Anaplasma platys (Ehrlichia platys) infection in a dog in France : description of the case, and characterization of the agent

ARTICLE ORIGINAL Anaplasma platys (Ehrlichia platys) infection in a dog in France : description of the case, and characterization of the agent J.P. BEAUFILS, H. INOKUMA, J. MARTIN-GRANEL, Ph. JUMELLE, M. BARBAULT-JUMELLE and P. BROUQUI Clinique Vétérinaire, Route de Salinelles, F-30250 Sommières, jpierre.beaufils@wanadoo.fr Unité des Rickettsies, Faculté de Médecine, Université de la Méditerranée, 27 bd Jean-Moulin, F-13385 Marseille cédex 5 (Present address of Prof. INOKUMA : Laboratory of Veterinary Internal Medicine, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan) SUMMARY A dog that presented fever and bleeding disorders after a routine surgery was infected by an intra-platelet organism and also suffered from hypoadrenocorticism. PCR and genetic studies showed that it was a strain of Anaplasma platys, (ex Ehrlichia platys), 99.8 % identical with A. platys identified in South China, and 99.6 % identical with A. platys identified in the USA. To our knowledge, this is the first description proving by genetic studies that A. platys exists in Europe. RÉSUMÉ Infection à Anaplasma platys (Ehrlichia platys) chez un chien en France : description du cas clinique et caractérisation de l agent responsable. Par J.P. BEAUFILS, H. INOKUMA, J. MARTIN-GRANEL, Ph. JUMELLE, M. BARBAULT-JUMELLE et P. BROUQUI. Une chienne qui présentait de la fièvre et des troubles de l hémostase après une ovario-hystérectomie était infectée par un organisme intra-plaquettaire, et présentait en outre un hypocorticisme. La PCR et des études génétiques ont montré que cet agent intra-plaquettaire appartenait à une souche d Anaplasma platys (ex Ehrlichia platys) identique à 99,8 % de l A. platys identifiée dans le sud de la Chine, et à 99,6 % et l A. platys identifiée aux USA. A notre connaissance, il s agit de la première description apportant la preuve génétique qu A. platys existe en Europe. KEY-WORDS : Anaplasma platys - Ehrlichia platys - Infectious cyclic thrombocytopenia - polymerase chain reaction. MOTS-CLÉS : Anaplasma platys - Ehrlichia platys - thrombocytopénie infectieuse cyclique - polymerase chain reaction. Introduction The agent of infectious cyclic thrombocytopenia (ICT), Ehrlichia platys, was first described in 1978 in Florida, USA, by HARVEY and others [18]. The organism appeared in blood platelets as morulae composed of one to eight subunits [12, 13, 18, 28]. Very recently, the genus Ehrlichia was reorganized on the basis of genetic studies, and E. platys, as well as organisms of the Ehrlichia phagocytophila group, now belong the genus Anaplasma [11]. Actually, A. platys is described in America, (different States of the USA [12, 27], Venezuela [36]), Europe, (France [3, 4]), Greece [26], Spain [34], and Germany as an imported parasite [14]), Middle- East, (Israel [16, 17]), Asia (Taiwan [9], Japan [21, 29], China [20], Thailand [36]), and Oceania (Australia [8, 22]). In the USA and Taiwan, natural or experimental infection by A. platys causes no or few clinical signs [5, 9, 12, 18, 28], although uveitis was described in one case [15], bleeding disorders in another [40], and lymph node enlargement in all experimentally infected dogs in a study [2]. Biological signs include cyclic thrombocytopenia and parasitemia, [5, 9, 13, 18], blood platelets dysfunction [13], and inconstantly anemia, leukopenia, hypoalbuminemia and hypergammaglobulinemia [1, 5]. In France, an organism resembling A. platys in both light and electronic microscopy was described in the 1980 s*. Dogs infected by this agent presented with severe clinical * Th Heaney, personnal communication.

