Armadillos (Dasypus novemcinctus) as a model to test antileprosy vaccines; a preliminary report.

The efficacy of two candidate leprosy vaccines, BCG and a mixture of BCG and killed Mycobacterium leprae, was tested in 62 armadillos caught in the wild. The abilities of the vaccines to convert lepromin-negative armadillos to a positive reaction were compared with a group of control animals. Both vaccines upgraded subsequent lepromin skin-test histopathology. The conversion results parallel the protection values obtained in some BCG vaccine trials against leprosy in humans. Before conducting expensive human trials with new antileprosy vaccines, it would be worthwhile first to evaluate them in the armadillo model.

Lepromin responses in recipients of a candidate antileprosy bacterin vaccine (WHO-IMMLEP Mycobacterium leprae killed preparation) in the USA.

BACKGROUND: Identification of the nine-banded armadillo as a potential source of massive numbers of Mycobacterium leprae led to the development of a candidate bacterin vaccine for possible immunoprophylaxis. METHODS: Volunteers were from a leprosy-hypoendemic, nonBCG-using area (USA). They had been vaccinated intradermally 3 years earlier with a candidate antileprosy bacterin vaccine of irradiated and autoclaved Mycobacterium leprae obtained from experimental nine-banded armadillos. They were tested for dermal responsiveness to standard lepromin A. RESULTS: Values for induration and erythema appeared slightly greater for the vaccinated group; however, the differences were not statistically significant, indicating no appreciable 'anamnestic' effect on either Fernandez (early) or Mitsuda (late) reactions after 3 years. CONCLUSIONS: Because previous studies had demonstrated that administration of this bacterin produced no humoral changes, it now appears less probable that laboratory methods will be of much help in assessing even possible effectiveness of such vaccination.

The pathogenesis of leprosy in the nine-banded armadillo and the significance of IgM antibodies to PGL-1.

Twenty-seven nine-banded armadillos captured from the wild and tested free of wild M. leprae infection were distributed into four groups. They were injected at the right hind footpad with saline suspensions of M. Leprae at doses of 10(3), 10(4), 10(5) and 10(6). PGL-1 antibody levels were estimated using an ELISA test, twice during six months before the infection and every two months after the infection. One animal from each group was sacrificed at 6, 12, 18, 24, and 30-month intervals and another eight at unspecified intervals. A thorough autopsy and histopathological examination were conducted on all of them. Of the 27 animals, 18 developed the infection. In 10, there were granulomas at the site of inoculation and in 17 the regional lymph nodes were infected. The disease spread extensively to other lymph nodes and to the liver and spleen and then to the other organs. Peripheral nerves were invaded by M. leprae in only five animals. PGL-1 antibody levels registered a positive reading in only six of the 18 animals with the infection. In armadillo leprosy, the lesions did not persist at the site of entry in all animals M. leprae multiplied in the macrophages at the site of inoculation and the reticuloendothelial cells of the lymph nodes before they spread to other organs. There was evidence of invasion of endothelial cells of capillaries and possible bacteraemia even at an early phase of the infection. Peripheral nerves were not the preferred sites of entry or multiplication of M. leprae. Progressive increase in PGL-1 antibodies was recorded in five lepromatous armadillos with disseminated infection and high bacterial load. However, PGL-1 antibodies response was not sensitive enough to detect early disease.

Comparison of polymerase chain reaction technique with other methods for detection of Mycobacterium leprae in tissues of wild nine-banded armadillos.

