Natural Infections of Leishmania and Trypanosomes Demonstrated by Skin Culture

Abstract

In the search for potential reservoirs of leishmaniasis, cultures were made from the skin of 61 wild-caught animals, of 17 genera, which showed no lesions or other external signs of infection. Leptomonad flagellates were obtained in culture from the skin of six of eight porcupines, Coendou rothschildi. These organisms were tested in hamsters and shown to be Leishmania sp. Sections of skin of positive porcupines showed L-D bodies in cells of the upper dermis. A few leptomonad flagellates were found in a small proportion of laboratory-reared Phlebotomus sandflies fed on infected porcupines. Trypanosomes were cultured from the skin of an opossum, Didelphis marsupialis, and of a marmoset, Saguinus geoffroyi. The search for the reservoir of cutaneous leishmaniasis in Panama has been carried on for a number of years at Gorgas Memorial Laboratory. Natural infections have been demonstrated in the spiny rats, Proechimys semispinosus (Tomes) and Hoplomys gymnurus Goldman (Ann. Rep. G.M.L. 1957-58), but in both cases only by culture of heart blood. There were no external signs of infection. Various members of the staff had examined by means of cultures and smears a great number of other animals, of at least 35 genera, from various parts of Panama, without isolating Leishmania or finding any external leishmanial lesion, until Thatcher et al. (1965) found an inconspicuous lesion containing L-D bodies on the ear of a kinkajou, Potos flavus (Schreber). In Peru it had been found (Herrer, 1948) that dogs naturally or experimentally infected with the Leishmania of Peruvian uta had skin lesions which were at best very inconspicuous, showing only slight depigmentation, and often had no external sign of infection. Nevertheless, smears from such lesions or the sites of inoculation often contained L-D bodies which could be revealed by prolonged examination. MATERIALS AND METHODS In March 1965 we began the examination of apparently normal skin of a variety of wildcaught Panamanian forest animals by means of stained smears. In one case smears were made Received for publication 8 April 1966. * The work reported here was supported in part by a research grant (AI-01251) from the NIAID, NIH, U. S. Public Health Service. from ample scrapings of sound skin on the rump of a porcupine, Coendou rothschildi Thomas. In one smear three L-D bodies were found. This positive finding was followed by the examination of other porcupines, in the course of which a biopsy-culture technique was developed. This method was based on previous work in Peru (Herrer, to be published elsewhere) in which tissue obtained by biopsy of the mucosa of Peruvian espundia (mucocutaneous leishmaniasis) patients was triturated in saline-plus-antibiotics, refrigerated for 24 hr, and then injected into dogs. Leishmania forms were recovered in culture from the developing papule. The technique finally developed for making cultures from the skin of porcupines and other animals is as follows: (1) After ether anesthesia, suitable areas are prepared by plucking out the spines of porcupines; hair of other animals is clipped and the skin shaved; (2) the skin is swabbed with iodine, followed by ether before the iodine has dried; (3) about 1.5 cm2 of skin is removed with a scalpel and dropped into 2.5 ml saline containing 500 units of potassium penicillin and 1 mg streptomycin sulphate per ml of saline; (4) the skin is triturated with scissors as finely as possible; (5) saline and tissue are transferred to a rubbertoppered test tube and refrigerated at 4 to 6 C for 24 to 72 hr; (6) cultures are made by inoculating each of five or six tubes with 0.3 to 0.4 ml of the saline containing the finer skin particles. The culture medium is prepared as follows: To 1,000 ml distilled water is added 25.0 g Difco Bacto-beef, which is boiled and filtered through filter paper. Distilled water is added to bring the m xture to its original volume. There is then added 20.0 g Difco Bacto-peptone, 5.0 g NaCl, and 30.0 g Difco Bacto-agar. The pH is adjusted to 7.2 to 7.4 and this stock medium is then autoclaved. Whole rabbit blood (no anticoagulant or defibrination) is added to the melted medium in the proportion of 10 to 13%, and the medium is tubed and slanted. The above differs from the modification of