Parasite load in the blood and skin of dogs naturally infected by Leishmania infantum is correlated with their capacity to infect sand fly vectors

AbstractThe sand fly Lutzomyia longipalpis is primarily responsible for the transmission of visceral leishmaniasis (VL) in the New World, and dogs are considered to be the main urban reservoir of this disease. In order to improve the efficacy of control measures, it is essential to assess the transmission capacity of Leishmania infantum to the sand fly vector by naturally infected dogs. The present study investigated the existence of correlations between canine clinical presentation and the intensity of parasite load in the blood, skin and spleen of naturally infected dogs. In addition, we also attempted to establish correlations between the intensity of parasite load in canine tissue and the parasite load detected in sandflies five days after feeding on naturally infected dogs. A total of 23 dogs were examined and classified according to clinical manifestation of canine VL. Blood samples, splenic aspirate and skin biopsies were collected and parasite DNA was quantified by qPCR. Canine capacity to infect Lu. longipalpis with parasites was evaluated by xenodiagnosis and parasite loads were measured five days after feeding. No significant differences were observed with respect to canine clinical manifestation and the parasite loads detected in the blood, skin and spleen samples obtained from naturally infected dogs. Regardless of clinical canine visceral leishmaniasis (CVL) presentation and the degree of parasite burden, almost half of the dogs successfully infected sandflies with parasites, albeit to a low number of sandflies with correspondingly low parasite loads. Parasite loads in both canine blood and skin were shown to be positively correlated with the canine infectiousness to the sand fly vector, and positive correlations were also observed with respect to these tissues and the sand fly infection rate, as well as the parasite load detected in sandflies following xenodiagnosis. In conclusion, this indicates that parasite loads in both blood and skin can function as potentially reliable markers of canine capacity to infect sand fly vector

Mathematical modelling using predictive biomarkers for the outcome of canine Leishmaniasis upon chemotherapy

Prediction parameters of possible outcomes of canine leishmaniasis (CanL) therapy might help with therapeutic decisions and animal health care. Here, we aimed to develop a diagnostic method with predictive value by analyzing two groups of dogs with CanL, those that exhibited a decrease in parasite load upon antiparasitic treatment (group: responders) and those that maintained high parasite load despite the treatment (group: non-responders). The parameters analyzed were parasitic load determined by q-PCR, hemogram, serum biochemistry and immune system-related gene expression signature. A mathematical model was applied to the analysis of these parameters to predict how efficient their response to therapy would be. Responder dogs restored hematological and biochemical parameters to the reference values and exhibited a Th1 cell activation profile with a linear tendency to reach mild clinical alteration stages. Differently, non-responders developed a mixed Th1/Th2 response and exhibited markers of liver and kidney injury. Erythrocyte counts and serum phosphorus were identified as predictive markers of therapeutic response at an early period of assessment of CanL. The results presented in this study are highly encouraging and may represent a new paradigm for future assistance to clinicians to interfere precociously in the therapeutic approach, with a more precise definition in the patient’s prognosis.This work was funded by the Brazilian agencies Bahia Research Foundation—FAPESB (Grant nº PRONEM 498/2011-PNE 0002/2011 to S.M.B-M), National Council for Scientific and Technological Development —CNPq (PQ scholarship nº 307813/2018-5 to SMBM, and nº 303621/2015-0 to HG) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior —CAPES (PDSE scholarship nº 88881.189587/2018-01 to R.S.G; Finance Code 001 and PV scholarship nº 23066.033859/2018-73 to R.S.). This work was supported by grants from CESPU (TramTap-CESPU-2016, Chronic-TramTap_CESPU_2017 and TraTapMDMA-CESPU-2018), from the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000013), funded by FEDER funds through COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI) and the Fundação para a Ciência e Tecnologia (FCT) (contract IF/00021/2014 to R.S.), Infect-Era (project INLEISH to R.S.) and Proyecto SNIP N◦ 292900 “Creación del Servicio de Laboratorio de Enfermedades Infecciosas y Parasitarias de Animales Domésticos de la Universidad Nacional Toribio Rodríguez de Mendoza de Amazonas.Instituto de Investigación en Ganadería y Biotecnología-IGBI. Universidad Nacional Toribio Rodríguez de Mendoza de Amazonas

