Geographical Distribution, Clinical Presentation, Treatment and Prevention of Canine Leishmaniosis in Portugal: a 2007 Field Survey

RPCV (2010) 109 (573-576) 21-29 REVISTA PORTUGUESA DE CIÊNCIAS VETERINÁRIAS

Geographical distribution, clinical presentation, treatment and prevention of canine leishmaniosis in Portugal: a 2007 field survey

Resultados de um inquérito realizado em 2007 sobre a distribuição geográfica, apresentação clínica, tratamento e prevenção da leishmaniose canina em Portugal

Ana M. Oliveira1*, Suraya Diaz2, Cristina Santos2, Patrick Bourdeau3, Isabel Pereira da Fonseca2

1Faculdade de Medicina Veterinária, Universidade Lusófona de Humanidades e Tecnologias, Campo Grande 376, 1749-024 Lisboa, Portugal 2CIISA, Faculdade de Medicina Veterinária, Pólo Universitário Alto da Ajuda, 1300-477 Lisboa, Portugal 3Ecole Vétérinaire de Nantes, Atlanpole, La Chantrerie, BP 40706, Nantes, France

Resumo: A leishmaniose canina, doença endémica em Portugal, clinical presentation, diagnosis, treatment and prevention é causada por Leishmania infantum, sendo transmitida por offered by the Portuguese clinicians throughout the country. Phlebotomus perniciosus e P. ariasi. Através de um inquérito Material and methods: 790 written questionnaires were sent, nacional, realizado em 2007, os autores pretenderam caracteri- one per clinic, to all licensed small animal clinics in Portugal. zar o panorama da doença, determinando as áreas geográficas From those 17.8% (141) clinicians answered the questionnaire. recententemente afectadas, e descrevendo a apresentação 93% of the clinicians had consulting patients with leishmanio- clínica, diagnóstico, tratamento e prevenção referidos pelos sis. The districts reported to be affected were Beja, Bragança, clínicos do País. Foram enviados 790 questionários, um para Castelo Branco, Coimbra, Évora, Faro, Guarda, Leiria, Lisboa, cada uma das clínicas de pequenos animais registadas em Madeira, Portalegre, Porto, Santarém, Setúbal, Viana do Portugal. Obtiveram-se 17,8% (141) de respostas, tendo 93% Castelo, Vila Real and Viseu. Several newly affected areas were dos veterinários referido casos de leishmaniose. A doença foi reported in these districts. The most common clinical signs mencionada nos distritos de Beja, Bragança, Castelo Branco, recognized were weight loss/muscle wasting, alopecia and lym- Coimbra, Évora, Faro, Guarda, Leiria, Lisboa, Madeira, phadenopathy. For the diagnosis indirect immunofluorescence Portalegre, Porto, Santarém, Setúbal, Viana do Castelo, Vila was commonly used and the first treatment option was allopuri- Real e Viseu. Nestes distritos foram referidas novas áreas onde nol which was very frequently associated with the meglumine recentemente surgiu leishmaniose canina. Os sinais clínicos antiamoniate. Leishmaniosis is still endemic in the same areas mais apontados foram perda de peso/atrofia muscular, alopécia as before and according to our data, it is likely that new areas e linfadenopatia. No diagnóstico, a técnica de imunofluores- are affected which would mean that the disease is spreading in cência indirecta foi comummente referida. A primeira opção Portugal. The clinical signs in the canine population are similar no tratamento foi alopurinol, frequentemente associado a to the typical leishmaniosis presentation and, in general, the antimoniato de meglumina. Em Portugal, a leishmaniose canina diagnostic tests, treatment protocols and prevention measures continua a ser endémica em áreas anteriormente conhecidas e offered by the veterinary clinicians are according to the scien- de acordo com os resultados agora obtidos, verificaram-se tific evidence available at the time of the survey. novas áreas afectadas devido à dispersão do parasita. Os sinais clínicos referidos são comuns na apresentação típica da leishmaniose e em geral, os testes de diagnóstico, os protocolos de tratamento e as medidas de prevenção preconizados pelos Introduction clínicos veterinários, estiveram de acordo com os conhecimentos científicos disponíveis à data do inquérito. Leishmaniosis are parasitic diseases caused by pro- Summary: Canine leishmaniosis (formerly leishmaniasis) is an tozoans of the genus Leishmania typically transmitted endemic disease in Portugal, caused by Leishmania infantum by phlebotomine insects (Thomaz-Soccol et al., and transmitted by Phlebotomus perniciosus and P. ariasi. We 1993). Depending on the Leishmania species they may performed a field survey with the following purposes: first, we infect humans and other animals (Maia et al., 2008; aimed to determine the geographical areas affected by Sastre et al., 2008), especially dog, that constitute the leishmaniosis including spreading of the parasite to areas not main reservoir for visceral zoonotic leishmaniosis due previously affected; second, we aimed to characterize the to L. infantum. Leishmaniosis is a common canine disease in the Mediterranean Basin, Latin America and an emergent disease in North America (Duprey *Correspondência: [email protected] et al., 2006). Human visceral leishmaniosis can be

