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Trypanosoma caninum
, a New Parasite Described in Dogs in Brazil: Aspects of
Natural Infection
Author(s): Maria F. Madeira, Arleana B. P. F. Almeida, Juliana H. S. Barros, Tatiana S. F. Oliveira,
Valeria R. F. Sousa, Andreia S. Alves, Luciana F. C. Miranda, Armando O. Schubach, and Mauro C. A.
Marzochi
Source: Journal of Parasitology, 100(2):231-234.
Published By: American Society of Parasitologists
DOI: http://dx.doi.org/10.1645/13-297.1
URL: http://www.bioone.org/doi/full/10.1645/13-297.1
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TRYPANOSOMA CANINUM, A NEW PARASITE DESCRIBED IN DOGS IN BRAZIL: ASPECTS
OF NATURAL INFECTION
Maria F. Madeira, Arleana B. P. F. Almeida, Juliana H. S. Barros, Tatiana S. F. Oliveira, Valeria R. F. Sousa, Andreia S. Alves,
Luciana F. C. Miranda, Armando O. Schubach, and Mauro C. A. Marzochi
Laborat
´
orio de Vigil
ˆ
ancia em Leishmanioses, Instituto de Pesquisa Cl
´
ınica Evandro Chagas, Funda¸ca˜o Oswaldo Cruz, Av. Brasil 4365, Rio de Janeiro 21045-
900. Correspondence should be sent to: fatima.madeira@ipec.fiocruz.br
ABSTRACT: Trypanosoma caninum constitutes the most recent trypanosomatid species infecting dogs in Brazil. Due to the limited
data available about this parasite, this study aims to disclose clinical and laboratory findings from 14 dogs naturally infected. The dogs
were diagnosed during a cross-sectional survey in Cuiaba
´
(Mato Grosso, Brazil) and followed up at an interval of 3, 6, and 12 mo in
order to evaluate the clinical evolution and to investigate the parasite, the DNA, or both in different biological samples (intact skin,
cutaneous scar, blood, bone marrow, and lymph node aspirate) by parasitological (culture and smear exam) and molecular (DNA-
based tests) methods. Specific anti-T. caninum and anti-Leishmania antibody production was also evaluated. Ten of 14 dogs infected by
T. caninum showed a good general state at the time of diagnosis, and this status did not vary during the follow-up. Anti-T. caninum and
anti-Leishmania IgG antibodies were detected by IFAT in 10 and 2 animals, respectively. Concomitant infection by Leishmania chagasi
was confirmed in 2 dogs, indicating an overlap of endemic areas in Cuiaba
´
. Trypanosoma caninum (parasite or DNA) was found only in
the intact skin in all animals examined. Our results suggest that T. caninum infection can be manifested as an asymptomatic case with
low humoral immune response.
Trypanosoma caninum was initially described in the state of Rio
de Janeiro (Madeira et al., 2009; Pinto et al., 2010) and later in
other states in Brazil. Although it is only a recently identified
species, 53 cases of natural infection in dogs were already reported
in areas where canine visceral leishmaniasis (CanL) is endemic
(Barros et al., 2012). Data related to the pathogenesis of this
parasite in dogs and the epidemiological aspects are still
unknown. Trypanosoma caninum is isolated exclusively from
intact skin fragments, an unusual feature of the Trypanosoma
genus; it also does not infect triatomine insects and it grows very
well in axenic cultures, with a predominance of epimastigote
forms (Madeira et al., 2009). Molecular analyses of partial SSU
ribosomal DNA sequences have shown that all isolates from T.
caninum described so far are genetically identical or closely similar
(Barros et al., 2012). Apparently, this parasite is not very
immunogenic for dogs, even though it is able to stimulate the
production of specific antibodies (Alves et al., 2012).
Visceral leishmaniasis (VL) is endemic in many Brazilian
regions, and the dog is the main domestic reservoir, playing an
important role in the epidemiological cycle, and is considered to
be an important target in the control of this parasite (Minist
´
erio
da Sa ´ude, 2006). The phenomenon of T. caninum parasitizing
domestic dogs in overlapping areas with VL is an important
consideration, mainly for its potential impact on the VL control
strategies that are being implemented in Brazil.
The acquisition of 14 dogs naturally infected by T. caninum
allowed a rare opportunity for clinical and laboratory study of
this disease.
MATERIALS AND METHODS
Animals and collection of biological samples
Fourteen dogs naturally infected by T. caninum were studied. The
animals were identified during the cross-sectional survey for CanL
conducted in Cuiaba
´
city, Mato Grosso, in 2009 (Almeida et al., 2011).