86 BEAUFILS (J.P.) AND COLLABORATORS signs including hyperthermia, lethargy, anorexia, algias, and sometimes digestive signs and bleeding disorders. Laboratory findings included thrombocytopenia and monocytosis [3, 4]. Several dogs infected by this organism, or presenting a thrombocytopenic purpura, were seropositive to A. platys (IFA test), with seroconversion in one case. All dogs were treated with oxytetracycline, doxycycline, or imidocarb dipropionate in the case of puppy with lacteal dentition : they all recovered dramatically, most often after a 48 hour delay [4]. Similar descriptions regarding clinical and laboratory signs and treatment were published in Greece [26] and Israel [16], suggesting that a strain of A. platys more pathogenic than in the USA and Taiwan might be present in mediterranean countries (except Spain where infection seems asymptomatic [34]). In the present study, infection of a dog by A. platys in France is confirmed by PCR, and the 16S rrna gene sequence of the agent is compared to those of the northamerican and chinese strains of A. platys. Case report A 17-month-old female mix-breed dog, weighing 20 kg, currently vaccinated for distemper, hepatitis and parvovirosis, was presented for an ovario-hysterectomy, due to an unwanted pregnancy. The dog was in a good condition before surgery. No abnormality was found on clinical examination. Surgery and awakening were uneventful. Eight hours after surgery, the dressing of the surgical wound was blood stained. The dressing was removed : there was an hematoma around the wound, but no more bleeding. The next day, (day 1), the dog appeared alert, but in the evening, bleeding recurred (photo 1). At that time, the dog was depressed, and rectal temperature was 39.7 C. Blood analysis performed in the clinic indicated a moderate leukocytosis (18,78.10 9 WBC/l ; usual range : 6,00-17,00.10 9 /l), lymphocytosis, (5,07.10 9 /l ; usual range : 1,00-4,80.10 9 /l), and a marked monocytosis (4,51.10 9 /l ; usual range : 0,05-1,35.10 9 /l). Red blood cells (RBC), hemoglobin, hematocrit, blood platelets (256.10 9 /l ; usual range : 200-500.10 9 /l), prothrombin time (PT) and total protein were within usual range [22]. Examination of the blood smear revealed the presence in blood platelets of granulations isolated, in pairs or in clusters, resembling Anaplasma platys (photos 2, 3). 58 % of blood platelets contained such inclusions. Many platelets had a large size, sometimes larger than RBC (photos 2, 3). We also noticed a strong monocytosis with many activated macrophages, some of them phagocytizing polynuclear neutrophils or blood platelets with morulae (photo 2, 4). No other blood parasite was detected. A rapid immunomigration test for detection of anti- Leishmania antibodies, (Witness Leishmana ND, Synbiotics), was negative. Oxytetracycline (TERRAMYCINE ND : 250 mg) and dexamethasone (TERESONE ND : 3,4 mg) were injected. The next day, the animal was in good form, and not bleeding any longer. Rectal temperature was 38.2 C. The dog was released to her owners with a prescription of doxycycline (DOXYLETS ND : 200 mg sid) for 15 days. They were followed by 15 days of minocycline) (ZACNAN ND : 200 mg sid), administered as an automedication by the owner. The dressing was removed after 4 days : no bleeding was noticed at that time, and hematoma was completely resorbed. According to the owners, it took around 12 days before the dog recovered completely. A control could be realized only 32 days after diagnosis and initiation of treatment. No hematoma persisted around the surgical scar. The dog was lethargic and coughed since a few hours. Rectal temperature was 38.1 C. A CBC revealed monocytosis (1,48.10 9 /l), and absence of thrombocytopenia (469.10 9 /l). No A. platys morula was found on a blood smear. Doxycycline (200 mg sid) was administered again. A tracheobronchitis developed in the two dogs of the household on following days. The other dog recovered rapidly, but the dog of this study was still lethargic after one week. It was presented in a very poor condition on day 50 with recumbency, hypothermia, bradycardia, (45 beats per minute ; P waves were absent on an electrocardiogram), and pre-renal azotemia. Strong hyperkalemia (9,4 meq/l) and hyponatremia (129 meq/l) supported a presumptive diagnosis of hypoadrenocorticism, confirmed later by a dosage of aldosterone (0 pmol/l). Recovery after administration of fluids, gluco and mineralocorticoids, was dramatic and uneventflul. Peripheral blood was collected with EDTA and heparin as anti-coagulants on days 1 and 32. They were sent to the National Center for Rickettsiae, Marseille, France, where molecular and serological analyses were performed. Materials and methods 1) DIAGNOSTIC PCR DNA was extracted from whole blood (200 µl) following the QIAamp Tissue Kit procedure (QIAGEN GmbH, Hilden, adjusted in 200 µl of TE buffer and storedat - 20 C until used. Species-specific forward primers CANIS, CHAFFEENSIS and EWINGII were used with the Ehrlichia genus-specific reverse primer of GA1UR to amplify Ehrlichia canis, Ehrlichia chaffeensis and Ehrlichia ewingii, respectively [38]. For the species-specific amplification of A. platys, PLATYS-F (5 -AAG-TCG-AAC-GGA-TTT-TTG- TC-3 ) and PLATYS-R (5 -CTT-TAA-CTT-ACC-GAA-CC-3 ) were used (INOKUMA et al. unpublished data). The condition of the PCR was the same as the method of PAROLA and others [32], except for the annealing temperature (55 C). 2) DETERMINATION AND ANALYSIS OF THE 16S rrnas GENE SEQUENCE Two other primer sets, fd1 / EHR16SR and EHR16SD / Rp2 were then used in PCR to amplify the majority of the 16S rrna gene sequence in the sample. The PCR products were extracted and sequenced by the method used in a previous paper [31]. Obtained nucleotide sequence was examinad for homology with registered sequences in GenBank by using a BLAST program [http://www.ncbi.nlm.nih.gov/blast]. Multiple alignment analysis and construction of a phylogenetic tree were performed with ClustalW program [37] version 1.8 in the DNA Data Bank of Japan (DDBJ ; Mishima, Japan