Modulação da infecção em modelo murino por moléculas de amastigota de Leishmania

Submitted by Repositório Arca ([email protected]) on 2019-08-06T11:28:05Z No. of bitstreams: 1 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2019-08-21T14:16:55Z (GMT) No. of bitstreams: 2 Daniela Farias Larangeira Modulação... 2003.pdf: 5748088 bytes, checksum: 2f0c750f59dfce5169687be1ea24272b (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Made available in DSpace on 2019-08-21T14:16:55Z (GMT). No. of bitstreams: 2 Daniela Farias Larangeira Modulação... 2003.pdf: 5748088 bytes, checksum: 2f0c750f59dfce5169687be1ea24272b (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2003Universidade Federal da Bahia. Faculdade de Medicina. Salvador, BA, Brasil / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil.Embora algumas espécies de Leishmania possam determinar várias formas de doença, existe usualmente uma relação entre a espécie infectante e a apresentação clínica da enfermidade e natureza da resposta imune ao parasito. A Leishmania Braziliensis, por exemplo, costuma ocasionar uma forte resposta imune celular e poucas lesões restritas à pele ou/e à mucosa, enquanto que a Leishmania Amazonensis pode causar uma forma muito grave da doença, conhecida como Leishmaniose cutâneo-difusa (LCD), na qual ocorre uma resposta imunológica ao parasito deficiente e lesões disseminadas pelo corpo do hospedeiro. É possível que diferenças moleculares entre estas duas espécies de Leishmania sejam responsáveis pela associação da LCD apenas com a infecção por L. Amazonensis. Neste trabalho, foi investigada a capacidade de moléculas de L. Amazonensis em potenciar a infecção por L. Braziliensis em camundongos BALB/c. A identificação e caracterização de moléculas parasitárias supressoras podem servir de base para o desenvolvimento de futuras estratégias de produção de vacinas e imunoterápicos.Although some species of Leishmania can determine several forms of illness, exists usually a relation between the sort infectante, and the clinical presentation of the illness and nature of the Immune answer to the parasito. To Leishmania braziliensis, by example, costuma cause a strong cellular immune answer and few restricted wounds to the skin or/and to the mucosa, while that to Leishmania amazonensis can cause a very grave form of the illness, known as leishmaniose cutaneous-diffuse (LCD), in the which occurs an answer imunológica to the parasito deficient and wounds disseminated by the body of the hospedeiro. It is possible that differences moleculares between these two species of Leishmania be responsible by the association of the LCD barely with the infection by L. amazonensis. In this work, was investigated the capacity of molecules of L. amazonensis in potenciar the infection by L. braziliensis in mice BALB/c. The identification and caracterização of molecules parasitárias supressoras can serve of base for the development of future vaccines output strategies and imunoterápicos

Evaluation of humoral and cellular immunity in dogs naturally infected with Leishmania (L.) chagasi and its correlation to the transmissibility to the vector

