Insights from animal models on the immunogenetics of leprosy: a review

A variety of host immunogenetic factors appear to influence both an individual's susceptibility to infection with Mycobacterium leprae and the pathologic course of the disease. Animal models can contribute to a better understanding of the role of immunogenetics in leprosy through comparative studies helping to confirm the significance of various identified traits and in deciphering the underlying mechanisms that may be involved in expression of different disease related phenotypes. Genetically engineered mice, with specific immune or biochemical pathway defects, are particularly useful for investigating granuloma formation and resistance to infection and are shedding new light on borderline areas of the leprosy spectrum which are clinically unstable and have a tendency toward immunological complications. Though armadillos are less developed in this regard, these animals are the only other natural hosts of M. leprae and they present a unique opportunity for comparative study of genetic markers and mechanisms associable with disease susceptibility or resistance, especially the neurological aspects of leprosy. In this paper, we review the recent contributions of genetically engineered mice and armadillos toward our understanding of the immunogenetics of leprosy.

Mycobacterium leprae in six-banded (Euphractus sexcinctus) and nine-banded armadillos (Dasypus novemcinctus) in Northeast Brazil

Human beings are the main reservoir of the causative agent of leprosy, Mycobacterium leprae. In the Americas, nine-banded armadillos (Dasypus novemcinctus) also act as a reservoir for the bacillus. In the state of Ceará (CE), which is located in Northeast Brazil and is an endemic area of leprosy, there are several species of armadillos, including D. novemcinctus and Euphractus sexcinctus (six-banded armadillo). Contact between humans and armadillos occur mainly through hunting, cleaning, preparing, cooking and eating. This study identified M. leprae DNA in the two main species of armadillos found in Northeast Brazil. A total of 29 wild armadillos (27 D. novemcinctus and 2 E. sexcinctus) were captured in different environments of CE countryside. Samples from the ear, nose, liver and spleen from each of these animals were tested by a nested M. leprae-specific repetitive element polymerase chain reaction assay. The samples that tested positive were confirmed by DNA sequencing. M. leprae was detected in 21% (6/29) of the animals, including five D. novemcinctus and one E. sexcinctus. This is the first Brazilian study to identify the presence of a biomarker of M. leprae in wild armadillos (D. novemcinctus and E. sexcinctus) in a leprosy hyperendemic area where there is continuous contact between humans and armadillos.

Coccidioidomycosis in armadillo hunters from the state of Ceará, Brazil

Coccidioidomycosis is a systemic mycosis with a variable clinical presentation. Misdiagnosis of coccidioidomycosis as bacterial pneumopathy leads to inappropriate prescription of antibiotics and delayed diagnosis. This report describes an outbreak among armadillo hunters in northeastern Brazil in which an initial diagnosis of bacterial pneumonia was later confirmed as coccidioidomycosis caused by Coccidioides posadasii. Thus, this mycosis should be considered as an alternative diagnosis in patients reporting symptoms of pneumonia, even if these symptoms are only presented for a short period, who are from areas considered endemic for this disease.

Search for Mycobacterium leprae in wild mammals

Leprosy is still a worldwide public health problem. Brazil and India show the highest prevalence rates of the disease. Natural infection of armadillos Dasypus novemcinctus with Mycobacterium leprae has been reported in some regions of the United States. Identification of bacilli is difficult, particularly due to its inability to grow in vitro. The use of molecular tools represents a fast and sensitive alternative method for diagnosis of mycobacteriosis. In the present study, the diagnostic methods used were bacilloscopy, histopathology, microbiology, and PCR using specific primers for M. leprae repetitive sequences. PCR were performed using genomic DNA extracted from 138 samples of liver, spleen, lymph nodes, and skin of 44 D. novemcinctus, Euphractus sexcinctus, Cabassous unicinctus, and C. tatouay armadillos from the Middle Western region of the state of São Paulo and from the experimental station of Embrapa Pantanal, located in Pantanal da Nhecolândia of Mato Grosso do Sul state. Also, the molecular analysis of 19 samples from internal organs of other road killed species of wild animals, such as Nasua nasua (ring-tailed coati), Procyon cancrivoros (hand-skinned), Cerdocyon thous (dog-pity-bush), Cavia aperea (restless cavy), Didelphis albiventris (skunk), Sphigurrus spinosus (hedgehog), and Gallictis vittata (ferret) showed PCR negative data. None of the 157 analyzed samples had shown natural mycobacterial infection. Only the armadillo inoculated with material collected from untreated multibacillary leprosy patient presented PCR positive and its genomic sequencing revealed 100 percent identity with M. leprae. According to these preliminary studies, based on the used methodology, it is possible to conclude that wild mammals seem not to play an important role in the epidemiology of leprosy in the Middle Western region of the São Paulo state and in the Pantanal of Mato Grosso do Sul state.

