M’ghirbi et al. Parasites & Vectors 2012, 5:180
http://www.parasitesandvectors.com/content/5/1/180
RESEARCH
Open Access
Anaplasma phagocytophilum in horses and ticks
in Tunisia
Youmna M’ghirbi1,2, Hèla Yaïch3,4, Abderazek Ghorbel3,4 and Ali Bouattour1,2*
Abstract
Background: Anaplasma phagocytophilum, the causative agent of granulocytic anaplasmosis, affects several species
of wild and domesticated mammals, including horses. We used direct and indirect methods to compare and
evaluate exposure to A. phagocytophilum in horses in northern Tunisia.
Methods: Serum from 60 horses was tested by IFA for antibodies to A. phagocytophilum, and whole blood was
tested for A. phagocytophilum 16S rRNA gene using a nested-PCR. To examine the risk of A. phagocytophilum
transmission, 154 ticks that had been collected from horses were examined for the presence of A. phagocytophilum
by nested-PCR targeting 16S rRNA gene.
Results: This is the first time that A. phagocytophilum has been detected in horses in Tunisia, with an overall
seroprevalence of 40/60 (67%). Six of the seroreactive samples (10%) had an IFA titer of 1:80, 14 (23%) of 1:160, 8
(13%) of 1:320 and 12 (20%) a titer 1 ≥ 640. The seroprevalence revealed no significant regional and sex differences.
In contrast, a significant difference was observed between breeds. Eight (13%) of the horses were positive for A.
phagocytophilum in the PCR, with no significant breed and age differences. Hyalomma marginatum was a
predominant tick species (130/154), and 3 were infected by A. phagocytophilum (a prevalence of 2.3%). The
concordance rate of A. phagocytophilum detection between IFA and PCR had a k value of −0.07.
Conclusions: The results presented in this study suggest that horses infested by ticks in Tunisia are exposed to A.
phagocytophilum.
Keywords: Anaplasma phagocytophilum, Horses, Ticks, 16S rRNA gene, nPCR, IFA, Tunisia
Background
Three former species of granulocytic bacteria, Ehrlichia phagocytophila, Ehrlichia equi and the agent of human granulocytic ehrlichiosis, are considered as one and the same
species and have been renamed as Anaplasma phagocytophilum following the reorganization of the Anaplasmataceae and Rickettsiaceae families in the order Rickettsiales
[1]. Anaplasma phagocytophilum, etiologic agent of the
formerly called equine granulocytic ehrlichiosis, is now
reported as equine granulocytic anaplasmosis (EGA), and
was first recognized as an equine disease in California [2].
The disease was subsequently found in other parts of the
US and in Europe [3-6]. The most common symptoms include fever, depression, lack of appetite, leg swelling,
* Correspondence: ali.bouattour@pasteur.rns.tn
1
Laboratoire d’épidémiologie et microbiologie vétérinaire, service
d’entomologie médicale, Institut Pasteur de Tunis, Tunis 1002, Tunisia
2
Université Tunis El-Manar, Tunis, Tunisia
Full list of author information is available at the end of the article
reluctance to move and yellowish gums. A. phagocytophilum
is maintained in a zoonotic cycle between infected mammalian host reservoirs and ticks, mainly (or perhaps only) those
of the Ixodes ricinus and I. persulcatus complex [7].
In North Africa (Morocco, Algeria and Tunisia),
Ixodes ricinus seems to be the main (or perhaps only)
vector, although Hyalomma detritum might also play a
role in transmitting A. phagocytophilum to cattle [8].
Moreover, A. phagocytophilum is likely to circulate in a
variety of ticks as they feed on dogs or reptiles, as
observed in Tunisia, South Africa and Ghana [9,10], although it is unknown whether they can transmit it.
To our knowledge, no information is available regarding the presence of A. phagocytophilum in horses and
ticks in Tunisia. The present study was undertaken to
further elucidate some epidemiological aspects of this
vectorial disease. We investigated the exposure of horses
in Tunisia to this bacterium by using an indirect
© 2012 M'ghirbi et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
M’ghirbi et al. Parasites & Vectors 2012, 5:180
http://www.parasitesandvectors.com/content/5/1/180
fluorescent antibody (IFA) test to detect reactive antibodies against A. phagocytophilum, and molecular methods (nested polymerase chain reaction, nPCR) to detect
the DNA. In addition, the latter assay was used to detect
A. phagocytophilum in Ixodid ticks infesting horses.
Methods
Study areas and blood samples
Between April and June 2008, sixty horses were identified
from 4 different localities all in a humid bioclimatic zone:
Sejnane (n = 15; 37°03'29.85"N 9°14'20.80"E; altitude
200 m), Tabarka (n = 5; 36°57'26.51"N 8°45'03.95"E; altitude
700 m), Aïn Draham (n = 10; 36°46'48.97"N 8°41'13.73"E;
altitude 800 m) and Ghardimaou (n = 30; 36°26'58.43"N 8°
26'10.59"E, altitude 250 m). Within each region, horses
were selected randomly based on availability due to previous contact to individual horse owners and clinical veterinary practices. Favorable to Ixodes ricinus tick, this
bioclimatic zone is characterized by oak forests (Quercus
faginea and Q. suber) with an undergrowth of ferns (predominantly Pteridium aquilinum) and tree heather (Erica
arborea) [11]. Local fauna is a mixture of domestic (cattle,
sheep, goats and horses) and wild animals, mainly lizards
(Psammodromus algirus), wild boar and several species of
birds (Erithacus rubecula, Turdus merula).
