The Armadillo (Dasypus novemcinctus): A Witness but Not a Functional Example for the Emergence of the Butyrophilin 3/Vγ9Vδ2 System in Placental Mammals.

1-5% of human blood T cells are Vγ9Vδ2 T cells whose T cell receptor (TCR) contain a TRGV9/TRGJP rearrangement and a TRDV2 comprising Vδ2-chain. They respond to phosphoantigens (PAgs) like isopentenyl pyrophosphate or (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMBPP) in a butyrophilin 3 (BTN3)-dependent manner and may contribute to the control of mycobacterial infections. These cells were thought to be restricted to primates, but we demonstrated by analysis of genomic databases that TRGV9, TRDV2, and BTN3 genes coevolved and emerged together with placental mammals. Furthermore, we identified alpaca (Vicugna pacos) as species with typical Vγ9Vδ2 TCR rearrangements and currently aim to directly identify Vγ9Vδ2 T cells and BTN3. Other candidates to study this coevolution are the bottlenose dolphin (Tursiops truncatus) and the nine-banded armadillo (Dasypus novemcinctus) with genomic sequences encoding open reading frames for TRGV9, TRDV2, and the extracellular part of BTN3. Dolphins have been shown to express Vγ9- and Vδ2-like TCR chains and possess a predicted BTN3-like gene homologous to human BTN3A3. The other candidate, the armadillo, is of medical interest since it serves as a natural reservoir for Mycobacterium leprae. In this study, we analyzed the armadillo genome and found evidence for multiple non-functional BTN3 genes including genomic context which closely resembles the organization of the human, alpaca, and dolphin BTN3A3 loci. However, no BTN3 transcript could be detected in armadillo cDNA. Additionally, attempts to identify a functional TRGV9/TRGJP rearrangement via PCR failed. In contrast, complete TRDV2 gene segments preferentially rearranged with a TRDJ4 homolog were cloned and co-expressed with a human Vγ9-chain in murine hybridoma cells. These cells could be stimulated by immobilized anti-mouse CD3 antibody but not with human RAJI-RT1Bl cells and HMBPP. So far, the lack of expression of TRGV9 rearrangements and BTN3 renders the armadillo an unlikely candidate species for PAg-reactive Vγ9Vδ2 T cells. This is in line with the postulated coevolution of the three genes, where occurrence of Vγ9Vδ2 TCRs coincides with a functional BTN3 molecule.

Cytokine responses to Mycobacterium leprae unique proteins differentiate between Mycobacterium leprae infected and naive armadillos.

New diagnostic tools for early detection of leprosy are necessary to help reduce its transmission and severity. M. leprae unique proteins have been used to assess differences in human T-cell responses in leprosy patients, household contacts and endemic controls. In this study, we examined the response of M. leprae-infected armadillos to a variety of M. leprae recombinant antigen candidates currently being examined for diagnostic efficacy in humans. Among recently M. leprae infected armadillos, IFN-gamma expression was enhanced after stimulation of PBMC with all M. leprae recombinant proteins except for ML2283 (mean: 2.65 Relative Quantification (RQ)). The group mean stimulation index for M. leprae proteins ML0009, ML1601, ML2478 and ML2531 averaged 35.2 RQ and was significantly higher (P < 0.05) than that measured among the non-infected, naive group (mean 6.2 RQ). Although ML0840 tended to enhance IFN-gamma levels, the mean IFN-gamma transcript levels of the currently experimentally inoculated group (20.1 RQ) was not significantly different statistically (P = 0.10) from the mean of the naive group (7.5 RQ). Also no statistically significant differences were observed in IFN-gamma transcript levels between the resistant and currently experimentally inoculated group (P > 0.05) or between the resistant and the naive group (P > 0.05) after stimulation of PBMCs with all M. leprae recombinant proteins. Only low levels of TNF-alpha were observed across all groups after in vitro stimulation with all the antigens examined. These data suggest that armadillos can be used effectively to help identify M. leprae specific proteins that may be applied for monitoring T-cell responses in M. leprae infected hosts as their disease progresses as well as for the early diagnosis of leprosy.

Characterization of langerhans cells in epidermal sheets along the body of Armadillo (Dasypus novemcinctus).

