301

Anaplasma phagocytophilum is an obligate 
intracellular zoonotic bacterium transmitted by 
Ixodid ticks which can cause high fever, neutropenia 
(due to unique tropism for granulocytes), reduced 
milk production and hemorrhagic diathesis in 
ruminants (Woldechiwet 2006, Giadinis et  al. 
2011). In Europe the Ixodes ricinus ticks is the most 
important vector. In a recent study the presence of 
A.  phagocytophilum infection in a bovine herd was 
associated with  depression, nasal and eye discharge, 
anorexia, coughing, diarrhea, recumbency, 
weakness, swelling of the hind limbs, decrease of milk 
production (Aktas and Ozubek 2015); co‑infection 
with other Anaplasma spp. did not seem to worsen 
the symptoms. The disease in ruminants is called 
tick‑borne fever (TBF). Reproductive disorders, such 
as impaired spermatogenesis and abortion have 
been observed in sheep but not in goats (Jones and 
Davies 1995, Garcia‑Perez et  al. 2003, Chianini et  al. 
2004, Lillini et al. 2006, Stuen 2007). 

In Greece, A.  phagocytophilum has been detected 
by PCR in humans (Psaroulaki et  al. 2008) and ticks 
(Kachrimanidou et al. 2011, Papa et al. 2017). By PCR  

A.  ovis has been also detected in small ruminants 
(Giadinis et  al. 2012), while serological evidence 
of the presence of A.  phagocytophilum has been 
recorded in an ill ram (Giadinis et al. 2011).

This manuscript describes sporadic abortions 
in two goat herds in Northern Greece, in which 
A. phagocytophilum was the only pathogen detected 
in placenta or aborted fetuses.

The 1st goat herd (Herd 1) was located in Chalkidiki 
Prefecture (Northern Greece) and consisted of 
800  dairy goats of local breeds that were reared 
under the semi‑intensive feeding system. In 2011, 
the farmer observed sporadic abortions in goats 
of different ages, and about 30 abortions were 
recorded during the last 45 days of pregnancy. Two 
aborted fetuses (from different mothers) without 
fetal membranes were submitted to the Farm Animal 
Clinic of the Aristotle University in Thessaloniki. Both 
fetuses were normal in appearance and no specific 
lesions were found by post mortem investigation. 
Whole blood from the mothers, brain tissue and 
stomach content from the fetuses were collected.

The 2nd farm (Herd 2) was situated in Thessaloniki 

1Laboratory of Clinical Microbiology and Microbial Pathogenesis, School of Medicine,
University of Crete, Heraklion, Crete, Greece.

2Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, Greece.
*Corresponding author at: Clinic of Farm Animals, Aristotle University of Thessaloniki, Greece.

Tel.: +30 2310 99 4509, Fax: +30 2310 99 4463, e‑mail: ngiadini@vet.auth.gr.

Keywords
Anaplasma 
phagocytophilum,
Abortion,
Goats,
Treatment.

Summary
Anaplasma phagocytophilum, transmitted by Ixodes ticks, is an intracellular pathogen of 
zoonotic interest. Regarding animals of veterinary importance, infection by this agent has 
been linked mainly to high fever, neutropenia, reduced milk production, but hemorrhagic 
diathesis, abortion and impaired spermatogenesis have also sporadically been reported. In 
Greece, A. phagocytophilum has been detected in dogs, ticks and humans, while so far only 
A. ovis had been detected in farm animals. Following the occurrence of multiple abortions in 
two goat farms in Northern Greece, samples were collected from aborted animals. Stomach 
contents and placental tissue from aborted animals tested positive for A.  phagocytophilum 
by molecular assays and negative for other infectious and parasitic agents. Treatment 
with oxytetracycline LA stopped the abortions. In tick risk areas clinicians should consider 
A. phagocytophilum as a cause of abortion in goats.

Dimosthenis Chochlakis1, Nektarios Giadinis2*, Evanthia Petridou2, George Filioussis2,
Yannis Tselentis1, Anna Psaroulaki1, Evi Ioannidou2, Vasiliki Papanikolopoulou2

and Harilaos Karatzias2

Molecular evidence of Anaplasma phagocytophilum 
in aborted goat fetuses and placenta 

Veterinaria Italiana 2020, 56 (4), 301‑303. doi: 10.12834/VetIt.1173.6516.2
Accepted: 12.12.2016  |  Available on line: 31.12.2020

SHORT COMMUNICATION



302

A. phagocitophila in goats Chochlakis et al.

Veterinaria Italiana 2020, 56 (4), 301‑303. doi: 10.12834/VetIt.1173.6516.2

Sequences revealed were 100% identical to each 
other and 100% identical to already published 
sequences (16S rRNA: Genbank accession number 
EU090186) and 99% identical to the 16S rRNA 
(M73220) and groESL (AF548385) of the already 
published variant known to cause a more severe 
form of the disease (Stuen et al. 2003).

In both herds, treatment with oxytetracycline LA 
of the rest pregnant animals (two injections with 
three days a part at a dose of 20 mg/kg) following 
A.  phagocytophilum detection, was effective 
and no other cases of abortion were observed. 
According to the owners, the animals in both herds 
were grazing during large periods of the year, 
consequently tick infestation was not unusual. At 
the intervention time, the animals didn’t have ticks, 
because few days earlier they had been treated for 
ectoparasites.

Abortions, which often are determined by zoonotic 
agents, are a serious economic threat  in goat herds, 
as they can reduce the meat and milk productions 
(Smith and Sherman 2009, Giadinis et al. 2013).