The age, sex and relative degree of tick infestation of each
horse were recorded. All horses studied were born in Tunisia and have not traveled outside the country. Five milliliters of venal blood were collected from a jugular vein of
each horse and an aliquot of 1 ml was placed in an EDTAtube, for nPCR analysis. The rest of the collected blood samples were placed in coagulant-free tubes and allowed to clot
at room temperature (25 °C). Samples were centrifuged at
2500 rpm for 15 min and sera removed and stored at −20 °
C for the IFA tests. Our survey did involve sampling of a
minimal number of horses according to internationally
recognized guidelines with the approval of the Human and
animal Ethics Committee of Pasteur Institute of Tunis.
Tick collection
Ticks were collected systematically from each horse, beginning at the head and neck region, then the pectoral,
axillary and inguinal regions, finishing at the tail. The
ticks were placed in a single container and preserved in
70% ethanol. Adult ticks were identified to the species
level using a published taxonomic key [12].
Serologic analysis
Samples were screened for IgG against A. phagocytophilum using a commercially available IFA assay from
Fuller Laboratories (US). This IFA assay utilizes A. phagocytophilum-infected HL60 cells. All samples were
tested at a 1:80 as starting dilution in phosphatebuffered saline solution (PBS), as per the manufacturer’s
Page 2 of 7
protocol. The resulting reactions were visualized using
standard fluorescence microscopy (400×), where a positive reaction appears as sharply defined apple-green
fluorescent inclusions (A. phagocytophilum morulae) in
the cytoplasm of infected cells. A negative reaction
appears as either red-counterstained cells or fluorescence unlike that seen in the positive control wells.
Horse seropositive samples were then analyzed to determine the titration end point and the antibody titers were
summarized into four different groups: 1:80, 1:160,
1:320, >1:640. A positive and a negative serum control,
provided in the kit, were added to each slide.
DNA extraction and nested-PCR
DNA was extracted from 200 μL of horse blood and unfed adult ticks, using Dneasy Tissue Kit (Qiagen Inc.,
Germany) as per the manufacturer’s instructions, eluted
with 90 μL of AE buffer and then stored at −20 °C until
further use. DNA yields were determined with a
NanoDropWND-1000 Spectrophotometer (NanoDrop
Technologies, DE, USA).
Genomic DNA of A. phagocytophilum was amplified by
nested-PCR (nPCR) using the 16S subunit rDNA gene primers described by Massung et al. [13]. The first PCR amplification of A. phagocytophilum agent was performed in a 25
μL reaction mixture containing 3 μL of each DNA template
with ge3a (5’-CACAATGCAAGTCGAACGGATTATTC3’) and ge10r (5’-TTCCGTTAAGAAGGATCTAATCTCC3’) to amplify 919 bp of the 16S rRNA gene of A. phagocytophilum complex. Reaction conditions were as follows:
0.2 μM each primer, 200 μM each deoxynucleoside triphosphate (Amersham Pharmacia biotech, UK), 1.25 U of Taq
polymerase (Amersham Pharmacia biotech, UK) and
1 × Taq buffer (1.25 mM MgCl2). The reactions were performed on an automated DNA thermal cycler (Perkin-Elmer
2400, CA) under the following conditions: initial denaturation at 93 °C for 1 min, annealing at 55 °C for 5 min and
extension at 72 °C for 30 s. The first PCR product from each
sample was then diluted 1:10 with distilled water and 3 μL
of the diluted solution were used as the template DNA for
the second PCR in a final volume of 25 μL. Species-specific
amplification primers ge9F (5’-AACGGATTATTCTTTA
TAGCTTGCT-3’) and ge2 (5’-GGCAGTATTAAAAG
CAGCTCCAGG-3’) were used to amplify a 546 bp portion
from the 5’ region of the 16S rRNA gene to identify A. phagocytophilum. The other PCR conditions were the same as
in the first PCR. Positive and negative quality controls were
also included with each experiment. DNA extracted from A.
phagocytophilum (kindly provided by D. Postic, Pasteur Institute of Paris) was used as positive control.
DNA sequencing and data analysis
To confirm the nPCR results, positive A. phagocytophilum amplified DNA was analyzed by sequencing using
M’ghirbi et al. Parasites & Vectors 2012, 5:180
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Page 3 of 7
species-specific primers as described previously [13].
The PCR products were purified using the ExoSAP
cleanup procedure (Amersham Biosciences). All nucleotide sequences were obtained using the Big Dye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems)
and the 3130 automated sequencer (Applied Biosystems). The sequences generated in this study have been
deposited in the GenBank database under accession
numbers JX121282-JX121291 (Table 1) and then edited
and aligned using the software programs BioEdit [14]
and ClustalW [15]. The BLAST program (http://www.
ncbi.nlm.nih.gov/BLAST) was used to compare and
analyze the data sequences.
Statistical analysis
IFA test results were compared with nPCR findings; the
two methods were assessed with a concordance test, and
a Cohel k value was calculated [16]. Chi-square test,
done with a commercially available statistical program
(EpiInfo 6.01), was used. Observed differences were considered to be significant when the resulting p value was
less than 0.05.
