Regulatory immune response in dogs with leishmaniosis at various clinical stages and undergoing different therapeutic protocols.

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Avaliação de duas técnicas serológicas (IFI e ELISA) e uma técnica molecular (qPCR em amostra de pele) no diagnóstico de infeção por Leishmania infantum em cães

Publication . Ribeiro, Ana Sofia Filipe Franco; Sampaio, Isabel Maria Soares Pereira da Fonseca de; Sampaio, Ana Teresa Severino Caldeira Reisinho de

A infeção por Leishmania infantum no cão tem adquirido ao longo dos anos uma maior importância no nosso País e a uma escala mundial, sendo Leishmaniose visceral zoonótica endémica em Portugal. Este estudo foi realizado numa amostra de 39 cães assistidos no Hospital Escolar da Faculdade de Medicina Veterinária da Universidade de Lisboa e teve como objetivos: 1. Comparação da eficácia de duas técnicas serológicas (IFI e ELISA) para a deteção de anticorpos anti-Leishmania em quatro grupos de cães, a saber: A - Cães com Leishmaniose e simultaneamente infetados com agentes de outras doenças caninas transmitidas por vetores; B - Cães doentes com outras afeções - Doenças dermatológicas, Alterações oculares, Infeções Bacterianas, Tumores, Doenças endócrinas e DCTV (Babesiose, Erlichiose, Ricketsiose, excluindo Leishmaniose); C - Cães clinicamente saudáveis; D - Cães com Leishmaniose (com ou sem outras afeções); 2. Pesquisa de DNA de L. infantum pela técnica molecular qPCR em amostras de pele, nos quatro grupos de cães acima referidos; 3. Avaliar se diferentes doenças e estados clínicos influenciam o resultado de técnicas de diagnóstico para deteção de L. infantum; 4. Determinação da influência de fatores de risco como o sexo, a idade, o porte, a pelagem, o local de habitação, o concelho de residência, entre outros, na positividade para L. infantum. Através do uso das técnicas serológicas de IFI e ELISA, no grupo A, obtiveram-se resultados positivos em ambas as técnicas para os 7 animais (7/7); nos grupos B e C, resultados negativos em ambas as técnicas para os 20 animais (20/20) e no grupo D, pela técnica de IFI, resultados positivos (7/12), negativos (4/12) e duvidosos (1/12). Para este mesmo grupo, pela técnica de ELISA, obtiveram-se resultados positivos (10/12) e negativos (2/12). Através da técnica molecular de qPCR, no grupo A obtiveram-se 6 resultados positivos (6/7); no grupo B, 8 resultados negativos (8/10); no grupo C, 9 resultados negativos (9/10) e no grupo D, 7 resultados negativos (7/12). O fator “época do ano” em que os animais se deslocava por Portugal, teve um valor de p<0,05, estatisticamente significativo na positividade para L. infantum, no grupo D e relativamente à técnica de qPCR.

2014-12-10Master thesis

Open access

Cytokine gene expression and cellular immune response in dogs with leishmaniosis before and under the two first-line treatment protocols : new insights into the animal disease

Publication . Santos, Marcos André Ferreira; Sampaio, Isabel Maria Soares Pereira da Fonseca de; Gomes, Gabriela Maria Santos

