Successful Treatment and Management of Canine Ehrlichiosis-Leishmaniosis-Heartworm Comorbidity

Background : Canine vector borne diseases (CVBD) are common in tropical countries where the climate favors arthropods abundance. Comorbidity with one or more CVBD are reported as clinical complication and worsen prognostic. Canine visceral leishmaniosis (CanL) is an endemic zoonotic disease in Brazil caused by Leishmania infantum , with several res-trictions to canine treatment and suggestion of reservoirs euthanasia for disease control. Heartworm (HW) is a helminthic disease caused by Dirofilaria immitis infection in dogs. It is a chronic heart disease, which can lead to death by congestive heart failure. Canine ehrlichiosis (CE) is caused by Ehrlichia canis bacterial infection with a zoonotic potential and fatal to dogs in acute and chronic presentations. Exposed the above, this study aims to describe a successful treatment and CanL, microfilaria, cytology Ehrlichia and microfilaria. to promote Th1 cytokine profile as INF-γ , IL-2, IL-12, and TNF-α . We used an immunostimulant protocol to favor polarization to the Th1 profile comprised by 30 days of domperidone protocol followed by a vaccine and an immunomodulator. Doxycycline was used successfully for Ehrlichia spp. and HE clearance after 2 treatment courses and 1 year of ivermectin every 15 days. The animal presented intermittent coughing episodes on the first treatment course, but no medical intervention was needed besides exercise restriction. Our report shows the successful management of one dog with CanL, CE and HE comorbidity. This success was possible due to early detection and good therapeutic choice.

Dogs with CanL or CE can be apparently healthy or present a broad of alterations including dermatitis, emesis, lymph nodes enlargement, and conjunctivitis. They are caused by Leishmania infantum and Ehrlichia canis infection, transmitted by Lutzomiya longipalpis sandfly and by Rhipicephalus sanguineus tick respectively [18,28].
Treatment of CanL needs assorting stage accordingly, since animals in early stages have good prognosis [6,28]. Additionally, dogs without clinical signs are related to lower transmission of the parasite to the vector [15]. Doxycycline is recommended to treat CE, it is efficient in clinical signs remission but can be repeated to clear infection [18,34].
Canine HW is caused by Dirofilaria immitis nematodes and transmitted by mosquito species, including Aedis, Anopheles, and Culex [9,29]. The adult worms parasitize pulmonary arteries and right heart chambers. Disease presentation will depend on parasite load, normally dogs remain without clinical signs with detectable microfilaria on blood smear, but cough and exercise intolerance can be observed [5]. Alternative slow killing treatment with macrocyclic lactone and doxycycline are reported [3,16].
These 3 CVBD cited can have silent infection or severe chronic diseases presentation. Therefore, the aim of this case report is to describe a successful treatment and management of a dog with CanL, CE, and HW comorbidity.

CASE
A 3-year-old male uncastrated black Labrador dog, weighing 35 kg, was admitted to the veterinary clinic due to immunochromatographic CanL positive test 1 (Dual Path Plataform ® ) performed by municipal zoonosis control center active surveillance in August 2014. Thereafter, dog was positive for CanL on IFAT (1:80), and ELISA, and for Ehrlichia spp. morulae and microfilaria on bone marrow cytology. Clinical exam showed a mild hair loss, popliteal lymph nodes enlargement, hyperemia mucous membranes, and intermittent eye discharge. Nonetheless the animal was active, normal temperature, and appetite. Total blood count, sera levels of creatinine, urea, and alanine aminotransferase were unremarkable, but microfilaria was observed on blood smear (Table 1 and Table 2).
An Ehrlichia spp. infection was reported one year before (2013), but no confirmatory diagnostic was done previously or after treatment. At the time, emesis, lack of appetites, alopecia, lymph node enlargement, and fever were detected on clinical exam. Dog was treated with intramuscular tetracycline injections for 21 days. Diagnostic and clinical management was done monitoring clinical improvement, platelet morphology and count (Table 1). Treatment was finished and the dog was healthy until August 2014.  No parasitological or laboratorial exams substantiate prescription of leishmanicidal drugs, despite positive in 3 serological tests (Table 2). Therefore, dog was categorized as CanL stage I and therapy was prescribed as follows: domperidone 4 [EMS ® -0.5 mg/kg, VO, SID for 30 days], allopurinol 3 [Medley ® -10 mg/kg, VO, SID until further recommendation]. Additionally, repellent collar 5 (Scalibor ® ), repellent spray 6 (Karflae Citronela ® ), and vitamin supplement 7 (Glicopan Pet ® ) were indicated. Dog showed intermittent emesis and coughs during this first protocol, even with exercise restriction.
In November 2014 (72 days of treatment), blood and bone marrow smear were negative for microfilaria (Table 2), but platelet count was lower compared to August 2014 and giant platelets were observed (Table 1). Serological tests for heartworm and CanL were positive, and bone marrow smear revealed  (Table 2).
After one year of clinical management, dog became consistently negative for all pathogens with persistent thrombocytopenia (Table 1 and 2). From 2015 until 2018, the patient did not show coughing, eye discharge or emesis, with exercise tolerance, appetite and healthy. In September 2018, dog was admitted to the clinic because of a lack of appetite and emesis. Acute pancreatitis was diagnosed by abdominal ultrasonography causing death of the animal with unremarkable total blood count and negative for all pathogens, including L. infantum whole blood PCR (Table 2).