Results
Horse population and tick collection
During the study, 60 horses from humid bioclimatic
zones were examined: 29 Arabian horses and 31 of the
Barb breed. The horses were 12 years old on average
(range of 2–28 years). Age was classified into four
groups of 1–4 years, 5–10 years, 11–20 years and over
21 years. The adult ticks collected from the horses
(n = 243) were classified as Hyalomma marginatum (144
males and 72 females), H. excavatum (17 males and 2
females) and Rhipicephalus bursa (4 males and 4
females). Only 11 of the 60 (18%) of the examined
horses were infested by at least one of these tick species.
Serological test
An indirect fluorescence assay showed that sera of 67%
of the analysed horse population reacted with A. phagocytophilum antigen. Horses (n = 30) from Sejnane, Aïn
Draham and Tabarka had a high rate of A. phagocytophilum antibodies (80%). In Ghardimaou, the seroprevalence was 53%. Titration endpoints for A.
phagocytophilum ranged from 1:80 to 1 ≥ 640. Six of the
positive samples (10%) had an IFA titer of 1:80, 14 (23%)
of 1:160, 8 (13%) of 1:320 and 12 (20%) of 1 ≥ 640. A.
phagocytophilum seroprevalence of horses revealed nonsignificant regional (χ2 = 4.8; ddl = 3; P > 0.05) and sex
(χ2 = 0; P = 1) differences but a significant breed difference (χ2 = 16.59; P < 0.05). In fact, 40% of the Barb
horses and 26.6% of the Arabian horses were positives.
nPCR amplification
A. phagocytophilum DNA was detected by nested PCR
in 8 blood samples among 60 tested horses (13%), including 2 horses from Sejnane, 4 from Ghardimaou and
two from Aïn Draham. These 8 horses were between 7
and 21 years old and included the two different breeds.
Breed (χ2 = 0.02; P = 0.89) and age (χ2 = 0.94; P = 0.3) differences were non-significant. Four of the 8 positive
horses were also seropositive for A. phagocytophilum,
but 4 horses infected with A. phagocytophilum were IFA
negative. The concordance rate of A. phagocytophilum
detection between IFA and nPCR was −0.07.
Table 1 Genetic variations detected in partial sequences of 16S rRNA gene of Anaplasma phagocytophilum detected in
horses and ticks
Nucleotide position2
1
Source of detection
11
23
28
73
118
130
234
246
249
276
284
287
302
397
JN990106
GenBank accession number
Tick
C
T
G
G
G
C
T
T
A
A
T
C
C
A
JX121282
Tick
*
*
*
*
*
*
*
*
*
*
*
*
*
*
JX121283
Tick
*
*
*
*
*
*
*
W
R
*
*
*
*
*
JX121284
Horse
*
*
*
*
*
*
C
*
*
*
*
*
*
*
JX121285
Horse
*
*
*
*
A
*
*
*
*
*
*
*
*
*
JX121286
Horse
*
*
*
*
*
*
*
*
*
*
*
*
T
*
JX121287
Horse
T
*
*
*
*
*
*
*
*
T
*
*
*
*
JX121288
Horse
*
C
*
R
*
*
*
*
*
*
*
*
*
*
JX121289
Horse
*
*
*
*
*
*
*
*
*
*
*
*
*
*
JX121290
Horse
*
*
A
*
*
T
*
*
*
*
*
T
*
*
JX121291
Horse
*
*
*
*
*
*
*
*
*
*
G
*
*
T
1
The sequences of the genotypes obtained in this work [GenBank: JX121282-JX121291] were compared to the sequence previously reported in China [GenBank
JN990106]. 2 Unique nucleotides are indicated with bold underlined letters. Conserved nucleotide positions are indicated with an asterisk.
M’ghirbi et al. Parasites & Vectors 2012, 5:180
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Only 154 unfed ticks (engorged specimens were discarded) were screened (130 H. marginatum, 16 H. excavatum and 8 R. bursa) for A. phagocytophilum DNA by
nPCR. Bacterial DNA was present in three male H. marginatum, detected as a single band of the appropriate
size by nPCR using primers specific for A.
phagocytophilum.
Sequencing data
Ten different variants in sequences of the partial 16S
rRNA gene of A. phagocytophilum are known to occur
[GenBank: JX121282-JX121291]. Only two sequences
were obtained from tick-derived amplicons [GenBank:
JX121282, JX121283]. The remaining DNA extracted
from H. marginatum male ticks led to a weak positive
signal in PCR and was not sequenced.
The two sequences for the tick-derived amplicons
[GenBank: JX121282, JX121283] differed from each
other by 2 nucleotide positions (Table 1). One sequence
[GenBank: JX121282] was 100% similar to the GenBank
JN990106 sequence derived from adult Ixodes persulcatus collected from grass in China [17]. The second sequence [GenBank: JX121283] was 99% identical to the
sequence identified in ticks in China [GenBank:
JN990106].
The partial sequences of the amplicons derived from
the 8 positive horses were submitted to the GenBank
database under accession numbers [GenBank:
JX121284-JX121291] (Table 1). BLAST search of the
horse derived sequences against GenBank revealed 99%
similarity with Anaplasma phagocytophilum isolate NE16S-2 [GenBank: JN990106] except one sequence [GenBank: JX121289] which is identical to JN990106
(Table 1). Sequences differed by 12 nucleotide positions
(Table 1).
