Three clinical cases of Anaplasma phagocytophilum infection in cats in Poland

Abstract

The purpose of this study was to describe Anaplasma phagocytophilum infection of three cats in Poland showing signs of fever, swollen and painful joints, pale mucous membranes and epistaxis. Morulae consistent with A phagocytophilum were present within the neutrophils of two of the cats. A polymerase chain reaction (PCR) was found targeting the 16S rRNA gene amplified DNA consistent with A phagocytophilum in the blood of all three cats. The sequence of the PCR product obtained showed 99.6–100% homology with the sequence of A phagocytophilum, gene number EU 090186 from Genbank. Applied therapy (including administration of tetracyclines for 3 weeks) resulted in a gradual clinical recovery.

Introduction

Granulocytic anaplasmosis (GA) is an infectious multi-organ disease of humans and animals accompanied by thrombocytopenia. The aetiological factors of the disease are the microorganisms previously classified within the family of Rickettsiaceae, genus Ehrlichia. Currently, they are classified within the order of Rickettsiales, family Anaplasmataceae. ¹ The 16S rRNA gene sequence studies made it possible to classify three pathogens considered, until recently, as separate species, and being aetiological factors of anaplasmosis in cats and dogs (Anaplasma phagocytophilum), horses (A phagocytophilum, Ehrlicha equi) and humans (human granulocytic ehrlichiosis agent) to the taxon of A phagocytophilum. ¹

Recently, some cases of animal granulocytic anaplasmosis have been reported in Poland. Serological monitoring carried out in dogs, cattle and pigs, for example, indicated contact with rickettsia, which proves indirectly the occurrence of Anaplasma species microorganisms in Poland.^2,3 The absolute confirmation of their presence was the detection of rickettsia genetic material in ticks and wild ruminants and horses coming from various regions of the country.^4–7 Before that, anaplasmosis had never been diagnosed in cats in Poland. In other regions of the world the disease is diagnosed rarely in this species.

The aim of this research was to present the first cases of A phagocytophilum infection in cats in Poland.

Materials and methods

The study was carried out on three cats, aged from 2 to 6 years, from the following regions: Lublin (case 1), Masovia (case 2) and Przemyśl (case 3). Two were male cats and one female. All the cats had contact with ticks, after which the clinical signs were observed: fever, swollen and painful joints, pale mucous membranes and epistaxis. In all three cases, the ticks from the body surface of the sick cats had been removed by their owners; hence, it was not possible to determine their species.

Case 1

Case 1 was a European shorthair male, aged 2 years and 5 months and weighed 5.2 kg. It was admitted to the clinic with the following signs: loss of appetite and thirst, apathy and paleness of mucous membranes. The internal body temperature was 39.8°C; the accessible lymph nodes were not enlarged, but palpation of the abdomen revealed a two-sided tenderness of the area below the ribs. Moreover, swelling and painfulness of both ankle joints was also observed. As reported in the anamnesis by the owners, the cat had suffered from periodic (occurring every 2 days) bleeding from the nose that had receded spontaneously. A radiographic examination demonstrated no lesions in the skeletal system of the problem joints. The observed signs had persisted for 5 days and had occurred approximately 3–4 days after the removal of the tick from the body surface of the animal.

Case 2

Case 2 was a European shorthair, non-neutered 3-year-old male and weighed 4.1 kg. Its owners had observed its difficulty in walking accompanied by painful wrist joints and ankle joints for about 2 weeks. A radiographic examination showed no lesions in the skeletal system of the problem joints. Ticks had been removed from the body surface of the cat about 3 weeks previously. The animal was apathetic, had reduced appetite and the accessible mucous membranes were pale. Its internal body temperature was 39.1°C; the accessible lymph nodes were not enlarged.

Case 3

A female 6-year-old European shorthair cat was admitted to the clinic. According to the owners, the animal had been apathetic and showed no desire to play for about 2 days. A tick had been removed from the body surface of the animal about a week previously. The internal body temperature was 39.6°C, appetite and thirst were reduced. The accessible lymph nodes were not enlarged. Except for the signs reported by the owners, clinical examination revealed no other abnormalities.

Blood for haematological tests, molecular tests for anaplasmosis and babesiosis, and for speed tests for feline leukaemia virus (FeLV), feline immunodeficiency virus (FIV) and feline coronavirus infection was obtained from the sick animals.

