The molecular detection of hemotropic Mycoplasma species in dogs

Abstract

Hemotropic mycoplasmas are uncultured bacteria that cause different degrees of hemolytic anemia in infected hosts. Molecular approaches are used to diagnose hemotropic mycoplasmas since they are difficult to identify using conventional methods. The aim of this study was to use molecular approaches to determine hemotropic mycoplasmas in dogs. Blood samples were collected in tubes (with EDTA) from 100 dogs. After DNA extraction, 100 DNA samples were investigated by PCR. Hemotropic mycoplasma was detected in 12 (12%) samples and positive samples were subjected to Sanger sequencing. Mycoplasma haemocanis was detected in 6 (50.0%) of the samples, Candidatus Mycoplasma hematoparvum in 5 (41.6%), and Candidatus Mycoplasma haemominutum in 1 (8.4%) of the samples after typing 12 PCR positive amplicons using Sanger sequencing. Anemia was found in 5 (83.3%) Mycoplasma haemocanis positive dogs while 3 (60.0%) were Candidatus Mycoplasma haematoparvum positive dogs. As a consequence of this research, hemotropic mycoplasmas were determined in dogs using molecular methods.

Full text

Thai J Vet Med. 2023. 53(2): 257-263.
The molecular detection of hemotropic Mycoplasma species
in dogs
Hafize Tuğba Yüksel Dolgun1* Şükrü Kırkan1
Abstract
Hemotropic mycoplasmas are uncultured bacteria that cause different degrees of hemolytic anemia in infected
hosts. Molecular approaches are used to diagnose hemotropic mycoplasmas since they are difficult to identify using
conventional methods. The aim of this study was to use molecular approaches to determine hemotropic mycoplasmas
in dogs. Blood samples were collected in tubes (with EDTA) from 100 dogs. After DNA extraction, 100 DNA samples
were investigated by PCR. Hemotropic mycoplasma was detected in 12 (12%) samples and positive samples were
subjected to Sanger sequencing. Mycoplasma haemocanis was detected in 6 (50.0%) of the samples, Candidatus
Mycoplasma hematoparvum in 5 (41.6%), and Candidatus Mycoplasma haemominutum in 1 (8.4%) of the samples after
typing 12 PCR positive amplicons using Sanger sequencing. Anemia was found in 5 (83.3%) Mycoplasma haemocanis
positive dogs while 3 (60.0%) were Candidatus Mycoplasma haematoparvum positive dogs. As a consequence of this
research, hemotropic mycoplasmas were determined in dogs using molecular methods.
Keywords: Anemia, dog, hemotropic mycoplasma, polymerase chain reaction, sequencing
1Department of Microbiology, Faculty of Veterinary Medicine, Aydin Adnan Menderes University, Türkiye
*Correspondence: tugba.yuksel@adu.edu.tr (H.T. Yüksel Dolgun)
Received May 3, 2023
Accepted July 30, 2023
https://doi.org/10.14456/tjvm.2023.28
Original Article

258 Dolgun Yüksel H.T. and Kırkan Ş. / Thai J Vet Med. 2023. 53(2): 257-263.
Introduction
Hemotropic mycoplasmas are small, pleomorphic
bacteria that do not have a cell wall and have been
detected in the blood of different kinds of animals (Soto
et al., 2017). Previously, hemotropic mycoplasmas were
classed as rickettsial organisms. Phenotypic
characteristics such as the difficulty of development
needs and the lack of a cell wall further supported this
relation. However, molecular sequencing and
phylogenetic studies indicated that these species are
more closely connected to the Mycoplasmataceae family
and a new classification has been renamed to reflect
their mycoplasmal origin (Tasker, 2010).
Mycoplasma haemocanis and Candidatus
Mycoplasma haematoparvum have been described as
hemotropic mycoplasma agents in dogs (Soto et al.,
2017). In addition, some studies have reported that
Candidatus Mycoplasma haemominutum has been
detected in dogs from China, France and Japan (Kenny
et al., 2004; Zhuang et al., 2009; Obara et al., 2011).
