Acta Tropica 180 (2018) 18–25

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Acta Tropica
journal homepage: www.elsevier.com/locate/actatropica

First molecular evidence of equine granulocytic anaplasmosis in Pakistan T

a a,⁎ a b
Sehrish Saleem , Muhammad Ijaz , Shahid Hussain Farooqi , Muhammad Imran Rashid ,
Amjad Khanc, Awais Masudd, Amjad Islam Aqiba, Kashif Hussaina, Khalid Mehmoode,
⁎
Hui Zhangf,
a
Department of Clinical Medicine and Surgery, University of Veterinary and Animal Sciences, 54600 Lahore, Pakistan
b
Department of Parasitology, University of Veterinary and Animal Sciences, 54600 Lahore, Pakistan
c
Department of Epidemiology and Public Health, University of Veterinary and Animal Sciences, 54600 Lahore, Pakistan
d
District Diagnostic Laboratory, Livestock and Dairy Development Department, 42200 Mianwali, Pakistan
e
University College of Veterinary and Animal Sciences, Islamia University of Bahawalpur, Pakistan
f
College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, People’s Republic of China

A R T I C L E I N F O A B S T R A C T

Keywords: Anaplasma phagocytophilum (A. phagocytophilum) is an obligate intracellular bacterium that causes equine
Equine granulocytic anaplasmosis granulocytic anaplasmosis (EGA) disease in equines. This pathogen has zoonotic potential, which makes it very
Anaplasma phagocytophilum important to be detected and controlled as early as possible. This study was aimed to assess the molecular
Phylogenetic analysis prevalence, associated risk factors of EGA along with its effects on various hematological parameters. This study
Risk factors
revealed an overall 10.67% prevalence in equine. Horses showed highest prevalence followed by mules and
Hematological parameters
donkeys presenting 11.86, 10.53 and 9.43% prevalence, respectively. The samples were confirmed for ana-
plasmosis through sequencing. The BLAST queries confirmed very high homology of our isolates with Chinese
and Japanese isolates of A. phagocytophilum (Accession no’s; KX505303, KY242456 and LC002836). The phy-
logenetic analysis found the study isolates clustered with each other and this cluster closely resembled Chinese
isolate of A. bovis (FJ169957), A. phagocytophilum (HQ872464) and A. phagocytophilum (NR_044762) human
isolate from northern Minnesota and Wisconsin. The key risk factors identified for occurrence of EGA in equine
species on the basis of univariable analysis were sex of animal, housing type, tick infestation, previous tick
history and tick control status, type of acaricides used, rearing system and farm hygiene, respectively. The
hematological parameters like Hemoglobin (Hb), Total Leukocyte Count (TLC), Total Erythrocytes Count (TEC),
and granulocytes were decreased in diseased animals. The mules showed no typical hematological variations
which make sense for its nature as carrier of infection to the susceptible species. This is the first molecular
evidence of EGA in Pakistan. The disease needs to be handled seriously as it has zoonotic potential. The animals
should be properly attended in disease conditions as leukopenia, neutropenia and lymphopenia can aggravate
the condition by making the animal prone to secondary infections.

1. Introduction
The equine population in Pakistan has risen from 4.8 million in
2006 to 5.7 million (asses 5.1, mules 0.2, and horses 0.4) as per the
economic survey report in 2015–2016. Over 60% of all horses and 95%
of all mules and donkeys are found in developing countries (Fielding,
1991), which plays a key role in their economics (Pearson et al., 2005).
In Pakistan, equines are raised as draught animal for transportation,
riding, racing and companion animal. However, in urban and rural
areas of Pakistan the role of equine as working animal is more crucial
(Javed et al., 2014).
Equines are susceptible to different medical ailments likewise other
animal species that are of great economic importance to the equine
owners (Goraya et al., 2013), which results in ill health and calamities
in case of severity (Chaudhry et al., 2014). Ticks are the most important
ectoparasites that are not only responsible for direct damage and blood
loss but also the transmission of various diseases such as equine ly-
meborreliosis, piroplasmosis and equine granulocytic anaplasmosis
(Sigg et al., 2010). Equine tick borne hemoparasitic diseases, affecting
mules, donkeys, horses and zebras are most commonly manifested as
acute hemolytic conditions in the affected species (Traub-Dargatz et al.,
2010).
Anaplasmosis caused by A. phagocytophilum, a pathogen infecting
many species of domesticated and wild mammals, including equines

