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Granulocytic anaplasmosis in captive ring-tailed lemur (Lemur catta) in Poland

Background Anaplasma are obligate intracellular bacteria and aetiological agents of tick-borne diseases of both veterinary and medical interest. The genus Anaplasma comprises six species: Anaplasma marginale, Anaplasma centrale, Anaplasma ovis, Anaplasma phagocytophilum, Anaplasma bovis and Anaplasma platys. They can infect humans, carnivores, ruminants, rodents, insectivores, birds and reptiles. The aim of this study was to present the first clinical case of granulocytic anaplasmosis in a captive ring-tailed lemur in Poland. Case presentation A 4-year-old female lemur presented anorexia, epistaxis and tick infestation. The microscopic examination of a blood smear revealed morulae in neutrophils. Polymerase chain reaction test and sequencing of obtained PCR product confirmed infection by the GU183908 Anaplasma phagocytophilum strain. Therapeutic protocol included doxycycline (2.5 mg/kg p.o., b.i.d.) for 3 weeks and the lemur recovered within 24 h. Conclusions This is the first report on granulocytic anaplasmosis in a ring-tailed lemur in Europe, indicating that A. phagocytophilum infection must also be considered in differential diagnosis in this animal species, especially in individuals with thrombocytopenia associated with Ixodes ricinus parasitism.


Background
Tick-borne diseases (TBD) constitute a diversified group of diseases of increasing importance in human and veterinary medicine [1]. As showed by the observation of many authors, in Europe ticks are considered the most important of the arthropod zoonotic vectors [2][3][4], that are able to transmit such pathogens as Anaplasma spp., Babesia/Theileria spp. or Borrelia spp. Anaplasma spp. are pathogenic for several animal host species [5], while Anaplasma phagocytophilum is an emerging human pathogen in the USA and Europe [6,7]. The clinical form of the disease is rarely reported in wild animals in captivity [8][9][10].
The aim of this study was to present the clinical case of granulocytic anaplasmosis in captive ring-tailed lemur (Lemur catta) in Poland.

Case presentation
The observation took place in March 2020. The animal concerned was a female ring-tailed lemur (Lemur catta), 4 years old, with a body weight of 4.2 kg, with signs of anorexia, weakness, epistaxis and uncoordinated gait. These clinical signs appeared four days before the animal was brought to the clinic. During a clinical examination two adult female Ixodes ricinus ticks were removed from the animal's body. The ticks were identified on the basis of morphology using taxonomic keys [11]. The lemur came from a zoo in eastern Poland. The animal lived in a group of 6 lemurs. Two months before she had received fenbendazole (50 mg/kg p.o. for 3 days) as deworming treatment, but no ectoparasite prophylaxis had been applied. The animal was clinically examined and blood samples were collected for biochemical, haematological and molecular tests for tick-borne diseases (babesiosis/theileriosis/anaplasmosis/ehrlichiosis). The ticks found on the animal's body were also tested for the above diseases using molecular methods.
DNA extractions from the blood samples and ticks for molecular tests were performed using a commercial DNA Genomic kit (A&A Biotechnology Gdańsk, Poland) following the manufacturer's instructions. Subsequent PCR tests were performed according to the methods described by Skotarczak et al. [12], Altay et al. [13] and Adaszek and Winiarczyk [14] ( Table 1). The final identification of tick-borne pathogens was performed by sequencing PCR products.
The haematological and biochemical test results did not reveal any abnormalities, except for thrombocytopaenia (PLT = 61 × 10 9 /l; range 165-685 × 10 9 /l) [15]. The microscopic examination of the stained blood smear (Giemsa method) revealed morulae in the cytoplasm of  circulating neutrophils suggestive of acute granulocytic anaplasmosis (Fig. 1). The PCR test revealed Anaplasma DNA in the lemur's blood and in I. ricinus ticks collected from the lemur's body (Fig. 2). The analysis of PCR product sequencing identified the Rickettsia species as A. phagocytuphilum GU183908 (100 % homology). Based on the microscopic blood smears and the molecular test results, the disease was caused by Anaplasma phagocytophilum infection. The treatment started with doxycycline (2.5 mg/kg p.o., b.i.d.) administered for three weeks. 24 h after the initiation of the treatment the lemur's condition improved significantly: her appetite increased and normal gait was restored. Three days later the epistaxis had subsided. Two weeks following the initiation of the antibiotic treatment a sample of the animal's blood was collected for a quick test to detect the presence of A. phagocytophilum antibodies (VetExpert, CaniV-4 Poland). The test result was positive. A control PCR test carried out after the next three weeks (according to the same procedure as previous) did not reveal genetic material of A. phagocytophilum in the animal's blood. Also, no DNA of bacteria was found in the blood of the other five lemurs from the same institution. Six months after the beginning of therapy the antibodies for A. phagocytophilum were still be present in the animal's blood.

Discussion and conclusions
This article presents the first clinical case of granulocytic anaplasmosis in a lemur in Europe. A. phagocytophilum is one of the most prevalent tick-transmitted animal and human pathogen [7]. The main clinical disorders observed in the course of granulocytic anaplasmosis are fever, thrombocytopenia and lameness. Ticks and wildlife are the main reservoirs of these bacteria [8], but clinical disease in free-ranging as well as in captive wild animals appears to be rare. However wildlife may play a role in the transmission and maintenance of granulocytic anaplasmosis, either acting as a reservoir of the bacteria or amplifying host for human or domestic animals [16]. Therefore, it is important to identify the potential hosts and characterise the role in the epidemiology of various animal species in this disease in order to adequately evaluate the potential risks and to design proper strategies of control.
The main vector of the microorganisms in Europe is the tick Ixodes ricinus [17]. There are only a few specific reports regarding A. phagocytophilum infection in lemurs. Specific antibodies against A. phagocytophilum were found in the serum of lemurs from St. Catherine's Island, Georgia, USA [18], whereas screening tests for infections, including A. phagocytophilum, conducted in the lemur population of Madagascar did not confirm a single case of the disease [19]. In Poland the disease was previously detected in horses [14], dogs [20] and cats [21], but never in exotic animals.
The definitive diagnosis of A. phagocytophilum infection in a ring-tailed lemur was confirmed by results of PCR and sequencing. The 16 S rRNA gene fragment of bacteria detected in the blood of the patient, as well as in the tick organism collected from the lemur's body showed 100 % similarity with GU183908 uncultured Anaplasma species clone Lublin-1 from previous studies [14]. This suggests an endemic occurrence of this microorganism strain in Poland. The description of the presented case indicates that A. phagocytophilum infection must also be considered in differential diagnosis in exotic animals living in Poland, especially in individuals with thrombocytopenia associated with Ixodes ricinus parasitism.