ANAPLASMA PLATYS (EHRLICHIA PLATYS) INFECTION IN A DOG IN FRANCE 87 [http://www.ddbj.nig.ac.jp/htmls/e-mail/clustalw-e.html]. The distance matrices for the aligned sequences with all gaps ignored were calculated using the KIMURA two-parameter method [25], and the neighbor-joining method was used for constructing a phylogenetic tree [35]. The stability of the tree obtained was estimated by bootstraps analysis for 1000 replication using the same program. Tree figures were generated using the Tree View program version 1.61 [30]. The sequence of the partial 16S rrna gene of A. platys Sommières has been deposited in GenBank data library under accession number AF303467. The GenBank accession numbers of the 16S rrna gene sequences of other species used to analyze data are as follows : A. platys M82801 and AF156784 ; A. phagocytophila, M73224 ; Anaplasma equi, M73223 ; HGE agent, U02521 ; Anaplasma bovis, U03775 ; Anaplasma marginale, M60313 ; Anaplasma centrale, AF283007 ; E. chaffeensis, M73222 ; E. canis, M73221 ; E. ewingii, M73227 ; E. muris, U15527 ; Ehrlichia found in Ixodes ovatus strain Yamaguchi, AF260591 ; Ehrlichia ruminantium, AF069758 ; Wolbachia pipientis, AF179630 ; Neorickettsia sennetsu, M73225 ; Neorickettsia risticii, M21290 ; Neorickettsia helminthoeca, U12457 ; and Rickettsia rickettsii, U11021. 3) INDIRECT IMMUNOFLUORESCENCE ASSAY (IFA) E. canis (Oklahoma strain) E. chaffeensis (Arkansas strain), HGE agent (Webster strain), A. equi (California strain), and N. sennetsu (Miyayama strain) were used as antigens in an IFA test as previously reported [7]. Each serum was diluted 1:25, 1:50 and 1:100 with PBS containing 3 % skim-milk for screening. Antibody levels for test samples were determined by comparison with positive and negative controls, and any positive reaction was recorded. Sera that reacted with any antigen in the screening test, were diluted again by two-fold dilution for titration. Results PCR was positive for A. platys but negative for E. canis, E. chaffeensis and E. ewingii on day 1 ; no positive reaction was detected for all these pathogens on day 32. Subsequent PCR using 2 sets of primers yielded a 1446 bp product from a DN on day 1. The obtained sequence was similar to that of A. platys. The blast search revealed that there were 3 nucleotide differences (1442/1445 (99.8 %) identical) with A. platys registered in South China (AF156784), and 6 difference (1437/1443 (99.6 %) identical) with A. platys registered in the USA (M82801). Table I shows the nucleotide differences in the 16S rrna gene sequence among these 3 strains. The phylogenetic relationships of these 3 A. platys strains and other ehrlichial species based on the 16S rrna gene nucleotide sequences are shown in Fig. 1. The rectangular cladogram with bootstrap analysis revealed that the 3 strains are closely related to each other ; however, A. platys strain Sommières found in this study has been significantly defined from the other two strains. The results of IFA tests on day 1 were all negative, while on day 32, IFA tests revealed positive results for A. equi (1/128) and the HGE agent (1/256). The sample on day 32 also reacted weakly with E. chaffeensis antigen (1/32), but no reaction was found with E. canis and N. sennetsu. Discussion We found interesting to report this case for both clinical and fundamental reasons. Recognizing an A. platys infection is important in everyday practice : in the present case, although only PT and a platelet count were performed, it is likely that the results of other common coagulation tests (activated partial thromboplastin time and thrombin time) would have been in the usual range. Finding an apparently normal coagulation screening in this case could have led to an inappropriate decision, such as a reintervention to look for a surgical hemostasis defect. Hence, research of blood parasites on a blood smear is important in bleeding disorders. Thrombocytopenia, (as parasitemia), is cyclis in A. platys infection [12, 13, 18]. In this case, blood was collected at a moment when parasitemia was high, and platelet number within the usual range. Although no laboratory test of platelet function was performed in this case, it is likely that the bleeding disorder may be explained by a thrombocytopathy : such platelet dysfunction is described in infections by A. platys [13] and E. canis [17, 24]. According to the owners, the dog was in a very good condition before surgery. The outbreak of the disease eight hours after an ovario-hysterectomy may be a coincidence : many dogs have ticks in August in the south of France, and this dog might have been infected during the previous days. Presence of ticks was not noted by the owners, but the dog escaped from her home one month before the disease, and spent several days in an animal protection kennel where cases of canine monocytic ehrlichiosis are frequent. It is also possible that surgery precipitated the outbreak of the disease if the dog was an asymptomatic carrier of A. platys : the IFA test of one dog remained positive 5 years after infection by A. platys [12], suggesting the possibility of a carrier state in untreated infected dogs. In E. canis infection, stress factors such as a major surgery, malnutrition, pregnancy, and concomitant diseases may precipitate clinical ehrlichiosis in a dog with sub-clinical disease [33]. The intra-platelet organisms described in France and in the USA are morphologically similar in both optic and electronic microscopy, and sera from some infected dogs in France are seropositive in IFA test for A. platys, with seroconversion in one case [4], indicating a strong proximity between the two organisms. Detection of A. platys DNA using A. platys specific primers, and a nucleotide sequence very close to those found in A. platys in the USA and China, show that the intraplatelet organism observed in France is a strain of A. platys. As indicated by the discovery in Oklahoma of a strain showing one nucleotide difference with the published sequence of A. platys [28], strain variations are possible for this organism. To our knowledge, it is the first time that presence of A. platys in a dog is assessed by genetic studies in Europe. Clinical signs in this case were more severe than those usually reported in A. platys infections in the USA or China.