Alignment of the ten partial 16S rRNA gene sequences
revealed 9 different sequence variants with 14 distinctions of nucleotide positions (Table 1). Two of these different strains revealed [GenBank: JX121283, JX121288]
mixed profiles of A. phagocytophilum strains with superposition of SNPs on the same sequence (Table 1). SNPs
were confirmed by sequencing twice the corresponding
amplicons by the ge9F and ge2 primers.
Discussion
A. phagocytophilum, the causative agent of granulocytic
ehrlichiosis, affects several species of wild and domesticated mammals, including horses. Our investigation
shows that A. phagocytophilum infects horses in Tunisia.
Of 60 horses screened by IFA, 40 were seroreactive
with A. phagocytophilum antigen, showing that horses in
Tunisia are exposed to A. phagocytophilum. The high
rate of A. phagocytophilum antibody prevalence in
healthy animals in the visited farms indicates that the
Page 4 of 7
horses may have developed subclinical A. phagocytophilum infection. A. phagocytophilum seroprevalence (67%)
in the present study was higher than in previous surveys
conducted in France (11.3%) [18], Spain (6.52%) [19],
Italy (7.79, 16.89%, 17.03, 9%) [20-23], Sweden (16.7%)
[24] and Denmark (22.3%) [25]. Other studies conducted
in the US and Asia also revealed lower seroprevalence
[26-29]. However, our result showed a significant seroprevalence difference between Arabian and Barb breeds.
This could be attributed to the fact that the latter horses,
used for several forest duties, were more exposed to
ticks.
Serological methods are often used to diagnose anaplasmosis mainly in investigations involving large numbers of animals. But these tests cannot provide sufficient
information for a diagnosis of the disease [30]. To overcome the limitation of the serological test, 16S rDNA
based nested-PCR was used to obtain molecular evidence of A. phagocytophilum infection. In addition, partial sequencing of the detected 16S rRNA gene of A.
phagocytophilum confirmed the nested-PCR results.
Thus PCR-based molecular assays are powerful tools for
diagnosing infectious diseases and for detecting bacterial
agents in clinical cases and arthropod vector samples
alike. These assays were instrumental in identifying A.
phagocytophilum [1,31].
Using nested-PCR, among 60 analyzed animals, A.
phagocytophilum DNA was detected in eight horses for
the first time in Tunisia. The rate of PCR-positive horse
samples (13%), drawn from the humid zone of Tunisia,
is similar to that on the island of Sardinia (15%) [6] but
is higher than previous surveys conducted in Central
Italy (8%) [22].
The comparison of serological and molecular results
shows that there is no concordance between the two
tests used. A serologically positive test against a PCRnegative result could correspond to a past infection.
Among PCR-Anaplasma positive horses (n = 8), only 4
had antibodies reactive with A. phagocytophilum. The
remaining horses may have been sampled very early in
the course of infection before antibody response had occurred or be related to a failure in responding to acute
Anaplasma infection. In naturally infected horses, immunity persists for at least two years and does not appear to depend on latent infection or carrier status [32].
It has also been demonstrated that indirect fluorescent
antibody titers to A. phagocytophilum persist for approximately 300 days after experimental infection [26]
and animals tend to become serologically negative progressively, unless reinfections intervene.
Regarding the entomological survey, ticks infesting
tested horses belong to three different species (H. marginatum, H. excavatum and R. bursa). H. marginatum
was the most abundant tick species in the collected
M’ghirbi et al. Parasites & Vectors 2012, 5:180
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samples. All of these three tick species are active in
spring [11], which was when we did our sampling. This
led us to hypothesize that H. marginatum can be
involved in transmitting A. phagocytophilum to horses.
Indeed, the PCR study of these specimens (n = 154)
revealed the presence of A. phagocytophilum in three H.
marginatum ticks, but does not of course prove that the
species can transmit the infection. This low number of
positive PCR results could depend on the vector competence of the tested tick species, the time of year, the geographical region and the limited bacteraemia of horses,
which might significantly reduce tick infectivity. In previous studies we also detected A. phagocytophilum infections in dogs and in 3% of Hyalomma detritum and 1%
of Ixodes ricinus ticks [8,9]. The latter tick species is
considered to be the main vector of A. phagocytophilum
in Europe. The absence of I. ricinus ticks on the horses
that we examined could be attributed to the collection
season, as this tick is active in Tunisia from September
to March, that is, during autumn and winter [11].
However, A. phagocytophilum is spread widely in the
northern hemisphere [33] and has been detected by PCR
in Ixodes ricinus in almost all countries in Europe [34].
In Germany, the overall prevalence ranges from 1.0% to
4.5% [35,36]. The overall prevalence in Europe ranges
from 0.25% in Scotland to 57.1% in Italy [37,38] with
great variation in every country: from 0.4% to 15% in
France [39,40], from 3% to 57% in Italy [38,41,42], from
0.25% to 2% in Scotland [37], 3% in Estonia [43], from
5.1% to 8.7% in Austria [44,45], from 0.5% to 2.2% in
Switzerland [46-48], up to 40.5% in Denmark [49] and up
to 14% in various Eastern European countries [50-54].