Armadillos are apparently important reservoirs of Mycobacterium leprae and an animal model for human leprosy, whose immune system has been poorly studied. We aimed at characterizing the armadillo's langerhans cells (LC) using epidermal sheets instead of tissue sections, since the latter restrict analysis only to cut-traversed cells. Epidermal sheets by providing an en face view, are particularly convenient to evaluate dendritic morphology (cells are complete), spatial distribution (regular vs. clustered), and frequency (cell number/tissue area). Lack of anti-armadillo antibodies was overcome using LC-restricted ATPase staining, allowing assessment of cell frequency, cell size, and dendrites extension. Average LC frequency in four animals was 528 LC/mm(2), showing a rather uniform non-clustered distribution, which increased towards the animal's head, while cell size increased towards the tail; without overt differences between sexes. The screening of antibodies to human DC (MHC-II, CD 1a, langerin, CD86) in armadillo epidermal sheets, revealed positive cells with prominent dendritic morphology only with MHC-II and CD86. This allowed us to test DC mobilization from epidermis into dermis under topical oxazolone stimulation, a finding that was corroborated using whole skin conventional sections. We hope that the characterization of armadillo's LC will incite studies of leprosy and immunity in this animal model.

Lepra en el armadillo salvaje / No disponible

Los armadillos salvajes de nueve bandas (Dasypus novemcinctus) de la zona central y sur de Estados Unidos son huéspedes naturales y endémicos de Mycobacterium leprae. Los estudios llevados a cabo durante los últimos 30 años o más en alrededor de 5.000 animales confirman que la infección está presente en Arkansas, Louisiana, Mississippi y Texas. Los índices de prevalencia más elevada se encuentran entre los animales de las zonas aluviales de menor altura y áreas costeras, principalmente en Lousiana y Texas. Tanto la densidad poblacional de animales como los factores locales pueden contribuir a la que se detecte lepra en el armadillo de cualquiera de estas zonas de los EE.UU. y sólo hay pocos casos que describan la infección entre animales de América del Sur o Central. Sin embargo, este tema ha suscitado escaso interés en otros países. Los armadillos son relativamente recientes en los EE.UU. y la lepra ya estaba presente en Texas y Lousiana antes de su llegada. No está muy clara la relación ecológica entre los humanos y los armadillos. Sin embargo, los armadillos infectados son un gran reservorio de M. leprae y pueden ser fuente de infección para algunos individuos de este país y quizás en otras partes comprendidas dentro del alcance de estos animales

Wild nine-banded armadillos (Dasypus novemcictus) in the south central United States are highly endemic natural hosts of Mycobacterium leprae. Surveys conducted over the lass 30 years on more that 5.000 animals confirm that the infection is present among armadillos in Arkansas, Louisiana, Mississippi and Texas. Highest prevalence rates are found among the animals in low-lying alluvial and coastal areas, primarily in Lousiana and Texas. Both animal density and local factors may contribute to the detectability of armadillo leprosy in those regions. Little evidence for M.leprae infection is found among armadillos elsewhere in the US range, and only a few reports relate finding the infection among animals in Central or South America. However, the issue has received only scant attention in other countries. Armadillos only recently expanded their range into the Us, and leprosy was present in Texas and Lousiana prior to the arrival of armadillos. The ecological relationship between humans and armadillos with M. leprae in this region remains unclear. However, infected armadillos constitute a large reservoir of M. leprae and they may be a source of infection for some humans in this country, and perhaps in other locations across the animal´s range

Expression of nine-banded armadillo (Dasypus novemcinctus) interleukin-2 in E. coli.

The nine-banded armadillo (Dasypus novemcinctus) is the only immunologically intact animal that regularly develops lepromatous-type leprosy when inoculated with Mycobacterium leprae. However, the ability to exploit this model for understanding the pathogenesis of leprosy has been limited by a lack of suitable immunological reagents. Recently, efforts began to sequence the entire armadillo genome, and this sequence information will help make possible the development of a wide array of new immunological reagents suitable for use with armadillos. Using the available sequence data, a region of high homology to interleukin-2 of other mammals was identified. Primers were designed to amplify the coding region corresponding to the mature peptide and its exact sequence was confirmed. cDNA was made from ConA-stimulated armadillo PBMC. The amplified coding region was sub-cloned into a pET expression vector and transformed into Escherichia coli for over-expression. The subsequent product was characterized by SDS-PAGE and bioassays. Tritiated thymidine incorporation by CTLL-2 and armadillo lymphoblasts confirmed functionality of the recombinant product. The advent of the D. novemcinctus genome sequence and subsequent generation of immunological tools will assist in advancing the armadillo as a translational model for leprosy.

