Methods of macrophages activation and their modulation for the prospection of new antileishmania drugs: a review

Leishmaniasis are a group of parasitic zoonoses provoked by protozoa from Leishmania genus and belonging to the group of neglected tropical diseases. The search and development for new drugs is necessary not only to investigate the activity against only the parasite, but also to investigate the possible synergistic effect of new drugs with the immune response of the host. In the present review, macrophages are pointed out as potential targets of the investigation of new antileishmanial drugs, and some methodologies in order to assess their activation as response to Leishmania-infected cells are presented. Macrophages are an important role in the cellular immune response, since they are cells from mononuclear phagocytic system, the first line of defense of the host, against parasites from Leishmania genus. Phagocytic capacity, lysosomal activity, increase of nitric oxide and intracellular calcium levels are parameters regarding assessment of macrophages activation which allow them to be more hostile in order to solve the infection and lead the patient to cure. In this context, we bring 19 substances already investigated and that activate macrophages, what makes them promising in the antileishmanial treatment. Therefore, assessment of macrophages activation, are important tools for discovery of immunomodulatory compounds which have potential to act in synergism with host immune response. Such compounds might be promising as monotherapy in the treatment of leishmaniasis, as well as being used as adjuvants in vaccines and/or in combination with conventional drugs.

Evaluation of the serum biochemistry and histopathology of kidney and bladder of dogs with Leishmania sp. in their urine / Avaliação da bioquímica sérica e histopatologia de rim e bexiga de cães com Leishmania sp. na urina

The visceral establishment of Leishmania infantum in dogs may result in kidney and bladder tissue injury, with L. infantum ending up in urine. This study therefore aimed at investigating the presence of Leishmania sp. in urinary sediments, and correlating the results with those from renal and bladder serum biochemistry and histopathology. Thirty dogs with negative Nested-Polymerase Chain Reaction (PCR) for E. canis were used in the experiment, and were divided into three groups: control group (10 dogs), neither leishmaniasis nor clinical changes; group I (15 dogs), leishmaniasis but no Leishmania sp. in urine; and group II (5 dogs), leishmaniasis, as well as Leishmania sp. in urine. All animals were submitted to clinical, serological, and parasitological diagnosis for leishmaniasis, biochemical exams, and kidney and bladder histopathology. The parasite was also detected in the bladder imprint of one group II dog. Group II dogs presented with very low albumin concentrations, low albumin/globulin ratios, and kidney and bladder lesions. In the kidneys, hydropic degeneration, thickened Bowman's capsule, and thickening of the tubular capsule were detected in all dogs with positive urinary sediment. However, no significant difference in these renal changes was observed between groups. The intensity and distribution of bladder inflammatory infiltrates were significantly (p-value < 0.05, Kruskal-Wallis' and Dunn's tests) higher in group II dogs, compared with those of the other groups. The presence of Leishmania sp.in the urine of infected dogs appeared to be related to low serum albumin concentrations and more severe bladder lesions

O estabelecimento visceral de Leishmania infantum em cães pode resultar em lesões nos tecidos dos rins e da bexiga, favorecendo a chegando do parasito até a urina. Portanto, este estudo teve como objetivo investigar a presença de Leishmania sp. em sedimentos urinários e correlacionar os resultados com os achados de quantificações bioquímicas séricas e histopatologia de rim e bexiga. Trinta cães com Nested-Reação em Cadeia da Polimerase (PCR) negativa para E. canis foram utilizados no experimento e foram divididos em três grupos: grupo controle (10 cães), negativos para leishmaniose e sem alterações clínicas; grupo I (15 cães), com leishmaniose, mas sem Leishmania sp. na urina; e grupo II (5 cães), com leishmaniose e com Leishmaniasp. na urina. Todos os animais foram submetidos a diagnóstico clínico, sorológico e parasitológico para leishmaniose, exames bioquímicos e histopatologia de rim e bexiga. O parasito foi detectado no imprimt de bexiga de um cão do grupo II. Os cães do grupo II apresentaram concentrações muito baixas de albumina, baixa relação albumina/globulina e lesões nos rins e na bexiga. Nos rins, foram detectadas degeneração hidrópica, espessamento da cápsula de Bowman e espessamento da cápsula tubular, em todos os cães com sedimento urinário positivo. No entanto, nenhuma diferença significativa nessas alterações renais foi observada entre os grupos. A intensidade e a distribuição dos infiltrados inflamatórios da bexiga foram significativamente (p-valor < 0,05, testes de Kruskal-Wallis e Dunn) maiores nos cães do grupo II, em comparação com a dos outros grupos. A presença de Leishmania sp. na urina de cães infectados parece estar relacionada a baixa concentração sérica de albumina e a lesões mais graves na bexiga.