Transmission can occur through contaminated
blood transfusions and dog fights. Blood-sucking
arthropods like fleas and ticks have been considered as
potential vectors (Zarea et al., 2022). Hemotropic
mycoplasma disease, which can vary from
asymptomatic infection to sudden death has clinical
signs such as acute hemolytic anemia, anorexia,
lethargy, dehydration and weight loss (Ravagnan et al.,
2017).
Cytological analysis of Romanowsky, Diff-Quick,
or filtered Giemsa-stained blood smears are used to
diagnose hemotropic mycoplasma infection. Single,
paired and chain organisms can be shown on the
surface of erythrocytes. Cytological diagnosis has a
low sensitivity and specificity. Due to the occurrence of
cross reactions, studies on serological diagnostic tests
have shown that they have not been efficient for the
diagnosis of hemotropic mycoplasma infections yet
(Tasker et al., 2018). Polymerase Chain Reaction (PCR)
exhibits higher sensitivity and specificity than
serological and cytological investigation for the
detection of hemotropic mycoplasma infections
(Tasker, 2010; Tasker et al., 2018).
The aim of this study was to determine hemotropic
mycoplasmas in dogs with molecular methods.
Materials and Methods
Specimen collection: Five ml EDTA-anticoagulated
blood samples were obtained from 100 (50 females, 50
males) dogs which were brought to veterinary clinics
and hospital for investigation of hemotropic
mycoplasma from Aydin province in Türkiye. The
experimental protocol was approved by the
Institutional Animal Care and Use Committee of
Aydin Adnan Menderes University
(64583101/2017/074). Table 1 shows the age and
gender distributions of male (n = 50) and female (n =
50) dogs whose blood was collected for investigation
(Table 1).
Table 1 Data on dog samples by age and gender
Whole Blood Samples
Age Ranges
Total
0-1 Age
2-5 Age
6-10 Age
11-15 Age
> 15 Years
F
M
F
M
F
M
F
M
F
M
Dog (n=100)
16
18
27
21
4
7
3
2
0
2
100
F: Female, M: Male
Table 1 shows that 32 % of female dog samples are
0-1 year old and 54 % are 1-5 years old, whereas 36 %
of male dog samples are 0-1 year old and 42 % are 1-5
years old.
Anemia was determined using hematological
analysis and the results were evaluated according to
Raskin and Wardrop (2010). According to Raskin and
Wardrop, reference values for RBC (red blood cell), Hb
(hemoglobin) and HCT (hematocrit) are stated as 5.5-
8.5 (1012/l), 12,0-18,0 (g/dl) and 37,0-55,0 (%)
respectively. Dogs with RBC, Hb and HCT values
below the reference range were evaluated and
diagnosed as having anemia. QuickGene DNA whole
blood kit S (Kurabo, Japan) was used to extract DNA
from all blood samples and until PCR analysis, DNA
samples were kept at -20°C.
PCR and sequencing: Extracted DNA samples were
subjected to PCR analysis. The 16S rRNA gene primers
(16S Myco322s 5'- GCC CAT ATT CCT ACG GGA
AGC AGC AGT -3 ', 16S Myco938as 5'- CTC CAC CAC
TTG TTC AGG TCC CCG TC -3') were used for the
detection of canine hemotropic mycoplasma (Varanat
et al., 2011). Positive control DNA was obtained from
the clinically infected dog blood sample, which was
validated using Sanger sequencing and BLAST (100%
similarity to M. haemocanis [MG594502]). As a negative
control, sterile deionized water was used. The PCR
mixture for each reaction contained 5 μl of extracted
DNA samples and 12.5 μl PCR master mix, 0.5 μl (30
μM) from each primer, 7 μl deionized water (Varanat
et al., 2011). PCR was performed with initial
denaturation at 94°C for 5 mins, followed by 55 cycles
of denaturation at 94 ° C for 15 secs, annealing at 68°C
for 10 secs, extension at 72°C for 15 secs and final
extension 72 ° C for 30 secs (Varanat et al., 2011). The
PCR products were detected by electrophoresis on a
2% agarose gel and stained with ethidium bromide.