21 Oliveira AM et al. RPCV (2010) 109 (573-576) 21-29 acquired through anthroponotic natural and artificial location and the number of patients consulted per cycles as well as through zoonotic cycle were the dog year; b) assessment of the origin and number of cases plays the main role (Marques et al., 2007). Due to the diagnosed with leishmaniosis; c) clinical presentation zoonotic nature of the disease infected dogs represent of the disease; d) diagnostic methods; e) treatment a problem for both veterinary and public health. protocols; f) prognosis; g) prevention; and h) zoonotic It has been estimated that at least 2.5 million dogs aspects. We also enclosed a prepaid envelope to be are infected in southwestern Europe alone (Moreno used to return the questionnaire. The deadline for and Alvar, 2002). In Portugal, leishmaniosis is caused reception of replies was October 2007. by several zymodemes of Leishmania infantum being MON-1 the most prevalent both in dogs and human beings (Baptista-Fernandes et al., 2007). The parasite Results is transmitted by females of Phlebotomus, especially of the species P. perniciosus and P. ariasi in Portugal A total of 141 (17.8%) answered questionnaires (Campino et al., 2006). The disease is endemic in were returned by mail. The results are presented in 3 several areas of the country such as Alto-Douro parts that cover different topics. (Abranches et al., 1992; Cardoso et al., 2004), Lisbon (Cortes et al., 2007), Setúbal (Abranches et al., 1984), Part 1: Distribution of leishmaniosis in Portugal Loulé (Campino et al., 1995) and Évora. In the first part of the questionnaire, we aimed to Canine leishmaniosis is a chronic systemic disease characterize the veterinary practices according to characterized by a variety of clinical signs such as skin location and the number of patients consulted. Results lesions, lymphadenopathy, weight loss, anorexia, show that most of the practices consulted 1000-2500 ocular lesions, epistaxis, renal failure and diarrhea dogs per year (Chart 1), with most of them consulting (Baneth et al., 2008). The pathogeny of the disease is fewer then 10 dogs per day (Chart 2). dependent on several factors such as the breed Clinicians were asked to give an average of the total (Solano-Gallego et al., 2000), virulence of the strain number of leishmaniosis cases per year in the last 2 (Baptista-Fernandes et al., 2007), and immune years. The total number includes new cases and ongoing response of the host against the parasite (Cabral et al., cases. The results revealed that 27% of the practices 1998). It is currently accepted that a specific were consulting between 1-5 leishmaniosis cases per anti-Leishmania cellular immune response is protec- year, 21% of the practices consulting 20-50 cases and tive and a humoral immune response is associated 19% practices consulting 10-20 cases (Chart 3). with development and severity of clinical disease Clinicians were also asked if in their opinion the (Cabral et al., 1998; Solano-Gallego et al., 2000). number of new and ongoing cases had "decreased", Several treatments can be used to clinically improve "increased", or "stabilized" in the last 5 years. Most of the patient but parasitological cure is rarely achieved the clinicians agreed that the number of new cases and (Noli and Auxilia, 2005). The aim of this survey was to characterize the Chart 1 - Number of dogs consulted at least once a year (total current status of leishmaniosis in Portugal. First, we of 137 answers) aimed to determine the geographical areas affected by leishmaniosis including spreading of the parasite to areas not previously affected. Progression to new areas is an important issue in any endemic disease. Second, we aimed to characterize the standard of care in diagnosis, treatment and prevention offered by the clinicians in the clinics throughout the country. The study had a similar design to a survey done in other European countries in order to obtain comparable results (Bourdeau et al., 2004). Chart 2 - Number of dogs consulted per day (total of 141 answers) Material and methods