Contact with armadillos increases the risk of leprosy in Brazil: a case control study.

BACKGROUND: Mycobacterium leprae infection has recently been detected in wild armadillos in Brazil. Leprosy is still endemic in Brazil and although its transmission is mostly by person-to-person contact, many cases report no history of previously known leprosy contact. It has been suggested that other sources of M. leprae may contribute to the transmission of leprosy in some areas. Aim: Our objective was to investigate whether contact with armadillos is associated with leprosy. METHODS: A case-control study was carried out in Brazil. Data was collected from 506 leprosy patients and 594 controls on exposure to armadillos and age, sex, place of birth and living conditions. Univariate analysis and unconditional logistic regression were conducted to investigate whether leprosy was associated with exposure to armadillos. RESULTS: Direct armadillo exposure was reported by 68% of leprosy cases and by 48% of controls (P < 0.001) roughly doubling the risk of leprosy, with odds ratio (OR) 2.0, 95% confidence interval (CI) [1.36-2.99]. CONCLUSION: The results of this study suggest that direct exposure to armadillos is a risk factor for leprosy in Brazil.

Experimental infections with Paracoccidioides brasiliensis obtained from armadillos: comparison to clinical isolates

Paracoccidioides brasiliensis causes paracoccidioidomycosis (PCM) that is one of the most prevalent systemic human mycoses in Latin America. Armadillos show a high incidence of PCM infection and could, therefore, be a natural reservoir for this fungus. In this study were compared the virulence profiles of isolates obtained from nine-banded armadillos (Dasypus novemcinctus) (PbT1 and PbT4) and isolates from PCM patients (Pb265 and Bt83). Pathogenicity was evaluated by fungal load and analysis of colony morphology. Immunity against the fungus was tested by delayed type hypersensitivity test (DTH) and antibody quantification by ELISA. The higher virulence of PbT1 and PbT4 was suggested by higher fungal load in spleen and lungs. Armadillo isolates and Bt83 presented a cotton-like surface contrasting with the cerebriform appearance of Pb265. All isolates induced cellular and humoral immune responses in infected BALB/c mice. DTH reactions were similarly induced by the four isolates, however, a great variability was observed in specific antibody levels, being the highest ones induced by Bt83 and PbT4. The present work confirms that armadillos harbor P. brasiliensis, whose multiplication and induced immunity in experimentally infected mice are heterogeneous, resembling the behavior of isolates from human PCM. This study reinforces the possibility that armadillos play an important role in the biological cycle of this pathogen.

Pesquisa de anticorpos anti PGL-I através de ELISA em tatus selvagens do Brasil / Research regarding anti-PGL-I antibodies by ELISA in wild armadillos from Brazil

Tatus têm sido envolvidos na transmissão da hanseníase e considerados como fonte de Mycobacterium leprae em muitas publicações. Médicos de partes dos EUA consideram o contato com tatus um fator de risco para hanseníase. Entretanto, há um desafio associado ao papel do tatu na perpetuação da hanseníase no Continente Americano. Foi pesquisada a presença de anticorpos anti-PGL-I em tatus selvagens de áreas endêmicas em hanseníase do Estado do Espírito Santo, Brasil, através de ELISA realizado em amostras de soro de 47 animais. Elisa positivo foi encontrado em 5 (10.6%) tatus. Tatus infectados podem ter algum papel na transmissão da hanseníase disseminando bacilos no meio ambiente, talvez tornando mais difícil a interrupção da cadeia de transmissão e redução do número de casos novos de hanseníase. A técnica de ELISA é um eficiente método para investigação soroepidemiológica da presença do Mycobacterium leprae em tatus.