Thirty, nine-banded armadillos weighing between 3 and 5 kilograms trapped from an area endemic for armadillo leprosy were collected at random; killed, autopsied and examined histopathologically. Also, one of the right inguinal lymph nodes was removed under sterile precautions and examined using PCR, direct smear examination, mouse footpad study, culture in laboratory media and histopathology with a view to detecting Mycobacterium leprae. Blood was collected at death and tested for IgM antibodies to PGL-1. According to the PCR study of the inguinal lymph nodes 16 of 30 armadillos (53.3%) had evidence of M. leprae. Significant levels of IgM antibodies to PGL-1 and identifiable lepromatous granuloma in inguinal lymph nodes were found in 2 animals (6.7%) with advanced disseminated disease. The prevalence of generalized leprosy according to autopsy study was 13.3% and according to histopathological examination of ear tissue 3.3%. The presence of M. leprae in the tissues evoked no special tissue reaction in the early stages. The pattern of spread of the disease in 2 animals closely resembled that found in experimental animals infected intracutaneously. Initiation of infection by inoculation of M. leprae through thorn pricks remains a distinct possibility.

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.

Isolation and characterization of an environmental acid-fast organism producing diphenoloxidase activity in vivo.

Water and soil samples were collected from natural habitats of the nine-banded armadillo and tested for the presence of acid-fast organisms by injection into the foot pads of experimental mice. Sixteen months post inoculation an acid-fast organism was isolated from the foot pad and spleen of one of the mice. The isolate exhibited diphenoloxidase activity as determined by its ability to convert D-3,4-dihydroxyphenylalanine to the corresponding quinone. The same organisms grown in vitro lacked detectable diphenoloxidase activity. However, diphenoloxidase activity was observed in acid-fast organisms harvested from spleen tissue of mice experimentally inoculated with a pure culture of the isolate. The environmental isolate was tentatively classed with the Mycobacterium avium-intracellulare complex.

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.

Lepromatous meningoencephalitis in the nine-banded armadillo (Dasypus novemcinctus).

The brains from 10 nine-banded armadillos with lepromatous leprosy were studied histopathologically. All of them showed evidence of lepromatous meningitis. In two there was invasion by Mycobacterium leprae into the brain tissue, with neuronal cells and glial cells containing intracellular bacilli. To our knowledge, this is the first report of meningoencephalitis in a lepromatous nine-banded armadillo.

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.

Our experiences with breeding of nine-banded armadillos (Dasypus novemcinctus) in captivity.

The nine-banded armadillo is considered the best animal model in the study of leprosy. Armadillos have never been successfully bred in the laboratory and therefore animals required for the experiments are captured from the wild and are likely to carry many diseases including leprosy. An attempt was made to breed them in captivity. Our attempt to house them in various combinations in their natural environment in the farmlands of Louisiana was successful. Although a significant increase in conception and delivery was recorded, consistent breeding of the animals was not obtained.

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.

Lepromatous placentitis and intrauterine fetal infection in lepromatous nine-banded armadillos (Dasypus novemcinctus).

Three pregnant lepromatous armadillos along with the three sets of four fetuses and their placentae were studied histopathologically. Mycobacterium leprae were present in the decidual tissue, trophoblastic cells which line the chorionic villi, and in the cells that form the internal structure of the villi. Acid-fast organisms were also seen in the spleens of three fetuses. Congenital infection is clearly possible in leprosy in the armadillo and may also occur in humans.

Beta-lactamase synthesis in Mycobacterium leprae.

Beta-lactam antibiotics are not active against Mycobacterium leprae. The enzyme beta-lactamase mediates the most common form of bacterial resistance to penicillins and cephalosporins. Cell-free extracts of purified suspensions of M. leprae were examined for beta-lactamase. The bacteria were prepared from the tissues of experimentally-infected nine-banded armadillos. Most of the suspensions were inactive. However, the bacteria obtained from the tissues of armadillos treated with penicillin G benzathine (bicillin) 6 months or more prior to sacrifice had beta-lactamase. If the organisms had been exposed to the antibiotic only for a few days, they were negative. Attempts to induce beta-lactamase in the bacteria in vitro did not succeed. Interestingly M. leprae separated from untreated armadillos, infected with the bacilli derived from treated animals contained the enzyme activity. Apparently, the M. leprae genome contains the operon for beta-lactamase, and once it is stimulated to express the enzyme, it continues to do so, even after the inducer is withdrawn.