The bands were visualized with UV trasilluminator
(Infinity™ VX2, Collégien, France) (Varanat et al.,
2011). ExoSAP-ITTM (AppliedBiosystems, CA, USA)
was used for the PCR positive amplicons' enzymatic
purification. Purified amplicons were subjected to
sequence PCR and the sequence PCR amplicons were
purified using by Sephadex (Merck, Darmstadt,
Germany) with spin columns (Sanger et al., 1977). The
ABI Prism 310 Genetic Analyzer (AppliedBiosystems,
FosterCity, CA, USA) was used to analyze purified
amplicons. The obtained sequences’ identity was
evaluated by checking against existing sequences
using by BLAST.

Dolgun Yüksel H.T. and Kırkan Ş. / Thai J Vet Med. 2023. 53(2): 257-263. 259
Table 2 Distribution of anemia findings by age and gender based on hemogram results
Sample No
Age/Gender
RBC*
Hb*
HCT*
1
2 Mo/ F
4,13
10,2
22,2
2
2 Mo/ F
3,58
10,1
20,2
3
3 Mo/ F
3,14
9,7
19,5
4
3 Mo/ F
5,15
11,8
34,1
5
6 Mo/ F
4,3
12
24,3
6
6 Mo/ F
5,14
11,3
32,1
7
7 Mo/ F
4,68
10,6
30,06
8
7,5 Mo/ F
5,29
11,5
33,16
9
9 Mo/F
4,49
11,1
28,5
10
1 A/ F
4,86
10,9
31,3
11
1 A/ F
4,67
10,6
29,2
12
1 A/ F
4,19
10,5
28,8
13
1 A/ F
5,23
11,8
36,1
14
1,5 A/ F
4,05
10,2
28
15
1,5 A/ F
5,39
11,9
32,3
16
2 A/ F
4,57
11,6
26,8
17
2 A/ F
3,76
8,1
23,5
18
2 A/ F
3,76
9,3
25,1
19
2 A/ F
2,18
5,5
17,5
20
3 A/ F
4,27
10,6
31,1
21
3 A/ F
3,79
9
29,3
22
3 A/ F
4,81
11,4
30,5
23
3 A/ F
5,24
11,8
34,8
24
3 A/ F
5,15
11,9
33,7
25
5 A/ F
4,93
11,7
36
26
5 A/ F
4,73
11,5
29,3
27
5 A/ F
4,47
11,2
27,9
28
5 A/ F
5,46
18
35,9
29
7 A/ F
2,18
5,4
16,4
30
10 A/ F
4,81
10,8
34,8
31
13 A/ F
3,4
6,6
21
32
2 Mo/ M
3,77
8,2
21,7
33
2 Mo/ M
1,43
4,6
7,6
34
2 Mo/ M
4,03
11
21,8
35
2 Mo/ M
2,68
8
14,7
36
2 Mo/ M
3,42
9,9
21,1
37
2 Mo/ M
3,47
9,6
19,2
38
4 Mo/ M
5,38
11
31,51
39
6 Mo/ M
3,15
9
19,7
40
10 Mo/ M
3,15
7,4
21,93
41
1 A/ M
4,04
11,7
25,7
42
1 A/ M
5,05
13,8
28,8
43
1 A/ M
3,85
10,7
22,2
44
1 A/ M
5,1
11,5
33,5
45
2 A/ F
4,2
10,9
28,4
46
2 A/ M
4,93
11,4
30,5
47
2 A/ M
4,47
9,9
29,43
48
2 A/ M
5,38
11,8
36,7
49
2 A/ M
3,2
8,4
24,7
50
3 A/ M
5,32
16
31,9
51
3 A/ M
5,36
10,2
36,8
52
4 A/ M
2,55
6,3
19,6
53
5 A/ M
4,61
8,6
26,19
54
5 A/ M
4,1
10,5
26,3
55
5 A/ M
4,11
10,9
25,5
56
7 A/ M
4,98
11,8
31,5
57
7 A/ M
5,12
11,7
36
58
8 A/ M
3,92
8,3
24,9
59
9 A/ M
3,5
6,5
23,7
60
15 A/ M
5,09
11,9
34,9
61
17 A/ M
3,22
8,8
23,13
62
18 A/ M
3,66
8,7
25,02
*Reference values= RBC: 5,5-8,5 1012/l; Hb: 12,0-18,0 g/dl; HCT: 37,0-55,0 (%); F: Female; M: Male; A: Age; Mo: Month
Results
Hematological Analysis: Hematological analysis was
performed to diagnose anemia (Abacus Junior Vet,
Diatron, Budapest, Hungary). The results were
compared to hematologic reference intervals for
healthy dogs and blood samples from dogs were
considered as anemic with low erythrocyte and