Seven hundred and ninety written questionnaires composed of 65 questions were sent to all licensed small animal clinics in Portugal including Madeira and Azores islands in June 2007. One questionnaire was sent per practice to avoid duplication of the data. The questionnaire covered the following topics: a) characterization of the veterinary clinic according to

22 Oliveira AM et al. RPCV (2010) 109 (573-576) 21-29

Chart 3 - Number of leishmaniosis cases per year (total of 140 ongoing cases had increased (Table 4). answers) The answers were grouped according to the 18 offi- cial Portuguese districts plus Azores and Madeira 27% Autonomous Regions instead of "Nomenclature for Territorial Units for Statistics" for easier comparison 0 with previous studies. Answers from all the districts 1-5 were received. Lisbon was the district with the higher 5-10 number of replies (25.95%), and Vila Real the district 10-20 with the lowest number of replies (0.71%). In each district clinicians were asked to name the areas 19% 20-50 ("localidades") where leishmaniosis was more preva- >50 lent (Table 5). Table 4 - Progression of the number of cases in the last 5 years Progression The number of cases No change in the number The number of cases Total of answers increased of cases decreased to this question Number of follow-ups 56 (51%) 43 (39%) 11 (10%) 110 Number of new cases 80 (71%) 26 (23%) 7 (6%) 113