Este estudo avaliou a imunidade humoral e celular em cães naturalmente infectados com L. (L.) chagasi correlacionando com a transmissibilidade para o vetor. Soros e biópsias de baço, linfonodo e pele foram coletados de 120 cães provenientes do Centro de Controle de Zoonoses do município de Araçatuba, São Paulo, Brasil. Os soros foram processados por ELISA para detecção de IgG, IgG1, IgG2 e IgE; e as biópsias foram processadas por técnicas histológicas usuais coradas pelo HE e imunoistoquímica para a detecção de parasito, macrófago e células T CD3. De acordo com os sinais clínicos, 65/120 (54%) cães foram classificados como sintomáticos e 55/120 (46%) como assintomáticos. O diagnóstico parasitológico foi confirmado em 71% dos sintomáticos e em 40% dos assintomáticos. A correlação dos sinais clínicos com parasitismo mostrou que a carga parasitária estava diretamente associada com cães sintomáticos (p<0.05). Em relação aos anticorpos específicos anti-L.(L.)chagasi, cães de área endêmica com diagnóstico parasitológico positivo mostraram maiores níveis de IgG total comparado com ambos os controles (p<0.05), sem diferença entre cães sintomáticos e assintomáticos. IgG1 esteve presente em baixos níveis e foi mais intensa no grupo sintomático parasito-positivo (p<0.05). Níveis mais elevados foram observados para IgG2 em cães de área endêmica (p<0.05), mas sem correlação com o parasitismo e sinais clínicos. IgE também esteve presente em baixos níveis, mas mostrou diferenças entre cães de área endêmica e cães de área não endêmica; e cães com diagnóstico parasitológico positivo mostrou níveis mais elevados que cães com diagnóstico parasitológico negativo (p<0.05). Histopatologicamente, linfonodos mostraram hiperplasia e hipertrofia de macrófagos na área medular e em muitos casos linfadenite granulomatosa. Na polpa branca do baço, hiperplasia folicular foi observada; e a polpa vermelha mostrou granulomas. As lesões de pele foram caracterizadas por infiltrado inflamatório crônico na derme formado por macrófagos, linfócitos e plasmócitos; variando de discreto a intenso, assim como de focal a difuso. Foi evidente a presença de granulomas epitelióides na pele de alguns animais. Imunoistoquímica mostrou presença de células marcadas pelo anticorpo anti-macrófago e anti-CD3 em 100% dos baços e linfonodos variando de intensidade entre discreto e intenso. Na pele, macrófagos foram positivos em 90% e células CD3 em 39% dos casos. Houve associação direta entre baixa expressão de células CD3 e alto parasitismo na pele. Animais assintomáticos mostraram baixa expressão de macrófagos junto com baixo parasitismo na pele. Em relação ao xenodiagnóstico, no 4o dia depois da alimentação, as fêmeas dos vetores foram dissecadas e examinadas para observação de parasitos no intestino. Formas promastigotas foram observadas em 27% das fêmeas que se alimentaram em cães sintomáticos e em 42% das fêmeas que se alimentaram em cães assintomáticos. A técnica da PCR foi também utilizada para avaliar as fêmeas positivas depois do xenodiagnóstico. DNA de Leishmania foi detectado em 24% das fêmeas que se alimentaram em cães sintomáticos e em 34% das fêmeas que se alimentaram em cães assintomáticos. Os dados mostraram que a imunidade humoral e celular não teve correlação direta com as formas clínicas de leishmaniose canina. A alta porcentagem de vetores infectados na alimentação em cães assintomáticos mostra a importância destes animais na transmissibilidade para o vetor.These studies evaluate humoral and cellular immunity in dogs naturally infected with L. (L.) chagasi correlating to the transmissibility to the vector. Serum and biopsy from spleen, lymph node and skin were collected from 120 dogs referred to the Center of Zoonosis Control of Araçatuba city, São Paulo, Brazil. The sera were processed by ELISA for IgG, IgG1, IgG2 and IgE detection; and the biopsies were processed usual histological techniques stained by HE and immunohistochemistry for parasite, macrophage and T CD3 cells detection. According to the clinical signs, 65/120 (54%) dogs were classified as symptomatic and 55/120 (46%) as asymptomatic. Parasitological diagnosis was confirmed in 71% of symptomatic and in 40% of asymptomatic dogs. The correlation of clinical signs and parasitism showed that parasite burden was directly associated with symptomatic dogs (p<0.05). Concerning to L.(L.)chagasi-specific antibodies, dogs from the endemic area with positive parasitological diagnosis showed high levels of total IgG compared to both controls (p<0.05), without difference between symptomatic and asymptomatic dogs. IgG1 was present at low levels and was more intense in the parasite-positive symptomatic group (p<0.05). More elevated levels were observed for IgG2 in dogs from endemic area (p<0.05), but with no correlation to parasitism and clinical signs. IgE was also present at low levels, but showed differences between dogs from non-endemic and endemic areas; and dogs with positive parasitological diagnosis showed higher levels than dogs with negative parasitological diagnosis (p<0.05). Histopathologically, lymph nodes showed macrophage hyperplasia and hypertrophy in the medullary area and in many cases granulomatous lymphadenitis. In the white pulp of the spleen, follicular hyperplasia was observed; and the red pulp showed granulomas. The skin lesions were characterized by dermal chronic inflammatory infiltrate formed by macrophages, lymphocytes and plasma cells; it varied between descreet to intense, as well as focal to diffuse. The epithelioid granulomas were evident in the skin of some animals. Immunohistochemistry showed presence of labeled cells by anti-macrophage and anti-CD3+ antibodies in 100% of spleen and lymph nodes varying the intensity between mild to intense. Macrophage was positive in 90% of the skin and CD3 cells in 39%. There was a direct association between lower CD3 cells expression and higher parasite burden in the skin. Asymptomatic animals showed lower macrophage expression together with lower parasitism in the skin. Concerning to the xenodiagnosis, on the 4th day after the blood meal, female flies were dissected and examined for visible parasites in the gut. Promastigotes forms were observed in 27% of female which fed in symptomatic dogs and in 42% of female which fed in asymptomatic dogs. PCR technique was also used to evaluate the positive females after the xenodiagnosis. Leishmania DNA was detected in 24% of female which fed in symptomatic dogs and in 34% of female which fed in asymptomatic dogs. The data showed that the humoral and cellular immune response not has direct correlation to the clinical form of canine leishmaniasis. The high percentage of sand flies female infected by feeding in the asymptomatic dogs show the importance these animals on the parasite transmissibility to the vector