Infection was diagnosed by parasite isolation and posterior identification
by PCR (18S rDNA), followed by amplicon sequencing for which results
have already been deposited in GenBank (Barros et al., 2012). For the
physical examination and collection of biological samples, the animals
were sedated with ketamine associated with acepromazine, as approved by
the Ethics Committee on the Use of Animals, Oswaldo Cruz Foundation
(CEUA, license number LW-01/10). The physical examination was based
on clinical signs compatible with CanL (e.g., weight loss, lymphadenop-
athy, conjunctivitis, alopecia, splenomegaly, and presence of ulcers or
cutaneous scars). Then the following biological samples were collected: (1)
Blood: obtained from the jugular vein with syringe and placed into 2
tubes, one for obtaining serum for the serological tests and the other
containing anticoagulant (EDTA) for culture and molecular assays. Also,
part of this material was smeared onto slides for cytological exam. (2)
Bone marrow: about 0.5–1.0 ml were collected from the sternum and
placed into a tube containing EDTA and processed in the same way as the
blood. Slides with bone marrow smears were also prepared. (3) Lymph
node: this material was obtained by puncture of the most-enlarged
popliteal lymph node with a Valery aspirator. Two slides with smears were
prepared with the punctured material and the remainder were used for
molecular analysis. (4) Tissue fragments: intact skin fragments were
collected from the scapular region after trichotomy, antisepsis, and local
anesthesia with lidocaine 2%. Two fragments were placed in physiological
solution for culture and 1 fragment was stored at 20 C for molecular
tests. Cutaneous scar fragments were processed in the same way as the
skin.
The animals were followed up at intervals of 3, 6, and 12 mo after the
initial diagnosis in order to evaluate the clinical evolution of the infection
by T. caninum and investigate the parasite or the DNA in different
biological samples by parasitological and molecular tools.
Serological tests
Anti-T. caninum and anti-Leishmania sp. IgG antibodies were assessed
in serum of all the animals by indirect fluorescence antibody test (IFAT),
immunoenzymatic assay (an ELISA), and an immunochromatographic
test DPPt (Dual Path Platform).
Anti-T. caninum IgG antibodies were researched using in-house tests
(IFAT and ELISA) with homologue antigens following protocols already
standardized by our group (Alves et al., 2012). For the tests to detect anti-
Leishmania IgG antibodies, IFAT (IFI-leishmaniose visceral canina),
ELISA (EIE-leishmaniose visceral canina), and the rapid immunochro-
matographic test (DPPt) kits were used. These commercial tests are
manufactured by Bio-Manguinhos/FIOCRUZ/MS and distributed to
public service offices for CanL diagnosis in Brazil (Minist
´
erio da Sa ´ude,
2006). The kits were used following the manufacturer’s instructions.
Culture procedure and smear examination
The culture of intact skin and scar fragments, blood, and bone marrow
was done according to the following steps. Tissue fragments were initially
immersed in saline solutions containing 1,000 U penicillin, 200 lg
Received 14 May 2013; revised 30 November 2013; accepted 11
December 2013.
DOI: 10.1645/13-297.1
231
J. Parasitol., 100(2), 2014, pp. 231–234
Ó American Society of Parasitologists 2014
streptomycin, and 50 lg5
0
fluorocytosine per milliliter and stored at 4 C
for 24 hr. Thereafter, each fragment was seeded into screw-cap tubes
containing blood-agar slants (NNN—Novy, MacNeal, and Nicolle)
overlaid with 1.5 ml of Schneider’s Drosophila medium supplemented
with 10% fetal calf serum. The blood and bone marrow (about 0.2–0.3 ml)
were seeded immediately after the collection in tubes containing the same
culture medium. All cultures were duplicated, processed, and kept at 27 C
(60.4 C) and examined weekly under optical microscopy for 40–50 days.
The isolated parasites were identified by PCR assay (epimastigote forms)
or by an isoenzyme electrophoresis technique (promastigote forms) as
previously described (Almeida et al., 2011; Barros et al., 2012).
The smears of blood, bone marrow, and lymph node aspirates were
fixed in methyl alcohol and later stained by Giemsa. The slides were
examined through optical microscopy scanning at least 2,000 microscopic
fields (31,000) looking for the presence of parasites.
