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Apr 4, 2009 - Animals in group 1 were treated with a combination of subcutane- ous meglumine antimoniate (Glucantime; Merial) at the standard dose.
SHORT COMMUNICATIONS Effects of therapy on haemostasis in dogs infected with Leishmania infantum, Ehrlichia canis, or both combined L. Cortese, A. Pelagalli, D. Piantedosi, A. Cestaro, A. Di Loria, P. Lombardi, L. Avallone, P. Ciaramella CANINE leishmaniosis and canine ehrlichiosis are well-recognised infectious diseases in the Mediterranean. In the same area, coinfection with Leishmania and Ehrlichia is very frequently seen too. Haemostatic disorders (epistaxis, haematuria and haemorrhagic diarrhoea) are common findings in both diseases (Bulla and others 2004). Ciaramella and others (2005) showed that haemostatic alterations during canine leishmaniosis were related to the severity of clinical signs and to thrombocytopenia, while Harrus and others (1996) observed platelet dysfunction in experimental acute canine ehrlichiosis. Recently, a reduction of the platelet aggregation response in dogs coinfected with Leishmania and Ehrlichia was reported; platelet aggregation was lower in comparison to healthy dogs and those with leishmaniosis and ehrlichiosis (Cortese and others 2006). Data are only available for haemostatic abnormalities during therapy for canine leishmaniosis. No data are available for haemostatic abnormalities during therapy for dogs with canine ehrlichiosis or those coinfected with Leishmania and Ehrlichia (Cortese and others 2008). The aim of the present study was to investigate platelet aggregation and secondary haemostasis in dogs naturally infected with L infantum, E canis or both combined during specific therapy. Thirty naturally infected male and female dogs of different breeds aged between four and nine years were included in the clinical trial. A control group of 10 healthy dogs was also studied. Sick animals had not received anti-Leishmania or anti-Ehrlichia therapy within the previous eight months and were randomly divided into three groups of 10 animals. Only dogs that showed a normal platelet count were included in the trial. All the animals in group 1 (canine leishmaniosis) showed typical signs of the disease (weight loss, plate mucous membranes, dry exfoliative dermatitis, splenomegaly and lymphadenomegaly), while two of the animals in group 2 (canine ehrlichiosis) showed splenomegaly and hyperthermia. Finally, Leishmania and Ehrlichia-coinfected dogs (group 3) exhibited the same clinical signs as those observed in group 1, plus epistaxis in three dogs.

Veterinary Record (2009) 164, 433-434 L. Cortese, DVM, PhD, D. Piantedosi, DVM, PhD, A. Di Loria, DVM, PhD, P. Ciaramella, DVM, Dipartimento di Scienze Cliniche Veterinarie – Sezione di Clinica Medica,

A. Pelagalli, PharmD, PhD, A. Cestaro, DVM, P. Lombardi, DVM, L. Avallone, DVM, Dipartimento di Strutture, Funzioni e Technologie Biologiche, Facoltà di Medicina Veterinaria, Università degli Studi di Napoli Federico II, Via F. Delpino 1, 80137 Napoli, Italy Correspondence to Mr Lombardi, e-mail: [email protected]

the VETERINARY RECORD | April 4, 2009

Animals in group 1 were treated with a combination of subcutaneous meglumine antimoniate (Glucantime; Merial) at the standard dose of 50 mg/kg twice daily for 30 days and 15 mg/kg allopurinol (Zyloric; GlaxoSmithKline) twice daily for 30 days. Animals from group 2 were treated with 10 mg/kg oral doxycycline monohydrate (Vibravet; Pfizer) once a day for 30 days. Animals in group 3 were treated for 30 days with a combination of the therapy given to group 1 and 2. A physical examination, complete blood cell count and biochemical analysis was performed for each dog. An immunofluorescent antibody test was used to confirm the clinical diagnosis of canine leishmaniosis (≥1/160) and canine ehrlichiosis (≥1/800). Leishmania was observed in Giemsa-stained fine-needle aspirates of bone marrow and/or lymph nodes. Blood was collected by jugular venipuncture from all dogs before therapy began (time 0), and from dogs in groups 1, 2 and 3 15, 30 and 60 days after therapy began. Blood samples were placed in plastic tubes containing a separator gel to obtain serum for clinical chemistry and in anticoagulant plastic tubes with sodium citrate (3·8 per cent) for haematological parameters, platelet aggregation assay and coagulation factors. Prothrombin time (Thromborel S; Dade Behring), activated partial thromboplastin time (Pathromtin SL; Dade Behring) and fibrinogen (Multifibren; Dade Behring), known as the Clauss method, were determined using a semiautomatic coagulometer (CoaData 1000; Labor BioMedical Technologies). Platelet aggregation was measured by a turbidimetric method, as reported by Lombardi and others (1999) using 0·5 to 10 µm adenosine 5’-diphosphate (ADP) and 5 to 200 µg/ml collagen type I calf skin as agonists. The Student-NewmanKeuls multiple comparisons test was used for statistical evaluation. P≤0·01 was considered significant. Dogs in groups 1 and 2 showed good clinical improvement two to three weeks after treatment, while all the dogs coinfected with Leishmania and Ehrlichia showed a transient, moderate clinical improvement. In fact, epistaxis and skin lesions regressed but splenomegaly and weight loss persisted in these dogs. All haematological parameters were in the normal range and showed no statistical differences. Table 1 shows the results of platelet aggregation obtained using the highest dose of agonists (ADP 10 µM and collagen 200 mg/ml) at day 0 and 60 days after therapy began. Results showed a significant improvement of the platelet aggregation profile at 60 days after the beginning of treatment in both group 1 and group 2. On the contrary, no significant effect was detected for group 3, the coinfected dogs. Platelet aggregation after 60 days of therapy was still significantly lower when compared with that of the control group and the collagen agonist seemed to be more sensitive in detecting this difference. Such a result is in agreement with previous studies that described differences within agonists due to specific receptor binding (Cortese and others 2008). The failure of combined therapy during Ehrlichia infection, and Leishmania and Ehrlichia coinfection in improving platelet aggregation is difficult to explain. A synergism between L infantum and E canis in altering platelet function could play an important role in the efficacy of the therapy for the dogs with dual infection. It has been shown that antiplatelet antibodies can be detected both in canine ehrlichiosis and canine leishmaniosis (Waner

TABLE 1: Percentage of platelet aggregation obtained using 10 µM adenosine 5’-diphosphate (ADP) and 200 µg/ml collagen at day 0 and day 60 after the beginning of therapy ADP 10 µM Control CL CE CL+CE

Day 0 73·0±2·4† 65·4±2·2† 58·8±3·6†

94·1±2·9 Day 60 87·4±2·7*† 75·0±3·2*† 61·7±3·3†

Collagen 200 µg/ml Day 0 67·3±2·1† 62·0±2·4† 51·3±2·9†

* P