Comparison between splenic and lymph node aspirations as sampling methods for the parasitological detection of Leishmania chagasi infection in dogs

The sensitivities of spleen and lymph node cultures for the diagnosis of canine visceral leishmaniasis were compared in 64 anti-Leishmania antibody positive dogs from an endemic area in Brazil. The sensitivity of spleen cultures for Leishmania detection was 97.9%; in lymph node cultures it was 25%. Positive spleen culture was more frequent (p = 0.048, Fisher's exact probability test) in symptomatic (28 out of 33 animals) than in asymptomatic animals (19 out of 31 animals). These results support the use of spleen instead of lymph node aspiration as the choice method for the parasitological diagnosis of the infection

A standardized cytological and immunochemical method for the analysis of fine-needle spleen aspirates: assessment of leukocyte population changes in canine visceral leishmaniosis

Submitted by Éder Freyre ([email protected]) on 2011-07-01T19:16:02Z No. of bitstreams: 1 Barrouin-Melo_Larangeira_Santos_etal.pdf: 337692 bytes, checksum: 085d3627daac4f4f547fcda56dfbede8 (MD5)Made available in DSpace on 2011-07-01T19:16:02Z (GMT). No. of bitstreams: 1 Barrouin-Melo_Larangeira_Santos_etal.pdf: 337692 bytes, checksum: 085d3627daac4f4f547fcda56dfbede8 (MD5) Previous issue date: 2006Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilUniversidade Federal da Bahia. Departamento de Patologia e Clínicas. Escola de Medicina Veterinária. Salvador, BA, BrasilUniversidade Federal da Bahia. Escola de Medicina Veterinária. Laboratório de Infectologia Veterinária. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilUniversidade Federal da Bahia. Escola de Medicina Veterinária. Laboratório de Infectologia Veterinária. Salvador, BA, BrasilUniversidade Federal da Bahia. Escola de Medicina Veterinária. Laboratório de Infectologia Veterinária. Salvador, BA, BrasilLaboratório Central do Estado da Bahia, Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilUniversidade Federal da Bahia. Departamento de Patologia e Clínicas. Escola de Medicina Veterinária. Salvador, BA, BrasilUniversidade Federal da Bahia. Escola de Medicina Veterinária. Laboratório de Infectologia Veterinária. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação para o Desenvolvimento das Ciências. Escola Bahiana de Medicina e Saúde Pública. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação para o Desenvolvimento das Ciências. Escola Bahiana de Medicina e Saúde Pública. Salvador, BA, BrasilA method for the evaluation of splenic cellularity using samples collected by fine-needle aspirative biopsy was standardized in this work. The procedure includes erythrocyte lysing, preparation of cytospin films and staining by histochemical and immunocytochemical techniques. The cellular profiles of spleen preparations were compared with those observed in peripheral blood samples subjected to the same procedure. Two groups were compared, one consisting of 14 healthy uninfected and the other of 15 polysymptomatic Leishmania chagasi/infantum-infected dogs, from an endemic area for visceral leishmaniosis. Cell populations were identified by conventional hematoxilin–eosin and Wright’ stainings, and by immunocytochemistry using monoclonal antibodies against canine CD45RA and CD45RB, phagocytes and a pan-leukocyte antigen. Larger neutrophil (P < 0.0001) and monocyte/macrophage (P = 0.0036) relative counts and lower lymphocyte relative counts (P < 0.0001) were found in the spleen, and not in the blood, of the animals with leishmaniosis than in those of the healthy animals. The proportions of CD45RB+ cells were higher, and of CD45RA+ cells were lower, both in the spleen and in the blood of animals with leishmaniosis than in those of healthy dogs (P < 0.05). Additionally, hematoxilin–eosin-stained cytospins of spleen aspirates from Leishmania-infected animals permitted the easy visualization of amastigote forms inside phagocytes, under light microscopy

Comparison of two commercial vaccines against visceral leishmaniasis in dogs from endemic areas: IgG, and subclasses, parasitism, and parasite transmission by xenodiagnosis.