Molecular assays
The blood, bone marrow, and tissue (intact skin and scar fragments)
samples were processed by 3 different PCR assays. Initially, the DNA of
all samples was extracted in accordance with the phenol-chloroform
method (Gomes et al., 2007). After this procedure the DNA was stored at
20 C until PCR assays.
DNA detection with targets directed to 18S gene (rDNA): The nested-
PCR protocol was used (Smith et al., 2008), and this same approach had
been used to identify all T. caninum isolates until then. External primers
TRY927F (5
0
-GAAACAAGAAACACGGGAG-3
0
) and TRY927R (5
0
-
CTACTGGGCAGCTTGGA-3
0
) were applied for the first round and
internal primers SSU561F (5
0
-TGGGATAACAAAGGAGCA-3
0
) and
SSU561R (5
0
-CTGAGACTGTAACCTCAAAGC-3
0
) were applied for
the second round. Each PCR run included T. caninum DNA (MCAN/BR/
2003/A27) and L. chagasi DNA (MHOM/BR/1974/PP75) as positive
controls.
DNA detection with targets directed to kDNA-minicircle: This followed
a protocol previously described using the primers 5
0
-(G/C)(G/C)(C/G)
CC(A/C)CTAT(A/T)TTACACAACCCC-3
0
and 5
0
-GGGGAGGGGCG-
TTCTGCGAA-3
0
(Degrave et al., 1994). Leishmania chagasi DNA was
used as a positive control.
DNA detection of the b-globin gene: This assay was used as an internal
control of PCR reactions in order to confirm the absence of inhibitors in
the samples. The primers 5
0
-CAA CTT CAT CCA CGT TCA CC-3
0
and
5
0
-ACA CAA CTG TGT TCA CTA GC-3
0
, which amplify a product of
118 bp, were used following the protocol according to Quaresma et al.
(2009).
All PCR products were run on a 1.5% agarose gel, stained with
ethidium bromide, and visualized under ultraviolet light.
RESULTS
Among the 14 dogs studied, 11 were male, 12 had non-defined
breed, and the ages ranged between 8 mo and 8 yr (median ¼ 2 yr).
The follow-up was carried out at 3, 6, and 12 mo after the T.
caninum infection diagnosis in 8, 3, and 1 dogs, respectively. The
other dogs could not be found or were taken to other regions. At
the moment of the initial diagnosis, 10 dogs were in good general
state and 4 showed 1 or 2 clinical signs such as weight loss (n ¼ 2),
lymphadenomegaly (1), and splenomegaly (2). One animal (no.
799) displayed a cutaneous scar on the scrotum. Only 1 dog (no.
732) had its clinical status changed at 6 mo after it developed
lymphadenopathy, splenomegaly, conjunctivitis, and hair loss,
compatible with the clinical signs of CanL, and infection by L.
chagasi was later confirmed by culture methods and isoenzyme
electrophoresis.
Anti-T. caninum antibodies were detected in 10 animals by
IFAT, with titers varying from 1:40 to 1:640, and in 8 animals by
ELISA. Employing the specific kits for CanL diagnosis, 2 animals
(732 and 799) were positive to IFAT, with titers of 1:40 and 1:80,
respectively. All animals were negative in EIE and only animal no.
732 showed a positive result in DPP.
Trypanosoma caninum infection was established through
culture of skin fragments. By this technique, cutaneous scar (only
dog 799), blood, and bone marrow collected at the same time were
negative for the presence of this parasite in all cases. Three
months later, T. caninum was re-isolated from skin fragments of
all the 8 dogs reexamined. The attempt to re-isolate T. caninum
from the skin in 6 mo (3 cases) and 12 mo (1 case) did not succeed.
Blood and bone marrow samples collected for culture at 3, 6, and
12 mo were negative in 3, 3, and 1 animal, respectively, except in 1
dog (732) in which L. chagasi was isolated from bone marrow at 6
mo.
The smear examinations of the blood, bone marrow, and lymph
node samples collected upon the occasion of initial diagnosis and
during the follow-up were negative except for dog no. 732 from
which amastigote forms were viewed in the bone marrow sample
in the 6th mo.
The PCR assay (18S rDNA) performed with skin fragments of
the 14 dogs, collected at the moment of the initial diagnosis,
showed positive results with amplification products similar to T.
caninum in 12 cases, and 2 animals showed negative results. The
blood, bone marrow, and lymph node samples collected at that
time were negative for T. caninum amplification pattern in all
cases. However, by this assay 1 animal (732) showed an
amplification product similar to the L. chagasi pattern in the
bone marrow sample. When we performed research of Leishman-
ia-specific DNA in these samples, only the bone marrow collected
from animal 732 was positive.