⁎
Corresponding authors.
E-mail addresses: mijaz@uvas.edu.pk (M. Ijaz), ahstuzh@sina.com (H. Zhang).

https://doi.org/10.1016/j.actatropica.2017.12.032
Received 27 October 2017; Received in revised form 29 December 2017; Accepted 29 December 2017
Available online 05 January 2018
0001-706X/ © 2018 Elsevier B.V. All rights reserved.
S. Saleem et al.
(Passamonti et al., 2010). Equine granulocytic anaplasmosis (EGA) is a
multi-host infectious disease in human and animal that is characterized
by thrombocytopenia (Chan et al., 2010). The disease prevails in high
tick activity season i.e. spring and autumn (Bown et al., 2003;
Rymaszewska and Grenda, 2008). The etiological agent is a gram-ne-
gative, small, pleomorphic or spheroid shaped bacteria which are re-
ported to reside principally inside thegranulocytes and especially neu-
trophils in infected animal (Bjöersdorff et al., 2002; McQuiston et al.,
2003).
To date, only few microscopic studies are conducted regarding the
prevalence and hematology of EGA in Pakistan (Javed et al., 2014;
Razzaq et al., 2015). PCR is considered as the gold standard for the
diagnosis of anaplasmosis (de Echaide et al., 2005) but unfortunately
PCR based diagnosis is still not developed in many areas of Pakistan.
Previously conducted studies regarding the risk factors associated with
equine anaplasmosis were confined to small areas in Pakistan (Razzaq
et al., 2015). It is very important to obtain correct information about
the prevalence and molecular diagnosis of equine anaplasmosis as the
etiological agent has zoonotic potential.
This study is the first molecular based evidence of EGA presence in
Pakistan. This study also focuses on association of various hypothesized
risk factors with EGA and effects of this malady on various hematolo-
gical parameters of equines.
2. Material and methods
2.1. Sampling strategy
This study was conducted from March to August 2017. The target
subjects were 150 diseased equines (59 Horses, 38 Mules and 53
Donkeys) from district Lahore located in the northeast of Punjab,
Pakistan (Fig. 1). Animals showing clinical signs such as; fever, anor-
exia, lethargy, icterus, petechiae, reluctance to move, distal limb edema
and ataxia were included in the study. Every 3rd diseased animal pre-
sented at various public and private hospitals was included in the study.
Fig. 1. GIS map of study area showing sampling regions of district Lahore.
19
Acta Tropica 180 (2018) 18–25
The majority of animals were originated from small holder farmers,
keeping equine as a draught animal.
The selected equines were examined clinically for the primary
screening as study subjects. Afterwards, thin blood smears were made
in triplets from the ear tips for the screening of animals through mi-
croscopy of Giemsa-stained blood smears as described by Moretti et al.
(2010) to observe intracellular bodies, resembling Anaplasma. Besides
smear microscopy 2 ml of blood was drawn into EDTA coated vacu-
tainers for DNA extraction using appropriate extraction kit. The blood
samples were transported to Medicine laboratory, University of Veter-
inary and Animal Sciences, Lahore maintaining the cold chain using
iceboxes. Each sample was accompanied by a piloted questionnaire for
capturing the information regarding animal, management and en-
vironmental factors.
2.2. DNA extraction
DNA was extracted from all the blood samples using genomic DNA
extraction kit (GeneAll®, Exgene™, Cat. No. 105-101). All the steps in
DNA extraction were carried out according to the manufacturer’s in-
structions. The extracted DNA from all the samples was checked by
using gel electrophoresis to monitor whether sufficient DNA has been
extracted. The DNA was stored at −20 °C till analysis by PCR.
2.3. Molecular screening for Anaplasmaspps
The molecular screening was based on amplification of 16S rRNA of
Anaplasma spps. The primers reported by Parola and Raoult (2001)
were utilized to amplify 345 bp fragment of 16S rRNA gene of Ana-
plasma spps. The primers set consisted of forward primer EHR 16SD: 5′-
GGTACCYACAGAAGAAGTCC-3′ and reverse primer EHR 16SR:
5′-TAGCACTCATCGTTTACAGC-3′. The PCR mixture was prepared in a
final volume of 20 μl consisting of 10 μl of TOP real™ GeneAll® (GeneAll
Biotechnology Co., Ltd.) qPCR 2x Pre MIX (containing 0.2 U of Taq/μl),
2 μl of DNA sample and 2 μl (10 pmol) of each primer. Reaction was
S. Saleem et al. Acta Tropica 180 (2018) 18–25

Table 1
Results of Univariable analysis by Mantel-Haenszel analysis of the possible risk factors associated with the risk of equine granulocytic anaplasmosis (EGA).

