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Doi:10.1016/j.tvjl.2007.09.014

Available online at www.sciencedirect.com The Veterinary Journal xxx (2007) xxx–xxx Use of domperidone in the treatment of canine visceral leishmaniasis: P. Go´mez-Ochoa a,*, J.A. Castillo a, M. Gasco´n a, J.J. Zarate a, F. Alvarez b, C.G. Couto b a Department of Animal Pathology, Veterinary Faculty of Zaragoza, c/ Miguel Servet 177, CP 50013 Zaragoza, Spain b Veterinary Teaching Hospital ,Department of Animal Pathology, College of Veterinary Medicine, The Ohio State University, 601 Vernon L. Tharp Street, Columbus, 43210 OH, United States The aim of this study was to evaluate the effects of domperidone, a dopamine D2 receptor antagonist, in dogs naturally infected by Leishmania infantum. Ninety-eight dogs were treated with single-agent domperidone at 1 mg/kg twice a day orally for 1 month. Clin-ical, serological, biochemical and immunological examinations were conducted for the following 12 months. Domperidone was effec-tive in controlling and reducing clinical signs and antibody titre. Significant decreases in reciprocal serum antibodies were seen in74.3% of the dogs with mild clinical signs and 40% of the dogs became seronegative. In dogs with several clinical signs and high anti-body titres, clinical improvement occurred in 86% of animals and the reciprocal serum antibody titres decreased in 38% of these dogs.
A significant increase was noted in the immune cellular status, as measured by the leishmanin skin test and a lymphocyte proliferationassay.
Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: Leishmaniasis; Immunomodulation; Domperidone the most frequently used treatment regime,supported by much clinical evidence, is long-term adminis- Canine leishmaniasis is widespread in the Mediterra- tration of allopurinol after an initial course of meglumine nean basin. It is transmitted by the sandfly Phlebotomus antimoniate. One suggested protocol consists of an initial perniciosus, and is a zoonosis considered by the WHO as treatment with both drugs for a minimum of 3 weeks fol- an emergent disease; indeed, every year some 500,000 lowed by long-term allopurinol treatment new cases arise in areas in which health services are poorly developed (). The clinical signs of leishmaniasis Virtually no treatment will completely eliminate para- in naturally infected dogs are variable, ranging from sites from the infected animal, and even if temporary clin- asymptomatic dogs with only mild lymphadenopathy or ical remission is achieved a relapse is to be expected weeks skin lesions, to dogs with ulceration, bleeding complica- tions, anemia, cachexia, and renal failure ( Although there are many protocols using several drugs The susceptibility of the leishmania parasite to antimonials such as aminosidine, amphotericin B, meglumine antimoni- ) and aminosidine ) decrease progres-sively ). The limitations of antimonialtherapy in dogs (induction of parasite resistance, lack of * Corresponding author. Tel.: +34 976761633; fax: +34 976761612.
parasitological cure, toxicity and expense) demand more 1090-0233/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tvjl.2007.09.014 Please cite this article in press as: Go´mez-Ochoa, P., et al., Use of domperidone in the treatment of canine visceral leishmaniasis.,The Veterinary Journal (2007), doi:10.1016/j.tvjl.2007.09.014 P. Go´mez-Ochoa et al. / The Veterinary Journal xxx (2007) xxx–xxx affordable alternatives with greater efficacy ( nopathy and skin lesions. Twenty-eight dogs were included in Group B; ). The adverse effects of existing drugs (antimo- the inclusion criteria were the presence of multisystemic clinical signs(exfoliative dermatitis, alopecia, lymphadenopathy, pale mucous mem- nials, amphotericin B and aminosidine) provide an addi- branes, weight loss, anterior uveitis, and onycogriphosis) and high anti- At the time of diagnosis, each dog underwent popliteal lymph node ment for parenteral administration and/or high cost may and bone marrow aspiration from the costochondral junction for culture; reduce the rate of compliance of the dogs’ owners during specimens were inoculated into Novy–MacNeal–Nicolle (NNN) culturemedium and incubated at 28 °C for 1 month. Culture tubes were examined every 10 days for the presence of motile promastigotes by ordinary light Leishmaniasis in humans and in dogs should be treated microscopy. Initially, 117 dogs positive for Leishmania were included in with drugs that act via separate mechanisms in order to the study but 19 were later excluded (11/19 were serologically positive but minimise the danger of generating resistant parasite strains Leishmania was not isolated in tissue culture; the other 8/19 showed concurrent diseases (six with ehrlichiosis and one with chronic otitis), orwere being treated with other drugs (one with corticosteroids and the other course of leishmaniasis are variable, and the host’s immune system plays a pivotal role in the establishment of infection All experiments were performed in accordance with the University of Zaragoza Ethics Committee guidelines and the owners gave prior informed consent. The dogs were fully monitored and treatment could be changed to conventional treatment could be a therapeutic key to suc- antimoniate meglumine/allopurinol at the owner’s request if clinical orbiochemical impairment was noticed. After inclusion, domperidone (EV- 4820 Esteve Veterinary, Dr. Esteve Labs. SA) was orally administered at a We have previously proposed a protective role of lacta- dosage of 1 mg/kg every 12 h for 1 month as the sole drug.