Table 5 - Distribution of the answers according to the district where the veterinary clinic was located and the most affected areas in each district District nº answers received Most affected areas ("localidades") in each district Azores 3 (2.13%) No areas were mentioned. Aveiro 8 (5.67%) No areas were mentioned. Beja 3 (2.13%) No areas were mentioned. Braga 5 (3.55%) Póvoa Lanhoso and Braga. Bragança 1 (0.71%) Bragança, Alfândega da Fé, Vinhais, Vimioso and Macedo de Cavaleiros. Castelo Branco 2 (1.41%) Alcains, Fundão, Covilhã, Tortosendo, Alcaide, Farela, Barroca and Alpedrinha. Coimbra 10 (7.09%) Póvoa Midões, Mouronho, Azere, Arganil, Penacova, Montemor-o-Velho, Coimbra, Góis, Lousã, Miranda do Corvo, Lousã, Góis, Vila Nova de Poiares, Taveiro, Ribeira de Frades, São Martinho do Bispo, Assafarge, Almalaguês, Ceira, Tavaro, Lamas, Semide, Rio de Vide and Vila Nova. Évora 6 (4.26%) Vendas Novas, Landeira, Cortiçadas de Lavre, Aldeia da Serra, Freixo, Montoito and Évora. Faro 6 (4.26%) Enxerim, Armação de Pêra, Algoz, Messines, Lagoa, Albufeira, Silves, Armação de Pêra, S.B. Messines, Bensafrim, Sargaçal, Portelas, Chinicato, Odiáxere, Pechão, Quelfes, Marim, Moncarapacho, Olhão, Santa Clara, Ribeira de Odeceixe, Monchique, Odemira, Faro, Lagos, Portimão, Loulé and Monchique. Guarda 2 (1.41%) Celorico da Beira, Guarda, Belmonte, Fig. Castelo Rodrigo and Pinhel. Leiria 2 (1.41%) No areas were mentioned. Lisbon 38 (25.95%) S. João do Estoril, S. Pedro Estoril, Parede, Monte Estoril, Alto dos Gaios, Pontinha, Odivelas, Moscavide, Brandoa, Magoito, Colares, Cacém, Azenhas, Qta Grande, Loures, Belas, Sintra, Cascais, Estoril, Loures, Odivelas, S. A.Tojal, São Julião de Loures, Á-das-Lebres, Bucelas, Alenquer, Arruda dos Vinhos, Azambuja, Vila Franca de Xira, Merciana, Carcavelos, Loures a Malveira, Queluz, Caneças, Casal de Cambra, Ramada, Montemor, Casal Novo, Torres Vedras, Ericeira, Sta Cruz, A-dos-Cunhados, Turcifal, Vale do Tejo, Linha Cascais, Alvalade, Restelo, Amadora, Margem Sul do Tejo, Azambuja, Vale Paraíso, Aveiras, Sacavém and Trancão river valley. Madeira 2 (1.41%) No areas were mentioned Portalegre 7 (4.96%) Avis, Fronteira, Alcôrrego, Cano, Casa Branca, Montargil, Ponte de Sor, Foros de Arrão, Mora, Ponte de Sor, Avis, Alter do Chão, Crato, Nisa, Castelo de Vide, Crato, Alter do Chão, Portalegre, Alter do Chão, Charça, Cunheira, Benavila and Ponte de Sôr. Porto 15(10.64%) Castelo de Paiva, Paredes, Penafiel, Lousada, Paços de Ferreira, Cinfães, Resende, Marco de Canaveses, Baião and Amarante. Santarém 8 (5.67%) Chamusca, Golegã, Entroncamento, Torres Novas, Vila Nova da Barquinha, Alvega, Mouriscas, Abrantes, Rossio ao sul do Tejo, Pego, Cartaxo, Rio Maior, Azambuja, Vale de Santarém and Pontével. Setúbal 13 (9.22%) Setúbal, Palmela, Alcochete, Montijo, Moita, Seixal, Almada, Costa Caparica, Azeitão, Setúbal, Seixal, Palmela, Azeitão, Setúbal, Alcácer, Grândola, Seixal, Verdizela, Aroeira, Belverde, Fernão Ferro, Moita, Alhos Vedros, Baixa da Banheira and Barreiro. Viana do Castelo 3 (2.13%) No areas were mentioned Vila Real 1 (0.71%) Régua, Lamego, Vila Real, Alijó and Vila Pouca. Viseu 6 (4.26%) Mortágua, Sta Comba Dão, Tondela, Viseu, São Pedro do Sul, Oliveira de Frades, Vouzela, Nelas, Folgosa do Douro, Fontelo, Vacalar, São Joaninho, Armamar, Orgens, Mangualde and Campo. TOTAL 141 (100%)