There have also been reports about the detection of A.
phagocytophilum in I. spinipalpis and I. dentatus in the
US [55,56]. In Asia, A. phagocytophilum has been
detected in the hard ticks I. persulcatus and I. ovatus
[57,58]. Apart from the genus Ixodes, A. phagocytophilum has also been detected in Dermacentor variabilis in
California and D. silvarum in China [59,60].
Seeking IgG against A. phagocytophilum provides an
excellent screening method to explore the circulation of
the bacteria; PCR therefore remains a critical tool for a
faster and earlier diagnosis. Positive serological testing
and DNA detection by PCR are both evidence of A. phagocytophilum circulation in Tunisia. Even clinical anaplasmosis in horses probably remains under-diagnosed
in Tunisia, as most horses recover spontaneously and
clinical signs resemble those caused by infections by
such other pathogens as Borrelia burgdorferi, Babesia
caballi, Theileria equi, equine herpesvirus, equine infectious anaemia virus, equine arteritis virus and Leptospiraceae [61]. Similarly, an earlier study of A.
phagocytophilum has indicated that up to 50% of seropositive horses, in endemic areas, have a subclinical
Page 5 of 7
infection [26]. It is assumed that clinical equine granulocytic anaplasmosis is an overlooked condition in most of
Europe, as most horses recover spontaneously and clinicians do not examine them [62].
Conclusions
This study marks the first report providing serological
and molecular evidence of A. phagocytophilum infection
in horses and ticks in Tunisia and should therefore be of
interest to veterinary clinicians regarding the possible
existence of clinical cases of equine anaplasmosis. Given
the zoonotic aspect of A. phagocytophilum, physicians
should also be informed of the presence of this bacterium in Tunisia. No serologic evidence of exposure to A.
phagocytophilum has been reported in humans in Tunisia to date and this bacterium has not yet been isolated
or detected by PCR in clinical cases in Tunisia. In
addition, the results show that more than one variant of
A. phagocytophilum, using partial 16S rRNA gene, seems
to be involved in equine granulocytic anaplasmosis in
Tunisia Furthermore, the zoonotic importance of A.
phagocytophilum lends support to the need to increase
the surveillance of the presence of this pathogen in
Tunisia.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
YM carried out the serological, molecular genetic studies, the sequence
alignment, the statistical analysis and drafted the manuscript. HY carried out
the serological assay. AG participated in the design of the study and sample
horse collection. AB conceived of the study, and participated in its design
and coordination, helped to draft the manuscript and sample horse
collection. All authors read and approved the final manuscript.
Acknowledgements
We are grateful to Dr. Eremeeva M. (CDC/OID/NCEZID), Dr. Uilenberg G., Dr.
Ghram A. (IP Tunis) and Glassman D. for constructive comments on early
drafts of the manuscript. We would like to thank Dr. Leila Saieh and Dr.
Ridha Ben Omrane for their help in field work. We also would like to express
thanks to Dr Postic D. (I.P.P.) for providing positive controls. This work was
supported by a grant from the Ministry for Higher Education, Scientific
Research, and Technology in Tunisia.
Author details
Laboratoire d’épidémiologie et microbiologie vétérinaire, service
d’entomologie médicale, Institut Pasteur de Tunis, Tunis 1002, Tunisia.
2
Université Tunis El-Manar, Tunis, Tunisia. 3Service pathologie médicale des
équidés et des carnivores, Ecole nationale de medicine vétérinaire de Sidi
Thabet, Tunis 2002, Tunisia. 4Université de la Manouba, Manouba, Tunisia.
1
Received: 21 June 2012 Accepted: 24 August 2012
Published: 30 August 2012
References
1. Dumler JS, Barbet AF, Bekker CP, Dasch GA, Palmer GH, Ray SC, Rikihisa Y,
Rurangirwa FR: Reorganization of genera in the families Rickettsiaceae
and Anaplasmataceae in the order Rickettsiales: unification of some
species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and
Ehrlichia with Neorickettsia, descriptions of six new species
combinations and designation of Ehrlichia equi and “HGE agent” as
subjective synonyms of Ehrlichia phagocytophila. Int J Syst Evol Microbiol
2001, 51:2145–2165.
M’ghirbi et al. Parasites & Vectors 2012, 5:180
http://www.parasitesandvectors.com/content/5/1/180
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
Gribble DH: Equine ehrlichiosis. J Am Vet Med Assoc 1969, 155:462–469.
Korbutiak E, Schneiders D: Equine granulocytic ehrlichiosis in the UK. Vet
Rec 1994, 135:387–388.
Bermann F, Davoust B, Fournier PE, Brisou-Lapointe AV, Brouqui P: Ehrlichia
equi (Anaplasma phagocytophila) chez un cheval adulte en France. Vet
Rec 2002, 150(25):787–788.
Von Loewenich FD, Stumpf G, Baumgarten BU, Röllinghoff M, Dumler JS,
Bogdan C: A Case of equine granulocytic ehrlichiosis provides molecular
evidence for the presence of pathogenic Anaplasma phagocytophilum
(HGE agent) in Germany. Eur J Clin Microbiol Infect Dis 2003, 22(5):303–305.
Alberti A, Zobba R, Chessa B, Addis MF, Sparagano O, Parpaglia MLP,
Cubeddu T, Pintori G, Pittau M: Equine and Canine Anaplasma
phagocytophilum Strains Isolated on the Island of Sardinia (Italy) are
phylogenetically related to pathogenic strains from the United States.