Anaplasma phagocytophilum has been demonstrated 
as a possible cause of reproductive failures in sheep 
flocks in Italy, Norway, Spain and UK (Jones and 
Davies 1995, Garcia‑Perez et  al. 2003, Chianini et  al. 
2004, Lillini et al. 2006, Giudice et al. 2011). However, 
this is the first time that A.  phagocytophilum has 
been associated to abortion cases in goats.

As regards the pathophysiology of abortions caused 
by A.  phagocytophilum, they seem to occur due to 
placental dysfunction (Chianini et  al. 2004). It has 
recently been found that transplacental transmission 
of A.  phagocytophilum can occur in sheep without 
abortion (Reppert et  al. 2013). It is interesting, that 
in the previous study, A.  phagocytophilum was 
detected in many tissues of an infected lamb during 
pregnancy. In the present study, the pathogen was 
detected in the stomach contents of the goat kids 
of the 1st herd and in placental tissue from the 2nd 
herd, while it was not found in other tissues of the 
aborted goat kids. This suggests that abortion 
might be a consequence of placental dysfunction. 
Unfortunately, histopathology was not conducted 
in the fetal membranes. In any case, it would 
be interesting to investigate the distribution of 
A. phagocytophilum in various tissues.

Since the number of examined herds and fetuses 
was low, these data should be treated with caution. 
However, in case of A. phagocytophilum infection in 
goat herds it is likely that abortion is a sporadic event 
(Giadinis et  al. 2011, Giadinis et  al. 2013). Abortion 
storm with ewe mortality has been reported once 
in sheep in UK (Jones and Davies 1995). However, 
this outbreak was not thoroughly investigated and 
oxytetracycline administration was not effective. In 

Prefecture (Northern Greece) and consisted of 
300  dairy goats of local breeds that were reared 
under the semi‑intensive feeding system. In 2014, 
the farmer observed sporadic abortions in goats of 
different ages, and referred about 15 abortions at 
the 3rd‑4th month of pregnancy. Two aborted fetuses 
(from different mothers) and fetal membranes were 
submitted to the Farm Animal Clinic of the Aristotle 
University in Thessaloniki. Both fetuses and fetal 
membranes were normal in appearance and no 
specific lesions were detected at autopsy. Whole 
blood from the aborting mothers along with fetal 
spleen, brain, liver, placental tissues and stomach 
contents were collected.

All samples were tested by PCR or cultures for 
Chlamydia  spp., Brucella  spp., Campylobacter  spp., 
Salmonella  spp., Escherichia coli, Listeria spp., 
Mycoplasma  spp., Toxoplasma spp., Neospora spp., 
and Schmallenberg virus at the Aristotle University 
and they were found all negative for the above 
pathogens (Giadinis et al. 2013).

DNA was extracted from the sampled tissues using a 
commercial kit (Tissue kit, Qiagen, Hilden Germany); 
portions of DNA samples were sent at the Laboratory 
of Clinical Bacteriology, Parasitology, Zoonoses and 
Geographical Medicine of the University of Crete, 
where they were tested by PCR for Babesia species 
(targeting 18s rRNA) and by a multiplex real‑time 
PCR for Coxiella burnetii (targeting com1), Anaplasma 
species (targeting msp4 of A.  centrale, A.  marginale 
and A. ovis) and A. phagocytophilum (targeting msp4). 
DNA from positive samples against the pathogens of 
interest was used as positive marker; sterile water was 
used as negative marker. All samples were negative 
for Babesia species, Anaplasma species and Coxiella 
burnetii, while DNA from the stomach contents of the 
1st herd and placental tissue from the 2nd herd were 
tested positive for A.  phagocytophilum. Also, blood 
samples from the examined mothers were found 
positive for A. phagocytophilum.

Positive samples were further amplified using 
primers ehr16SR and ehr16SD (targeting a 345 
bp portion of the 16s rRNA (Parola et  al. 1998) and 
HS1 and HS6 (targeting a 1300‑1450 bp portion of 
groesl) (Sumner et al. 1997).

PCR amplicons (345 bp for 16s rRNA and 1350 bp 
of groesl) were purified using a commercial kit (QIA 
quick PCR purification spin kit, Qiagen, Germany). 
Sequencing was performed twice using the 
above described primers at a CEQ 8000 Beckman 
Coulter sequencer. The sequences revealed were 
processed using Chromas v1.49 and Lasergene 
ver.7.1 software for viewing the chromatograms and 
editing of the retrieved nucleotide sequences, and 
nucleotide BLASTn for investigating the homology 
of the obtained sequences compared to the 
NCBI databases.



303

Chochlakis et al.  A. phagocitophila in goats

Veterinaria Italiana 2020, 56 (4), 301‑303. doi: 10.12834/VetIt.1173.6516.2 Veterinaria Italiana 2020, 56 (4), xxx‑xxx. doi: 10.12834/VetIt.1173.6516.2

(Stuen et al. 2003, Teglas and Foley 2006, Granquist 
et  al. 2010). Anaplasma phagocytophilum infection 
may persist in different animal species, depending 
on individual variations, genetic variants and host 
species involved. However, persistent infection 
in goats has not been reported yet (Stuen 2007, 
Thomas et al. 2012). 

the present study, oxytetracycline administration 
was very effective in controlling the abortions. 

Regarding the responsible strain, the sequences 
revealed a 99% similarity with those of 
A.  phagocytophilum variant 1 (Stuen et  al. 2003). 
The incidence and severity of the disease depend 
on geographic area, species and host breed, as 
well as the variant of A.  phagocytophilum involved 

Aktas M. & Özübek S. 2015. Bovine anaplasmosis in 
Turkey: first laboratory confirmed clinical cases 
caused by Anaplasma phagocytophilum. Vet Microbiol, 
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Chianini F., Adams C. & Buxton D. 2004. Neuropathological 
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Garcia‑Perez A.L., Barandika J., Oporto B., Povedano J. 
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