Estudos de infecção canina por Leishmania no Alto Douro
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2004
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Cardoso, Luís Miguel Martins
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A leishmaniose visceral causada por Leishmania infantum é uma zoonose endémica nos países do sul da Europa. O cão é o reservatório principal do parasita, que tem comovectores insectos flebotomíneos. A leishmaniose canina (LCan) representa um importante problema médico-veterinário. Simultaneamente, a informação sobre a epidemiologia da infecção por L. infantum nos cães é essencial para a definição de medidas eficazes de controlo da leishmaniose zoonótica.
A investigação apresentada nesta tese tem como objectivo fundamental a aquisição de conhecimento actualizado sobre aspectos epidemiológicos da infecção canina por Leishmania, na região endémica do Alto Douro, e também a validação de meios de diagnóstico simples, rápidos e sensíveis. Com este propósito, foram empreendidos diversos trabalhos, descritos nos capítulos seguintes:
Capítulo 1 – Introdução geral. Revisão da literatura científica sobre Leishmania, leishmanioses humanas, a LCan no sul da Europa, o diagnóstico, o controlo e a situação da leishmaniose em Portugal.
Capítulo 2 – Rastreio serológico de infecção por Leishmania em cães do concelho de Peso da Régua (Alto Douro, Portugal) com o teste de aglutinação directa (DAT) e o teste rápido de aglutinação directa (FAST). Pretendeu-se definir níveis de seroprevalência actualizados e utilizar, pela primeira vez, o FAST num rastreio de infecção canina por Leishmania. Amostras de soro foram obtidas de 294 cães simultaneamente examinados para a presença de sinais clínicos de LCan. A seroprevalência de infecção foi de 20,4%, após o rastreio da população com o FAST e a confirmação pelo DAT. Apenas 15,0% dos cães seropositivos apresentavam sinais clínicos de LCan e a prevalência geral de doença foi de 3,1%. Observou-se elevado nível de concordância (88,4%; κ = 0,71) entre o DAT e o FAST.
Os resultados demonstraram que o FAST pode ser utilizado como teste simples, rápido e sensível em áreas de elevada endemicidade e, juntamente com o DAT, é um instrumento valioso para avaliar a prevalência de infecção canina por Leishmania.
Capítulo 3 – Estudo seroepidemiológico de infecção canina por Leishmania no concelho de Alijó (Alto Douro, Portugal). A prestação do DAT foi avaliada em 205 amostras de sangue em papel de filtro (S-PF) e de soro dos mesmos cães. Observou-se elevado nível de concordância (97,6%; κ = 0,83) entre os resultados dos dois tipos de amostras. O DAT foi então utilizado para testar mais amostras de S-PF de outros 1335 cães. A seroprevalência detectada foi de 18,7% (288/1540), com valores oscilando entre 0,0% e 81,1% em cada uma das freguesias de Alijó. Três zonas geográficas com distinta seroprevalência média foram assim definidas: zona noroeste (2,5%), intermédia (11,4%) e sul (49,9%). Não foi observada diferença estatisticamente significativa (p = 0,560) entre a seroprevalência nos machos (19,1%) e nas fêmeas (17,8%). À excepção dos animais com 9-11 anos de idade (28,4%), todos os outros grupos etários (0-2, 3-5, 6-8 e 12-17 anos) apresentaram valores de seroprevalência (15,0% a 22,3%) sem diferença significativa em relação à média de toda a população estudada. A idade e a zona geográfica foram factores de risco para a infecção canina. Apenas 5,9% dos animais seropositivos tinham sinais clínicos de LCan e a prevalência geral de doença foi de 1,1%. Este estudo valida a utilização de amostras de S-PF e confirma o DAT como teste serológico simples e sensível, para avaliar o nível de infecção canina por Leishmania em áreas de elevada seroprevalência.