Appl Env Microbiol 2005, 71:6122–6418.
Thomas RJ, Dumler JS, Carlyon JA: Current management of human
granulocytic anaplasmosis, human monocytic ehrlichiosis and Ehrlichia
ewingii ehrlichiosis. Expert Rev Anti Infect Ther 2009, 7:709–722.
Sarih M, M’ghirbi Y, Bouattour A, Gern L, Baranton G, Danièle P: Detection
and identification of Ehrlichia spp. in Ticks collected in Tunisia and
Morocco. J Clin Microbiol 2005, 43(3):1127–1132.
M’ghirbi Y, Ghorbel A, Amouri M, Nebaoui A, Haddad S, Bouattour A:
Clinical, serological, and molecular evidence of ehrlichiosis and
anaplasmosis in dogs in Tunisia. Parasitol Res 2009, 104(4):767–774.
Nowak M, Cieniuch S, Stańczak J, Siuda K: Detection of Anaplasma
phagocytophilum in Amblyomma flavomaculatum ticks (Acari: Ixodidae)
collected from lizard Varanus exanthematicus imported to Poland. Exp
Appl Acarol 2010, 51(4):363–371.
Bouattour A, Darghouth MA, Daoud A: Distribution and ecology of ticks
(Acari: Ixodidae) infesting livestock in Tunisia: an overview of eight years
field collection. Parassitologia 1999, 41:5–10.
Bouattour A: Clé dichotomique et identification des tiques (Acari:
Ixodidae) parasites du bétail au Maghreb. Arch Inst Pasteur Tunis 2002,
79:43–50.
Massung RF, Slater K, Owens JH, Nicholson WL, Mather TN, Solberg VB,
Olson JG: Nested PCR assay for the detection of granulocytic ehrlichiae. J
Clin Microbiol 1998, 36:1090–1095.
Hall TA: BioEdit: A user-friendly biological sequence alignment editor and
analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 1999,
41:95–98.
Thompson JD, Higgins DG, Gibson TJ: ClustalW: improving the sensitivity
of progressive multiple sequence alignment through sequence
weighting, position-specific gap penalties and weight matrix choice.
Nucleic Acids Res 1994, 22:4673–4680.
Cohen J: A coefficient of agreement for nominal scales. Edu Psychol Meas
1960, 20:37–46.
Li Y, Zuo S-Y, Tang K, Pu G-Q: Investigation on the infection of ticks with
Anaplasma phagocytophilum in the Northeast China forest region from
2010 to 2011. Chin J Vector Biol and control 2012, 23(2):111–117.
Leblond A, Pradier S, Pitel PH, Fortier G, Boireau P, Chadoeuf J, Sabatier P:
An epidemiological survey of equine anaplasmosis (Anaplasma
phagocytophilum) in southern. Rev Sci Tech 2005, 24(3):899–908.
Amusategui I, Sainz A, Tesouro MA: Serological evaluation of Anaplasma
phagocytophilum infection in livestock in northwestern Spain. Ann N Y
Acad Sci 2006, 1078:487–490.
Torina A, Vicente J, Alongi A, Scimeca S, Turla R, Nicosie S, Di Marco V,
Caracappa S, de la Fuente J: Observed prevalence of tick-borne
pathogens in domestic animals in Sicily, Italy during 2003–2005.
Zoonoses Santé Publ 2007, 54(1):8–15.
Ebani V, Cerri D, Fratini F, Ampola M, Andreani E: Seroprevalence of
Anaplasma phagocytophilum in domestic and wild animals from central
Italy. New Microbiol 2008, 31(3):371–375.
Passamonti F, Veronesi F, Cappelli K, Capomaccio S, Coppola G, Marenzoni
ML, Piergili FD, Verini SA, Coletti M: Anaplasma phagocytophilum in horses
and ticks: a preliminary survey of Central Italy. Comp Immunol Microbiol
Infect Dis 2010, 33(1):73–83.
Giudice E, Giannetto C, Furco V, Alongi A, Torina A: Anaplasma
phagocytophilum seroprevalence in equids: a survey in Sicily (Italy).
Parasitol Res 2012, 111(2):951–955.
Egenvall A, Franzén P, Gunnarsson A, Engvall EO, Vågsholm I, Wikström UB,
Artursson K: Cross-sectional study of the seroprevalence to Borrelia
Page 6 of 7
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
burgdorferi sensu lato and granulocytic Ehrlichia spp. and demographic,
clinical and tick-exposure factors in Swedish horses. Prev Vet Med 2001,
49(3–4):191–208.
Hansen MG, Christoffersen M, Thuesen LR, Petersen MR, Bojesen AM:
Seroprevalence of Borrelia burgdorferi sensu lato and Anaplasma
phagocytophilum in Danishhorses. Acta Vet Scand
2010, 18:52–53.
Madigan J, Hietala A, Chambers S: Seroepidemiologic survey of antibodies
to Ehrlichia equi in horses in northern California. J Am Vet Med Assoc 1990,
196:1962–1964.
Bullock PM, Ames TR, Robinson RA, Greig B, Mellencamp MA, Dumler JS:
Ehrlichia equi infection of horses from Minnesota and Wisconsin:
detection of seroconversion and acute disease investigation. J Vet Int
Med 2000, 14:252–257.