Transmission of Leishmania infantum from cats to dogs / Transmissão de Leishmania infantum de gatos para cães

Abstract Leishmania infantum infection in cats has been reported in several countries, including Brazil. However, the transmission of visceral leishmaniasis (VL) from cats to another host has not been proven yet. Therefore, the aim of this study was to verify the possibility of L. infantum transmission from cats to dogs. In order to verify the possibility of VL transmission from the cat to the dog, xenodiagnosis was carried out in a VL-positive cat, using 55 female Lutzomyia longipalpis. Five days later, 40 insects were dissected to verify Leishmania infection. The remaining 15 females were fed in a healthy dog. The potential infection of the dog was verified through clinical, serological, parasitological examinations, and PCR, at three, six, and twelve months post-infection. All 55 L. longipalpis females became visibly engorged. Leishmania promastigotes were detected in 27.5% of the dissected insects. Leishmania infection in the dog was confirmed upon first evaluation. DNA sequencing of the parasite isolated from the cat confirmed L. infantum infection and showed 99% similarity with the L. infantum DNA sequences from the dogs. Through this study, it was possible to confirm the L. infantum experimental transmission from a domestic cat to a domestic dog through its biological vector L. longipalpis.

Resumo A infecção por Leishmania infantum em gatos tem sido relatada em vários países, incluindo o Brasil. No entanto, a transmissão da leishmaniose visceral (LV) de gatos para outro hospedeiro ainda não foi comprovada. Portanto, o objetivo deste estudo foi verificar a possibilidade de transmissão de L. infantum de gatos para cães. Para verificar a possibilidade de transmissão da LV do gato para o cão, foi realizado xenodiagnóstico em um gato com LV, utilizando-se 55 fêmeas de Lutzomyia longipalpis. Cinco dias depois, 40 insetos foram dissecados para verificar a infecção por Leishmania. As 15 fêmeas restantes foram alimentadas em um cão saudável. A possível infecção no cão foi verificada por meio de exames clínicos, sorológicos, parasitológicos e PCR, três, seis e doze meses após a infecção. Todas as 55 fêmeas de L. longipalpis ficaram visivelmente ingurgitadas. Promastigotas de Leishmania foram detectadas em 27,5% dos insetos dissecados. A infecção por Leishmania no cão foi confirmada na primeira avaliação. O sequenciamento do DNA do parasito isolado do gato confirmou a infecção por L. infantum e apresentou 99% de similaridade com sequências de DNA de L. infantum de cães. Através deste estudo, foi possível confirmar a transmissão experimental de L. infantum de um gato doméstico para um cão doméstico através do seu vetor biológico L. longipalpis.

Atractis thapari (Nematoda, Atractidae) parasitizing Chelonoidis carbonarius and C. denticulatus (Testudinidae) in the state of Piauí, Brazil / Atractis thapari (Nematoda, Atractidae) parasitando Chelonoidis carbonarius e C. denticulatus (Testudinidae) no estado do Piauí, Brasil

Abstract Atractis thapari Petter, 1966, an atractid nematode, was collected parasitizing the large intestine of tortoises of the species Chelonoidis carbonarius (Spix, 1824) (Cc) and C. denticulatus (Linnaeus, 1766) (Cd) (Testudinidae) in the Zoobotanical Park of the municipality of Teresina, state of Piauí, Brazil. Taxonomic identification was based on morphological and morphometrical features, and using bright-field and scanning electron microscopy. The present study adds new observations on the morphology of A. thapari, mainly relating to mouth papillae, the excretory pore, deirids, and male and female posterior ends. The parasitic indices of prevalence (P), mean intensity (MI), mean abundance (MA) and range of infection (RI) for A. thapari in these two tortoise species were: P = 100%, MI = 154,667, MA = 154,667, RI = 5,500-588,500 (Cc); P = 100%, MI = 93,639, MA = 93,639, RI = 1,000-224,500 (Cd). This report confirms the occurrence of A. thapari in Neotropical region, South America, Brazil, and extends its occurrences to a new host, the tortoise C. carbonarius. Adjustment of host management with the aim of improving hygiene and health conditions is suggested.