Capítulo 4 – Desenvolvimento de um teste "dipstick" para a detecção de anticorpos específicos anti-Leishmania em cães. Após a determinação das condições óptimas para o seu uso, a prestação do "dipstick" foi comparada com a de outros dois testes serológicos, o DAT e o FAST. O "dipstick" revelou uma sensibilidade de 99,2% e uma especificidade de 87,9%. O DAT mostrou sensibilidade de 97,7% e especificidade de 95,2%, enquanto o FAST revelou sensibilidade também de 97,7% e especificidade de 93,0%. Elevados níveis de concordância foram observados entre o "dipstick" e o DAT (93,7%; κ = 0,86), entre o "dipstick" e o FAST (91,8%; κ = 0,82), e entre o DAT e o FAST (95,2%; κ = 0,90). A elevada sensibilidade, a rapidez e a facilidade de utilização tornam o teste "dipstick" apropriado para rastreio serológico.
Capítulo 5 – Comparação de métodos parasitológicos e serológicos para diagnóstico da infecção por Leishmania em cães. Avaliou-se o desempenho de um protocolo de reacção em cadeia da polimerase (PCR) como método de diagnóstico parasitológico da infecção canina por Leishmania. Em animais suspeitos de LCan ou assintomáticos, foram recolhidas amostras de medula óssea e de sangue, para tubos com EDTA e para papel de filtro. Os resultados obtidos foram comparados com os da observação microscópica de esfregaços de medula óssea e da pesquisa de anticorpos específicos anti-Leishmania, com vista à caracterização clínica, parasitológica e serológica dos cães analisados. A PCR proporcionou boa sensibilidade com as amostras de medula óssea em EDTA ou em papel de filtro e de sangue em EDTA; a sensibilidade da prova com sangue em papel de filtro foi menor. Em alguns animais suspeitos e seropositivos, a PCR foi negativa na medula óssea e/ou no sangue.
Os dados indicam a PCR como técnica mais útil para confirmar casos clinicamente suspeitos de LCan e a serologia mais sensível para a detecção de cães infectados assintomáticos. Em áreas endémicas, a combinação destes meios poderá aumentar a sensibilidade do diagnóstico.
Capítulo 6 – Análise de respostas imunitárias anti-Leishmania em cães sintomáticos e assintomáticos da região endémica do Alto Douro. Pesquisou-se a presença de respostas imunitárias humorais das subclasses de imunoglobulinas G1 (IgG1) e IgG2 específicas e a sua relação com a resposta imunitária celular de hipersensibilidade do tipo retardado (HTR), em grupos de cães caracterizados clínica e serologicamente. A resposta imunitária humoral nos cães seropositivos consistiu principalmente na produção elevada de IgG2. Esta subclasse não constituiu um marcador de infecção assintomática, pois foram observados níveis de anticorpos IgG2 significativamente mais elevados nos cães sintomáticos do que nos assintomáticos. Verificou-se forte correlação entre os níveis de IgG2 e o título do DAT, sendo mais baixa a correlação entre IgG1 e IgG2 e entre IgG1 e o DAT. Isto poderá indicar a IgG2 como a principal subclasse na resposta humoral detectada pelo DAT. Por outro lado, foi registada uma resposta de IgG2 mais reduzida, embora não significativa, entre os animais com imunidade celular específica demonstrada por reacção de HTR positiva. Adicional-mente, não foi encontrada correlação entre a resposta humoral, quantificada pelo título do DAT ou pelos níveis de IgG1 e IgG2, e a resposta celular determinada por HTR. A avaliação da imunidade celular anti-Leishmania pode ser útil quando considerada em conjunto com a resposta de anticorpos específicos. Combinar a serologia e o teste intradérmico de HTR constitui um meio clinicamente prático de avaliar respostas imunitárias em cães assintomáticos.
Capítulo 7 – Conclusões. Incluem-se as principais conclusões decorrentes do trabalho experimental e são apontados alguns aspectos que devem ser alvo de investigação adicional.
Admitindo que nem todas as infecções são detectáveis através dos testes serológicos, o controlo da LCan dificilmente será alcançado sem a aplicação de medidas eficazes aos cães seropositivos.