tion against leishmaniasis in the Syrian Hamster model Animals were checked on days 0, 15, 30, 60, 90, 120, 150, 180, 270 and 360 after the treatment had started, and clinical signs and lesions associ- ability to kill the parasite seems to be related to the hyper- ated with leishmaniasis were evaluated and monitored. A complete bloodcount, biochemistry profile (liver, kidney and electrolytes), and serum prolactinaemic status during lactation. Prolactin has a cen- protein electrophoresis were performed on all dogs. DAT were also per- tral role in the immune reaction, but its mechanism of formed, with 1/800 used as the cut-off titre. To check the cellular immunity action is largely unknown. The hormone, whose main func- status the leishmanin skin test reaction (LST) was used on days 0, 90 and tion is to stimulate milk production in mammals, is now 360 and an in vitro lymphocyte proliferation assay (LPA) (cell prolifera- classified as a proinflammatory lymphocyte-derived cyto- tion detection kit III, Boehringer/Roche) on days 0, 30, 180 and 360 afterthe treatment commenced.
The LST was performed as described elsewhere ). Briefly, 0.1 mL of a suspension of 3 · 108 inactivated Leishmania CD4+Th1 subsets, and in interleukin (IL)-2, IL-12, inter- infantum (MHOM/FR/78/LEM75) promastigotes per millilitre diluted in feron (INF)-c and tumour necrosis factor (TNF)-a release, 0.4% phenol–saline were injected intradermally (ID) in the skin of the right leading to a natural killer (NK) cell and macrophage acti- groin. The same amount of 0.4% phenol–saline was injected ID in the skinof the left groin as a negative control. An induration diameter of >5 mm vation, followed by a decrease in CD4+Th2 subsets and LPA was performed using the whole blood microassay technique previously described for dogs In short, 200 lL of The main goal of the present study was to evaluate the heparinised blood were diluted in 3 mL of RPMI medium supplemented effects of a hyperprolactinaemic drug in dogs with leish- with 2 mM glutamine, 100 lg of streptomycin and 100 IU penicillin/mL.
One hundred microlitres of diluted blood were placed in triplicate in 96 maniasis. We tested domperidone (EV-4820 Esteve Veteri- well flat-bottomed culture plates to which Leishmania antigen was added nary, Dr. Esteve Labs. SA), a gastric prokinetic and anti- at concentrations of 1.0, 1.25 and 5 lg/mL (100 lL/well) to standardise emetic drug that is also a dopamine D2 receptor antagonist the test. The results of the optimal concentration (1 lg/mL) were carried that results in the release of serotonin, which in turn stim- out in duplicate. The cells were incubated at 37 °C in 5% O2. The prolif- ulates prolactin production. It has been well documented eration was checked using 5-bromo-20-deoxyuridine (BrdU) () added over 18 h on day 4 after incubation. Cell proliferation was that this drug significantly increases serum prolactin con- determined using an ELISA technique according to the manufacturer’s instructions (BrdU, cell proliferation detection kit III, Boehringer/Roche).
The cells that had incorporated BrdU to the DNA were recognised using a monoclonal antibody against BrdU and measured by an enzyme conju-gated second antibody. Cell proliferation was expressed as a stimulation Ninety-eight dogs (males and females of various breeds and ages index (SI), which represents differences in optical density (OD) between ranging from 2–13 years) admitted to the Veterinary Faculty Hospital of the stimulated and non-stimulated cultures; (SI) = (OD stimulated the University of Zaragoza and naturally infected with leishmaniasis were cells À OD background)/(OD non-stimulated cells À OD background).
included in the study. Sixty-two were housed outdoors and 36 indoors.
The absorbance was read using a plate reader at 405 nm, considering Deltamethrin-impregnated collars were fitted on all dogs, which were kept in the same conditions throughout the study period.
The data were submitted to statistical analysis using the Instat pro- Due to the broad spectrum of clinical signs, haematological and bio- gram (GraphPad). Statistical methods were chosen according to the data chemical abnormalities that are found in dogs with leishmaniasis, we features. The Gaussian distribution of the data was assessed with the divided the affected dogs into two groups. Group A included 70 dogs with Kolmogorov–Smirnov test. A one-way analysis of variance for repeated- low antibody titres (1/800–1/1600 as measured with a Direct Agglutina- measures was used to assess differences between males and females, and to tion Test [DAT]); of these 70 dogs, 34 (48.57%) had no obvious clinical compare differences in all the variables between day 0 and the rest of the signs, and the remaining 36 animals had clinically relevant lymphade- Please cite this article in press as: Go´mez-Ochoa, P., et al., Use of domperidone in the treatment of canine visceral leishmaniasis.,The Veterinary Journal (2007), doi:10.1016/j.tvjl.2007.09.014 P. Go´mez-Ochoa et al. / The Veterinary Journal xxx (2007) xxx–xxx Table 1Induration diameter in millimetres (mean ± SD) 72 h after intradermalinjection. A diameter >5 mm was considered positive No differences were found between males and females in any of the variables analysed so all the dogs in each group were analysed together. At the fifth check point (day 90) all animals from group A were normal in their physical exam- ination and no lymphadenopathy or skin lesions werefound. An increase in antibody titre was not seen in any icant increase in this group comparing day 0 to days 30, dog. There was a decrease in titre in 52/70 (74.3%) of the 180 and 360 after treatment had started (P < 0.001).