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Most of clinicians (65%) considered their practice Part 2: Clinical presentation, diagnostic tests, to be located in an endemic area (Chart 6). In addition, treatment and prevention we asked if there were new areas affected by leishmaniosis. In seven districts, new areas were mentioned All the clinicians answered the question concerning (Table 7). In the other districts no areas were reported the clinical signs present in patients with leishmanio- to be affected. sis: weight loss/muscle atrophy (11%), alopecia (9%), lymphadenopathy (9%), fine scaling (8%), exfoliation Chart 6 - Percentage of clinics localized in an endemic area (8%), onichogriphosis (7%), nasal lesions (7%), anaemia (6%), renal insufficiency (4%), prostration (4%), skin ulceration (4%), pioderma (4%), eye lesions (4%), footpad lesions (3%), skin nodules (2%), epistaxis (2%) and arthritis (2%). Very rarely (<1%) diarrhea, fever, depigmentation and bone lesions were noticed. Some clinicians also referred to the presence of vomit, hematuria and spleen enlargement. Most of the clinicians suspect leishmaniosis based on the history and clinical signs. When leishmaniosis is suspected, specific tests are employed in order to confirm the diagnosis. The specific tests/analyses frequently employed for the diagnosis of this condition Table 7 - Distribution of the answers to the question "If you are in an endemic zone, which are the newly affected areas in your were indirect immunofluorescence assay (27%), district?" immunomigration (24%), ELISA (11%), proteino- District Newly affected areas gram (11%), lymph node fine needle aspirate (11%), Azores No area mentioned bone marrow fine needle aspirate (6%), PCR (6%), Aveiro No area mentioned histopathologic examination (1.7%), skin cytology Beja Moura (1.7%), immunohistochemistry (0.3%) and formogeli- Braga Prado, Amoras,Terras Bravas and Braga fication (0.3%). Most of the clinicians send samples to Bragança No area mentioned external laboratories (83%) although the immunomi- Castelo Branco No area mentioned gration testing is available commercially in Portugal Coimbra Vale de Miranda do Corvo, Ponte do Sótão as an in-house test (Witness Leishmania, Symbiotics). (Góis county), Pegos (Lousã county), When the samples were sent for an external laborato- Coimbra, Condeixa, Pampilhosa and Arganil ry, the majority of the clinicians preferred a veterinary Évora No area mentioned laboratory (77%) as opposed to a human laboratory. Faro No area mentioned Non-specific testing as an aid to diagnose leishmanio- Guarda Guarda sis were rarely used and these included total blood Leiria No area mentioned cell count, proteinogram, clinical biochemistry and Lisbon Óbidos urianalysis. Madeira No area mentioned For the treatment of disease the clinicians use Portalegre Mora allopurinol (84%) most of the time associated with Porto Amarante, Marco Canaveses and Paredes meglumine antiamoniate (68%). The protocols varied Santarém No area mentioned from 10-20 mg/kg/SID for allopurinol and when asso- Setúbal No area mentioned ciated with meglumine antiamoniate (100 mg/kg/SID Viana do Castelo No area mentioned for 1 month). Allopurinol was administered during a Vila Real No area mentioned minimum of 3 months to a life-long treatment and was Viseu No area mentioned considered a safe drug. Meglumine antiamoniate side effects were occasionally noticed (36%) and included vomiting, diarrhea, anorexia, lethargy and pain at the injection site. The other treatments (anfotericine B, For the clinicians not localized in an endemic area lomidine, ketoconazole, metronidazole, antibiotics) we asked the origin of the cases. The cases were orig- were rarely prescribed and miltefosine was not reported inated from the following areas: Trás-os-Montes, to be used by the clinicians. The occurrence of side Coimbra, Alijó, Serra da Estrela, Lisboa, Vale do Tejo, effects was a reason to stop the treatment for 89% of the Arrábida, Cascais, Estoril, Alentejo, Algarve. Some clinicians. Some clinicians (41%) associate the treat- cases were originated from Spain. If the practice was ment of leishmaniosis with other drugs like localized in Azores or Madeira islands the cases were vitamins, essential fatty acids, glucocorticoids and coming from from Portugal mainland. The reasons antibiotics. The glucorticoids are used in case of renal mentioned were traveling during holidays, weekends failure and/or lameness due to immune complex and hunting purposes. deposition and antibiotics are prescribed in case of con-