Canine leishmaniosis (CanL) caused by Leishmania infantum is a zoonotic visceral disease of worldwide concern. The drugs used for its treatment improve the animal’s clinical condition, although, in most cases, the parasites are not completely destroyed. The current study aimed to evaluate the immune response of the dog with leishmaniosis before and during treatment with first-line drugs, by analyzing the profile of cytokines and subsets of CD4+ and CD8+ T-cells in peripheral blood, lymph node and bone marrow. Two groups of six dogs diagnosed with CanL were treated with either miltefosine or meglumine antimoniate in combination with allopurinol. Simultaneously, another group of ten clinically healthy dogs was used as a control group. Upon diagnosis and during the following three months of treatment, clinical signs, hematological and biochemical parameters, urinalysis results and anti-Leishmania antibody titers using IFAT were recorded. Furthermore, peripheral blood, popliteal lymph node and bone marrow mononuclear cells were collected to evaluate the gene expression of IL-2, IL-4, IL-5, IL-10, IL-12, TNF-α, TGF-β and IFN-γ by qPCR. In parallel, these cells were also immunophenotypically analyzed be flow cytometry, using surface monoclonal antibodies anti-CD45, CD3, CD4, CD8, CD25 and intracellular monoclonal antibody anti-nuclear factor FoxP3. Both treatment protocols promoted the remission of clinical signs, normalization of hematological and biochemical parameters and urinalysis values. Sick dogs showed a generalized increase in IFN-γ gene expression and a decrease of IL-2, IL-4, and TGF-β. The expression of IL-12, TNF-α, IL-5, and IL-10 showed variations between groups of dogs and the tissue analyzed. CanL also resulted in an overall increase in the percentage of CD8+ T-cells in all tissues. In the peripheral blood there was also a decrease in CD4+ T-cells and an increase of CD4+CD25+FoxP3+ and CD8+CD25+FoxP3+ T-cells, with the latter also increasing on the bone marrow. CD4+CD25-FoxP3- T-cells showed a marked decrease in blood and bone marrow. During treatment, a trend towards normalization of cytokine gene expression and T-cell subsets was observed. However, high levels of IFN-γ gene expression were still observed in all tissues. In turn, the treatments caused an increase in the percentage of CD4+CD25+FoxP3+ and a decrease in CD8+CD25-FoxP3- T-cells, leading to normalization of CD4+ and CD8+ T cells in all tissues. Furthermore, the effect of treatment on gene expression of cytokines that were not significantly altered by infection indicates that these combined treatment protocols directly affect cytokine production. Both combined treatments are effective in remitting clinical sings and appear to influence the dog’s immune response, sustaining a pro-inflammatory immune environment while promoting the normalization of T-cell subsets. These findings indicate that L. infantum may be able to manipulate elements of the dog's immune system to avoid differentiating an efficient protective response, preventing the rapid development of severe pathology while ensuring the parasite’s survival and securing the possibility of several transmission cycles. Allied to these results, other studies carried out in collaboration with the working group on the role of neutrophils, hepatocytes and Kupffer cells in CanL, as well as the evaluation of treatment in feline leishmaniosis, have allowed to enhance the knowledge in the area of animal leishmaniosis.

2020-10-01Doctoral thesis

Open access

Estudo observacional transversal de parasitas em cães errantes no concelho de Vila Franca de Xira, Portugal

Publication . Santos, João Pedro Gibert Alvarez Bettencourt dos; Sampaio, Isabel Maria Soares Pereira da Fonseca de; Pissarra, Hugo Abel Sampaio Monteiro Martins; Protozoa; Ectoparasites

Os animais errantes são uma população particularmente importante em medicina veterinária, permitindo a manutenção dos agentes no biótopo e a dispersão para áreas não endémicas. Esta população tem vindo a aumentar, por todo o mundo, sendo fácil o seu contacto, quer directo quer indirecto com o Homem. Este trabalho foi elaborado com a intenção de pesquisar parasitas de vários órgãos, sistemas e localizações, na população de cães errantes do concelho de Vila Franca de Xira, Portugal, utilizando várias técnicas de diagnóstico. Relativamente a parasitas do sistema macrofágico fagocítico e de células do sangue, este estudo detetou uma seroprevalência de 28,75% (23/80) de Leishmania infantum, 57,5% (46/80) de Babesia canis/B. vogeli/B.rossi, 34% (28/80) de Anaplasma phagocytophilum, 34% (29/80) de Ehrlichia canis e 76,2% (61/80) de Rickettsia conorii. No entanto, não foram detetados hemoparasitas através de esfregaços sanguíneos. No que se refere a parasitas dos aparelhos cardiovascular, respiratório e serosas, foi detetada, pelo teste Speed Diro uma prevalência de 12,2% (10/80) de Dirofilaria immitis e, por pesquisa de microfilárias pela técnica de Knott, foram observadas prevalências de 10% de D. immitis (8/80) e 11,25% (9/80) de Acanthocheilonema reconditum. Pela técnica de Baermann não foram detetadas larvas de Angiostrongylus vasorum. Os parasitas intestinais identificados pela técnica de Willis foram: Toxocara canis (15%), Strongyloides stercoralis (7,5%), Ancylostoma caninum (15%), Uncinaria stenocephala (16,25%), Trichuris vulpis (11,25%), Taenia spp. (10%). Pela técnica de Ziehl-Neelsen, foram observados quistos de Giardia spp. (32,5%) e oocistos de Cryptosporidium spp. (11,25%). Foram ainda, identificados proglotes de Dipylidium caninum nas fezes de 3,75% dos animais. Relativamente a ectoparasitas, foram observados ácaros, ixodídeos e pulgas, sendo: 30% (6/20) de Sarcoptes scabiei e 10% (2/20) de Demodex canis, apenas em animais suspeitos e, 23,75% (19/80) de Rhipicephalus sanguineus e 10% (8/80) de Ctenocephalides felis. Não foram observados ácaros pelas técnicas de colheita de cerúmen auricular e escovagem do pêlo. Foram ainda avaliadas co-infeções parasitárias e analisados diversos fatores que podem influenciar as prevalências obtidas para os diferentes agentes. A importância do estado sanitário da população canina errante no que se refere à Saúde Pública e à Saúde Animal foi salientada.