88 BEAUFILS (J.P.) AND COLLABORATORS * Numbers refer to position in the 16S rrna gene of A. platys China (AF156784). ** Between 1020 and 1021 TABLE I. Comparison of 16S rrna gene nucleotide sequence of Anaplasma platys in France, China and USA. FIG. 1. Phylogenetic relationship of 3 Anaplasma platys strains and other ehrlichial species based on the 16S rrna gene nucleotide sequences. (Nomenclature of Ehrlichial genera anterior to 2001 is still used in this picture). The neighbor-joining method was used to construct the phylogenetic tree using ClustalW program. The numbers at nodes are the proportions of 1000 bootstrap resamplings that support the topology shown. The rectangular cladogram was drawn by the Tree View program, ant it provided tree topology only. The lengths of both the vertical lines and the horizontal lines are not significant.

ANAPLASMA PLATYS (EHRLICHIA PLATYS) INFECTION IN A DOG IN FRANCE 89 PHOTO 1. Aspect of the surgical wound at day 1 : bleeding and hematoma. PHOTO 3. Many inclusions of Anaplasma platys in a giant platelet (blood, diff quik, x 1000, bar = 10 µm). PHOTO 2. Mononucleosis, and several platelets containing Anaplasma platys morulae ; one of the platelets is as large as RBC (arrow) (blood, diff quik, x 1000, bar = 10 µm). PHOTO 4. A parasitized platelet being digested in a macrophage : Anaplasma platys inclusions are visible in the phagocytosis vacuole (blood, diff quik, x 1000, bar = 10 µm). When more cases of infection by the french strain of A. platys will be diagnosed by PCR, it will be inte-resting to compare the severity of clinical signs with descriptions from other countries. As A. platys is not cultivated, we could not perform an IFA test for this organism. A seroconversion was noticed between days 1 and 32 for the agent of HGE and for A. equi. As these agents are closely related to A. platys (fig. 1) [11, 41], this seroconversion probably results from a serologic cross-reaction, although it is reported that A. platys usually does not cross-react with A. equi [38]. Another seroconversion, for E. chaffeensis, obviously results from a cross-reaction, as PCR for this agent was negative at both days 1 and 32. Another possibility would be that the dog was acutely infected at the same time by both A. platys and an organism of the A. equi- A. phagocytophila-hge group. This is possible, as co-infections by several «Ehrlichieae» are common in the dog [6, 19, 27], but unlikely as it seems that A. platys and the HGE group agents are transmitted by different species of ticks [9, 21, 31]. A clinical improvement happened within 12 hours after initiation of doxycycline therapy, with resolution of fever and bleeding disorders. Nevertheless, it took 12 more days before the dog recovered completely, and there were apparent «relapses» later : this unusual evolution is probably due to the intercurrent hypoadrenocorticism, as on day 32, after 15 days of doxycycline therapy, A. platys DNA could not be detected by PCR, while retrospective dosages on stored sera revealed that a high kalemia was already present. Negativation of PCR on day 32 is in agreement with a recent publication [10], indicating that unlike in infection by E. canis, short doxycycline treatments (8 days) seem to completely eliminate A. platys. In this case, fever and bleeding disorders were certainly due to A. platys alone, while persistent tiredness and so-called relapses were caused by hypoadrenocorticism. As for many endocrinologic diseases, evolution of hypoadrenocorticism is often chronic, and the condition remains sub-clinical as long as compensating mechanisms exist in the dog. Surgery and an A. platys infection could have upset this balance, and precipitated a clinical hypoadrenocorticism un this case.

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