24 Oliveira AM et al. RPCV (2010) 109 (573-576) 21-29 comitant pyoderma. Vitamins and essential fatty acids The clinicians were asked to assess the frequency in are prescribed to improve the body condition and skin. which euthanasia was elected instead of treatment The decision to stop the treatment was based on using the words "never", "occasionally", "frequently" criteria like clinical examination, serology, proteino- or "always" (total of answers 132). The results gram, complete blood cell count, clinical chemistry, revealed that euthanasia was elected occasionally immunomigration and bone marrow smears. Most of (58%), frequently (23%), never (14%) or always (5%) the clinicians (80% from a total of 124 answers) used instead of the therapy. In most cases (64%, 119/141) more than one of the above criteria (total cumulative the owner suggested the euthanasia to the clinician. of answers 296). The criteria that the clinician found The reasons for euthanasia included renal insufficiency to be most important for the decision were clinical (26%), the cost of the treatment (22%), the presence of examination (32%), serology results (26%) and children in the household (15%), the presence of other complete blood cell count/clinical biochemistry (24%) dogs (10%) or elderly people (8%) at home. Less followed by less important criteria like proteinogram common reasons (<19%) included presence of (15%), immunomigration (2%) and bone marrow immunosupressed and HIV infected people, problems aspirate (1%). Serology testing for this purpose with treatment administration, blindness, poor quality included indirect immunoflourescence (49%, 27/55), of life, poorly informed owner, concomitant condi- immunomigration (38%, 27/55), ELISA (9%, 27/55) tions or no response to the treatment. and PCR (4%, 27/55). We also asked if other conditions were associated Our survey also included the assessment of the with the leishmaniosis. A percentage of 52% of the survival time for patients that received treatment. The clinicians (69/141) replied that other conditions were clinicians answered that most of the patients (58%, commonly diagnosed with patients with leishmaniosis. 117/141) that received treatment survived between Most of clinicians reported more than one disease 2 and 5 years and less frequently would live more than (total cumulative answers 109). The condition most 5 years (Chart 8). commonly mentioned was ehrlichiosis (40%) fol- We also asked the clinicians to refer the percentage lowed by dirofilariosis (19%) (Chart 10). of dogs that suffered euthanasia after receiving treatment for leishmaniosis. Most of clinicians (44/102) Chart 10 - Conditions associated with leishmaniosis (percent- ages based on a cumulative total of 109) answered that they are performing euthanasia in less then 10% of the treated patients (Chart 9).

Chart 8 - Survival time of the patients after receiving treatment (total 117 answers)

Part 3: Prevention of leishmaniosis and zoonotic aspects

Prevention with insecticides or insect repellents was recommended by 96% (135/141) of the clinicians if the owner lived in an endemic area and by 95% (134/141) of the clinicians if the owner was travelling Chart 9 - Percentage of euthanasia in cases that received to an endemic area. Other preventive measures recom- treatment (total of 102 answers) mended included avoiding walking the dog before dark or near wet areas and keeping the dog indoors during the night. The clinicians agreed that they would recommend a vaccine if there was one available. The vaccine would be used if the owner lived in or was travelling to an endemic area. The clinicians were asked if the owners were aware that leishmaniosis is a zoonotic condition. The clinicians answered that 60% (83/137) of the owners were rarely to occasionally aware of the situation and only 40% (54/137) were informed. The information was likely to be provided by the veterinarian (96%,