260 Dolgun Yüksel H.T. and Kırkan Ş. / Thai J Vet Med. 2023. 53(2): 257-263.
hemoglobin values (Raskin and Wardrop, 2010). The
age ranges and gender of dogs with anemia are
indicated in Table 2 as a result of hemotological study
performed on canine blood samples.
Low red blood cell rates were found in 62 (62 %) (32
% female, 30 % male) samples as a result of
hematological analysis performed on 100 whole blood
samples and these samples were considered as positive
for anemia. Ninety percent of female dogs and 76.6 %
of male dogs found to be anemic were between the ages
of 0 and 5.
Canine Hemotropic Mycoplasma 16S rRNA PCR
Results: As a result of the PCR analysis, 12/100 (12%)
samples were detected positive at 600 bp on the
agarose gel.and these samples were considered as
hemotropic mycoplasma (Fig. 1). In 16S rRNA PCR,
88/100 (88%) of samples were found to be negative for
hemotropic mycoplasma.
Figure 1 Canine hemotropic mycoplasma 16S rRNA PCR agarose gel image.
M: 1000 bp DNA ladder; N: Negative control (Sterile deionized water); P: Positive control (sequenced M.haemocanis DNA;
Accession Number MG594502.); B2, B9, B45, B63, B67, B77, B80, D1, D2, D6; Hemotropic mycoplasma PCR positive
samples (600 bp); B55, D3: Hemotropic mycoplasma PCR negative samples.
PCR analysis revealed that 10 (83.3%) of the 12
hemotropic mycoplasma positive dogs were aged from
2 to 5, one (8.3%) was aged 0 to 1 and one (8.3%) was
aged from 6 to 10 (Table 3).
It was seen in canine hemotropic mycoplasma 16s
rRNA PCR that 3 (25%) of positive dogs were female
and 9 (75.0%) of positive dogs were male after PCR
analysis. Anemia was found in 8/12 (66.7%) positive
PCR samples but it was not found in the remaining
4/12 (33.3%) samples, according to the hemogram
analysis.
Sequence Results: The Sanger sequencing method was
used to identify 12 canine hemotropic mycoplasma 16S
rRNA PCR positive samples. Mycoplasma haemocanis
was found in 6/12 (50.0%) samples; Candidatus
Mycoplasma hematoparvum was found in 5/12
(41.6%) samples; and Candidatus Mycoplasma
haemominutum was found in 1 (8.4%) sample. The
distribution of canine hemotropic mycoplasma species
identified by Sanger sequence method are shown in
Table 4 according to gender and age ranges. It was
determined that 25.0% (n=3) of the 12 isolates that were
detected were female and 75.0% (n=9) were male
(Table 4).