Resumo Atractis thapari Petter, 1966, um nematoide atractídeo, foi coletado parasitando o intestino grosso de jabutis, Chelonoidis carbonarius (Spix, 1824) (Cc) e C. denticulatus (Linnaeus, 1766) (Cd) no Parque Zoobotânico, município de Teresina, estado do Piauí, Brasil. A identificação taxonômica foi baseada nos caracteres morfológicos e morfométricos usando microscopias de campo claro e eletrônica de varredura. O presente estudo adiciona novas observações na morfologia, principalmente relacionadas as papilas bucais, poro excretor, deirídeos, e extremidade posterior de machos e fêmeas. Os índices parasitários de prevalência (P), intensidade média (IM), abundância média (AM) e amplitude de variação de infecção (AI) de A. thapari em ambos jabutis foram P = 100%, IM = 154.667, AM = 154.667, AI = 5.500-588.500 (Cc); P = 100%, I M= 93.639, AM = 93.639, AI = 1.000-224.500 (Cd). Este registro confirma a ocorrência de A. thapari na região Neotropical, América do Sul, Brasil, e expande a um novo hospedeiro, o jabuti C. carbonarius. É sugerido um ajuste no manejo dos hospedeiros objetivando melhora das suas condições higiênico sanitárias.

Serological tests fail to discriminate dogs with visceral leishmaniasis that transmit Leishmania infantum to the vector Lutzomyia longipalpis

Abstract INTRODUCTION The control of reservoirs for Leishmania infantum -induced zoonotic visceral leishmaniasis requires the identification of dogs posing a population risk. Here, we assessed the performance of several assays to identify Lutzomyia longipalpis infectious dogs. METHODS We evaluated 99 dogs that were positive for visceral leishmaniasis based on parasite identification. Serological analyses were performed using an enzyme-linked immunosorbent assay, immunofluorescence antibody tests in 1:40 and 1:80 dilutions, rapid dual path platform tests, immunochromatographic assay with a recombinant rK39 antigen, fast agglutination screening tests, and direct agglutination tests. We also performed PCR to analyze peripheral blood and xenodiagnosis. RESULTS Forty-six dogs infected at least one L. longipalpis specimen. Although the serological test sensitivities were above 85% for detecting L. longipalpis infectious dogs, none showed a satisfactory performance, as both specificity (0.06 to 13%) and the area under the receiver operating characteristic curve (45 to 53%) were low. The PCR results were also weak, with a sensitivity of 30%, specificity of 72%, and an area under the receiver operating characteristic curve of 51%. The infected L. longipalpis proportion was higher among asymptomatic dogs than symptomatic dogs. Among the symptomatic dogs, those with ulceration-free skin diseases were more infectious, with an odds ratio of 9.3 (confidence interval of 1.10 - 428.5). The larger the number of insects fed, the greater the detected infectiousness. CONCLUSIONS Our study supports the imperative to develop novel technologies for identifying the infectious dogs that transmit L. infantum for the benefit of public health.

The performance of serological tests for Leishmania infantum infection screening in dogs depends on the prevalence of the disease