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132/137) and mainly clarified the mode of transmis- except Aveiro, Beja, Leiria, Viana do Castelo, Azores sion. Some clinicians also informed clients that and Madeira. Some of these districts have been children, immunosupressed patients or elderly people reported before as endemic areas: Alto-Douro inclu- can be susceptible. The veterinarian tended to inform ding Alijó (Abranches et al., 1992; Cardoso et al., the owner verbally. Rarely written information was 2004), Lisbon urban area (Cortes et al., 2007), Setúbal provided. district including Arrábida (Abranches et al., 1984), The risk of direct contact transmission was not a Loulé county (Campino et al., 1995) and Évora concern for the clinician or practice staff (68%, (Semião-Santos et al., 1995). 90/132), but was more a concern for the owner (68%, In our survey, according to some clinicians’ pers- 91/134). Finally, the clinicians were asked if they were pective, there are several newly affected areas in seven aware of cases in which the owner and the dog were districts. It is interesting to note that most of these simultaneously affect by leishmaniosis. Eight clini- areas are localized in the north of Portugal (Table 7). cians reported that they were aware of one case in the Dispersion of the disease was already recognized in last two years and two clinicians reported two cases 2005 since the leishmaniosis was spreading north into since practicing veterinary medicine. the foothills of the Alps and the Pyrenees (Ferroglio et al., 2005). More recently, northern continental Italy has been classified has an endemic area (Maroli et al., 2008). Spreading of the disease also has been demons- Discussion trated in France (Bourdeau et al., 2004). The most common clinical signs reported in this The authors considered this survey to add important survey were weight loss/muscle atrophy, alopecia, data regarding the distribution and clinical manage- lymphadenopathy, fine scaling, exfoliation, onicho- ment of leishmaniosis in Portugal caused by L. infan- griphosis, nasal lesions, anemia, renal insufficiency, tum. In this survey, answers were obtained from clini- prostration, skin ulceration, eye lesions and footpad cians working in all districts including Madeira and lesions which were also reported by other authors Azores. Furthermore, 93% of the clinicians that (Koutinas et al., 1999; Baneth et al., 2008). In spite answered the questionnaire were consulting several that arthritis is a less representative clinical sign, a cases of leishmaniosis which means that most of the case of polyarthritis associated with visceral clinicians had field experience with many aspects of leishmaniosis in an 8-month-old male Boxer that was the disease. In fact, some of them (21%) were con- successfully treated with a multistep combination of sulting between 20-50 cases of leishmaniosis per year meglumine antimoniate, aminosidine and allopurinol which was a high number of cases taking into account was described in Portugal (Santos et al., 2006). that most of the practices (65%) consult less then 10 Serological tests were widely used by the dogs per day. This survey was performed also in Portuguese clinicians. It is well known that the Greece and France. In Greece the percentage of specific humoral response in canine leishmaniosis is, answers obtained was 17.5% (Saridomichelakis, per- in general, very intense with high levels of sonal communication) and in France was 17.9% immunoglobulins although it understimates the (Bourdeau et al., 2004). Those results are very similar infection rate of Leishmania in endemic areas (Alvar to the one obtained in our survey (17.8%), although a et al., 2004). Serology of infected and symptomatic higher rate of answers would provide a more represen- dogs reveals increasing antibodies over time but, the tative study. production of antibodies is low in the early and late The districts with the highest number of answers phase or in asymptomatic infections (Oliva et al., were Lisbon, Porto and Setúbal. This was likely a 2006). The most used diagnostic test by the clinicians reflection of the large number of inhabitants, the high was indirect immunoflourescence assay which is number of veterinary practices and the fact the leish- considered the "gold standard" of the serologic tests maniosis is endemic in those areas. In fact, a canine (Maia and Campino, 2008). This test is useful for epi- survey performed during 2003 in the urban area of demiological testing, clinical practice and treatment Lisbon demonstrated a high prevalence (19.2%) of follow-up (Mancianti et al., 2002). Immunomigration leishmaniosis (Cortes et al., 2007). When results were testing was the second option for the clinicians compared with a previous survey by Abranches et al. because it is rapid, in-house and easy to use. The relia- (1983), an increase of the canine leishmaniosis preva- bility of these tests can be questionable as different lence in Lisbon area was noticed. The increase in the commercial kits will have different a sensitivity and prevalence was associated with the larger number of specificity (Mancianti et al., 2002). The high sensitivity family homes with gardens and the deficient sanitary was likely one of the reasons why most of the clini- conditions in certain areas which may provide macro- cians prefer indirect immunofluorence assay. habitat for the development of the sand flies (Cortes et For the clinicians, the first treatment option is al., 2007). allopurinol which is very frequently associated with According to our results, all the districts were affected meglumine antiamoniate. On a systematic review, the