Armadillos have been involved in leprosy transmission and are considered a source of Mycobacterium leprae in numerous reports. Clinicians from certain areas of the USA consider contact with armadillos a risk factor for leprosy. However, there is a challenge associated with the role of wild armadillos perpetuating human leprosy in the American Continent. The presence of anti-PGL-I antibodies was investigated in wild nine-banded armadillos from leprosy-endemic areas in State of Espirito Santo, Brazil, by ELISA performed on serum samples from 47 armadillos. Positive ELISA was obtained from 5 (10.6%) armadillos. Infected armadillos may play some role in leprosy transmission, disseminating bacilli in the environment, perhaps making it more difficult to interrupt transmission and reduce the number of new leprosy cases. ELISA is an efficient tool for seroepidemiological investigations of Mycobacterium leprae in armadillos.

On the unknown ecological niche of Paracoccidioides brasiliensis: our hypothesis of 1989: present status and perspectives

In 1989, CONTI DÍAZ & RILLA published a hypothesis concerning the as yet unknown ecological niche of Paracoccidioides brasiliensis. The hypothesis proposed a highly efficient paracoccidioidal ecological strategy based on an important natural reservoir of the parasite, probably in heterothermic animals from fresh water environments. Further, the hypothesis proposed, a transient and variable residence in the soil with a wide aleuriospore dispersion throughout the environment together with an elevated capability of infecting humans, and domestic and wild animals. This paper analyzes scientific publications from the IX International Meeting on Paracoccidioidomycosis held in Aguas de Lindoia, São Paulo, Brazil from 2-5 October 2005, providing a comparative study among this articles and with other recently published papers and the hypothesis' postulates. Since various findings and observations appear to agree with the postulates, the pursuit of novel, specific research projects in the supposed reservoirs is recommended partially or fully to confirm the hypothesis using classical laboratorial methods and modern molecular biology techniques.

En 1989 CONTI DÍAZ & RILLA publicaron una hipótesis sobre el aún hoy desconocido nicho ecológico de Paracoccidioides brasiliensis proponiendo esencialmente en ella la existencia de una estrategia ecológica altamente eficiente del agente. al estrategia estaría basada en una importante reserva del parásito en animales heterotérmicos de fuentes naturales de agua dulce, una estadía sólo transitoria y de variable duración en el suelo, con una amplia dispersión de aleuriosporos en el ambiente exterior con capacidad de infectar al hombre y a los animales. El presente trabajo analiza comunicaciones científicas del último Encuentro Internacional sobre Paracoccidioidomicosis celebrado en Aguas de Lindoia, San Pablo, Brasil, entre el 2 y 5 de octubre del 2005, comparando diferentes conceptos y resultados allí emitidos como también de otros trabajos al respecto, con los postulados de la hipótesis. Desde el momento en que se comprueba una aparente clara correlación entre unos y otros, se insiste en la conveniencia y necesidad de llevar a cabo nuevos proyectos de investigación al respecto centrados fundamentalmente en la búsqueda del agente en los probables animales reservorios, mediante métodos micológicos clásicos y técnicas de biología molecular tan bien manejadas en varios laboratorios de la región.

Detection of Mycobacterium leprae infection in wild nine-banded armadillos (Dasypus novemcinctus) using the rapid ML Flow test

Mycobaterium leprae infection was investigated in armadillos from the State of Espírito Santo, Brazil. The ML Flow test was performed on 37 nine-banded armadillos and positive results were found in 11 (29.7 percent). The ML Flow test may be used to identify possible sources of Mycobaterium leprae among wild armadillos.

Tem sido pesquisado infecção pelo Mycobaterium leprae em tatus provenientes do estado do Espírito Santo-Brasil. O teste rápido ML Flow, foi realizado em 37 tatus selvagens, tendo sido positivo em 11 (29,7 por cento). O teste de ML Flow pode ser utilizado para identificar possíveis fontes de Mycobaterium leprae em tatus selvagens.

Virulence attenuation and phenotypic variation of Paracoccidioides brasiliensis isolates obtained from armadillos and patients

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, the most important systemic mycosis in Latin America. The virulence profiles of five isolates of P. brasiliensis were studied in two different moments and correlated with some colonial phenotypic aspects. We observed a significant decrease in the virulence and an intense phenotypic variation in the mycelial colony. The recognition of all ranges of phenotypic and virulence variation of P. brasiliensis, as well as its physiological and genetic basis, will be important for a better comprehension of its pathogenic and epidemiological features.

