Occurrence of Dermatophytes in Fresh Bat Guano1

KAJIHIRO, EDWIN S. (Wayland Baptist College, Plainview, Tex.). Occurrence of dermatophytes in fresh bat guano. Appl. Microbiol. 13:720-724. 1965.-Evidence is presented in support of the hypothesis that fresh bat guano serves as a means of pathogenic fungi dissemination in caves. A total of 371 guano samples were collected from caves in southeastern New Mexico. Each sample was agitated in sterile saline and sand. The supernatant fluid was treated with an antibiotic and streaked on differential media. Cultures were incubated at 25 and 37 C and examined at intervals over a 4-week period. For animal inoculation, highly concentrated inoculum was injected intraperitoneally into white Swiss mice. Animals were sacrificed 4 weeks later, and portions of their lung, liver, and spleen were cultured on selective media, incubated at 25 C, and examined at intervals over a 4-week period. Microsporum gypseum was isolated at all 10 collecting stations with an incidence of 22.4%, Trichophyton mentagrophytes at 7 stations with an incidence of 5%, T. rubrum at 3 stations with an incidence of 3%, and T. terrestre at 1 station with an incidence of 0.5%. From a total of 60 pools of liver-spleen-lung suspensions, 6 pools yielded positive cultures of Histoplasma capsulatum and 1 pool yielded T. mentagrophytes. No significant difference was found among the different selective media with respect to recovery of dermatophytes. Among the human pathogenic fungi isolated were Candida sp., Cladosporium sp., Coccidioides immitis, Cryptococcus neoformans, H. capsulatum, M. gypseum, T. mentagrophytes, T. rubrum, T. terrestre, and Sporotrichum sp. The dermatophytes discussed in this paper were isolated during a search for Histoplasma capsulatum in fresh bat guano. The findings provide information on their distribution and incidence in caves infested with bats. The first report of a dermatophyte growing saprophytically in nature was of one isolated from the mud of watercourses in the park of the University of Peco (Szathmary, 1936). One year later a colony of Trichophyton sp. was found growing on horse dung (Muende and Webb, 1937). While investigating the cause of "Cave Disease," T. mentagrophytes (asteroides type) was isolated from the soil of Johnson's Pothole and Makapan caves (Lurie and Borok, 1955). This was believed to be the first record of the isolation of T. mentagrophytes from soil. T. mentagrophytes and MDicrosporum gypseum were isolated from the atmosphere of the Makapan caves by exposure of laboratory animals (Lurie and Way, 1957). Cultures of the livers and spleens of some of the sacrificed mice resulted in the growth of dermatophytes. This was believed to be I Presented at the Spring Meeting of the Texas Branch of the American Society for Microbiology, Fort Worth, Tex., 9-10 April 1965, and at the 65th Annual Meeting of the American Society for Microbiology, Atlantic City, N.J., 25-29 April 1965. the first record indicating that the spores of these fungi may be found in the atmosphere. In April, 1963, the workers at Carlsbad Caverns were reported as being predominantly reactors to histoplasmin sensitivity tests. With the knowledge that environmental factors may intensely affect the occurrence of human pathogenic fungi (Zeidberg an Ajello, 1954), attempts were made to explore the relationship of H. capsulatum to its environment of Carlsbad Caverns. In view of the recent reports (Emmons, 1958, 1961; McDonough et al., 1961) of the isolation of H. capsulatum from soils enriched with bat or bird guano, samples of fresh bat guano from Carlsbad Caverns were examined for the presence of H. capsulatum. A large number of dermatophytes were found, and, since the recovery of these organisms from bat guano has not been reported, an investigation of the occurrence of dermatophytes in bat guano resulted. The description of the isolation methods used and the occurrence of dermatophytes isolated from fresh bat guano from four caves in the southeastern region of New Mexico are reported in this paper. MATERIALS AND METHODS The mycological study of fresh bat guano was begun on 6 July 1964. A total of 371 fresh guano 720 on S etem er 6, 2017 by gest ht://aem .sm .rg/ D ow nladed fom DERMATOPHYTES IN FRESH BAT GUANO samples were collected on three occasions (July, August, and October, 1964) from Carlsbad, Sitting Bull, McKittrick, and Cottonwood caves, all of which were infested with bats. Due to road hazards, it was not possible to visit Mudgetts Cave. The samples were scooped up directly into sterile 8-oz screw-cap bottles and were placed in a dry-ice chest to reduce the activity of the microflora. Immediately upon return to the laboratory, 1 g of each sample was suspended in 100 ml of sterile physiological saline solution with sterile sand and vigorously agitated for 30 sec. After several hours of settling at room temperature, 10 ml of the supernatant fluid were pipetted into a test tube. For each milliliter of the supernatant fluid, 0.25 ml of an antibiotic solution (2 mg of streptomycin and 5 mg of penicillin per ml of water) was added; 1 ml of this inoculum was streaked on two plates, each composed of Sabouraud Dextrose, Mycobiotic, Littman Oxgall, and Dextrose (all Difco products) agar medium. One set of the cultures was left at room temperature (25 C), and the duplicated set at incubated temperature (37 C). The culture plates were examined at intervals over a 4-week period. For selective isolation of dermatophytes and other keratinophilic fungi, the hair-bait technique was also used (Vanbreusegham, 1952). Sterile petri dishes of moistened guano were baited with short strands of sterilized human hair and incubated at room temperature. Mycelium growth on hair filaments was examined microscopically in lactophenol cotton blue and also cultured on Sabouraud Dextrose medium containing streptomycin and penicillin. For animal inoculation, 1 ml of the antibiotic-treated inoculum was injected intraperitoneally into white Swiss mice. Animals were sacrificed 4 weeks later, and the lungs, livers, and spleens of five mice were pooled and made into a homogeneous suspension with the aid of sterile sand, mortar, and pestle. The supernatant fluid from this suspension was then inoculated onto five plates of Brain Heart Infusion Dextrose (Difco) agar with 6% human blood, 20 units of penicillin, and 40 units of streptomycin per ml. The petri plates were incubated at 37 C and examined at intervals over a 4-week period. The remainder of the original guano suspension was kept at room temperature for further examination if necessary. Identification of fungi recovered was made by direct examination of the colony characteristics (Fig. 1) and microscopic examination of the spores.