Variable Variable levels Positive (%) Negative (%) Odds Ratio (OR) and 95% CI p-Value

Horses
Sex Male 05 (12.82) 34 (87.18) 1 –
Female 02 (10.00) 18 (90.00) 1.324 (0.23–7.51) 0.551
Age 1–3 Years 04 (10.26) 35 (89.74) 1 –
4–6 years 03 (15.00) 17 (85.00) 0.648 (0.13–3.22) 0.444
Tick Infestation Yes 03 (08.82) 31 (91.18) 1 –
No 04 (16.00) 21 (84.00) 0.508 (0.10–2.50) 0.328
Previous Tick History Yes 04 (12.12) 29 (87.88) 1 –
No 03 (11.54) 23 (88.46) 1.05 (0.21–5.20) 0.635
Rearing System Single Specie 02 (20.00) 08 (80.00) 1 –
Mixed Sp. 05 (10.20) 44 (89.80) 2.20 (0.36–13.37) 0.333
Tick Control Status Yes 04 (16.00) 21 (84.00) 1 –
No 03 (08.82) 31 (91.18) 1.96 (0.39–9.71) 0.328
Type of acaricide use Topical 03 (15.00) 17 (85.00) 1 –
Parenteral 01 (20.00) 04 (80.00) 3.84 (0.13–109.4) 0.430
NA 03 (08.82) 31 (91.18) 0.32 (0.11–0.981) 0.512
Housing type Concrete 03 (15.79) 16 (84.21) 1 –
Muddy 04 (10.00) 36 (90.00) 1.688 (0.33–8.43) 0.400
Shrubs around the housing Present 01 (06.67) 14 (93.33) 1 –
Absent 06 (13.64) 38 (86.36) 0.452 (0.05–4.09) 0.423

Donkeys
Sex Male 04 (10.53) 34 (89.47) 1 –
Female 01 (06.67) 14 (93.33) 1.65 (0.16–16.07) 0.561
Age 1–3 Years 03 (08.82) 31 (91.18) 1 –
4–6 years 02 (10.53) 17 (89.47) 0.82 (0.12–5.41) 0.596
Tick Infestation Yes 02 (08.70) 21 (91.30) 1 –
No 03 (10.00) 27 (90.00) 0.85 (0.13–5.60) 0.627
Previous Tick History Yes 01 (06.67) 14 (93.33) 1 –
No 04 (10.53) 34 (89.47) 0.60 (0.06–5.92) 0.561
Tick Control Status Yes 02 (12.50) 14 (87.50) 1 –
No 03 (08.11) 34 (91.90) 1.61 (0.24–10.76) 0.480
Type of acaricide used Topical 01 (11.11) 08 (88.89) 1 –
Parenteral 01 (14.29) 06 (85.71) 0.70 (0.06–1.70) 0.77
NA 03 (08.11) 34 (91.89) 0.52 (0.04–5.97) 0.52
Farm hygiene Satisfactory 01 (20.00) 04 (80.00) 1 –
Unsatisfactory 04 (08.33) 44 (91.67) 2.75 (0.24–30.88) 0.403
Housing type Concrete 02 (18.18) 09 (81.82) 1 –
Muddy 03 (07.14) 39 (92.86) 2.88 (0.41–19.91) 0.750

Mule
Age 1–3 Years 03 (09.68) 28 (90.32) 1 –
4–6 years 01 (14.29) 06 (85.71) 0.64 (0.05–7.29) 0.574
Tick Infestation Yes 02 (10.53) 17 (89.47) 1 –
No 02 (10.53) 17 (89.47) 1.00 (0.12–7.94) 0.698
Previous Tick History Yes 02 (11.76) 15 (88.24) 1 –
No 02 (09.52) 19 (90.48) 1.26 (0.15–10.07) 0.613
Tick Control Status Yes 01 (07.14) 13 (92.86) 1 –
No 03 (12.50) 21 (87.50) 0.53 (0.05–5.74) 0.520
Housing type Concrete 01 (20.00) 04 (80.00) 1 –
Muddy 03 (09.09) 30 (90.91) 2.50(0.20–30.21) 0.446
Shrubs around the housing Present 01 (08.33) 11 (91.67) 1 –
Absent 03 (11.54) 23 (88.46) 0.69 (0.06–7.48) 0.625