Pesquisa de Mycobacterium leprae e outras micobactérias em tatus selvagens / Search of Mycobacterium leprae and other mycobacteria in tatus

A hanseníase é, ainda hoje, um problema global de saúde pública. O Brasil e a Índia são os países com maior prevalência da doença. A existência de tatus Dasypus novemcinctus naturalmente infectados por Mycobacterium leprae foi relatada nos Estados Unidos, México, Argentina e no Brasil, no Estado do Espírito Santo. Micobactérias potencialmente patogênicas e algumas ambientais já foram isoladas destes animais. Aidentificação do bacilo é dificultada, particularmente pela sua incapacidade de crescimento in vitro. Portanto, o uso de ferramentas moleculares representa uma alternativa mais rápida e sensível para diagnóstico de micobacterioses. Neste trabalho foram feitas baciloscopia, cultura e PCR com primers específicos para sequências repetitivas de M. leprae, a partir de DNA extraído de amostras de fígado, baço, linfonodose pele de 21 tatus D. novemcinctus, Euphractus sexcintus e Cabassous tatouay provenientes da região Centro-Oeste do Estado de SP, dos quais 17 eram provenientes do Banco de DNA do Laboratório de Biologia de Fungos do Departamento de Microbiologia e Imunologia - IBB. Para a padronização da metodologia foram utilizados hamsters experimentalmente infectados por M. avium e o modelo experimental para hanseníase (D. novemcinctus) do Biotério de Experimentação Animal do Instituto Lauro de Souza Lima, Bauru-SP. Com exceção de dois tatus, cujas amostras fezes foram positivas pela PCR, nenhum tatu selvagem apresentou infecção natural por micobactérias. Somente o tatu inoculado com biópsia de paciente virchowiano foi positivo, tanto na PCR, quanto no sequenciamento genômico, que revelou 100% de identidade com o M. leprae. Os resultados preliminares deste estudo, com base na metodologia empregada, sugerem que existe um indicativo de que os tatus selvagens podem apresentar papel de reservatório de M. leprae na cadeia epidemiológica da doença, na região estudada.

Inquérito epidemiológico sobre a paracoccidioidomicose utilizando a gp43 em dois municípios do noroeste do Paraná, Brasil / Paracoccidioidomycosis epidemiological survey using gp43, in two cities of northwestern region of Paraná, Brazil

Foi realizado um inquérito epidemiológico sobre paracoccidioidomicose com gp43, em 118 operários no Noroeste do Paraná. A positividade foi de 43 por cento, o que aliado às condições ambientais favoráveis ao desenvolvimento do fungo reforçam que essa região seja endêmica para paracoccidioidomicose e também reservárea de Paracoccidioides brasiliensis.

Pesquisa de Mycobacterium leprae em tatus selvagens da espécie Dasypus Novencintus do Estado do Espírito Santo / Mycobacterium leprae research in wild armadillos of the Dasypus novemcinctus specie from Espirito Santo state

Introducao: Realizou-se um estudo de prevalencia em 52 tatus selvagens da especie Dsypus novemcinctus, capturados no estado do Espirito Santo, no periodo de junho de 2000 a julho de 2001. Objetivos: Estudar a prevalencia da infeccao pelo Mycobacterium leprae e compara-la aos trabalhos publicados na literatura, bem como avaliar e comparar os metodos utilizados para deteccao da infeccao. Metodos: Apos anestesiados, os tatus foram pesados, determinado o sexo, submetidos a exame clinico e coleta de fragmento de orelha para estudos histopatologicos (hematoxilina-eosina e Ziehl-Neelsen), imunohistoquimicos com anti-BCG, e fragmentos de orelha, pele, pata e lesao cutanea para a tecnica de PCR. Vinte tres tatus foram necropsiados e fragmentos de figado, baco, cerebro, pulmao, rim e intestino foram obtidos para a tecnica de PCR. Resultados e conclusoes: Nao foram evidenciadas lesoes de hansenomas e apenas um tatu apresentou linfadenomegalia palpavel na regiao inguinal dentre os 52 tatus examinados. Nao foi evidenciado quadro histopatologico compativel com nenhuma das formas de hanseniase pela coloracao de hematoxilina-eosina, e nao foi evidenciado presenca de BAAR pela tecnica de Ziehl-Neelsen, nem imunoexpressao do marcador BCG compativel com M. leprae. Foi detectada 10,6 porcento de soro-prevalencia de anticorpos IgM anti-PGL-I, e 11,9 porcento de positividade nas amostras de sangue, atraves da tecnica de PCR. Foi detectada 52,8 porcento de positividade na pesquisa de DNA de M. leprae nos fragmentos de tecidos (pele, orelha, lesao, pata, figado, baco, pulmao, cerebro, rim e intestino) atraves da tecnica de PCR. Dos tecidos testados, o cerebro foi o orgao onde mais se encontrou positividade pela tecnica de PCR (40,9 porcento), seguido da pele (22,2 porcento), orelha (20 porcento), pulmao (13,6 por cento), baco (13 porcento), pata (11,5 porcento), lesao cutanea (11,1 porcento), figado (8,7 porcento), rim (8,7 porcento) e intestino (4,5 porcento). O metodo diagnostico mais sensivel para deteccao de M. leprae foi o PCR dos tecidos, seguido do PCR do sangue e pesquisa de anticorpos anti-PGL-I atraves da tecnica de ELISA.

