A simple method for quantifying Leishmania in tissues of infected animals

Lima, Hermenio; Bleyenberg, Julie A.; Titus, Richard G.

doi:10.1016/s0169-4758(96)40010-2

Cited by 149 publications

(118 citation statements)

References 13 publications

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“…The assay was performed according to the method of Titus et al (14) and Lima et al (15). Skin fragments from the inoculation site between the heel and toes were taken from the footpad on the 7th and 30th day of infection, weighed and cut into several pieces in Schneider's medium (Gibco Laboratories, Grand Island, NY, USA), supplemented with 10% fetal calf serum, 2% normal human urine, 5 µg/ml phenol red, 5 mM HEPES, pH 7.3, 50 µg/ml gentamicin, and 100 IU/ml penicillin.…”

Section: Limiting Dilution Assay For Parasite Quantificationmentioning

confidence: 99%

The role of complement in the early phase of Leishmania (Leishmania) amazonensis infection in BALB/c mice

Laurenti

Örn

Sinhorini

et al. 2004

Braz J Med Biol Res

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Complement-depleted and -non-depleted BALB/c mice were inoculated with Leishmania (Leishmania) amazonensis promastigotes into the hind footpad to study the role of the complement system in cutaneous leishmaniasis. Total serum complement activity was measured by hemolytic assay and C3 fragment deposit at the inoculation site was determined by direct immunofluorescence in the early period of infection, i.e., at 3, 24, 48 h and 7 days post-infection. The inflammatory reaction and the parasite burden were evaluated in the skin lesion at 7 and 30 days post-infection. Total serum complement activity decreased in the early phase of infection, from 3 to 24 h, in non-depleted mice compared to non-infected and non-depleted mice. C3 fragment deposit at the site of parasite inoculation was present throughout the period of infection in non-depleted mice. In contrast, no C3 fragment deposit was observed at the inoculation site in complement-depleted mice. Complement-depleted mice showed a significant decrease in the inflammatory response and a significant increase in the number of parasites (70.0 ± 5.3 vs 5.3 ± 1.5) at 7 days of infection (P < 0.05). A higher number of parasites were also present at 30 days of infection at the inoculation site of complement-depleted mice (78.5 ± 24.9 vs 6.3 ± 5.7). These experiments indicate that complement has an important role at the beginning of experimental cutaneous leishmaniasis caused by L. (L.) amazonensis by controlling the number of parasites in the lesion.

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Section: Limiting Dilution Assay For Parasite Quantificationmentioning

confidence: 99%

The role of complement in the early phase of Leishmania (Leishmania) amazonensis infection in BALB/c mice

Laurenti

Örn

Sinhorini

et al. 2004

Braz J Med Biol Res

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“…Os dados numéricos foram obtidos a partir da contagem do número de poços nos quais houve crescimento de promastigotas de leishmânia, verificado pela leitura em microscópio de luz invertida, e foi calculada a média dos valores obtidos para os dois grupos pelo programa Elida ® . 13 Foram usados, também, os testes estatísticos t de Student e Qui-quadrado.…”

Section: Materiais E Métodosunclassified

“…Porém, estudo clíni-co realizado em Manaus com a azitromicina em 26 pacientes teve baixa taxa de cura e muitos efeitos colaterais. 13 As diferenças entre a leishmaniose experimental e em humanos, bem como a variedade de espécies de Leishmania que causam a LTA, podem ser causa dessa disparidade de resultados.…”

Section: Agradecimentosunclassified

O uso da associação azitromicina e N-metil glucamina no tratamento da leishmaniose cutânea causada por Leishmania (Leishmania) amazonensis em camundongos C57BL6

Sampaio

Lucas

Filho

2009

An. Bras. Dermatol.