2014-04-14Master thesis

Open access

Meglumine antimoniate and miltefosine combined with allopurinol sustain pro-inflammatory immune environments during canine leishmaniosis treatment

Publication . Santos, Marcos André Ferreira; Alexandre-Pires, Graça; Pereira, Maria A.; Marques, Cátia Filipa Saraiva; Gomes, Joana; Correia, Jorge Manuel de Jesus; Duarte, Ana; Gomes, Lídia; Rodrigues, Armanda; Basso, Alexandra; Reisinho, Ana; Meireles, José; Santos-Mateus, David; Villa Brito, Maria Teresa; Tavares, Luis; Santos-Gomes, Gabriela M.; Pereira da Fonseca, Isabel

Canine leishmaniosis (CanL) caused by Leishmania infantum is a zoonotic disease of global concern. Antileishmanial drug therapies commonly used to treat sick dogs improve their clinical condition, although when discontinued relapses can occur. Thus, the current study aims to evaluate the effect of CanL treatments in peripheral blood, lymph node, and bone marrow cytokine profile associated with clinical recovery. Two groups of six dogs diagnosed with CanL were treated with miltefosine combined with allopurinol and meglumine antimoniate combined with allopurinol (MT+A and MG+A), respectively. At diagnosis and after treatment, during a 3-month follow-up, clinical signs, hematological and biochemical parameters, urinalysis results and antileishmanial antibody titers were registered. Furthermore, peripheral blood, popliteal lymph node, and bone marrow samples were collected to assess the gene expression of IL-2, IL-4, IL-5, IL-10, IL-12, TNF-alpha, TGF-beta, and IFN-gamma by qPCR. In parallel, were also evaluated samples obtained from five healthy dogs. Both treatment protocols promoted the remission of clinical signs as well as normalization of hematological and biochemical parameters and urinalysis values. Antileishmanial antibodies returned to non-significant titers in all dogs. Sick dogs showed a generalized upregulation of IFN-gamma and downregulation of IL-2, IL-4, and TGF-beta, while gene expression of IL-12, TNF-alpha, IL-5, and IL-10 varied between groups and according to evaluated tissue. A trend to the normalization of cytokine gene expression was induced by both miltefosine and meglumine antimoniate combined therapies. However, IFN-gamma gene expression was still up-regulated in the three evaluated tissues. Furthermore, the effect of treatment in the gene expression of cytokines that were not significantly changed by infection, indicates that miltefosine and meglumine antimoniate combined therapy directly affects cytokine generation. Both combined therapies are effective in CanL treatment, leading to sustained pro-inflammatory immune environments that can compromise parasite survival and favor dogs' clinical cure. In the current study, anti-inflammatory and regulatory cytokines do not seem to play a prominent role in CanL or during clinical recovery.

2019-10-18Journal article

Open access