The p-value based on Wald Statistic; Multivariable model was not developed because no single variable was found significantly (P ≤ 0.05) associated with occurrence of equine
anaplasmosis.

cycled 35 times after initial denaturation at 95 °C for 5 min with de-
naturation at 95 °C followed by annealing at 58 °C and extension step at
72 °C, each step was given 30 s, a final elongation step at 72 °C for
10 min was performed. A positive control (A. marginale USA isolate)
and a negative control (sterile distilled water), were included in each
PCR run. The PCR products were observed for positive band at 345 bp
position against a 100 bp molecular weight marker on ethidium bro-
mide stained 2% agarose gel using UV-illuminator.
2.4. Sequencing
The positive samples were subjected to gel purification kit
[Invitrogen® PureLink™ Quick Gel Extraction Kit (Catalog no.
K210012)], after slicing the bands from gel using sterilized cutter on
UV-illuminator. All the steps were performed according to the
manufacturer’s instructions. The samples were sent to TSINGKE biolo-
gical technology, China for sequencing. The phylogenetic analysis was
performed using the Geneious software.
2.5. Hematological analyses
Hematological analyses were carried out using automatic hema-
tology analyzer (Abacus Junior Vet®) for a total of 12 equines (n = 4
horses, n = 4 donkeys and n = 4 mules) positive for anaplasmosis. For
comparative purpose 12 healthy animals (n = 4 horses, n = 4 donkeys
and n = 4 mules) were included as negative control. Hb, TEC, TLC,
Differential Leukocyte Count (DLC) and platelets count were assessed in
diseased and healthy animals. The results were further compared with
reference values of hematological parameters mentioned in Schalm’s
veterinary hematology (Weiss and Wardrop, 2011).