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Resumo: FUNDAMENTOS: O tratamento de primeira escolha da leishmaniose tegumentar americana é a Nmetil-glucamina que tem alta toxicidade, exige administração parenteral e nem sempre cura. A azitromicina mostrou ação in vitro e resultado contraditório na doença humana. OBJETIVO: Verificar se a associação N-metil glucamina+azitromicina é mais eficaz do que N-metil glucamina no tratamento da leishmaniose experimental. MÉTODOS: 25 camundongos inoculados com a cepa C57BL/6 de L. (L.) amazonensis foram divididos em dois grupos. Um foi tratado com 400mgSbV/kg/dia de N-metil-glucamina associado a 200mg/kg/dia de azitromicina durante 20 dias, e o outro com N-metil glucamina, na mesma dose, durante o mesmo tempo. Foi feita avaliação clínica e parasitológica com análise estatística. RESULTADO: Na avaliação clínica, pesquisa de amastigotas e das culturas, não houve diferença estatística. Verificou-se, entretanto, diferença significante no resultado das culturas realizadas através de diluição limitante, que desfavoreceu a associação NMG+ azitromicina. CONCLUSÃO: A associação N-metil glucamina e azitromicina não demonstrou mais eficácia do que o N-metil glucamina em uso isolado. Palavras-chave: Azitromicina; Leishmaniose cutânea; Leishmaniose cutânea/terapia Abstract: BACKGROUND: The first choice treatment for cutaneous Leishmaniasis is N-methyl glucamine: it has high toxicity, requires parenteral administration and cure is not always reached. Azythromycin showed in vitro action and controversial results in humans with the disease. OBJECTIVE: To verify if the association of N-methyl glucamine -azythromycin is more effective than N-methyl glucamine alone for the treatment of experimental Leishmaniasis. METHODS: Twenty-five C57BL/6 mice were inoculated with L. (L.) amazonensis strain and divided into two groups. One group was treated with 400mgSbV/kg/day of N-methyl glucamine and 200mg/kg/day of azythromycin for 20 days and the other group received the same dose of N-methyl glucamine alone during the same period of time. Clinical and parasitological evaluations were submitted to statistical analyses. RESULTS: There was no statistical difference in clinical analysis, in amastigotes investigation and in cultures. There were significant differences in cultures using limiting dilution, which showed lower efficacy of the association N-methyl glucamine -azythromycin. CONCLUSION: N-methyl glucamine-azythromycin association was not more effective than N-methyl glucamine alone.

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“…Similarly, isolation of Leishmania (Leishmania) infantum chagasi using laboratory animals, such as hamsters 9 , is a very slow and costly method. Several techniques have been described to obtain isolations in vitro, including: cultivation on semi-solid Novy-McNeal-Nicolle medium (NNN) 15 , on RPMI-1640 medium 13 or on Schneider medium enriched with urine 14 , but all fail to perpetuate leishmanias after several passages, thereby limiting large-scale production.…”

Section: Introductionmentioning

confidence: 99%

Reproduction of Leishmania (Leishmania ) infantum chagasi in conditioned cell culture growth medium

Nogueira

Galati

2006

Rev. Inst. Med. trop. S. Paulo

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SUMMARYLeishmanias can be produced by inoculation in conditioned McCoy cell culture growth medium (CGM). Leishmania (Leishmania) infantum chagasi (100 parasites) grown in NNN medium was inoculated in 2.5 mL CGM, kept in plates (24 wells) and its multiplication was observed for five days (120 hours). After day 5, the medium was saturated with the flagellate forms of the parasite (promastigotes). The reproduction of the leishmanias was observed every 24 hours and the number of parasites was calculated by counting the parasites in a drop of 10 µL and photomicrographied. So the number of Leishmanias was adjusted to 1 mL volume.The advantage of the technique by isolation of Leishmania in CGM demonstrated in this study is its low cost and high efficacy even with a small quantity of parasites (10² promastigotes) used as inoculum. Additionally, isolation of the leishmania can be obtained together with an increase in their density (180 times) as observed by growth kinetics, within a shorter time.These results justify the use of this low-cost technique for the isolation and investigation of the behavior and multiplication of Leishmania both in vertebrates and invertebrates, besides offering means of obtaining antigens, whether whole antigens (leishmanias) or the soluble antigens produced by the parasites which may be useful for the production of new diagnostic kits.

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A simple method for quantifying Leishmania in tissues of infected animals

Cited by 149 publications

References 13 publications

The role of complement in the early phase of Leishmania (Leishmania) amazonensis infection in BALB/c mice

The role of complement in the early phase of Leishmania (Leishmania) amazonensis infection in BALB/c mice

O uso da associação azitromicina e N-metil glucamina no tratamento da leishmaniose cutânea causada por Leishmania (Leishmania) amazonensis em camundongos C57BL6

Reproduction of Leishmania (Leishmania ) infantum chagasi in conditioned cell culture growth medium

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