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.

Evaluation of monitoring antibodies to PGL-I in armadillos experimentally infected with M. leprae.

An enzyme-linked immunosorbent assay (ELISA) for IgM antibodies to the phenolic glycolipid-I antigen of Mycobacterium leprae was evaluated for efficacy in monitoring armadillos experimentally infected with the bacillus. IgM antibodies were detected in armadillos from 186 days after experimental infection until the animals were sacrificed. The ELISA demonstrated the establishment of infection earlier and more reliably than the histologic methods previously applied. Satisfactory yields of M. leprae from individual armadillos could be predicted 97% of the time, and the technique may be useful in identifying appropriate harvest times or resistance among armadillos. The ELISA seems to be a valuable adjunct for managing experimental infections of M. leprae in armadillos.

Evaluation of the origin of Mycobacterium leprae infections in the wild armadillo, Dasypus novemcinctus.

An enzyme-linked immunosorbent assay (ELISA) using the phenolic glycolipid-1 (PGL-1) antigen of Mycobacterium leprae and cross-reactive antisera specific for human IgM was developed to detect IgM antibodies to M. leprae in the nine-banded armadillo (Dasypus novemcinctus). Statistical definitions for positive and negative interpretations in the ELISA were developed by screening animals recently captured and experimentally inoculated with M. leprae. The ELISA was shown to have high sensitivity and specificity. Modern day armadillos of central Louisiana were observed to have a PGL-1 antibody prevalence rate as high as 20%, and a clinical disease rate as high as 5%. A retrospective serological survey of 182 armadillos taken in the years 1960-1964 and predating the use of armadillos in leprosy research was used to evaluate the 1968 environmental contamination hypothesis for the origin of M. leprae infections in the wild armadillo. Antibodies to the apparently species-specific PGL-1 antigen were detected in 17 of the samples taken in 1960-1964. Absorption with whole M. leprae, M. intracellulare, M. terrae, M. rhodesiae, M. scrofulaceum, M. diernhoferi, M. kansasii, M. phlei, M. avium, BCG, and 2 new armadillo-derived mycobacterial species showed these antibody reactions to be specific for PGL-1. Apparently, M. leprae was enzootic in armadillos as early as 1961, and original infection of these animals could not have occurred in 1968.

Serologic evidence of natural togavirus infections in Panamanian sloths and other vertebrates.

Plasmas of sloths and other Central Panamanian wild vertebrates were tested for plaque-reduction neutralizing (PRN) antibodies against four flaviviruses and one alpha-virus. Forty percent of 97 two-toed sloths, Choloepus hoffmanni, and 8% of 168 three-toed sloths, Bradypus variegatus, were specifically positive against St. Louis encephalitis (SLE) virus. The prevalence of antibody against SLE virus was considerably higher in sloths than in any other group of wild vertebrates tested, including birds, and was found mainly in adult sloths. Specific PRN antibody against yellow fever (YF) virus was found only in monkeys. A high prevalence of PRN antibody against Ilheus and Mayaro viruses was detected in agoutis, Dasyprocta punctata, and against Mayaro virus in howler monkeys, Alouatta villosa. No plasma was specifically positive against Bussuquara virus. The results are interpreted as evidence that sloths are probably not important hosts in jungle YF cycles, but may be significant amplifying hosts in tropical SLE virus cycles.

Intraspecies differences of resistance against leprosy in nine-banded armadillos.

Infection of nine-banded armadillos with decreasing doses of armadillo-passaged leprosy bacilli show that most individuals of this species are susceptible and only about 20 percent are resistant regardless of the size of the infecting bacterial dose. It is pointed out that the intraspecies distribution of resistance to leprosy in human beings and in nine-banded armadillos differs because most individuals of the former species are resistant. Attention is drawn to the possibility that the results of anti-leprosy vaccination in armadillos might not apply to vaccination of human beings because the differences in distribution of resistant individuals might also reflect different mechanisms of susceptibility in the two species.

Formation of antibody against Mycobacterium leprae antigen 7 in armadillos.

A radioimmunoassay developed to measure antibody against Mycobacterium leprae antigen 7 in man was applied to the nine-banded armadillo (Dasypus novemcinctus). Normal armadillo sera had low but significant antibody activity in the test. Fourteen of 17 armadillos with systemic mycobacterial infection after inoculation with M. leprae showed increased antibody activity in the assay, and in some instances the activity was higher than in a pool of sera from patients with lepromatous leprosy. Crossed immunoelectrophoresis with armadillo serum in the intermediate gel revealed antibodies against five distinct antigenic components of M. leprae. Development of systemic mycobacterial infection after inoculation with M. leprae is thus associated with a distinct humoral immune response. The use of radioimmunosassay for selection of animals for inoculation and for following the development of the infection is discussed.

Leucocyte migration inhibition with Armadillo lepromin in human lymphocytes of healthy donors.

Armadillo lepromin activates human lymphocytes from normal donors to release leucocyte inhibiting factor. The above activity was expressed optimally when leucocytes were incubated with lepromin at 37 degrees C, and only partially when incubation was carried out at 30 degrees C or at 35 degrees C. The possible mechanism of the in vitro production of lymphokine from lymphocytes stimulated by armadillo lepromin is discussed.

A study of cellular and humoral immunity in three species of armadillos. Part I.

In the present study the membrane receptors of immunocompetent cells and immunoglobulins in three varieties of armadillos were explored for determining, in later studies, the possible differences in inoculated animals developing leprosy. The studies of cellular immunity were performed in five Chaetophroctus villosus (Ch.v), one Dasypus hibridus septecinctus (DHS) and one Zaedus Pichei (ZP), while the humoral immunity was studied with a serum pool of 17 Ch.v and 6 DHS. The results obtained demonstrate that the lymphocytes of the three species studied have receptors for SRBC, C3 and Ig-s, and no receptors for Fc segment of immunoglobulins. With reference to immunoglobulins no definite alteration of the humoral immunity was observed with the exception that DHS presents increased IgG levels and Ch.v increased IgM.

Immunological characteristics of the armadillo, Dasypus sabanicola.

The immunological responses of the armadillo are of interest because of its susceptibility to generalized lepromatoid infection with Mycobacterium leprae. In this study, specimens of Dasypus sabanicola were found to have a typical mammalian distribution of lymphoid cells in thymus, spleen, lymph nodes and blood. Their complement was active in bactercidal, protozoan immobilization and haemolytic systems. Blood lymphocytes responded to phytohaemagglutinin and to pokeweed mitogen. Sensitization with ovalbumin in CFA resulted in the production of circulating precipitins; strong Arthus reactions were detectable in the sensitized animals. Responses of cell-mediated immunity to DNCB and to M. tuberculosis were very discrete. Heat-killed M. leprae elicited granulomatous reactions characterized by microscopic necrosis, but without abundant lymphocytic infiltration; skin tests and lymphocytic transformation were generally negative in the animals injected with M. leprae.

Comparison of reactions to human and armadillo lepromins in leprosy.

To assess the usefulness of Mycobacterium leprae-infected armadillo tissue as a substitute for human lepromas for the manufacture of lepromin, we compared skin reactions to preparations from these two sources in 115 leprosy patients. The patient sample represented all the primary clinical forms of leprosy. Lepromin derived from the armadillo (lepromin-A) provoked the same pattern of responses as human derived lepromin (lepromin-H), i.e., lepromatous patients gave the weakest reactions. Lepromin-A reactions were consistently more intense than those to lepromin-H. We conclude that lepromin-A is a promising alternative to lepromin-H and may make the worldwide distribution of a standardized skin testing reagent feasible.