Partial characterization of phagocytic activity in neutrophils of the nine-banded armadillo Dasypus novemcinctus.

Though the armadillo is important as a research model in leprosy studies, the activity of armadillo's neutrophils is an aspect of little research. The aim of this study was carried out to partially characterize the chemotaxis, endocytosis and bacteriocidal ability of the neutrophils found in the nine-banded armadillo (Dasypus novemcinctus). Results showed that the chemotactic activity of the neutrophils, evaluated by the movement of the neutrophils through a nitrocellulose membrane (5 microm) in response to a chemo-attractive substance, was greater towards the armadillo serum (5.16+/-1.35 migration index, p<0.05) than towards the formil methionyl leucil phenylalanine (fMLP, 1.43+/-0.18 migration index) or human serum (0.56+/-0.18 migration index). Regarding endocytic capacity of the neutrophils and the monocytes against Escherichia coli was evaluated by a flow cytometry and using opsonized and non-opsonized E. coli-FITC at the following incubation times: 5, 10, 20, 30 and 60 min. The largest percentage of endocytosis by the neutrophils was 92.32+/-0.12% with opsonized bacteria and 77.73+/-14.33% with non-opsonized bacteria at 10 min incubation time, while the largest percentage of endocytosis by monocytes was 89.94+/-1.40% with opsonized bacteria and 73.07+/-15.6% with non-opsonized bacteria at 20 min incubation time. Evaluation of the bacteriocidal capacity of neutrophils using the methyl-thiazol-tetrazolium salts (MTT) reduction color-measurement assay showed an 89.0+/-10% mortality rate of non-opsonized E. coli and 89.0+10% of opsonized E. coli. In conclusion, the armadillo neutrophils show a good phagocytosis and bacteriocidal activity; however, a deficiency in the migration towards the fMLP was observed. This deficiency could be a cause so that the armadillo neutrophils do not respond quickly to invading microorganism.

Nine-banded armadillo and leprosy research

In this presentation an attempt has been made to describe the nine-banded armadillo as an animal model, probably the only one in which lepromatous leprosy similar to that found in humans can be experimentally produced. Some unique features of the physiology of the animal are mentioned. The pathology and the microbiology of leprosy in the armadillo are described in detail. The discovery of lepromatous leprosy in the wild armadillos in the southern parts of United States, the transmission of disease among them through trauma and thorn pricks and the pathogenesis of the disease are presented. The impact of leprosy in the wild animals may have on human leprosy is discussed.

Immune response of armadillos (Dasypus novemcinctus). I. Use of lectins to identify lymphocyte subpopulations and to evaluate cell proliferation.

Lectins have been used to study populations and discrete differentiation stages of lymphocytes. Likewise, lectins have been of practical importance in promoting mitogenic stimulation of lymphocytes in numerous species. In this research project, we took advantage of these tools in an attempt to identify specific subsets of peripheral blood lymphocytes obtained from healthy nine-banded armadillos (Dasypus novemcinctus). The same cell source served to evaluate mitogenic stimulation. Twelve FITC-labeled lectins were used; 5 (ConA, LcH, RCA, WGA and UEA) reacted with almost 100% of the lymphocytes and 7 (PNA, DBA, SBA, PCA, PHA-L, PWM and VVA) recognized variable percentages (< 100% of these cells). This latter group of lectins may be useful in the identification of armadillo lymphocyte subsets, or may correlate with discrete stages of differentiation of these cells. The same lectins served to evaluate mitogenic stimulation in an aliquot of the same peripheral blood mononuclear cells. Of the 12 lectins studied, 5 (ConA, PHA-L, PWM, DBA and SBA) had the capacity to induce mitogenic stimulation in the whole mixture of mononuclear cells, giving rise to variable degrees in the corresponding mitogenic index obtained for each of the 5 lectins. Those lectins that gave an indication of selective identification of lymphocytes, that is, the percentages at or below 75%, may prove useful in the evaluation of the immune response of healthy armadillos as well as the evolution of progression stages of lepromatous leprosy in armadillos inoculated with the same strain of Mycobacterium leprae that affects humans.