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2014-05-29T16:52:55Z No. of bitstreams: 1 Fernandes CB Comparison of two....pdf: 1344041 bytes, checksum: 2122a7970195d89d9565fe8291d03648 (MD5)Made available in DSpace on 2014-05-29T16:52:55Z (GMT). No. of bitstreams: 1 Fernandes CB Comparison of two....pdf: 1344041 bytes, checksum: 2122a7970195d89d9565fe8291d03648 (MD5) Previous issue date: 2014Federal University of Bahia (UFBA). Laboratory of Veterinary Infectious Diseases. Hospital of Veterinary Medicine (HOSPMEV). Salvador, BA, BrasilFederal University of Bahia (UFBA). Laboratory of Veterinary Infectious Diseases. Hospital of Veterinary Medicine (HOSPMEV). Salvador, BA, BrasilFederal University of Bahia (UFBA). Laboratory of Veterinary Infectious Diseases. Hospital of Veterinary Medicine (HOSPMEV). Salvador, BA, BrasilLaboratory of Cellular and Molecular Biology. HOSPMEV. UFBA. Salvador, BA, BrasilFederal University of Bahia (UFBA). Laboratory of Veterinary Infectious Diseases. Hospital of Veterinary Medicine (HOSPME V). Salvador, BA, Brasil / UFBA. Departament of Veterinary Anatomy. Pathology and Clinics of the School of Veterinary Medicine and Zootechny. Salvador, BA, BrasilUFBA. Departament of Preventive Veterinary Medicine and Animal Production of the School of Veterinary Medicine and Zootechny. Salvador, BA, Brasil / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, BrasilFederal University of Bahia (UFBA). Laboratory of Veterinary Infectious Diseases. Hospital of Veterinary Medicine (HOSPMEV). Salvador, BA, Brasil / UFBA. Departament of Preventive Veterinary Medicine and Animal Production of the School of Veterinary Medicine and Zootechny. Salvador, BA, Brasil / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, BrasilBACKGROUND: The incidence of zoonotic canine visceral leishmaniasis (CVL) would decrease if dogs were effectively vaccinated; however, additional data on the efficacy of canine vaccines are required for their approved preventative use. PURPOSE: To prospectively evaluate vaccination outcomes using two products commercially available in Brazil, with respect to adverse reactions (reactogenicity), humoral response, disease signs, parasitism, and parasite infectiousness in naturally exposed pet dogs in an endemic area of visceral leishmaniasis (VL). METHODS: From 2010 to 2012, healthy dogs were vaccinated with Leishmune(®) (50 animals) or Leish-Tec(®) (50 animals). Each dog was examined to identify clinical signs during peri- and post-vaccination procedures every 2 months for 11 months to identify the presence of parasites or parasite DNA in splenic samples using culturing or PCR, respectively. Levels of anti-Leishmania IgG, IgG1, and IgG2 were quantified in sera by ELISA and infectiousness was assessed by xenodiagnosis. RESULTS: Adverse effects occurred in 2.2% (1/45) and 13.0% (6/46) of the animals in the Leishmune(®) and Leish-Tec(®) groups, respectively. IgG levels peaked on the 21st day following the first dose of Leishmune(®) and on the 21st day after the second dose of Leish-Tec(®). The final seropositivity rate for IgG was 32.5% (13/40) and 30.9% (13/42) in the Leishmune(®) and Leish-Tec(®) groups, respectively. The Leishmune(®) group presented higher levels of IgG1 and IgG2 compared to the Leish-Tec(®) group (p<0.001), and ELISA reactivity in both vaccinated groups was significantly lower (p<0.001) than in infected positive control dogs. Parasitism was observed in 12.2% (5/41) of the Leishmune(®) group, and 7.9% (3/38) of the Leish-Tec(®) group, with xenodiagnostic transmission rates of Leishmania to Lutzomyia longipalpis of 5.1% (2/39), and 5.4% (2/37), respectively. CONCLUSIONS: No significant differences were observed in dogs vaccinated with Leishmune(®) or Leish-Tec(®), with respect to LVC clinical aspects, parasitism, IgG seropositivity, or dog infectiousness. The Leishmune(®)-vaccinated animals presented higher levels of IgG, IgG1, and IgG2. The animals vaccinated with Leish-Tec(®) exhibited adverse reactions with greater frequency and severity