Of the skin fragments collected from 8 dogs after 3 mo, 4 were
PCR positive for T. caninum (nos. 527, 798, 799, and 808),
whereas the research of Leishmania DNA was negative in all 8
cases. Both T. caninum and Leishmania DNA were negative in
blood, bone marrow, and lymph node samples of the 3 dogs
examined in this interval. After 6 mo, T. caninum DNA was not
detected in skin, blood, bone marrow, and lymph node samples of
the 3 animals reevaluated. Nevertheless, a pattern of amplification
similar to L. chagasi was detected in the bone marrow and lymph
node samples of animal 732 and in the cutaneous scar and lymph
node samples of animal 799. This result was later confirmed by
PCR using specific primers for the Leishmania donovani complex,
and the expected 800-bp product was obtained in the lymph node
sample (data not shown). By PCR specific assay, Leishmania
DNA was detected in these samples, and the other samples
showed negative results. After 12 mo, only 1 animal (604)
evaluated showed negative results for both PCR assays in all the
samples analyzed.
In the PCR assay for b-globin, all samples were positive,
showing an expected product of about 118 bp.
All the results can be found in detail in Table I, and the pattern
of amplification of T. caninum and Leishmania by PCR assays can
be observed in Figure 1.
DISCUSSION
Trypanosoma caninum is a recently described parasite, and
many aspects of this agent are unknown—among them is accurate
diagnosis. In all cases reported so far, T. caninum infection has
been established from the culture of dog skin fragments; however,
in all of these cases the aim was the CanL diagnosis (Madeira et
al., 2009; Silva et al., 2011). Using culturing success as a
diagnostic tool, a prevalence of 3.19% and 5.23% of T. caninum
232 THE JOURNAL OF PARASITOLOGY, VOL. 100, NO. 2, APRIL 2014
infection has been reported (Pinto et al., 2010). Using the same
test in Cuiaba
´
city, Almeida et al. (2011) found a prevalence of
3.25%. These data suggest that the prevalence of T. caninum may
be low or that such rates are influenced by other factors. In fact,
both the test and the biological sample analyzed can have an
impact on the sensitivity and specificity of the diagnostic test and,
consequently, on the infection prevalence rates.
Of the 14 animals studied, 71% were in good general state, even
during the follow-up. Relevant clinical signs were found in only 1
animal that was co-infected by L. chagasi and then attributed to
CanL. The results obtained herein, associated to previous
observations, suggest that T. caninum infection can be asymp-
tomatic, without specific clinical signs, and occurs with low
parasitemia, making its detection difficult by the methods used up
to the moment. The lack of knowledge about the biological cycle
of T. caninum restricts the choice of biological samples to be
analyzed and of more appropriate tools for its diagnosis.
Considering t he molecular tools applied i n this study, we
confirmed that this parasite is found exclusively in intact skin.
It is interesting that the first case of T. caninum infection was
described in a dog co-infected by Leishmania braziliensis (Madeira
et al., 2009), and now we report the co-infection by T. caninum
and L. chagasi, demonstrating that T. caninum and Leishmania
parasites can co-exist in concomitant infections.
Species-specific molecular targets are not available for T.
caninum. Here, the use of targets for the region of the ribosomal
gene (18S rDNA), despite being general, allowed the detection of
Leishmania DNA in 2 dogs, and these results were confirmed by
other tests. This approach shows that a combination of methods
may be useful when the parasites are rarely studied, such as in co-
infections with T. caninum.
The humoral immune response in the infection by T. caninum
has been low or absent (Alves et al., 2012), and the results
obtained herein corroborate this observation, showing that of 14
dogs naturally infected by this parasite 43% were seronegative,
even when the homologous antigen was used in the tests. This
may be related to the characteristic of T. caninum in which its
presence appears to be limited and restricted to skin. Unlike
CanL, the presence of L. chagasi in different organs and tissues is
considered as 1 of the factors responsible for the high humoral
immune response. The low humoral response observed in the T.
caninum infection may on one hand limit the variety of options for
its diagnosis but, on the other hand, is a positive issue considering
that serology constitutes the recommendation for CanL diagnosis
TABLE I. Serological, parasitological, and molecular assays performed with different biological samples obtained from dogs naturally infected by
Trypanosoma caninum.Tc(Trypanosoma caninum), Lc (Leishmania chagasi), () negative results, (þ) positive results; nd (not done).
Dog
no.