20
S. Saleem et al.
2.6. Statistical analysis
Data from questionnaires were entered into Epi Data software 3.1.
Data were validated through crosschecking. Statistical analysis of the
data was conducted using the SPSS statistical software version 20.00.
Several categorical variables were changed to binary form to avoid
problems of the linearity. Categorical variables having more than 2
levels were presented as dummy variables for inclusion in analysis. For
each predictor with missing data, a dummy/indicator variable was
created to indicate whether or not data are missing on that predictor.
All such dummy/indicator variables were included as predictor in re-
gression. Multicollinearity was checked through Pearson’s correlation
and variables “interval between the acaricides usage” and “type of
acaricide used” were found significantly correlated more than the cut
off value (0.80). Therefore, “interval between the acaricides usage” was
dropped. “Rearing system” and “shrubs around the house” in donkey’s
data analysis were dropped because of having “0” count in one cell.
While, in case of mule the data variables i.e. “rearing system”, “type of
acaricide used”, “farm hygiene”, and “interval between the acaricides
usage”.
Univariable analysis was conducted and all the variables having p
value ≤0.05 were retained in the final model to test the association of
outcome with the predicting variables. Blood parameters mean values
were compared in positive and negative sampled equines through in-
dependent sample t-test at 95% confidence interval.
3. Results
3.1. Epidemiology of EGA in equines
This study showed an overall prevalence of 10.67% (16/150) of
EGA in and around Lahore district, Punjab, Pakistan. Among the PCR
positive samples 7 were reported negative based on stained smear mi-
croscopy. The point prevalence was highest in horses followed by mules
and least in donkeys presenting 11.86%, 10.53% and 9.43%, respec-
tively.
The assumed risk factors like sex, age, tick infestation, previous tick
history, rearing system, tick control status, type of acaricide used,
housing type and shrubs around the housing etc. for their association
with EGA infection in equines were statistically analyzed (Table 1).
Their effect on disease dynamics was evaluated on the basis of chi-
square test ‘p-value’ (< 0.05). To identify the key risk factors univariate
analysis for odds ratio (OR) was performed.
Certain factors were explored as potential risk factor based on
univariate logistic regression affecting the disease dynamics in horses
(Table 1). The first factor studied was the sex of the animal which was
found as a potential risk for the occurrence of EGA in horses and
donkeys with odds ratios (OR = 1.324; CI = 0.23–7.51; OR = 1.65;
CI = 0.16–16.07). Where, male showed a higher prevalence than fe-
male animals. Previous tick history proved as a risk factor for the oc-
currence of EGA (OR = 1.05; CI = 0.21–5.20; OR = 1.26;
CI = 0.15–10.07) in horses and mules, respectively. The animals having
previous history of tick infestation were at higher risk as compared to
those with no such previous history. Another factor confirmed as risk
for EGA was the rearing system of horses (OR = 2.20;
CI = 0.36–13.37). However, in donkeys and mules this factor was not
found a significant determinant. Horses, reared with other species were
at 2.5 time higher risk than those kept as single species. Tick control
status was found to have significant association with disease dynamics
in horses and donkeys (OR = 1.96; CI = 0.39–9.71: OR = 1.61;
CI = 0.24–10.76). The animals protected from tick infestation were less
affected than the unprotected. Type of acaricide used were also de-
clared as key risk factors in horses giving odds ratio (OR = 3.84;
CI = 0.13–109.4). The animal treated topically for tick control were at
the same risk as unprotected animals while these were three times more
affected than animals treated parenteral. Housing type was recorded a
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Acta Tropica 180 (2018) 18–25
major risk factor affecting disease dynamics in all the three species i.e.
horses, donkeys and mules giving odds ratios greater than ‘1’. Animals
kept in muddy housing were found at higher risk than those kept in
concrete housing. In donkeys the farm hygiene was found as a risk
factor for anaplasmosis (OR > 1). The animals kept in unhygienic
environment were at 4 time higher risk than those kept in hygienic
conditions.
3.2. Anaplasma 16S rRNAgene analysis
This study is the first evidence for the presence of A. phagocyto-
philum in equines of Pakistan. The PCR products for 345 bp fragment of
Anaplasma 16S rRNA gene were subjected to sequencing. Sequencing
was carried out for the16S rRNA (345 bp) of local isolates collected
from different hosts and locations. These sequences were analyzed
using BLAST and CLUSTAL W alignment tools. Furthermore, for com-
parative purposes, DNA sequences of 16S rRNA sequences from
GenBank database were included. The amplicons showed high
homology with the nucleotide sequences for this gene deposited in
GenBank. BLAST queries of the resulted sequenced nucleotides in-
dicated the sequence identity up to 99% with 16S rRNA of A. phago-
cytophilum isolates which was isolatedin China, Japan and USA.
Local isolates were identified and compared for variation among the
isolates of this study based on BLAST alignment. These sequences were
also compared with other closely resembling (98–99%) isolates from
the GenBank database.
The comparison between the isolates of this study revealed sub-
stitutions in 16SR DNA/Pakistan/2017/E29 isolate at seven positions
i.e. 10, 95, 113, 306, 316, 317 and 318 respectively. The 16SR DNA/
Pakistan/2017/E16 isolated also showed substitutions at seven posi-
tions i.e. 1, 2, 7, 306, 316, 317 and 318 respectively. The 16SR DNA/
Pakistan/2017/E23 isolate presented substitutions at five positions i.e.
2, 95, 131, 304 and 306, respectively. The fourth isolate 16SR DNA/
Pakistan/2017/E2 revealed substitutions at four positions i.e. 1, 4, 137
and 306 respectively (Fig. 2).
The comparison of local isolates with closely resembling isolates
(Accession no’s; KX505303, KY242456 and LC002836) from GenBank
database revealed insertions in isolates sequences of this study at po-
