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Sistema de Información Científica
Red de Revistas Científicas de América Latina y el Caribe, España y Portugal
Arch Med Vet
46, 333-336 (2014)
Real-time PCR-based study of haemotrophic mycoplasmas in dogs
from Ribeirão Preto, Brazil
Estudio de micoplasmas hemotróﬁcos en perros de Ribeirão Preto, Brasil,
basado en la PCR en tiempo real
, SA Faggion
, EV Santos
, PG Roberto
, SC França
, AL Fachin
, M Marins
Unidade de Biotecnologia, Universidade de Ribeirão Preto, Ribeirão Preto, Brazil.
Heranza-Biotecnologia, Ribeirão Preto, Brazil.
Candidatus Mycoplasma haematoparvum
son hemoparásitos caninos asociados a la anemia en perros. La garrapata
está indicada como el principal vector de transmisión, pero una clara asociación de la exposición de canes a garrapatas e
infección no fue establecida. El objetivo de este estudio fue la aplicación de la PCR en tiempo real para estudiar la prevalencia de haemoplasmas
perros de la ciudad brasileña de Ribeirão Preto, donde
es un vector común para hemoparásitos, tales como
. ADN de
fue detectado en 3 perros y de
Candidatus Mycoplasma haematoparvum
en 1 perro, entre 154 analizados,
indicando una baja prevalencia de esos hemoparásitos.
Candidatus Mycoplasma haemocanis
PCR en tiempo real.
Candidatus Mycoplasma haematoparvum
are canine haemoparasites associated with anemia in dogs. The tick
has been pointed as the main vector of transmission, but a clear association between the exposure of dogs to ticks and
infection has not been established. The aim of this study was to apply real-time PCR to study the prevalence of haemoplasmas
in dogs from the
Brazilian town of Ribeirão Preto, where
is a common vector for other haemoparasites such as
was detected in 3 and
Candidatus Mycoplasma haematoparvum
in 1 out of 154 tested dogs, indicating a low prevalence of
Candidatus Mycoplasma haemocanis
; real-time PCR.
order) forms a
group of bacteria which are obligate parasites of seve-
ral species of animals and are associated with anemia,
arthritis, infertility and respiratory disorders (Chalker
2007). Generically called mycoplas-
mas, they have a very small genome and are devoid of
a cell wall. Among the pathogenic species, the hemotro-
phic mycoplasmas (haemoplasmas) are receiving more
attention in recent years due to increased infections in
human patients, especially those with compromised im-
mune systems (Santos
2008). They are characterised
by parasitism of the surface of erythrocytes of different
mammalian species in which they cause anemia with va-
riable intensity, from asymptomatic cases to the potentia-
lly fatal (Chalker 2005, Willi
are associated with
anemia in dogs (canine hemoplasmosis), but the establis-
hment of more severe clinical cases seem to be dependent
on other factors including immune status, co-infection
and splenectomy (Chalker 2005). The prevalence in dogs
is considered low; however, the use of more sensitive
methods of detection may change this picture. The in-
fection of animals that may or may not develop clinical
signs of disease, with varying degrees of severity has
been descrbed worldwide (Kenny
2010). The tick
has been poin-
ted as transmission vector (Messick 2003), but a clear as-
sociation of dog exposure to ticks and infection has not
been established. Other modes of transmission would be
by oral ingestion and passage from mother to fetus via
the placenta (Krakowka 1977).
distribution in tropical countries and is the vector of seve-
ral other hemoparasites of veterinary importance such as
, causative agent of canine
ehrlichiosis, and the protozoan
agent of canine babesiosis (Dantas-Torres 2008). The-
se two diseases are quite common in dogs in Brazil and
thrombocytopenia is a common laboratory ﬁnding after
infection by these parasites, which also occurs in infec-
2004). In addition, co-infection with these parasites,
followed by a state of immunosuppression, can lead to
hemoplasmosis (Chalker 2005).
Due to the speciﬁc culture requirements, which have
not yet been fully established, the identiﬁcation of myco-
plasmas is based on clinical signs and visualization in
blood smears, using the methods of Romanowsky stai-
ning or acridine orange. Although they should not be
excluded, cytological methods have low sensitivity and
speciﬁcity to establish a precise diagnosis for clear asso-
ciation with clinical symptoms.