Haematological tests

Blood for the haematological tests was placed into 3-ml test tubes with ethylenediamine tetra-acetic acid (EDTA), shaken and tested (about 300 µl) in an Exigo (Boule) analyser.

Blood smear tests

Blood smears were made on a degreased microscopic glass, stained with the Diff Quick (POCH Lublin) method and viewed under an Olympus CH 20 (Olympus Optical) microscope when dry. Blood smears were screened for presence of Babesia, Anaplasma and Mycoplasma species.

Tests for FeLV, FIV and feline coronavirus infection were performed according to the manufacturer’s instructions (BioVeto Test).

DNA isolation

DNA for the analysis was extracted from 100 µl of fresh blood. DNA isolation was carried out with a DNA blood mini kit (DNA Gdan’sk).

Polymerase chain reaction

Polymerase chain reaction (PCR) amplification was performed using a programmable thermal cycler (Biometra). The PCR reaction for Babesia/Theileria species was carried out using a pair of primers, ie, RLB R2 and RLB F2, which made it possible to amplify the DNA section with a 390–430 base-pair-long fragment of the 18S rRNA conservative gene. ⁸ The PCR reaction for Anaplasma species was carried out according to a method described by Adaszek et al ⁹ using the following primers: EHR 521: 5’-TGT AGG CGG TTC GGT AAG TTA AAG-3’; EHR 747: 5’ GCA CTC ATC GTT TAC AGC GTG-3’. This limited the DNA section to a length of 247 bp of the conserved part of the 16S rRNA gene. The positive control was the DNA of A phagocytophilum from human blood (National Reference Center for Borreliae of Max von Pettenkofer Institute of Ludwig Maximilian University Munich) and the negative control was DNA from the blood of a healthy cat. Each reaction was composed of 35 cycles with the denaturation stage at 94°C for 30 s, primers were attached at a temperature of 56°C for 30 s and the threads were elongated at a temperature of 72°C for 45 s.

Electrophoresis

The PCR products were analysed with the electrophoresis method in a 1% agarose gel and Tris-borate-EDTA (TBE) buffer at a voltage of 10 V/cm for 50 mins.

Sequencing

The PCR reaction products purified with the QIAquick PCR Purification Kit (Qiagen) were sequenced by the DNA Sequencing and Synthesis Service of the Institute of Biochemistry and Biophysics at Polish Academy of Science in Warsaw. The sequencing results were received via email and developed using the computer software Lasergene DNA Star. The same software was used to analyse the sequence of A phagocytophilum isolates and compare them with sequences available in the National Center for Biotechnology Information (NCBI) Genebank.

Results

Table 1 demonstrates low content of haemoglobin in the erythrocytes, decreased haematocrit and thrombocytopenia. Each cat was negative for FeLV p27 antigen, and antibodies against FIV and feline coronavirus. Babesia protozoa and Mycoplasma species bacteria were not found in the erythrocytes in the blood smears, whereas in two cats (cases 1 and 3) morulae of Anaplasma species were observed in neutrophils (Figure 1a, b).

Table 1.

Results of haematological examination of the blood sampled from sick cats on the day after onset of clinical signs

Investigated parameters	RBC (1012/l)	WBC (109/l)	HGB (mmol/l)	HCT (%)	PLT (109/l)	Morulae in the blood smears
Case 1 (Lublin)	5.12	4.6	7.7	23	34	+
Case 2 (Masovia)	4.31	5.0	5.31	21	52	+
Case 3 (Przemyśl)	3.95	4.2	4.80	21	47	−
Range	6.5–10	5.5–19	4.96–9.31	24–45	100–400	−

HCT = haematocrit; HGB = haemoglobin; PLT = platelets; RBC = red blood cells; WBC = white blood cells

Figure 1.

(a, b) Presence of Anaplasma species morulae (arrows) in neutrophils

The PCR test for Babesia species did not show the presence of the genetic material of protozoa in the blood of the diseased animals. However, the presence of the genetic material of Anaplasma species was observed in all three samples.

The sequences of Anaplasma species products with a length of 247 bp obtained from the PCR reaction and developed in the Lasergene DNA Star showed a high level of similarity (99.6–100%) to the 16S rRNA gene section sequence of A phagocytophilum strain Lublin, isolated in an earlier study from a horse in Poland, listed in the Genbank under the number GU183908.