Table 3 Distribution of canine hemotropic mycoplasma 16S rRNA PCR positive samples by gender and age ranges
PCR positive samples
Age Ranges
Total
0-1 Age
2-5 Age
6-10 Age
11-15 Age
> 15 Years
F
M
F
M
F
M
F
M
F
M
Dog (n=12)
1
-
2
8
-
1
-
-
-
-
12
F: Female; M: Male
Table 4 Overall results for canine hemotropic mycoplasma
Sample
Age/Gender
Anemia
PCR
Sanger Sequence
Accesion Number
Similarity
Rate(%)
B2
3 A / M
-
+
CMhp
MG594500
97,56
B9
3 A/ M
+
+
Mhc
MG594502
97,03
B20
6 Mo/F
+
+
CMhp
MG594500
98,41
B38
7 A / M
+
+
CMhp
MG594500
98,74
B45
3 A / M
+
+
Mhc
MG594502
100,00
B50
5 A / M
+
+
CMhp
MG594500
97,17
B67
2 A / M
+
+
Mhc
MG594502
99,46
B77
5 A / M
+
+
Mhc
MG594502
100,00
D2
2 A / F
-
+
Mhc
MG594502
99,23
D6
2 A / M
+
+
Mhc
MG594502
99,74
D12
2 A / M
-
+
CMhp
MG594500
97,72
D18
4 A / F
-
+
CMhm
KU852583
97,72
Mhc: Mycoplasma haemocanis; CMhp: Candidatus Mycoplasma haematoparvum; CMhm: Candidatus Mycoplasma haemomunitum; (-):
Negative; (+): Positive; F: Female; M: Male; A: Age; Mo: Month

Dolgun Yüksel H.T. and Kırkan Ş. / Thai J Vet Med. 2023. 53(2): 257-263. 261
Discussion
Increasing knowledge of bacterial genomic
sequences and phylogenetic relationship has led to the
reclassification of these organisms as hemotropic
mycoplasmas in the genus Mycoplasma (Neimark et al.,
2001). Because of the difficulties in growing these
agents in vitro, research into hemotropic mycoplasmas
is limited. In addition, for the sensitive detection of
canine hemotropic mycoplasmas, species-specific
conventional and real-time TaqMan PCR analysis have
been developed. These analyses allow research further
into the pathophysiology and epidemiology of
hemotropic mycoplasma infections in dogs (Willi et al.,
2010).
Hemotropic mycoplasma prevalence was reported
in dogs as follows; 1.2% in Switzerland (Wengi et al.,
2008), 15.4% in France (Kenny et al., 2004), 9.5% in Italy
(Novacco et al., 2010), in Spain 14.9% (Roura et al.,
2010), 10.6% in Greece (Tennant et al., 2011), 12.3% in
Romania (Hamel et al., 2012) , 7.7% in Nigeria (Aquino
et al., 2016), 6.9% in Brazil (Valle et al., 2014), 12.2% in
India (Abd Rani et al., 2011), and in the USA 1.3%
(Compton et al., 2012), 22.9% (Guo et al., 2017) and
15.3% (Aktas and Ozubek, 2018) in Türkiye. The
prevalence of hemotropic mycoplasma was detected to
be 12% in this study, which is similar to the prevalence
values observed by Guo et al. (2017), and Aktas and
Ozubek (2018).
Aktaş and Ozubek (2018) conducted research in
Elazig, Erzurum, Ankara, Nevsehir, Adapazar, Izmit,
Mersin, Giresun, and Izmir, whereas Gou et al. (2017),
conducted study in Konya province. Aktas and
Ozubek (2018) reported the prevalence of M.
haemocanis is 2.5% in Elazig; 5.9% in Erzurum; 4.1% in
Ankara; 2.0% in Nevsehir; Adapazarı 3.4%; 1.8% in
Izmit; 6.7% in Mersin; 8.0% in Giresun and 6.7% in
Izmir and prevalence of Candidatus Mycoplasma
haematoparvum in accordance with the provincial
order indicated above by 3.3%; 2.0%; 2.0%; 0%; 1%; 0%;
9.4%; 6.0% and 15.0%. In this study, hemotropic
mycoplasma isolates were classified as 6 (6.0%)
Mycoplasma haemocanis, 5 (5.0%) Candidatus
Mycoplasma haematoparvum and 1 (1.0%) Candidatus
Mycoplasma haemominutum and our results were
similar to the studies previously obtained in Türkiye.
Other studies reported that there is no substantial
link between anemia and hemotropic mycoplasma
infections (Wengi et al., 2008; Roura et al., 2010; Tennant
et al., 2011; Ravagnan et al., 2017). In this study, 8
(66.6%) of the 12 positive samples were determined to
be anemic, whereas the remaining 4 (33.4%) samples
were not anemic. Unlike previous studies, anemia was
detected in animals infected with the hemotropic
mycoplasma as a result of this research.
In this study, it was determined that the age
distribution of hemotropic mycoplasma positive dogs
with anemia (n=8) was between 6 months and 7 years,
and 7 of them were male and 1 was female. In
hemotropic mycoplasma positive dogs without anemia
(n=4), the age range was between 2 and 4 years, and 2
were female and 2 were male (Table 4). The average
age of hemotropic mycoplasma positive dogs is
approximately 3 years old.