Visceral leishmaniosis caused by Leishmania infantum is an endemic zoonosis in southern European countries. Dogs are the main reservoirs of the parasite, which is transmitted by phlebotomine sand flies. Canine leishmaniosis (CanL) represents an important veterinary medicine problem. Simultaneously, information on the epidemiology of Leishmania infection in dogs is essential in order to define effective control measures for zoonotic leishmaniosis. The aim of the research presented in this thesis was the acquisition of up-to-date knowledge about epidemiological aspects of canine Leishmania infection in the region of Alto Douro, where it is endemic, and also the validation of simple and sensitive tools for its diagnosis. With this purpose, several studies were carried out as described in the following chapters: Chapter 1 – General introduction. A review of the scientific literature concerning Leishmania, the human leishmaniosis, CanL in southern Europe, diagnosis, control and the current state of leishmaniosis in Portugal. Chapter 2 – Serological survey of Leishmania infection in dogs from the municipality of Peso da Régua (Alto Douro, Portugal) using the direct agglutination test (DAT) and fast agglutination screening test (FAST). The objectives were to define updated levels of seroprevalence and to use FAST, for the first time, in a survey of canine Leishmania infection. Serum samples were obtained from 294 dogs examined for clinical signs of CanL. The seroprevalence of infection was 20.4%, after screening the study population by FAST and with subsequent confirmation by DAT. Only 15.0% of the sero-positive dogs had clinical signs of CanL and the overall prevalence of the disease was 3.1%. A high degree of agreement (88.4%; κ value = 0.71) was found between DAT and FAST. This study further demonstrates that FAST can be used as a simple, rapid and sensitive screening test in areas of high endemicity and, together with DAT, is a valuable tool in the assessment of canine Leishmania infection. Chapter 3 – Sero-epidemiological study of canine Leishmania infection in the municipality of Alijó (Alto Douro, Portugal). Performance of DAT was assessed in 205 matching samples of blood collected on filter paper and serum. A high degree of agreement (97.6%; κ value = 0.83) was found between the results obtained from both types of samples. DAT was then used to test more blood on filter paper (B-FP) samples from a further 1335 dogs. The detected seroprevalence was 18.7% (288/1,540), with values ranging from 0.0% to 81.1% in each of the 19 parishes of Alijó. Three geographical zones of distinct mean seroprevalence could be defined: north-western (2.5%), intermediate (11.4%) and southern (49.9%). No statistically significant difference was observed between male (19.1%) and female (17.8%) seroprevalences (p = 0.560). With the exception of dogs aged 9-11 years (28.4%), all the other age-intervals (0-2, 3-5, 6-8 and 12-17 years) presented values of seroprevalence (15.0%-22.3%) not significantly different from the mean of the whole study population. Risk factors for canine infection were age and geographical zone. Only 5.9% of the sero-positive animals had clinical signs of CanL and the overall prevalence of the disease was 1.1%. This study validates the use of B-FP samples and confirms DAT as a simple and sensitive serological test to evaluate the level of canine Leishmania infection in areas of high seroprevalence. Chapter 4 – Development of a dipstick assay for detection of Leishmania-specific canine antibodies. After determination of optimal dipstick test conditions, the test performance was compared with two existing serological tests, i.e., DAT and FAST. The dipstick test had a sensitivity of 99.2% and a specificity of 87.9%. DAT had a sensitivity of 97.7% and a specificity of 95.2%, whereas FAST also had a sensitivity of 97.7% and a specificity 93.0%. High degrees of agreement were observed between the dipstick test and DAT (93.7%; κ = 0.86), between the dipstick test and FAST (91.8%; κ = 0.82), and between DAT and FAST (95.2%; κ = 0.90). The high sensitivity and ease of performance make the disptick test very suitable for surveys. Chapter 5 – Comparison of parasitological and serological methods for the diagnosis of Leishmania infecion in dogs. A polymerase chain reaction (PCR) protocol was assessed for the parasitological diagnosis of canine Leishmania infection. Samples of bone marrow and blood were collected, into tubes with EDTA or onto filter papers, from CanL suspect or asymptomatic animals. The results were compared with those obtained by microscopic examination of bone marrow smears and by detection of Leishmania-specific antibodies, with a view to a complete definition of dogs’ clinical, parasitological and serological status. The