IFAT titers
(T. caninum)
Parasitological tests Molecular assays—PCR
Culture Smear exam 18S rDNA Leishmania-kDNA
Blood
Bone
marrow Skin Blood
Bone
marrow
Lymph
node Blood
Bone
marrow Skin
Lymph
node Blood
Bone
marrow Skin
Lymph
node
527* 1:640 – – þ (Tc) – – – – – þ (Tc) – – – – –
527† nd nd nd þ (Tc) nd nd nd nd nd þ (Tc) nd nd nd – nd
531* þ(Tc) þ(Tc)
531† nd nd nd þ (Tc) nd nd nd nd nd nd nd nd nd
534* 1:80 þ(Tc) þ(Tc)
534† nd nd nd þ (Tc) nd nd nd nd nd nd nd nd nd
576* þ(Tc) þ(Tc)
577* 1:160 þ(Tc) þ(Tc)
604* 1:80 þ(Tc)
604
§
nd
627* þ(Tc) þ(Tc)
669* 1:80 þ(Tc) þ(Tc)
669† nd nd nd þ (Tc) nd nd nd nd nd nd nd nd nd
732* 1:80 þ(Tc) þ(Lc) þ(Lc)
732‡ nd þ(Lc) þ þ(Lc) þ(Lc) þ(Lc) þ(Lc)
769* þ(Tc) þ(Tc)
769† nd nd nd þ (Tc) nd nd nd nd nd nd nd nd nd
784* 1:160 þ(Tc) þ(Tc)
798
*
1:40 þ(Tc) þ(Tc)
798† nd þ(Tc) þ(Tc)
798
‡
nd
799* 1:160 þ(Tc) þ(Tc)
799† nd þ(Tc) þ(Tc)
799
‡
nd þ(Lc) þ(Lc)
808* 1:160 þ(Tc) þ(Tc)
808† nd þ(Tc) þ(Tc)
* Biological samples collected at the same occasion as the T. caninum diagnosis.
† After 3 mo.
‡ After 6 mo.
§ After 12 mo.
MADEIRA ET AL.—ASPECTS OF NATURAL INFECTION BY T. CANINUM 233
in Brazil. Among the dogs studied, only the animals co-infected
by Leishmania reacted to specific tests for CanL.
The follow-up of dogs in endemic areas is a hard task. Dogs are
usually taken from 1 area to another, which constitutes 1 of the
reasons for the geographic expansion of some zoonoses that have
the dog as a dispersion element; for instance with visceral
leishmaniasis (Marzochi et al., 2009). Although T. caninum is
apparently not pathogenic to dogs, it may represent another agent
in this complicated context. Thus, it is important that the
infection dynamics in this host be understood so that specific
diagnostic tools can be applied.
In conclusion, this study supports a biological feature of T.
caninum and suggests that this parasite is not pathogenic or not
very virulent to dogs. Moreover, the infection course is
asymptomatic and transitory with low humoral immune response.
ACKNOWLEDGMENTS
This study was funded in part by grants from PAPES VI (CNPq,
process no. 407700/2012-9), Funda ¸ca
˜
o de Amparo a Pesquisa do estado
do Rio de Janeiro (FAPERJ, 26102321-2009), and Funda ¸ca
˜
o de Amparo
a Pesquisa do estado de Mato Grosso (FAPEMAT). M. C. A. Marzochi
and A. Schubach are the recipients of fellowships from CNPq.
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FIGURE 1. Agarose gel electrophoresis showing the amplified products
with representative samples of the study. (A) PCR assay that targeted a
partial sequence of the 18S rDNA gene. Lane 1: DNA ladder, 100 bp;
Lane 2: DNA control of Trypanosoma caninum (MCAN/BR/2003/A27);
Lanes 3–5 (positive samples with T. caninum pattern): skin of dogs 527,
531, and 534, respectively; Lanes 6–8 (positive samples with L. chagasi
pattern): bone marrow of dog 732, cutaneous scar of dog 799, and lymph
node of dog 799, respectively; Lane 9: DNA control of Leishmania chagasi
(MHOM/BR/1974/PP75); Lane 10: negative control. (B) PCR assay that
the conserved region of Leishmania kDNA minicircles. Lane 1: DNA
ladder, 100 bp; Lane 2: DNA control of Leishmania chagasi (MHOM/BR/
1974/PP75); Lane 3: bone marrow sample of dog 732; Lane 4: cutaneous
scar sample of dog 799; Lane 5: lymph node sample of dog 799; Lane 6:
lymph node sample of dog 732; Lanes 7–9: skin of dogs 527, 531, and 534,
respectively; Lane 10: negative control.
234 THE JOURNAL OF PARASITOLOGY, VOL. 100, NO. 2, APRIL 2014