Di Cataldo et al. (2021), reported that they found
hemotropic mycoplasma at a rate of 17.8% in male dogs
and 5.6% in female dogs. Moreover, when the age
ranges of the dogs in the study group were evaluated,
14.7% positivity was found in adult dogs that are 1 year
old and older and 5.5% positivity was found in young
dogs under 1 year old. Di Cataldo et al. (2021),
attributed that the high risk of exposure to the
pathogen as they age and the high contact with the
external environment increased the rate of hemotropic
mycoplasma infection in male and adult dogs.
Barbosa et al. (2021), stated in their study on
hemotropic mycoplasma that gender and presence of
wild animals were not statistically significant in the
transmission of infection to dogs. However, they
reported that ticks, tick bites and dogs fighting play an
important role in the contagion of hemotropic
mycoplasma infections. Ravagnan et al. (2017), stated
that identified both Mhc and CMhp were in free-range
dogs in northern and northeastern Italy and there was
not significant relationship among hemoplasma
infection and gender, age or habitat. Cortese et al.
(2020), reported that male sex and tick infestations are
high risk factors for Mhc, while adult and large-sized
dog breeds are high risk factors for CMhp. In this
study, it was shown that hemotropic mycoplasma
positivity was higher in male and adult dogs (3 years
and older). As a conclusion of this study, it was
inferred that gender and age factors cannot be
considered as risk factors in hemotropic mycoplasma
infections.
Studies have shown that Mycoplasma haemocanis
was more common than Candidatus Mycoplasma
haematoparvum in Nigeria (Aquino et al., 2016), in
Australia (Barker et al., 2010), in Spain (Roura et al.,
2010), in Greece (Tennant et al., 2011), in Iran (Torkan
et al., 2013). In studies conducted in France (Kenny et
al., 2004), the Sudan (Inokuma et al., 2006), and the
United States (Compton et al., 2012), Candidatus
Mycoplasma haematoparvum was found to be the
most common canine hemotropic mycoplasma. The
prevalence of Mycoplasma haemocanis and Candidatus
Mycoplasma haematoparvum was found to be similar
in this study.
Guo et al. (2017), used the PCR test to detect vector-
borne pathogens in different dog groups in Türkiye. As
a result of the sequence analyses performed on 4 PCR
positive samples, Mycoplasma haemocanis similarity
was detected at the rates of 99.67%-99.83%. Aktas and
Ozubek (2018) stated that the prevalence of hemotropic
mycoplasma in dogs was 15.3%, after PCR analysis in
Türkiye. While Mycoplasma haemocanis was found to be
99.6% similar to the isolate previously described in
Türkiye, Candidatus Mycoplasma haematoparvum was
found to be 99.7% similar to the isolate previously
described in Türkiye. In this study, 6 obtained
sequences show similarities with Mycoplasma
haemocanis between rates 93.7%- 100% in the Genbank
database, 5 sequences were detected 91.17%- 98.74%
homology to Candidatus Mycoplasma haematoparvum
and 1 sequence was similar to Candidatus Mycoplasma
haemominutum with 97.72% homology.
In conclusion, the clinical manifestations of
hemotropic mycoplasmas in dogs can range from
asymptomatic to anemia, lethargy and sudden death.

262 Dolgun Yüksel H.T. and Kırkan Ş. / Thai J Vet Med. 2023. 53(2): 257-263.
Since the identification of these organisms cannot be
achieved through conventional methods, molecular
techniques have become increasingly crucial.
Consequently, in this study, hemotropic mycoplasmas
were detected in apparently healthy dog samples using
molecular techniques. These findings highlight the
importance of employing sensitive and specific
diagnostic tools for the timely identification and
intervention of hemotropic mycoplasma infections,
even in asymptomatic animals. Furthermore, it is
recommended that veterinary professionals be trained
in the agents of hemotropic mycoplasma infection and
that new epidemiological studies be conducted to
better understand the prevalence and distribution of
hemotropic mycoplasma agents in light of the results
of this study.
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