Herrera et al., 2004; Savani
et al., 2005). TESA-ELISA and epi-ELISA yielded positivity rates for
T. cruzi infected dogs in this region of 10.6% and 32%, respectively;
diagnosed with canine Chagas disease by TESA-blot was confirmed
by xenodiagnosis followed by isolation in culture and molecular
diagnosis of parasites. These results are of particular significance in
the reemergence of human Chagas disease and importance of dogs
Brazil. The two T. cruzi isolates obtained from dogs were assigned
to TCIIc, a lineage associate to terrestrial transmission cycles. This
is the first report of TCIIc in Central Brazil, and it is the first time
that TCIIc is found in Brazilian dogs, although this lineage has been
described infecting dogs in Paraguay and Argentina (Martins et al.,
2008; Cardinal et al., 2008; Marcili et al., in preparation).
To date domestic dogs infected with neither T. cruzi nor L. cha-
gasi have been detected in the State of Rondonia, in the Brazilian
Amazonia (Shaw et al., 2007). TESA-blot, TESA-ELISA and epi-ELISA
were positive for 4.5%, 6.8% and 4.5%, respectively, of dogs exam-
ined in this region, and two out of 44 dogs tested were confirmed
to be infected with T. cruzi. The lower positivity using epi-ELISA
observed in this region compared with the higher indices in Cen-
tral Brazil is very probably due to the absence of canine visceral
leishmaniasis in this area. To our knowledge, this is the first report
of domestic dogs infected by T. cruzi in Brazilian Amazonia (Monte
Negro, Rondonia state). The dogs examined in this study circulate
freely inside houses and in the neighboring forest, where they are
exposed to the zoonotic transmission cycle of T. cruzi because of
their habit of hunting and eating the uncooked meat of wild mam-
mals. In addition, Monte Negro is a small village with abundant
palm trees inhabited by sylvatic triatomines infected with T. cruzi
reported in this village (Marcili et al., in preparation).
epidemiological role in the emergence of human Chagas disease
with Amazonia colonization. Canines act as source of T. cruzi-
infected blood for triatomines and can attract infected vectors from
nearby palms to peridomiciliary and domiciliary environments,
thus enhancing the risk of both human infection and triatomine
domiciliation (Cardinal et al., 2007). This study disclosed T. cruzi-
infected dogs in Brazilian regions endemic or non-endemic for
human Chagas disease. The results show that dogs may play an
important role in the emergence, reemergence and surveillance of
human Chagas disease by connecting T. cruzi from enzootic sylvatic
urally infected with T. cruzi in Brazilian rural areas, where these
animals circulate between sylvatic and domestic environments,
warrants further investigation.
mission routes of dogs infected with T. cruzi is essential for an
understanding of the role of these animals in the epidemiology of
human Chagas disease and can be helpful for designing and mon-
itoring control measures. TESA-blot is currently the only method
that has been standardized for diagnosis of canine Chagas disease
that takes into account cross-reactivity with other canine diseases,
especially visceral leishmaniasis and T. evansi infection. The find-
ings of the present study suggest that if conventional methods are
used to detect T. cruzi infection in dogs, the prevalence could be
significantly overestimated because of cross-reactivity, particularly
with sera from dogs infected with Leishmania. In conclusion, TESA-
blot proved to be an excellent assay for serological diagnosis in
epidemiological surveys of T. cruzi infection in dogs in Brazil and
Leishmania spp. and T. evansi.
This study was supported by grants from LIM49-FMUSP and
nical help, and Professor E. Camargo for his valuable comments on
the manuscript. V. Pinedo-Cancino and A. Marcili are recipients of
fellowships from CNPq.
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