Algunos aspectos de la bacteriología de Mycobnacterium leprae / Some aspects regarding Mycobacterium leprae bacteriology

Se brinda una información de algunos aspectos de la baceteriología de Mycobacterium leprae, a través de la historia del descubrimiento del agente causal, sus características, estructura, modelos experimentales (ratón y armadillo), antígenos carbohidratos y genética

Viability of acid-fast bacilli from gamma-& UV-irradiated lepromatous armadillo tissues infected with Mycobacterium leprae.

Gamma-irradiated splenic homogenates of armadillos infected with M. leprae proved sterile by conventional tests and media. However, on media for chemoautotrophy, these could repeatedly grow as a single type of acid-fast nocardioform bacterium like the unirradiated specimens, although with a much reduced count. In the slide culture, transition from the initial AFB/coccoid bodies, to sporulating mycelia and granules in the final stage, could be observed sequentially. The gamma-irradiated tissue specimens failed to yield any other mycobacterium/corynebacterium tested according to standard protocols.

An attempt to produce experimental tuberculoid leprosy in the nine-banded armadillo.

In an attempt to produce experimental tuberculoid leprosy, three nine-banded armadillos, two with borderline tuberculoid lepromin reaction, and one with tuberculoid lepromin reaction, were chosen. They were injected subcutaneously in a four square centimetre area in the abdominal skin with saline suspension of 6.5 x 10(7) M. leprae. Induration of skin at the injected site appeared in 24 hours and persisted for 6 months in one and for 18 months in the other two animals. Histopathological examination of the infected site at 6 weeks, 18 and 20 months showed progressively decreasing granulomatous inflammation; but the cutaneous nerves were uninvolved. Autopsy examination of the three animals failed to show disseminated disease. Since there was no evidence of nerve involvement, experimental transmission of tuberculoid leprosy to armadillos could not be established in this study.

Early infection with M. leprae and antibodies to phenolic glycolipid-I in the nine-banded armadillo.

Nine-banded armadillos were intravenously infected with 10(9) M. leprae. IgM antibodies to PGL-I were evaluated three times during the six months before and every two months after the infection. A thorough autopsy examination was done on animals that died or were sacrificed at intervals of 3, 4, 6, 12, 15 and 18 months after the infection. Three animals which had acquired the infection in the wild and one experimentally infected animal showed significant increases in antibody levels corresponding to their high bacterial load. In the other five experimentally infected animals, M. leprae infection was established in the cells of the reticulo endothelial system (RES) long before the IgM antibody levels to PGL-I became positive. It is possible that in human leprosy also M. leprae may enter and multiply in the RES initiating antibody production during the incubation period before clinical disease with neuritis becomes manifest.

Effect of temperature, cholesterol and nerve tissue on multiplication of armadillo M. leprae.

The effect of temperature, nerve tissue and certain constituents of the medium on multiplication of armadillo M. leprae was studied using Hanks BSS. An equal or better growth was seen at 30 degrees C and 10 degrees C compared to 37 degrees C. Multiplication was also seen at -20 degrees C. Adding cholesterol, foetal calf serum, cystine-HCl, sodium thioglycollate or nerve suspension and covering medium with liquid paraffin each showed beneficial effect. Hanks containing foetal calf serum, cholesterol with sodium thioglycollate or cystine-hydrochloride showed maximum multiplication. These combinations may be used for testing additional factors for further improvement of the medium.