Analysis of vaccines prepared from armadillo-derived M. leprae; results of an inter-laboratory study coordinated by the World Health Organization. Immunology of Mycobacterial Diseases (IMMYC) Steering Committee, World Health Organization.

Preparations of armadillo-derived Mycobacterium leprae used in vaccine trials were analyzed using a combination of morphological, chemical and immunological criteria. When compared to more recent preparations, vaccine lots prepared in 1984 and 1985 were found to contain fewer intact bacilli and lower amounts of M. leprae antigens. These differences may be characteristic of the original preparations, or alternatively, may have arisen during prolonged storage. The early vaccine lots were those used in the recently published Venezuela trial.

Identification and purification of armadillo (Dasypus novemcinctus) immunoglobulins: preparation of specific antisera to evaluate the immune response in these animals.

In this work we describe the purification and characterization of armadillo immunoglobulins. The IgM was precipitated using low-strength ionic solution and further purified by filtration through Sephadex G-200. The IgG was obtained in pure form by precipitation of serum with ammonium sulfate and DEAE-cellulose ion exchange chromatography. The purity of these immunoglobulins was evaluated by polyacrylamide gel electrophoresis. The results showed 28-kDa light chains and 55-kDa and 70-kDa heavy chains for IgG and IgM, respectively. The rabbit antibodies against these molecules were used to prepare fluorescein (FITC) and peroxidase conjugates. The FITC conjugate was used to quantify IgM-bearing lymphocytes. An average of 17% of peripheral blood lymphocytes were sIgM+ from 14 healthy animals. Additionally, in the same animals we quantified lymphocytes with the capacity to form rosettes with sheep red-blood cells; the average for this marker was 10%. Also, the production of crossreacting antibodies to BCG was evaluated in healthy and Mycobacterium leprae-inoculated animals using the peroxidase conjugates. All animals with active infection recognized BCG antigens.

The mycobacterial secreted antigen 85 complex possesses epitopes that are differentially expressed in human leprosy lesions and Mycobacterium leprae-infected armadillo tissues.

The granulomatous skin lesions in leprosy are thought to be initiated by the immune response to certain antigens of the causative agent, Mycobacterium leprae. The antigen 85 complex is one of the major targets in the immune response to M. leprae infection. In the present study, a panel of previously characterized monoclonal antibodies (MAbs) (3A8, Rb2, A4g4, A2h11, Pe12, and A3c12) reacting with different epitopes of the 85 complex proteins of Mycobacterium tuberculosis and M. leprae was employed in a comparative immunohistological analysis to demonstrate the in situ expression of 85 complex antigenic epitopes in leprosy lesions across the clinical spectrum and in M. leprae-infected armadillo liver tissues. These MAbs showed a heterogeneous staining pattern in a given leprosy lesion. In highly bacilliferous borderline and lepromatous leprosy lesions, MAbs Rb2, A4g4, A2h11, and Pe12 stained clear rod-shaped M. leprae bacilli within macrophages, and the degree of staining correlated with the bacillary index of the lesion. On the other hand, MAbs 3A8 and A3c12 staining was mostly seen as a diffuse staining pattern within interstitial spaces and on the membranes of the infiltrated cells but not the bacilli. In paucibacillary borderline and tuberculoid leprosy lesions, only 3A8, Rb2, and A3c12 showed distinct staining in association with infiltrates in the granuloma. None of these MAbs showed any detectable reaction with control nonleprosy skin lesions, while MAb A3c12 positively stained the granulomas of both leprosy and control specimens. In situ reactivity of these MAbs with M. leprae-infected armadillo liver tissues also showed a heterogeneous staining pattern. Interestingly, a clear difference in expression of these epitopes was observed between armadillo tissues and human leprosy lesions. By immunogold ultracytochemistry, we further showed the differential localization of these MAb-reactive epitopes on the cell surface, in the cytosol, and at the vicinity of M. leprae within Kupffer cells of armadillo liver tissues. Our results indicate that these antigenic epitopes of the antigen 85 complex are differentially expressed in leprosy lesions and infected armadillo tissues and that they could be target determinants in the immunopathological responses during M. leprae infection.