sition 9 in 16SR DNA/Pakistan/2017/E29. All the isolates sequences
presented insertions at 18 and 19 positions, at position 31 in 16SR
DNA/Pakistan/2017/E29 and E23 and at position 298 in 16SR DNA/
Pakistan/2017/E23 and E2 respectively (Fig. 2).
3.3. Phylogenetic analysis
A phylogenetic tree based on 16S rRNA gene fragment sequences of
Pakistani isolates, Chinese isolates, South African isolates, German
isolates and others (Fig. 3) was generated using neighbor joining
bootstrapping method at 1000 replications. In the phylogenetic tree,
isolates of this study clustered with each other and this cluster showed
resemblance with the Chinese isolates of A. bovis (FJ169957), A. pha-
gocytophilum (HQ872464) and A. phagocytophilum (NR_044762) human
isolate from northern Minnesota and Wisconsin.
3.4. Effect of EGA on hematological parameters
In this study the comparison of hematological parameters in dis-
eased and healthy animals was conducted. In horses the hematological
picture showed significant difference in the monocytes and granulo-
cytes among the diseased and healthy horses. However, TLC was found
raised in diseased compared healthy animals, similarly, lymphocytes
were also found raised in diseased as compared to healthy (Table 2).
Among the donkeys Hb, TLC, TEC, and granulocytes counts varied
significantly (p < 0.05) in healthy and diseased animals. However, the
other parameters like lymphocytes and monocytes showed increasing
pattern in diseased as compared to healthy animals. While the platelets
S. Saleem et al.
Fig. 2. BLAST alignment of local isolates with reported isolates of A. phagocytophilum.
count was almost similar among the two groups (Table 3). In case of
mules only the TEC varied among the diseased and healthy animals
(Table 4). All the other parameters showed a non-significant difference
among the healthy and diseased animals. However, TLC, lymphocytes
showed increasing while granulocytes reflected decreasing pattern in
diseased animals.
4. Discussion
Anaplasmosis in equine is caused by A. phagocytophilum, and it has
worldwide distribution and of great economic importance (Goraya
et al., 2013). The infectious diseases are serious threat for animal health
and productivity (Elhaig et al., 2016; Wen et al., 2016; Yilmaz et al.,
2016). The epidemiology of anaplasmosis has not been fully explored in
different districts of Pakistan, despite the fact that the etiological agent
has zoonotic potential (Razzaq et al., 2015). To date, only few micro-
scopic studies are conducted regarding the prevalence and hematology
of equines in Pakistan (Javed et al., 2014; Razzaq et al., 2015). PCR is
considered as the gold standard for the diagnosis of anaplasmosis (Strik
et al., 2007), but unfortunately PCR based diagnosis is still not devel-
oped in many areas of Pakistan. Therefore, current study was designed
to incorporate PCR testing for direct evidence for the presence of A.
phagocytophilum within the surveyed population. To check the pre-
valence along with the associated risk factors in district Lahore, Paki-
stan.
4.1. Epidemiology of EGA in equines
In the current study, the overall presence of anaplasmosis in equines
was 10.67% based on PCR. However, the seroprevalence of A. phago-
cytophilum in Danish horses was 22.3% reported by Hansen et al.
(2010). Maurizi et al. (2009) conducted seroprevalence in center-wes-
tern and eastern regions in France and found overall seroprevalence
16.0% and 20.1% respectively. Torina et al. (2007) compared ser-
ological testing and PCR amplification and reported that 7.8% horses
and 8.9% donkeys were positive for A. phagocytophilum by ELISA. High
false positive results were obtained as none of ELISA positive samples
22
Acta Tropica 180 (2018) 18–25
were positive by PCR. This study proves that seroprevalence is less
reliable tool as compared to PCR (Ashraf et al., 2013). PCR based di-
agnosis conducted by Razzaq et al. (2015) in Southern Punjab of Pa-
kistan revealed 4.3% of horses positive with A. phagocytophilum but, the
current study tells us the prevalence in horses to be 11.86% in district
Lahore, Pakistan.
In the present study, male showed a higher prevalence (12.82%)
than female (10.00%) horses. The results were not in accordance with
the results of Maurizi et al. (2009), who reported female (23.1%)
showing a higher prevalence than male (15.8%) horses. Javed et al.
(2014) showed slightly higher prevalence in male horses (20.97%) than
in female horses (20.19%) which support the findings of this study. The
higher prevalence in male could be attributed to the trend of high male
rearing in Pakistan for polo, racing and draught purposes while fewer
female are kept for breeding purposes. In case of donkeys the pre-
valence came out to be 9.43% which showed a moderate circulation of
A. phagocytophilum among donkeys which was congruent to another
study, in which different tick borne pathogens were studied and pre-
valence of A. phagocytophilum came out to be 7.4% (Passamonti et al.,
2010). It also became clear by Giudice et al. (2012) who examined 100
donkeys and prevalence came out to be 6%. The results obtained from
the present showed that A. phagocytophilum is more prevalent in horses
(11.86%) than in donkeys (9.43%), as recently reported in Sicily and
countries like Denmark and Sweden (Hansen et al., 2010; Engvall and
Engvall, 2002). In this study type of housing was found significantly
associated (OR > 1) with EGA in all the three species of equine
(Table 1). This study found higher prevalence in equines kept in muddy
houses. The significant association is attributed to cracks and crevices
present in muddy houses that provide hiding space for the ticks. Be-
sides, rodents find their hiding spaces in muddy houses, which are
known to possess this pathogen (Zhan et al., 2010a,b). In this study the
animals previously having tick infestation history showed high pre-
valence than those previously not exposed, these findings are in line
with the findings of (Zhang et al., 2014; Leiby and Gill, 2004) relating
the higher prevalence with direct or indirect contact to ticks. Another
factor found significantly associated (OR > 1) with occurrence of
anaplasmosis was tick control status. These findings are in agreement
S. Saleem et al. Acta Tropica 180 (2018) 18–25