DISCUSSION
This study shows a dog that lived for years after CanL, HW, and CE comorbidity remission after treatment according to the literature. These diseases have severe chronical presentations but favorable prognosis in early diagnosis [6, 28]. Hence, a mammal infected by one organism can be more susceptible to infection with the other. Dirofilaria immitis can immunomodulate the host by increasing Th2 profile which downregulates Th1 response, favoring an infection by intracellular pathogens, such as Ehrlichia spp. and Leishmania spp. [17]. On our report, the order of events cannot be stablished, but certainly these CVBD are favored by climate, vector/pathogen abundance and/or host immunomodulation.
The therapeutic protocol iniciated by staging the CanL which the patient can be classified in 4 or 5 stages. According Leishvet [28] and Brasileish group [6], our patient had good prognosis classified as stage I and stage II, respectively. Recommended treatment ranges from scientific neglect to allopurinol, immunotherapy, immunomodulation, and miltefosine [6,28]. Since the patient had clinical signs, allopurinol was prescribed as a well-established drug for CanL.  Marbofloxacin was added in therapeutic as an alternative drug for CanL due to its high safety drug in clinical improvement of infected dogs with and without renal disease [22,25]. Furthermore, in vitro effectiveness against L. infantum strains was reported [1,11]. Domperidone was used on recommended lowsecure dose [0.5 mg/kg, VO, SID, for 30 days] for its results on clinical remission and prevention of CanL. This drug increases prolactin serum level, and this hormone has been related to promote Th1 cytokine profile as INF-γ, IL-2, IL-12, and TNF-α [26,33]. Immunotherapeutic approaches are reported as efficient on preventing disease progression in CanL stages I and II [30]. These protocols are based on switching Treg or Th2 to a Th1 profile, enhancing parasite clearance [23,24]. Here we used an immune stimulant protocol to polarize Th1 patient response comprised by 30 days of domperidone protocol followed by a vaccine with A2 Leishmania antigen conjugated with saponin (Leishtec ® ), an immunomodulator (Infervac ® ), with Escherichia coli lipopolysaccharide and inactivated cells of Propionibacterium acnes. This protocol was repeated annually.
Prognostic of CE and HW is good when detected early and doxycycline is a recommended drug for CE and HW, thus was prescribed on the initial treatment. Doxycycline is efficient for CE in clinical improvement but may not clear infection in one treatment course. Hence, repeating the treatment is suggested before using alternative drug since Ehrlichia spp. resistance to tetracycline derivatives was not reported [18]. On our study, prescribed protocol for Ehrlichia spp. did not clear the bacterial infection in one course, therefore it was repeated after two months being successful despite lowering the dose.
A long-term alternative treatment for HW is association of doxycycline with ivermectin [21]. Here we report a patient diagnosed with D. immitis by visualization of microfilaria on blood smear (Table  2). Echodopplercardiography was done in September 2014, but no adult worms were visualized. The alternative treatment chosen suggests ivermectin continuous use until seroconversion, since microfilaremia clearance occurs before death of adult worms.
On our case, doxycycline was used successfully for Ehrlichia spp. and HE clearance after two treatment courses and one year of ivermectin every 15 days. The animal presented intermittent coughing episodes on the first treatment course, but no medical intervention was needed besides exercise restriction. It is noteworthy the persistent thrombocytopenia after detection and treatment of the comorbidity (Table 1). Low platelet count is reported in E. canis, D. immitis, and L. infantum infection and several mechanisms are described such as immune mediated destruction, dysfunction, splenic sequestration via complement biding and vasculitis [7,12,20,27]. The exact mechanism that occurred on this report was not investigated. Regardless of the low platelet count, as the dog became clinically healthy and negative on all tests used, therapeutic intervention with ivermectin or allopurinol were not supported after one year.
Our report shows the successful management of one dog with CanL, CE and HE comorbidity. This success was possible due to early detection and good therapeutic choice. The authors also reinforce the importance of performing differential diagnosis for CVBD in endemic areas, regardless of one infection detected, other pathogens may infect the same animal, and treatment success will depend on commitment of veterinary practitioner to find these pathogens. Additionally, prevent vectors on an infected/suspected animal is ethically fundamental to avoid zoonotic transmission.