The evolution of antibody response in armadillos inoculated with Mycobacterium leprae.

Plasma from 30 armadillos (Dasypus novemcinctus) was collected prior to inoculation and at approximately 3-month intervals for a period of 1-3 years. These animals were inoculated intravenously with 6.1 x 10(8) +/- 2 x 10(8) (x +/- SD) armadillo-derived Mycobacterium leprae. These samples were analysed for antibodies of IgM and IgG class to phenolic glycolipid-I (PGL-I) and to sonicated M. leprae components using ELISA and immunoblotting techniques, respectively. We had previously observed among a group of 11 armadillos, that some animals produced and maintained a high IgG antibody response to PGL-I. In this study, an animal's ability to produce and maintain an elevated IgG anti-PGL-I response was significantly correlated with their ability to delay dissemination of the infection and their ability to survive longer. When the animals were moribund, a significant decrease in the IgG anti-PGL-I absorbance value was observed. The detection of PGL-I in the plasma samples collected from moribund armadillos suggested that high concentrations of PGL-I in the plasma may have contributed to a drop in absorbance values by the formation of non-lattice-type immune complexes in vivo. As detected by immunoblotting, the IgM and IgG response to antigens derived from sonically disrupted M. leprae was directed towards molecules with broad bands of immunoreactivity ranging from 21- to 45-kDa. There were no distinguishing features of these antibody responses among armadillos as was evident with the IgG anti-PGL-I responses.

Antibodies to the phenolic glycolipid-1 antigen for epidemiologic investigations of enzootic leprosy in armadillos (Dasypus novemcinctus).

Other than man, nine-banded armadillos (Dasypus novemcinctus) are the only known natural hosts of leprosy with high rates of disease. The origin, range and risk of their infection is not yet clear and a better description of the rate of leprosy over the armadillo's range is needed. Both histopathological examination of armadillo ear tissues and serologic screening for IgM antibodies to the phenolic glycolipid-1 (PGL-1) antigen of Mycobacterium leprae are good relative indices of enzootic prevalence. A survey of 216 armadillos from Louisiana and Florida detected infection only among Louisiana animals. Average antibody prevalence (12.5%) was five times higher than the fully disseminated disease rate described histopathologically (2.7%). The differences in antibody and histopathological prevalence are due to the sensitivity of the methods for detecting early infection. Histopathological examinations describe an advanced disease. The higher antibody prevalence of wild armadillos is not likely to be the result of false positive serologies from self-healing infections or other casual encounters with M. leprae as might be mimicked by lepromin injection. The environmental reservoir of M. leprae represented by infected armadillos is greater than could be previously estimated.

Prevalence and significance of positive Mitsuda reaction in the nine-banded armadillo (Dasypus novemcinctus).

One hundred two armadillos captured from the wild were lepomin tested. Nine of them (8.8%) showed a positive Mitsuda reaction. The histopathological appearance of the reaction had a spectrum showing tuberculoid, borderline tuberculoid, borderline lepromatous, and lepromatous histology. It is possible that armadillos can develop all the different types of leprosy seen in humans. The armadillo is a good animal model to test protective vaccines against leprosy.

Ability of the phenolic glycolipid-I antigen of M. leprae to elicit a positive Mitsuda response in the armadillo (Dasypus novemcinctus).

Three lepromin-positive armadillos and three lepromin-negative armadillos were tested intradermally with 100 micrograms of phenolic glycolipid-I (PGL-I) in 0.1 ml of normal saline. Positive delayed-hypersensitivity granulomas at 21 days in the lepromin-positive animals and negative responses in the lepromin-negative animals were obtained. These observations suggest that purified PGL-I is capable of eliciting cell-mediated immune or delayed-hypersensitivity responses in animals sensitized to Mycobacterium leprae.