Fig. 3. Phylogenetic tree showing relationship of study isolates of Anaplasma with reported isolates.

with results of Kocan (1995), who reported the similar findings. It is
obvious from the literature that tick is the principle vector for trans-
mission of EGA in equine population. Hence, controlling the vector will
definitely reduce the disease occurrence in susceptible population. In
this study type of acaricide used was only studied in horses because the
donkey and mule’s owners are usually of lower socio-economic status
and do not giving much attention to the tick control measures in their
animals. The parenteral route of acaricide administration was found
more efficacious compared to topical route. These findings are in line
with those of Kocan (1995) who also dictated the similar findings.
These findings are not in line with the those of Kerr et al. (2009) who
found cypermethrin as the better solution for tick control in filed. The

Table 2
Comparison of Horse blood parameters mean values based on independent sample t-test.

Variable Level Mean Standard Deviation Levene’s Test for equality of Var. (F-value) Mean difference 95% CI of the difference p-value

HGB Positive 13.3750 1.48183 51.229 1.150 07.59–09.89 0.759

Negative 14.5250 6.99494
TEC Positive 15.5500 13.70681 7.830 6.150 10.65–22.95 0.405
Negative 09.4000 0.88318
TLC Positive 04.8250 0.57373 10.00 0.727 02.23–03.69 0.570
Negative 05.5525 2.35588
Lymphocyte Positive 53.7500 8.35883 0.903 10.750 02.02–23.52 0.085
Negative 43.0000 6.26152
Monocyte Positive 11.3750 1.90153 7.682 2.875 0.340–05.40 0.032
Negative 08.5000 0.81650
Granulocyte Positive 34.8750 8.75952 0.085 30.700 47.42–13.97 0.004
Negative 65.5750 10.49964
Platelet Positive 2.62502 88.78626 0.026 70.250 67.04–207.5 0.257
Negative 3.32752 68.62640

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S. Saleem et al. Acta Tropica 180 (2018) 18–25

Table 3
Comparison of Donkey blood parameters mean values based on independent sample t-test.

Variable Level Mean Standard Deviation Levene’s Test for equality of Var. (F-value) Mean difference 95% CI of the difference p-value

HGB Positive 09.07 1.228 01.38 6.275 08.91–03.63 0.001

Negative 15.35 1.705
TEC Positive 14.85 4.064 03.78 8.625 02.33–14.91 0.021
Negative 06.22 0.935
TLC Positive 02.90 0.419 00.56 1.920 02.58–01.25 > 0.001
Negative 04.82 0.340
Lymphocyte Positive 36.30 10.38 00.13 19.15 02.02–40.32 0.068
Negative 55.45 13.49
Monocyte Positive 09.50 0.663 07.06 1.925 0.360–04.21 0.086
Negative 07.57 01.75
Granulocyte Positive 35.30 14.51 0.887 32.67 54.41–10.93 0.013
Negative 67.97 07.78
Platelet Positive 2.615 90.34 0.063 41.25 97.60–180.10 0.486
Negative 2.202 63.23

Table 4
Comparison of Mules blood parameters mean values based on independent sample t-test.