Effect of repeated lepromin testing on experimental nine-banded armadillo leprosy.

Twenty-eight armadillos were lepromin tested and infected with M. leprae; 18 intravenously and 10 intradermally. The lepromin test was repeated after 3 months and at intervals of 6 months thereafter until their death or sacrifice up to 30 months. The one animal with tuberculoid lepromin was resistant and 14 of the 16 with lepromatous lepromin developed generalized disease. Of the 11 with borderline lepromin, 6 developed disseminated disease and 5 were resistant. There is a definite relationship between resistance and tuberculoid lepromin in the armadillo. Repeated lepromin testing had no effect in the rate of infection and the course of the disease in animals infected intravenously. In the intradermally infected animals the results were inconclusive. Whereas all the 20 animals with disseminated disease showed lesions in the liver, spleen and lymph nodes, only 4 animals had sciatic nerve involvement. Peripheral nerve trunk is not necessarily the preferred site in the armadillo. Lung lesions were an important cause of death in lepromatous armadillos.

Manifestations of experimental leprosy in the armadillo.

Three experiments, using different routes and doses of infection, were conducted using 42 armadillos. Thirty-six of them developed generalized disease. There is no significant sex or age difference in susceptibility. Route and dose of infection make very little difference in the disease prevalence except that the intravenous administration of a large dose reduces the period of development of generalized disease. It is quite possible that in armadillos the resistance to the disease is partly genetic. Although a majority of the armadillos developed lepromatous disease, borderline leproma is fairly common. In skin nodules large colonies of extracellular bacilli are demonstrated. Bacilli are also demonstrated in liver parenchymal cells.

First lesion in experimental armadillo leprosy.

Eighteen armadillos were infected intravenously with 10(8) M. leprae and 10 intracutaneously with 10(7) M. leprae. Among those which developed disseminated disease, a nodule at the site of inoculation was the first lesion noticed in 14 of the 16 infected intravenously and 4 of the 4 infected intradermally. It is possible that in human leprosy the first sign of infection is localized proliferation of M. leprae at the site of entry, and even nodule formation in lepromatous patients. It may be important to search for asymptomatic swelling or keloid-like lesions in skin or in nasal mucosa while screening a population for early leprosy.

Attempts to breed the nine-banded armadillo (Dasypus novemcinctus) in captivity--a preliminary report.

Three pairs of nine-banded armadillos were successfully kept in wire-fenced pens in natural surroundings with light, temperature, and weather as in the farms of Louisiana, a natural habitat of armadillos. One female delivered quadruplets after being in the experimental pen for 10 months and 11 days. It is possible that in this experiment breeding of nine-banded armadillos in captivity has been achieved under controlled conditions for the first time.

Variable lepromin response to Mycobacterium leprae in resistant armadillos.

Eight armadillos resistant to the infection of Mycobacterium leprae were lepromin tested. The tissue response was tuberculoid in 5, borderline in 2, and lepromatous in 1, thus showing a wide variation. It is seen that although cell-mediated immunity as evidenced by a tuberculoid granulomatous response to killed M. leprae is associated with resistance to the disease, there may be other yet unknown factors which protect armadillos from the infection. Lepromin responses were recognized histologically even at a low dose of 10(3) organisms, and the response increased with the dose up to 10(7) organisms. The tissue reaction to live organisms was the same as that to killed ones, and autoclaving of M. leprae produced no change in the tissue response to the antigens of M. leprae.

Borderline leprosy in an experimentally infected armadillo.

Out of six armadillos infected intravenously with 4.66 x 10(8) M. leprae, one developed a delayed hypersensitivity tissue response with the formation of an epithelioid cell granuloma. It is suggested that, although 80% of armadillos are susceptible to infection with M. leprae, some among these susceptible ones will exhibit varying degrees of delayed hypersensitivity reactions. These animals may go through a borderline phase before ultimately undergoing lepromatous degeneration.