More recently, some techniques based on DNA am-
pliﬁcation are becoming an alternative for the detection
and identiﬁcation of parasites in veterinary practice. In
this regard, the detection of haemoplasmas
polymerase chain reaction (PCR) is currently considered
the most accurate technique (Willi
tional and quantitative real-time PCR assays based on the
ampliﬁcation of 16S ribosomal RNA genes are among
the most commonly used to differentiate between species
Gentilini et al 2009, Santos
The development of cheaper and less complex procedu-
res associated with these techniques will facilitate their
large-scale application in veterinary laboratories.
The aim of this study was to conduct a molecular sur-
vey and characterisation of
tus .M. haemocanis
in dogs from Ribeirão Preto, Brazil,
a country with increasing population of pet dogs, high
in rural and urban areas and
still with few data available about canine haemoplasmas.
MATERIAL AND METHODS
CLINICAL SAMPLES AND DNA EXTRACTION
EDTA-anticoagulated blood samples from 154 dogs
presenting to a private veterinary clinic were collected
throughout a one year period between August 2009 and
DNA was extracted from 200 ml blood using
the Illustra blood Miniprep kit (GE Health care).
The samples were analysed by a published Real Time
PCR assay using primers and TaqMan probes targeted
to the 16S ribosomal RNA gene of
Candidatus. M. haematoparvum
Positive control reactions were performed with primers
and TaqMan probe targeted to the canine gene GAPDH
(Canine) and for each combination of primers and pro-
bes a negative reaction was also performed using sterili-
zed water instead of DNA. Basically each reaction tube
contained a general solution of
12.5 µL 1X Master Mix
(Roche Real time PCR Mix), 0.5 µL of each primer (200
nM), 0.5 µL TaqMan probe (100 nM), 10 µL DNA (0.5-
1.0 ug), and sterile pure water in a ﬁnal volume of 15 µL.
After a brief centrifugation tubes were taken to the ther-
mal cycler (Stratagene MXp3500P) where they were in-
cubated under the following conditions: 95 °C for 15 min
followed by 45 cycles with a ﬁrst step of 95 °C for 15 s
and a second step of 60 °C for 60 s. In the second step the
ﬂuorescence data was collected.
DETECTION THRESHOLD OF THE REAL-TIME PCR
The detection threshold of the assay was evaluated using
plasmid containing a cloned fragment of the target genes
obtained by conventional PCR. After measuring the plas-
mid DNA concentrations with the Genequant 1300 system
(GE Healthcare, USA), the corresponding copy numbers
were calculated. The plasmids were diluted to contain 10
copies/µL and 10-fold serial dilutions were then prepared.
One microliter was used in each reaction mixture for eva-
luation of the detection threshold of the assay which could
detect a minimum of 10 copies of the plasmid.
Positive samples in real-time PCR were subjected to
conventional hemi-nested PCR using primers directed
to the 16S rDNA of these species. In the ﬁrst reaction,
forward primer MHCFWD1 5´ TCGAACGGACCTT-
GGTTTCGGCC 3´ and MHCREV1 5´ TCCTATCCGA-
ACTGAGACGAA 3´were used to generate a fragment of
1187 bps encompassing position 1 to 1187 of the
gene, based in the reference sequence EF416568.
For ampliﬁcation of the
Candidatus M. haematoparvum
primers CMHFWD1 5´ GGAGAATAGCAATCCGAA-
AGG 3´ and CMHREV2 5´ CAGCCCGAGGCATAA-
GGGGC 3´were used to generate a fragment of 1062 bps
encompassing position 110 to 1171, based in the referen-
ce sequence EF416569. In the second reaction, primers
MHCFWD2 5´- GCAAGCGCAGGCGGATGTG 3´ and
MHCREV1 were used to generate a fragment of 705 bps
encompassing position 483 to 1187 of the
Candidatus M. haematoparvum
primers CMHFWD1 and CMHREV2 5´ CCACGCTTT-
CAAGCCTCAGCGT 3´were used to generate a fragment
of 635 bps encompassing position 110 to 744, based in
the reference sequence.
For the ﬁrst PCR, a general so-
lution was prepared containing the reagents in sufﬁcient
amounts for each reaction tube to contain 400 nM of each
primer, 200 mM of dNTPs, 1.8 mM magnesium chloride,
; REAL-TIME PCR
1X PCR buffer (100 mM Tris HCl, pH 8.5, and 500 mM
KCl), 1.5 units Taq polymerase (LGCBio), and sterile
pure water in a ﬁnal volume of 25 μL. A 10-μL aliquot
of puriﬁed DNA (0.5-1.0 μg) was added to each tube.