Variable Level Mean Standard Deviation Levene’s Test for equality of Var. (F-value) Mean difference 95% CI of the difference p-value

HGB Positive 13.47 4.21 2.329 1.65 7.9–4.6 0.17

Negative 15.12 1.87
TEC Positive 10.97 3.11 4.059 4.27 0.46–9.01 0.09
Negative 6.70 1.02
TLC Positive 4.88 2.77 6.541 0.515 4.8–3.8 0.04
Negative 5.40 0.577
Lymphocyte Positive 56.17 4.22 1.092 23.77 13.0–34.5 0.33
Negative 32.40 7.14
Monocyte Positive 9.87 1.65 0.145 1.37 1.2–4.0 0.71
Negative 8.50 1.33
Granulocyte Positive 33.95 5.11 1.262 28.67 40.6–16.0 0.30
Negative 62.62 7.86
Platelet Positive 2.675 154.73 2.313 18.75 255.3–217.8 0.17

lower efficacy of cypermethrin could be attributed to the faulty usage of
this preparation due to the lack of knowledge regarding proper use of
acaricide. Secondly this might be due to lack of resistance to ivermectin
in our study area, which make it the better choice in controlling the tick
population and ultimately reducing the occurrence of EGA and pir-
oplasms. In this study horses kept mixed with other animal species such
as cattle, buffaloes, sheep and goats appeared to be affected more than
horses kept as single specie these findings are supported by Zhan et al.
(2010a,b) who stated that infections of A. phagocytophilum have been
reported in rodents, sheep, goats, cattle and buffaloes. Hence, they can
act as a source of infection to the ticks and horses kept there.
4.2. Molecular characterization of A. phagocytophilum 16S rRNA gene
In this study clinical cases from equine population showing clinical
symptoms and microscopic observation of inclusion bodies resembling
Anaplasma were included from various stud farms, polo club, race club
and veterinary hospitals. The samples were processed for PCR based
detection on the bases of 16S rRNA gene fragment amplification using
primers reported by Parola and Raoult (2001). The samples revealed
345 bp of 16S rRNA gene upon electrophoresis on ethidium bromide 2%
agarose gel. The positive samples were preceded to sequencing and the
results were compared to other isolates submitted to GenBank database
using BLAST. The BLAST showed the local isolates resembled closely
(98–99%) with A. phagocytophilum isolates (Accession no’s; KX505303,
KY242456 and LC002836) originally published by Zhang et al. (2016),
and others two unpublished from China and Japan.
Equine infection with A. phagocytophilum has not been reported yet
in Pakistan; and this is the first occurrence report of an infection with
Anaplasma spp. in equines of Pakistan. In our research, Giemsa-stained
thin blood smears were examined under light microscopy, and several
samples revealed inclusion bodies resembling Anaplasma in the blood.
Keeping in view the previous studies reporting Anaplasmosis in equines
based on microscopic examination. The samples were preceded for
molecular identification of underlying etiological species. PCR, DNA
sequencing and phylogenetic analysis confirmed the microscopy results
as well as seven samples negative on microscopy also showed positive
results on PCR. The results indicated the existence of A. phagocyto-
philum, in the blood of the sick equines. The presence of the A. phygo-
cytophilum isolates in horses is considered to be transmitted through
ixodid ticks as reported by Yaxue et al. (2011) from china stating its
presence in ixodid ticks.
4.3. Effect of EGA on hematological parameters
In our study only monocytes and granulocytes varied significantly
while, the other parameters showed non-significant variations.
However, a slight decreasing pattern was observed in Hb, TEC and
platelets count, while, increasing pattern was seen in TLC and lym-
phocytes. Similar findings were observed by Rashid et al. (2009). In
donkeys, a significant decrease in Hb, TEC, lymphocytes and Granulo-
cytes was observed in diseased animals. These findings are in line with
Franzén et al. (2005) who reported marked lymphopenia, neutropenia
while contrary to his finding no leukopenia was observed in donkeys.
Mules showed a decrease in erythrocyte count which could be due to
mixed infection because the characteristic decrease in granulocytes and
leukocytes were not observed in mules (Franzén et al., 2005). Fur-
thermore, the mules can be carrier of infection for the susceptible
equine species.
5. Conclusion
Here we identified A. phagocytophilum as a principle etiological
agent for causing anaplasmosis in equines. Factors like sex of animal,

24
S. Saleem et al.
housing type, tick infestation, previous tick history, tick’s control status,
type of acaricide used, rearing system and farm hygiene were found as
potential risk factors associated with the occurrence of EGA in equines.
The hematological parameters like Hb, TLC, TEC, granulocytes were
recorded to decrease in the diseased animals. These findings will be
helpful in designing better control strategies in future.
Conflict of interest statement
The authors declare no conflict of interest in submission/publication
of this data.
Acknowledgments
The author is thankful to Higher Education Commission Pakistan,
Molecular Parasitology laboratory, and Medicine laboratory, University
of Veterinary and Animal Sciences for the provision of laboratory and
technical support during the study.
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