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188 PCR BASED ANALYSIS OF HAEMOBARTONELLOSIS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS
Hosseini, Sekhavatmandi and Khamesipour
plasma in Giemsa staining of blood smears (Murray
et al., 1995; Lumb, 2001).Mode of transmission has not
been clearly identi ed but bloodsucking arthropods like
ticks were the suspected vectors. Another possible mode
of transmission is close ghting among dogs. Intrauter-
ine and lactation-related transmission was also reported
(Neimark et al., 2002; Jane et al., 2005).Acute disease
presents with fever, anorexia, weight loss, jaundice,
apathy, adenopathy, motor incoordination and spleno-
megaly (North, 1978; Sykes et al., 2004). Chronic dis-
ease has atypical symptoms like anaemia, weight loss,
paraplegia, dehydration, hyperesthesia and depression
(Saykes et al., 2008). Latent form of the infection has
also been described (Pitulle et al., 1999).Diagnosis of
haemobartonellosis depends on clinical and hematologi-
cal ndings together with microscopic examination of
blood smears and speci c serological and PCR testing
for the pathogen (Lappin et al., 2006). Various antibiot-
ics were reported to be effective in the treatment of hae-
mobartonellosis. Haemobartonellosis was rst described
in 1953 in the United States (Splitter et al., 1956) but the
number of studies about incidence and prevalence of the
disease and the risk factors in transmission remains lim-
ited after 50 years (Kemming et al., 2004). In addition,
studies examining Candidatus mycoplasma haematopar-
vum and Mycoplacma haemocanis infection have not
been came across in this country except for one study
(Foley et al., 2001).Therefore, this study was planned to
investigate prevalence of Candidatus mycoplasma hae-
matoparvum and Mycoplacma haemocanis infection in
dogs.
MATERIAL AND METHODS
Study population consisted of 294 dogs (140 females,
154 males). To determine molecular and haematological
parameters, 2 ml blood with and without anticoagulant
were taken according to technique from V. cephalica. The
dogs were clinically examined and blood samples with
and without anticoagulant were drawn into tubes for
haematological and molecular analysis. Prepared blood
smears were stained with Geimsa method and examined
under light microscope according to the literature (Foley
et al., 2001).
Blood was processed for DNA extraction as described
by d’Oliveira et al. (1995). Brie y, 200: L of thawed
blood was washed 3-5 times by mixing with 0.5 mL PBS
(137mM NaCl, 2.6 mM KCl, 8.1mM Na2HPO4, 1.5 mM
KH2PO4, pH 7.4), each time followed by centrifugation
at maximum speed (13,000 rpm) for 5 min. After the
nal wash, the cell pellet was resuspended in 100: L of
lysis mixture (10mM Tris-HCl, pH 8.0, 50 mM KCl, 0.5%
Tween 20, and 100: g/mL of proteinase K). This mixture
was incubated overnight at 56ºC, followed by 10 min of
boiling to inactivate the Proteinase K and kept at -20ºC
until needed for PCR for ampli cation of the 16S rRNA
gene (Dean et al., 2008).
The DNA samples from cattle blood were used in PCR
reactions (Reverse line blot-PCR) to amplify any Ana-
plasma (or even any Ehrlichia) 16S rRNA gene present.
One primer set was used to amplify a 309-328 bp frag-
ment being part of the V1 region of the16S rRNA gene.
The forward primer was (EF416566) (5’-GAAACTAA-
GGCCATAAATGACGC -3’) for Mycoplacma haemocanis
and (HQ918288) (5’-ACGAAAGTCTGATGGAG-
CAATAC-3’) for Candidatus mycoplasma haematopar-
vum whereas the reverse primer was (5’- ACCTGTCAC-
CTCGATAACCTCTAC-3’) for Mycoplacma haemocanis
and (5’-TATCTACGCATTCCACCGCTAC-3’) for Candida-
tus mycoplasma haematoparvum. The 1xPCR reaction
constituents in a nal volume of 25: L were as follows:
1xPCR buffer (Invitrogen), 3.0 mM MgCl2 (Invitrogen),
200: M each dATP, dCTP, dGTP, 100: M dTTP (ABgene)
and 100: M dUTP (Amersham), 1.25 U of Taq polymerase
(Invitrogen), 0.1U of UDG (Amersham), 25 pmol of each
primer and 2.5: L of template DN A. This was over laid
with about 12.5: L of mineral oil. Positive control DNA
(E. canis) from Molecular Biology Laboratory, Makerere
University and negative control (reaction constituents
without DNA) tests were included. The reactions were
performed using a three phase touchdown program as
previously described (Barker et al., 2009; Brinson et al.,
2001). The possible presence of ectoparasites on the dogs
was also looked for carefully. Statistical analyses were
done using SPSS for Windows.
RESULTS AND DISCUSSION
Clinical signs including temperature, pulsation and
respiration rates were in normal ranges. Some of the
infected cats had anorexia and weight loss. Microscopic
examination revealed Candidatus mycoplasma haema-
toparvum and Mycoplacma haemocanis in 26 (8.82%)
cats. The animals had no ectoparasites on them. Baseline
haematological and biochemical ndings did not differ
after the treatment. Appearance of Candidatus myco-
plasma haematoparvum and Mycoplacma haemocanis in
Geimsa staining of blood smears is presented. According
to the results sex had no effect on the infection of the
dogs (P > 0.05) (Tables1, 2 and 3).
In this study, we used histological methods and novel
molecular techniques to determine the regional preva-
lence and identity of Candidatus mycoplasma haema-
toparvum and Mycoplacma haemocanis. Of 294 samples,
66 supported ampli cation of parasite rRNA by PCR. 25
samples were then examined in parallel by microscopic