In the second PCR reaction, the same general solution
but a 1-μL aliquot of the ﬁrst reaction was used as tem-
plate. In both reactions, sterile water replaced DNA in
negative control reactions. The tubes were brought to the
thermocycler (Bioer) and incubated with the following
times and temperatures: 95 °C for 5 min followed by
35 cycles with three steps of 95 °C for 30s, 60 °C for
30s and 72 °C for 60 s. The reactions were completed
with a ﬁnal step of 72 °C for 10 min and stored at 10 °C.
PCR products were checked by agarose gel electropho-
resis, stained with ethidium bromide and visualized un-
der ultraviolet light. Positive nested PCR products were
puriﬁed using the Illustra GFX PCR DNA and Gel Band
Puriﬁcation kit (GE Healthcare) and cloned with the
PCR Cloning kit (Qiagen) following the protocols of the
manufacturers. After cloning the plasmid containing the
fragments of interest were extracted and puriﬁed with
aid of Plasmid Miniprep GeneJET kit (Fermentas). After
checking the clones with inserts by DNA enzyme diges-
tion, they were submitted to automatic DNA sequencing
using a MegaBace 1000 DNA Analysis System (GE
Healthcare). Sequence identity was checked by similarity
search with the BLASTN software (Altschul
A published PCR protocol was also used for detection of
in order to compare
infection with Mycoplasma species (Santos
RESULTS AND DISCUSSION
In Ribeirão Preto, the prevalence of canine haemo-
was considered low. Among the 154 dogs tested,
DNA was detected in 3 dogs (1.9%) and
DNA in 1 (0.6 %). Co-
infection of both hemoplasma species was not detected.
is the vector for two other canine
hemoparasites, the Gram-negative bacteria
which cause the canine monocytic ehrlichiosis agent,
, the canine babesiosis agent. Among
the 154 samples tested, 32 were positive for
(20.7%) and 5 were positive for
Co-infection with these parasites, which have considera-
ble prevalence in Brazil, and canine haemoplasmas were
also not detected. The PCR fragments of 16S rRNA gene
of the canine haemoplasma isolates were cloned and se-
quenced (GeneBank HQ918287, HQ918288). Similarity
search with the BLASTN program showed 99% similarity
M. haematoparvum (EF416569) published sequen-
is pointed as the biological transmis-
sion vector of canine haemoplasmas
and studies conduc-
ted in Europe indicate a correlation between the presence
and infection of dogs. For example, a
study conducted in Switzerland, where
not common, indicated rare infection of dogs by hemo-
2008) while in France, where the tick
is common, a 3.3 %
and 9.6% rate of infection was found
2004). In Brazil,
has a wide
distribution and is the most important canine tick in urban
areas (Labruna and Pereira 2001). Studies conducted with
conventional PCR detected infection of dogs by canine
but with variable rates of prevalence in
different regions of the country. In Belo Horizonte, a town
with a tropical climate located in the southeast region of
prevalence varied between 5.8%
(6/104) for dogs living in urban areas to 11.3% (20/176)
for dogs in rural areas (Biondo
2009). In Londrina,
located in the south region and with a subtropical clima-
te, prevalence of
was of 1.4% (2/147)
2009). Ribeirão Preto is also located in the
southeast, 522 km far from Belo Horizonte and 491km
from Londrina, and presents a tropical climate. Using
real-time PCR, we found a 1.9%
(3/154) prevalence for
and 0,6% (1/154) for
. Until now, detection of
only described for Londrina, with 7.5 % (11/147) pre-
valence. Remarkably, while the isolate described in this
study showed 99% similarity to published sequences of
, after DNA sequencing the detec-
ted isolates from Londrina were found to be more similar
2009). The low number of hemoplasma infection found
in the dogs of Ribeirão Preto does not allow a safe corre-
lation between infection and gender, age, race or clinical
signs. A possible explanation for this low infection could
be a low exposition of the studied canine population to
but though the dogs infected by the two stu-
species were not co-infected by
, 21 % of the samples were positive for
DNA, indicating that the population is exposed to
the tick vector. Moreover, the studies conducted in other
Brazilian towns also do not indicate these associations.
We conclude that, at least in Brazil, it is still not possible
to establish the risk factors for infection, including regio-
nal climate variation and presence of the potential vector
. Therefore, the impact of haemoplasmas
in canine health needs further study. The application of
molecular assays would help by improving veterinary
diagnosis. With the increasing world dog population, the-
se assays should be applied more often to monitor canine
and other hemoparasites in order to keep
a healthy population of dogs.
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