Teglas M, Matern E, Lein S, Foley P, Mahan SM, Foley J: Ticks and tickborne disease in Guatemalan cattle and horses. Vet Parasitol 2005,
131(1–2):119–127.
Chahan B, Jian Z, Xuan X, Sato Y, Kabeya H, Tuchiya K, Itamoto K, Okuda M,
Mikami T, Maruyama S, Inokuma H: Serological evidence of infection of
Anaplasma and Ehrlichia in domestic animals in Xinjiang Uygur
Autonomous Region area, China. Vet Parasitol 2005, 134(3–4):273–278.
Rikihisa Y: The tribe Ehrlichiae and Ehrlichial diseases. Clin Microbiol Rev
1991, 4:286–308.
Chen SM, Dumler J, Bakken J, Walker D: Identification of a
granulocytotropic Ehrlichia species as the etiologic agent of human
disease. J Clin Microbiol 1994, 32:589–595.
Sellon DC: Miscellaneous parasitic diseases. In Equine infectious diseases.
Edited by Sellon DC, Long MT. St. Louis: Saunders Elsevier; 2007:
473–480.
Teglas MB, Foley J: Differences in the transmissibility of two Anaplasma
phagocytophilum strains by the North American tick vector species,
Ixodes pacificus and Ixodes scapularis (Acari: Ixodidae). Exp Appl Acarol
2006, 38:47–58.
Strle F: Human granulocytic ehrlichiosis in Europe. Int J Med Microbiol
2004, 293(Suppl 37):27–35.
Hartelt K, Oehme R, Frank H, Brockmann SO, Hassler D, Kimmig P:
Pathogens and symbionts in ticks: prevalence of Anaplasma
phagocytophilum (Ehrlichia sp.), Wolbachia sp., Rickettsia sp., and
Babesia sp. in Southern Germany. Int J Med Microbiol 2004,
293(Suppl 37):86–92.
Leonhard S: Untersuchungen zur Häufigkeit von. Borrelia burgdorferi
sensu lato, Anaplasma phagocytophilum und Babesia spp. In Ixodes
ricinus aus Bayern und BadenWürttemberg. Dissertation. Germany: LudwigMaximilians-University München; 2005.
Alberdi MP, Walker AR, Paxton EA, Sumption KJ: Natural prevalence of
infection with Ehrlichia (Cytoecetes) phagocytophila of Ixodes ricinus ticks
in Scotland. Vet Parasitol 1998, 78(4):203–213.
Mantelli B, Pecchioli E, Hauffe HC, Rosà R, Rizzoli A: Prevalence of Borrelia
burgdorferi s.l. and Anaplasma phagocytophilum in the wood tick Ixodes
ricinus in the Province of Trento, Italy. Eur J Clin Microbiol Infect Dis 2006,
25:737–739.
Ferquel E, Garnier M, Marie J, Bernède-Bauduin C, Baranton G, Pérez-Eid C,
Postic D: Prevalence of Borrelia burgdorferi sensu lato and
Anaplasmataceae members in Ixodes ricinus ticks in Alsace, a focus of
Lyme borreliosis endemicity in France. Appl Environ Microbiol 2006,
72:3074–3078.
Halos L, Vourc’h G, Cotte V, Gasqui P, Barnouin J, Boulous HJ, Vayssier TM:
Prevalence of Anaplasma phagocytophilum, Rickettsia sp. and Borrelia
burgdorferi sensu lato DNA in Questing Ixodes ricinus Ticks from France.
Ann N Y Acad Sci 2006, 1078:316–319.
Cinco M, Padovan D, Murgia R, Maroli M, Frusteri L, Heldtander M,
Johansson KE, Engvall EO: Coexistence of Ehrlichia phagocytophila and
Borrelia burgdorferi sensu lato in Ixodes ricinus ticks from Italy as
determined by 16S rRNA gene sequencing. J Clin Microbiol 1997,
35:3365–3366.
Piccolin G, Benedetti G, Doglioni C, Lorenzato C, Mancuso S, Papa N, Pitton
L, Ramon MC, Zasio C, Bertiato G: A Study of the Presence of B.
burgdorferi, Anaplasma (Previously Ehrlichia) phagocytophilum, Rickettsia,
and Babesia in Ixodes ricinus Collected within the Territory of Belluno,
Italy. Vector Borne Zoonotic Dis 2006, 6:24–31.
M’ghirbi et al. Parasites & Vectors 2012, 5:180
http://www.parasitesandvectors.com/content/5/1/180
43. Mäkinen J, Vuorinen I, Oksi J, Peltomaa M, He Q, Marjamäki M, Viljanen MK:
Prevalence of granulocytic Ehrlichia and Borrelia burgdorferi sensu lato in
Ixodes ricinus ticks collected from southwestern Finland and from Vormsi
Island in Estonia. APMIS 2003, 111:355–362.
44. Sixl W, Petrovec M, Marth E, Wüst G, Stünzer D, Schweiger R, Avsic-Zupanc
T: Investigation of Anaplasma phagocytophila Infections in Ixodes ricinus
and Dermacentor reticulatus ticks in Austria. Ann N Y Acad Sci 2003,
990:94–97.
45. Polin H, Hufnagl P, Haunschmid R, Gruber F, Ladurner G: Molecular
Evidence of Anaplasma phagocytophilum in Ixodes ricinus Ticks and Wild
Animals in Austria. J Clin Microbiol
2004, 42:2285–2286.