ABSTRACT Dogs are considered the main reservoir of Leishmania infantum. This protozoan causes visceral leishmaniasis (VL), an uncontrolled urban zoonosis in Brazil. Serological tests and polymerase chain reaction (PCR) on peripheral blood were performed to identify infected dogs in scenarios of higher and lower prevalence of the disease (Teresina and Vitória). One-hundred infected and 57 non-infected animals from Teresina and 100 non-infected animals from Vitória were studied. Animal selection was not dependent on previous serology. The sensitivity (Teresina) and specificity (Teresina and Vitória) were as follows: indirect antibody fluorescence (IFAT) cut-off of 1:40 (IFAT 1:40): 96%, 18%, and 76%; IFAT 1:80: 90%, 33%, and 93%; direct agglutination test (DAT): 96%, 33%, and 98%; fast agglutination screening test (FAST): 93%, 68%, and 100%; immunochromatographic assay with a recombinant rK39 antigen (rK39): 88%, 74%, and 98%; enzyme linked immunosorbent assay (ELISA): 91%, 79%, and 98%; rapid dual-path platform test (TR DPP®): 98%, 60%, and 98%; and blood PCR: 29%, 93%, and 97%, respectively. In the high transmission area, none of the tests adequately discriminated L. infantum-infected from non-infected dogs. However, in the high transmission city, the area under the receiver operating characteristic (ROC) curve of FAST, DAT, ICrK39, ELISA and TR DPP® was high.

Leishmania (infantum) chagasi in canine urinary sediment / Leishmania (infantum) chagasi em sedimento urinário canino

Canine visceral leishmaniasis (CVL) is difficult to diagnosis, mainly due to the presence of asymptomatic animals, the diversity of clinical symptoms and the difficulty in obtaining diagnostic evidence of high sensitivity and specificity. The purpose of this study was to diagnose CVL in urinary sediment of 70 dogs of different breeds, sexes and ages from the veterinary hospital of the Federal University of Piauí and Zoonosis Control Center of Teresina, Brazil. The serological tests were TR DPP® for CVL and enzyme-linked immunosorbent assay (ELISA) for CVL, parasitological exams of bone marrow and lymph nodes and urine sediment cultures. Leishmania was detected in the bone marrow and/or lymph node of 61.0% of the animals (43/70), and urine sediment culture was positive in 9.30% (4/43) of these animals. In the serological exams, 70.0% (49/70) were reactive using the DPP and 78.2% (55/70) were reactive using ELISA. The goal of this study was to diagnose the presence of L. (infantum) chagasi in a culture of urinary sediment.

A leishmaniose visceral canina (LVC) é uma doença de difícil diagnóstico. Principalmente devido à presença de animais assintomáticos, a diversidade da sintomatologia clínica apresentada e também pela dificuldade em se obter uma prova diagnóstica que reúna alta sensibilidade e especificidade. O objetivo deste trabalho foi relatar a presença de L. (infantum) chagasi em meio de cultura, utilizando-se sedimento urinário. Foram utilizados neste experimento, 70 cães provenientes do Hospital Veterinário Universitário da Universidade Federal do Piauí e do Centro de Controle de Zoonoses de Teresina, com raça, sexo e idade variada. Foram realizados exames sorológicos: TR DPP® Leishmaniose Visceral Canina (DPP) e Ensaio Imunoenzimático Leishmaniose Visceral Canina (ELISA), exames parasitológicos de amostras de medula e/ou linfonodo e cultura de sedimento urinário. Em 61,0% (43/70) dos animais estudados, observou-se presença de Leishmania em medula e/ou linfonodo, e destes 9,30% (4/43) foram positivos na cultura de sedimento urinário. Nos exames sorológicos, 70,0% (49/70) dos animais apresentavam-se reativos no DPP e 78,2% (55/70) no ELISA. Pode-se concluir, neste estudo, que é possível diagnosticar a LVC por meio da cultura de sedimento urinário.

Avaliação da infestação de piolhos mastigadores (Struthiolipeurus rheae) em emas (Rhea americana) mantidas em criadouro comercial no estado do Piauí / Evaluation of biting lice infestations (Struthiolipeurus rhea) in rheas (Rhea americana) kept in commercial breeding in the state of Piauí