PCR showed good sensitivity with the bone marrow samples in EDTA or filter paper, and also with the blood samples in EDTA; the sensitivity of the test was lower with the blood samples in filter paper. In some suspect animals, the PCR was negative in bone marrow and/or blood. The obtained data suggest the PCR as a very useful technique to confirm CanL suspect cases and serology more sensitive to detect asymptomatic infected dogs. In endemic areas, the combination of both methods may increase the sensitivity of diagnosis. Chapter 6 – Analysis of Leishmania-specific immune responses in symptomatic and asymptomatic dogs from the endemic region of Alto Douro. Humoral immune responses of specific immunoglobulin G subclasses (IgG1 and IgG2) were measured and compared with the delayed type hypersensitivity (DTH) cellular immune response, in groups of dogs clinically and serologically characterised. The humoral immune response in seropositive animals mainly consisted of an increased production of IgG2. This subclass was not a marker of asymptomatic infection, since significantly higher levels of IgG2 were recorded in the symptomatic dogs. A strong correlation was observed between the levels of IgG2 and DAT titre; the correlations between IgG1 and IgG2, and between IgG1 and DAT were weaker. This may indicate IgG2 as the main subclass in the humoral response detected by DAT. An IgG2 reduced response, although not significantly different, was registered among the animals with cellular immune responses detected by an HTR positive reaction. Furthermore, no correlations were observed between cellular response determined by HTR and humoral responses quantified by DAT titre or IgG1 and IgG2 levels. The evaluation of anti-Leishmania cellular immunity may be useful when considered in conjunction with the measurement of specific antibodies. Combining serology and the HTR skin test is a practical method to evaluate immune responses in asymptomatic dogs. Chapter 7 – Conclusions. The main conclusions resulting from the experimental work reported above are included. Some aspects requiring further investigation are additionally mentioned. Not all infections are detected using serologic tests, but eradication of CanL will hardly be achieved without the application of effective control measures on the seropositive dogs.
Visceral leishmaniosis caused by Leishmania infantum is an endemic zoonosis in southern European countries. Dogs are the main reservoirs of the parasite, which is transmitted by phlebotomine sand flies. Canine leishmaniosis (CanL) represents an important veterinary medicine problem. Simultaneously, information on the epidemiology of Leishmania infection in dogs is essential in order to define effective control measures for zoonotic leishmaniosis. The aim of the research presented in this thesis was the acquisition of up-to-date knowledge about epidemiological aspects of canine Leishmania infection in the region of Alto Douro, where it is endemic, and also the validation of simple and sensitive tools for its diagnosis. With this purpose, several studies were carried out as described in the following chapters: Chapter 1 – General introduction. A review of the scientific literature concerning Leishmania, the human leishmaniosis, CanL in southern Europe, diagnosis, control and the current state of leishmaniosis in Portugal. Chapter 2 – Serological survey of Leishmania infection in dogs from the municipality of Peso da Régua (Alto Douro, Portugal) using the direct agglutination test (DAT) and fast agglutination screening test (FAST). The objectives were to define updated levels of seroprevalence and to use FAST, for the first time, in a survey of canine Leishmania infection. Serum samples were obtained from 294 dogs examined for clinical signs of CanL. The seroprevalence of infection was 20.4%, after screening the study population by FAST and with subsequent confirmation by DAT. Only 15.0% of the sero-positive dogs had clinical signs of CanL and the overall prevalence of the disease was 3.1%. A high degree of agreement (88.4%; κ value = 0.71) was found between DAT and FAST. This study further demonstrates that FAST can be used as a simple, rapid and sensitive screening test in areas of high endemicity and, together with DAT, is a valuable tool in the assessment of canine Leishmania infection. Chapter 3 – Sero-epidemiological study of canine Leishmania infection in the municipality of Alijó (Alto Douro, Portugal). Performance of DAT was assessed in 205 matching samples of blood collected on filter paper and serum. A high degree of agreement (97.6%; κ value = 0.83) was found between the results obtained from both types of samples. DAT was then used to test more blood on filter paper (B-FP) samples from a further 1335 dogs. The detected