46. Leutenegger CM, Pusterla N, Mislin CN, Weber R, Lutz H: Molecular
evidence of coinfection of ticks with Borrelia burgdorferi sensu lato and
the human granulocytic ehrlichiosis agent in Switzerland. J Clin Microbiol
1999, 37:3390–3391.
47. Pusterla N, Leutenegger CM, Huder JB, Weber R, Braun U, Lutz H: Evidence
of the human granulocytic ehrlichiosis agent in Ixodes ricinus ticks in
Switzerland. J Clin Microbiol 1999, 37:1332–1334.
48. Liz JS, Anderes L, Sumner JW, Massung RF, Gern L, Rutti B, Brossard M:
PCR detection of granulocytic ehrlichiae in Ixodes ricinus ticks and wild
small mammals in western Switzerland. J Clin Microbiol 2000,
38:1002–1007.
49. Skarphédinsson S, Lyholm BF, Ljungberg M, Sogaard P, Kolmos HJ, Nielsen
LP: Detection and identification of Anaplasma phagocytophilum, Borrelia
burgdorferi, and Rickettsia helvetica in Danish Ixodes ricinus ticks. APMIS
2007, 115:225–230.
50. Petrovec M, Sumner JW, Nicholson WL, Childs JE, Strle F, Barlic J,
LotricFurlan S, Avsic ZT: Identity of ehrlichial DNA sequences derived
from Ixodes ricinus ticks with those obtained from patients with
human granulocytic ehrlichiosis in Slovenia. J Clin Microbiol 1999,
37:209–210.
51. Derdáková M, Halanova M, Stanko M, Stefancikova A, Cislakova L, Pet’ko B:
Molecular evidence for Anaplasma phagocytophilum and Borrelia
burgdorferi sensu lato in Ixodes ricinus ticks from eastern Slovakia.
Ann Agric Environ Med 2003, 10:269–271.
52. Hulínská D, Langrová K, Pejcoch M, Pavlásek I: Detection of Anaplasma
phagocytophilum in animals by real-time polymerase chain reaction.
APMIS 2004, 112:239–247.
53. Stańczak J, Gabre RM, Kruminis-Lozowska W, Racewicz M, Kubica-Biernat B:
Ixodes ricinus as a vector of Borrelia burgdorferi sensu lato, Anaplasma
phagocytophilum and Babesia microti in urban and suburban forests.
Ann Agric Environ Med 2004, 11:109–114.
54. Koči J, Movila A, Taragel’ová V, Toderas I, Uspenskaia I, Derdáková M, Labuda
M: First report of Anaplasma phagocytophilum and its co-infections with
Borrelia burgdorferi sensu lato in Ixodes ricinus ticks (Acari: Ixodidae) from
Republic of Moldova. Exp Appl Acarol
2007, 41:147–152.
55. Zeidner NS, Burkot TR, Massung R, Nicholson WL, Dolan MC, Rutherford JS,
Biggerstaff BJ, Maupin GO: Transmission of the agent of human
granulocytic ehrlichiosis by Ixodes spinipalpis ticks: evidence of an
enzootic cycle of dual infection with Borrelia burgdorferi in Northern
Colorado. J Infect Dis 2000, 182:616–619.
56. Goethert HK, Telford SR: Enzootic transmission of the agent of human
granulocytic ehrlichiosis among cottontail rabbits. AmJTrop Med Hyg
2003, 68:633–637.
57. Cao WC, Zhoa QM, Zhang PH, Dumler JS, Zhang XT, Fang LQ, Yang H:
Granulocytic Ehrlichiae in Ixodes persulcatus Ticks from an Area in
China Where Lyme Disease is Endemic. J Clin Microbiol 2000,
38:4208–4210.
58. Ohashi N, Inayoshi M, Kitamura K, Kawamori F, Kawaguchi D, Nishimura Y,
Naitou H, Hiroi M, Masuzawa T: Anaplasma phagocytophilum-infected
ticks, Japan. Emerg Inf Dis 2005, 11:1780–1783.
59. Holden K, Boothby JT, Anand S, Massung RF: Detection of Borrelia
burgdorferi, Ehrlichia chaffeensis, and Anaplasma phagocytophilum in
ticks (Acari: Ixodidae) from a coastal region of California. J Med Entomol
2003, 40:534–539.
60. Cao WC, Zhan L, He J, Foley JE, DE Vlas SJ, Wu XM, Yang H, Richardus JH,
Habbema JD: Natural Anaplasma phagocytophilum infection of ticks and
rodents from a forest area of Jilin province China. AmJTrop Med Hyg 2006,
75:664–668.
Page 7 of 7
61. Salvagni CA, Dagnone AS, Gomes TS, Mota JS, Andrade JM, Baldani CD,
Machado RZ: Serologic evidence of equine granulocytic anaplasmosis
in horses from central West Brazil. Rev Bras Parasitol Vet 2010,
19(3):135–140.
62. Butler CM, Nijhof AM, Jongejan F, Van der Kolk JH: Anaplasma
phagocytophilum infection in horses in the Netherlands. Vet Rec 2008,
162:218–219.
doi:10.1186/1756-3305-5-180
Cite this article as: M’ghirbi et al.: Anaplasma phagocytophilum in horses
and ticks in Tunisia. Parasites & Vectors 2012 5:180.
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