Avaliou-se a infestação de piolhos mastigadores (Struthiolipeurus rheae) em emas (Rhea americana) mantidas em um criadouro comercial no estado do Piauí. Foram utilizadas 16 emas, sendo 12 adultas, das quais seis eram machos e seis eram fêmeas. Apenas quatro emas analisadas eram jovens. Coletaram-se, aleatoriamente, duas penas das asas e duas da base do pescoço de cada animal, as quais foram colocadas em sacos plásticos fechados. Posteriormente, os piolhos foram contados, preservados em frascos com álcool etílico 70ºGL, e enviados ao Laboratório de Sanidade Animal da Universidade Federal do Piauí. Nas amostras obtidas das emas adultas, encontrou-se um total de 77 piolhos, sendo a incidência nas penas colhidas das asas, 84,4% (65), significativamente maior do que nas penas da base do pescoço, 15,6% (12). Não foram encontrados piolhos nos animais jovens nascidos em chocadeira. As emas adultas apresentaram um alto grau de infestação por S. rheae. A incubação artificial dos animais evitou a infestação das emas jovens por S. rheae. A alta infestação por S. rheae inviabiliza o aproveitamento comercial das penas de emas. Este é o primeiro registro da ocorrência de S. rheae em emas no estado do Piauí.

We evaluated the level of infestation of biting lice (Struthiolipeurus rheae) in emus (Rhea americana) kept in a breeding business in the state of Piaui. We used 16 emus, 12 adults, six of them were males and six females. Only four of emus analyzed were young. Were collected randomly, two wings and two feathers of the base of the neck of each animal, which were placed in plastic bags. Later, lice were counted, preserved in jars with alcohol 70ºGL and sent to the Laboratory of Animal Health of Federal University of Piauí for identification. In samples obtained from adult emus, met a total of 77 lice, and the incidence of the wing feathers collected, 84.4% (65), significantly higher than in the feathers of the lower neck, 15.6% (12 ). No lice were found in young animals born in brooder. The adult rheas showed a high degree of infestation by S. rhea. The artificial incubation of the animals prevented the infestation of young emus S. Rhea. The high infestation S. rhea prevents the commercial use of emu feathers. This is the first record of occurrence of S. rhea in rheas in the state of Piaui.

Infecção natural de Lutzomyia longipalpis por Leishmania sp. em Teresina, Piauí, Brasil / Natural infection of Lutzomyia longipalpis by Leishmania sp. in Teresina, Piauí State, Brazil

No Brasil, o controle do vetor Lutzomyia longipalpis é uma das principais estratégias utilizadas para limitar a expansão da leishmaniose visceral americana. Entretanto, poucos são os estudos sobre a infecção natural destes flebotomíneos por espécies de Leishmania. Um estudo sobre a infecção natural de Lu. longipalpis por Leishmania sp. foi realizado no bairro Bela Vista, Teresina, Piauí, um dos principais focos urbanos da leishmaniose visceral americana no Brasil. Entre fevereiro de 2004 e janeiro de 2005 realizaram-se 180 capturas usando-se armadilhas luminosas do tipo CDC. Foram dissecadas e examinadas, em média, dez fêmeas por captura para detecção de formas evolutivas de Leishmania sp., identificando-se 1.832 exemplares de Lu. longipalpis e seis de Lu. whitmani. Vinte (1,1 por cento) espécimes, todas de Lu. longipalpis, estavam infectadas com as formas procíclica e nectomonada de Leishmania sp., localizadas, principalmente, na porção posterior do trato digestivo. O maior percentual de insetos infectados foi encontrado quatro meses após o período de maior precipitação pluviométrica, sugerindo que variáveis climáticas podem contribuir para a predição não apenas da abundância destes dípteros, mas também do seu grau de infecção natural.

In Brazil, control of the vector Lutzomyia longipalpis is one of the main strategies used to limit the expansion of American visceral leishmaniasis. However, studies on the ecology of this sand fly are rare, especially regarding its natural infection with species of Leishmania. A study of the natural infection of Lu. longipalpis by Leishmania sp. was carried out in the Bela Vista neighborhood in the city of Teresina, Piauí State, Brazil, an important area of American visceral leishmaniasis transmission. From February 2004 to January 2005, sand flies were captured with CDC light traps. Approximately 10 female sand flies in each capture were dissected and examined for the presence of evolutionary forms of Leishmania sp. Two sand fly species were identified: 1,832 were Lu. longipalpis and six Lu. whitmani. Twenty female sand flies (1.1 percent), all Lu. longipalpis, were infected with procyclic and nectomonad forms of Leishmania sp., found mostly in the hindgut. Higher proportions of infected sand flies were found four months after the rainy season, suggesting that environmental factors may predict not only vector abundance (as already known) but also their level of infection.