seroprevalence was 18.7% (288/1,540), with values ranging from 0.0% to 81.1% in each of the 19 parishes of Alijó. Three geographical zones of distinct mean seroprevalence could be defined: north-western (2.5%), intermediate (11.4%) and southern (49.9%). No statistically significant difference was observed between male (19.1%) and female (17.8%) seroprevalences (p = 0.560). With the exception of dogs aged 9-11 years (28.4%), all the other age-intervals (0-2, 3-5, 6-8 and 12-17 years) presented values of seroprevalence (15.0%-22.3%) not significantly different from the mean of the whole study population. Risk factors for canine infection were age and geographical zone. Only 5.9% of the sero-positive animals had clinical signs of CanL and the overall prevalence of the disease was 1.1%. This study validates the use of B-FP samples and confirms DAT as a simple and sensitive serological test to evaluate the level of canine Leishmania infection in areas of high seroprevalence. Chapter 4 – Development of a dipstick assay for detection of Leishmania-specific canine antibodies. After determination of optimal dipstick test conditions, the test performance was compared with two existing serological tests, i.e., DAT and FAST. The dipstick test had a sensitivity of 99.2% and a specificity of 87.9%. DAT had a sensitivity of 97.7% and a specificity of 95.2%, whereas FAST also had a sensitivity of 97.7% and a specificity 93.0%. High degrees of agreement were observed between the dipstick test and DAT (93.7%; κ = 0.86), between the dipstick test and FAST (91.8%; κ = 0.82), and between DAT and FAST (95.2%; κ = 0.90). The high sensitivity and ease of performance make the disptick test very suitable for surveys. Chapter 5 – Comparison of parasitological and serological methods for the diagnosis of Leishmania infecion in dogs. A polymerase chain reaction (PCR) protocol was assessed for the parasitological diagnosis of canine Leishmania infection. Samples of bone marrow and blood were collected, into tubes with EDTA or onto filter papers, from CanL suspect or asymptomatic animals. The results were compared with those obtained by microscopic examination of bone marrow smears and by detection of Leishmania-specific antibodies, with a view to a complete definition of dogs’ clinical, parasitological and serological status. The PCR showed good sensitivity with the bone marrow samples in EDTA or filter paper, and also with the blood samples in EDTA; the sensitivity of the test was lower with the blood samples in filter paper. In some suspect animals, the PCR was negative in bone marrow and/or blood. The obtained data suggest the PCR as a very useful technique to confirm CanL suspect cases and serology more sensitive to detect asymptomatic infected dogs. In endemic areas, the combination of both methods may increase the sensitivity of diagnosis. Chapter 6 – Analysis of Leishmania-specific immune responses in symptomatic and asymptomatic dogs from the endemic region of Alto Douro. Humoral immune responses of specific immunoglobulin G subclasses (IgG1 and IgG2) were measured and compared with the delayed type hypersensitivity (DTH) cellular immune response, in groups of dogs clinically and serologically characterised. The humoral immune response in seropositive animals mainly consisted of an increased production of IgG2. This subclass was not a marker of asymptomatic infection, since significantly higher levels of IgG2 were recorded in the symptomatic dogs. A strong correlation was observed between the levels of IgG2 and DAT titre; the correlations between IgG1 and IgG2, and between IgG1 and DAT were weaker. This may indicate IgG2 as the main subclass in the humoral response detected by DAT. An IgG2 reduced response, although not significantly different, was registered among the animals with cellular immune responses detected by an HTR positive reaction. Furthermore, no correlations were observed between cellular response determined by HTR and humoral responses quantified by DAT titre or IgG1 and IgG2 levels. The evaluation of anti-Leishmania cellular immunity may be useful when considered in conjunction with the measurement of specific antibodies. Combining serology and the HTR skin test is a practical method to evaluate immune responses in asymptomatic dogs. Chapter 7 – Conclusions. The main conclusions resulting from the experimental work reported above are included. Some aspects requiring further investigation are additionally mentioned. Not all infections are detected using serologic tests, but eradication of CanL will hardly be achieved without the application of effective control measures on the seropositive dogs.
Descrição
Tese de Doutoramento em Ciências Veterinárias
Palavras-chave
Cão , Leishmania , Doenças infecciosas , Região do Alto Douro (Portugal)