dogs, and 28/70 (40%) of the dogs seroconverted (i.e.
became negative) at the end of the study (Thisdecrease was very significant (P < 0.001) when comparing day 0 to days 90, 120, 150, 180, 270 and 360 after treatmenthad commenced. The results of clinicopathological testing Due to the current lack of an effective therapy for canine leishmaniasis, new drugs, delivery systems and treatment A significant increase in diameter induration was found strategies are necessary to achieve a cure in infected dogs in group A for LST (P < 0.001) between days 0 and 90.
However no differences were noted between days 90 and ing domperidone against canine leishmaniasis. Prior to this 360. The LPA showed a significant increase (P < 0.001) study, we evaluated the dosage scheme in an experimental in the SI in this group comparing day 0 against days 30, dog population (20 Beagle dogs) and, using a commercial 180 and 360 after treatment had started. This response ELISA kit, demonstrated a significant increase in serum remained stable during all the year and no differences were prolactin concentration (P. Go´mez-Ochoa et al., unpub- found comparing days 30, 180 and 360.
In animals from group B, two dogs suffering from Dogs from group A showed better results than those in chronic renal failure died, being euthanased at the owner’s group B, probably because they were at an earlier stage of request 15 and 21 days, respectively, after entering the the disease. In addition, anti-leishmanial antibody levels study. Clinical signs remitted in 24/28 (85.7%) dogs whose did not increase in the majority of the dogs in the 3– clinicopathological abnormalities improved during the 5 month period post-treatment, indicating a regression of study period. Antibody titre increases in 2/28 (7.2%) of the dogs remained stable in 16/28 (57.1%), and decreased in 10/28 (35.7%) by the end of the year (). This associated with the dissemination of the parasite to differ- decrease was significant (P < 0.05) when comparing day 0 to days 90, 120, 150, 180, 270 and 360 after treatment ), therefore it might be hypothesised that the absence of this rise in antibody levels implies there is no Significant differences were found in the induration diameter in group B between days 0 and 360, but no differ- An evident increase in cellular immunity status was ences were found when comparing day 0 to day 90, nor found in both groups. This did not lead to a complete cure when comparing day 90 to day 360 (means ± SD are in all cases, implying that cellular stimulation is necessary shown in ). The SI index from LPA showed a signif- but not sufficient alone to cause improvement (). LST and LPA, the testused to check immune status, are more feasible in a clinicaltrial and, together with the IFN-c cytopathic effect inhibi-tion bioassay (IFNB), are considered to be the most suit-able tests for checking cellular immunity in canineleishmaniasis ). The resultsfound using LPA were in agreement with recent studiesin which no significant correlation was found between spe-cific response and clinical score ). The LST, reported to be the most useful test toevaluate specific cellular immunity ) may however cause interference. Moreover, therepeated use of LST could affect the humoral and cellularresponses in Leishmania-infected dogs and studies assessingthe effect of multiple doses of LST in dogs are necessary.
Fig. 1. Graph showing the percentage of antibodies titre tendency at theend of the year in both groups. Negative titres were considered <1/400 In the present study we did not include a positive control using the Direct Agglutination test.
group. We felt that its inclusion in this kind of clinical assay Please cite this article in press as: Go´mez-Ochoa, P., et al., Use of domperidone in the treatment of canine visceral leishmaniasis.,The Veterinary Journal (2007), doi:10.1016/j.tvjl.2007.09.014 P. Go´mez-Ochoa et al. / The Veterinary Journal xxx (2007) xxx–xxx was not ethically admissible, since there are several refer- clinical trials are needed to determine the ideal dose sche- ences regarding to leishmaniasis disease progression ( dule and to investigate whether the dogs remain infectious by factors such as the virulence and susceptibility to drugsof the strain parasite and the genetic susceptibility of indi- vidual dogs (). A long-term study hasrevealed that 15% of all infected dogs were able to circum- Pablo Go´mez would like to thank the Government of vent the establishment of disease or demonstrate spontane- Arago´n for a research grant and Dr. Esteve Labs. SA for their continuous support since the beginning of this problem without a control group, it was decided to include Project. Parts of this work have received the VI 3M Foun- in the study a large number of animals which were serolog- dation Innovation award in the Public Health field and the ically (DAT) and parasitologically (NNN cultures) positive.
Francisco Ferna´ndez Lo´pez Research award.
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