26 Oliveira AM et al. RPCV (2010) 109 (573-576) 21-29 association of these two drugs was recommended as that Leishmune®, the first licensed vaccine against there is good clinical evidence of good efficacy and L. chagasi in Brazil, allowed the absence of symptoms reduced relapse rate (Noli and Auxilia, 2005). When and of evidence of Leishmania DNA and parasites in meglumine antimoniate is used alone, the drug was 32 vaccinated animals one year after the administra- not efficient to treat dogs with severe renal dysfunc- tion, indicating the non- infectious conditions of those tion and the side effects observed were pain at the site animals (Nogueira et al., 2005). of injection and the probable transient hepatotoxicity In conclusion, more than 20 years after the first evidenced only on clinical chemistry (Ikeda-Garcia et epidemiological study, leishmaniosis is still endemic al., 2007a). Nevertheless, the same author reported in the same areas as before and according to our data, that in a few cases treatment with 75 mg/kg meglu- likely new areas are affected which would mean that mine antimoniate subcutaneously every 12 h for 21 the disease is spreading in Portugal. The clinical signs days, and followed-up for a period of 6 months pro- in the Portuguese canine population studied were moted clinical cure but did not eliminate the parasites similar to the typical leishmaniosis presentation and, completely (Ikeda-Garcia et al., 2007b). Considering in general, the diagnostic tests, treatment protocols the periodic administration of allopurinol at the daily and prevention measures offered by the veterinary dose of 20 mg/kg, for one week per month, during the clinicians were according to the scientific evidence period of sandfly activity, a study performed in available at the time of the survey. The present work endemic areas, showed that this procedure does not opens a wide range of opportunities to study and to prevent the infection of non-infected dogs by L. quantify several issues related to zoonotic visceral infantum, and does not help in the elimination of the leishmaniosis in Portugal, namely the role of cats in parasite from dogs with asymptomatic infections the epidemiology of the disease as well as the role of (Saridomichelakis et al., 2005). The systematic review climatic changes in the spreading of Leishmania also pointed out that lethargy, gastro-intestinal signs infantum vectors. and local pain and are usually seen in 20% of the treated animals (Noli and Auxilia, 2005; Manna et al., 2008). Meglumine antiamoniate side effects can be a burden and in our study they were one of the reasons Acknowledgements to stop the treatment. It should be taken under consideration that when our The authors would like to acknowledge Dr survey was performed, miltefosine (Milteforan®, Francisco Ferraz for the technical support. Virbac), was just starting to be commercialized in Portugal and therefore none of the clinicians reported to use it. Therapy with miltefosine and allopurinol has been reported to drastically reduce L. infantum load in Bibliography lymph node aspirates (Manna et al., 2008) and to be equally effective when compared to meglumine antia- Abranches P, Conceiçao-Silva FM and Silva-Pereira MC moniate-allopurinol treatment (Miro et al., 2008). (1984). Kala-azar in Portugal. V. The sylvatic cycle in the In general, the decision to stop treatment is based enzootic endemic focus of Arrabida. Journal of Tropical mainly on clinical examination and in many cases is Medicine and Hygiene, 87, 197. accompanied by serology. The serological test most Abranches P, Lopes FJ, Silva FM, Ribeiro MM and Pires CA (1983). Kala-azar in Portugal. III. Results of a survey on common used for this purpose was indirect immuno- canine leishmaniasis performed in the Lisbon region. fluorescence assay which has been suggested to be a Comparison of urban and rural zones. Annales de useful tool to monitor response to treatment with anti- Parasitologie Humaine et Comparee, 58, 307-315. amonial drugs (Gradoni et al., 1987). PCR has been Abranches P, Sampaio-Silva ML, Santos-Gomes GM, reported to be more reliable for determining the Avelino IC, Pires CA, Conceição-Silva FM, Seixas-Lopes presence and identification of the parasite not only in A, Silva-Pereira MCD and Janz JG (1992). Kala-azar in active cases, but also for monitoring parasitological Portugal. VII. Epidemiological survey in Alijó (endemic cure after treatment (Maia and Campino, 2008), but region of Alto-Douro). Research and Reviews in this test was rarely used by the clinicians likely due to Parasitology, 52, 121-124. its relatively high cost. 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