The treatment of the cats involved oxytetracycline (5 mg/kg IM: Oxyvet; Biowet Puławy) administered intramuscularly twice daily (q12h) for 2 days, then changed to doxycycline (Ronaxan; Merial) administered for 3 weeks at a dose of 10 mg/kg of body mass, and dexamethasone (0.2 mg/kg SC: Dexasone; ScanVet) for 3 days. The applied therapy led to a significant improvement in the state of health of all cats 48 h after application of the drugs. Complete recovery of the infected animals was observed within 3 weeks.

Haematological tests performed a month after the initiation of the treatment did not show any abnormalities.

Discussion

Anaplasma phagocytophilum is a microorganism able to infect many animal species and humans. The main vector of the microorganisms in Europe is the tick Ixodes ricinus.^10,11 The reservoirs of rickettsias are rodents and wild ruminantas.^7,12 Feline anaplasmosis is a relatively rare disease, as evidenced by the many clinical reviews on this subject. There are only a few specific reports that include detailed case descriptions regarding the disease in domestic cats.^13–17 In Poland the disease was previously detected only in horses and dogs,^2,6 never in cats.

It ought to be noted that A phagocytophilum infection is diagnosed more rarely in cats than in dogs, possibly as a result of the specific behaviour of cats and the mechanical removal of ticks from their body surface while they groom. ¹⁸

In Europe, the DNA of these pathogens was detected occasionally in the blood of naturally infected cats from Sweden, Finland, Italy, UK, Ireland and Denmark, which showed signs of lethargy, anorexia, fever and tachypnoea.^{12,14,17,19,20}

In the cases described the diagnosis was established on the basis of haematological tests (thrombocytopenia and presence of morulae in neutrophils in two of the infected cats), molecular tests and applied therapy efficiency.

One of the most characteristic signs of anaplasmosis is thrombocytopenia,^11,14 which may lead to bleeding from the mucous membranes. Thrombocytopenia might have been the reason why one of the patients developed bleeding from the nose — a clinical sign that has not been described previously in cats, but which has been observed in the course of anaplasmosis in dogs. ²¹ The mechanisms that cause the platelet count to drop are not explained fully and may involve immunological destruction of thrombocytes, their increased phagocytosis by macrophages and reduced production by the bone marrow as a result of its hypoplasia. ¹² For the cases studied here, the elimination of rickettsia from the body by tetracyclines also contributed to the withdrawal of this haematological irregularity. Tetracyclines appear to be the most efficacious in the treatment of anaplasmosis in cats, although in case of poor tolerance of this group of antibiotics, ampicillin or amoxicillin with clavulanic acid may be also used to combat the infection. ¹² We also used dexamethasone in the therapy. An immune-mediated mechanism is partially responsible for the thrombocytopenia and decreased platelet function. The rationale for corticosteroid therapy has some scientific basis. Some clinicians prefer to use glucocorticoids and tetracycline in combination initially because of the difficulty in distinguishing between anaplasmosis and immune-mediated thrombocytopenia. Even though the use of corticosteroids in granulocytic anaplasmosis (GA) is ambiguous, it can also be indicated as a therapeutic mean in primary and secondary immune mediated haemolytic anaemia (IMHA) owing to stabilisation of erythrocyte membrane. ²²

In one cat, the analysis of the blood smears stained using the Giemsa method showed no inclusions characteristic of A phagocytophilum in the leukocytes; similar observations were made by Lappin et al. ¹² Out of the six cats in the bodies of which DNA of A phagocytophilum was detected using molecular biology techniques, the presence of morula in blood was shown only in one animal. Later, a cytological test did not reveal any of these structures in its blood cells. As the genetic material of the rickettsia was shown using the PCR method at an early stage of infection, one may assume that this technique is more sensitive than others (including serological methods), especially while detecting the acute phase of the infection.

Anaplasma phagocytophilum infection in the cats was confirmed by molecular tests. The analysed 16S rRNA gene fragment of rickettsia showed 99.6–100% similarity to the corresponding sequence GU183908 Uncultured Anaplasma species clone Lublin-1, which was obtained in earlier studies of a clinical case of equine anaplasmosis. ⁹ This suggests an endemic occurrence of this microorganism strain in Poland.

Conclusions

The fact that Anaplasma species infection has been detected in cats demonstrates the danger of this disease in areas of eastern Poland. This infection must be considered when severe pathological clinical signs follow contact between animals and ticks, and are accompanied by thrombocytopenia in a haematological test.