3
420 CAN. J. MICROBIOL. VOL. 20, 1974 tableau 1). Cette variation peut Ctre expliqute par un certain nombre de facteurs. Le mode de fixation des cellules, A l'alcool pour les coupes de foie de rat et a l'acetone pour les fibroblastes, les effets engendrts par la mise en culture des fibroblastes, l'origine des cellules, les dilutions diffkrentes des serums (8), sont la autant de variantes pouvant expliquer en partie les modi- fications morphologiques de la fluorescence. Remerciements Les auteurs dtsirent remercier les Docteurs B. Rose et M. Mandl, The Harry Webster Thorp Laboratories, H6pital Royal Victoria, Montrtal, QuC. 1. ARANJO, F. G., E. V. BARNETT, L. 0. GENTRY et J. S. REMINGTON. 1971. False-positive anti-Toxo- plasma fluorescent-antibody tests in patients with antinuclear antibodies. Appl. Microbiol. 22: 27&275. 2. HANSHAW, J. B. 1969. Congenital Cytomegalovirus infection: laboratory methods of detection. J. Pediatr. 75(6): 1179-1 185. 3. HANSHAW, J. B., H. J. STEINFELD et C. J. WHITE. 1968. Fluorescent antibody test for Cytornegalovirlrs macroglobulin. N. Engl. J. Med. 279: 566-570. 4. JACK, I., et K. C. Mc AULIFFE. 1968. Sero-epidemio- logical study of Cytotnegalovirirs infection in Mel- bourne children and some adults. Med. J. Aust. 1: 206213. 5. KRAUS, S. J., J. R. HASERICH et M. A. LANTZ. 1970. Fluorescent treponemal antibody-absorption test reactions in lupus erythematosus. N. Engl. J. Med. 282: 1287-1290. 6. KURSTAK, E., S. BELLONCIK, P. A. ONJI, S. MONT- PLAISIR et B. MARTINEAU. 1972. Localisation par I'immunoperoxydase des antigenes du virus cytome- galique en culture cellulaire de fibroblastes humains. Arch. Gesamte Virusforsch. 38(1): 67-76. 7. LANGENHUYSEN, M. M. A. C., T. H. THE, H. 0. NIEWEG et J. G. KAPSENBERG. 1970. Demonstration of IgM Cytomegalooirus-antibodies as an aid to early diagnosis in adults. Clin. Exp. Immunol. 6(3): 387-393. 8. MONIER, J. C., et J. THIVOLET. 1972. Patterns of antinuclear antibody fluorescence in leucocytes and hepatocytes. Rev. Eur. Etud. Clin. Biol. 17: 582-591. 9. MONTPLAISIR, S., et B. MARTINEAU. 1972. Infection causee par le virus Cytornigaliqire (VCM) dans la region de Montreal: Ctude CpidCmiologique. Can. J. Public Health, 63: 333-341. 10. MONTPLAISIR, S., S. BELLONCIK, N. P. LEDUC, P. A. ONJI, B. MARTINEAU et E. KURSTAK. 1972. Electron microscopy in the rapid diagnosis of Cytornegalovirus: ultrastructural observation and comparison of methods of diagnosis. J. Infect. Dis. 125(5): 533-538. The growth of Mycobactevium lepvae in snakes1 J. B. G. KWAPINSKI, E. H. KWAPINSKI, AND N. M. MCCLUNG Utriversity of Manitoba, Winnipeg, Manitoba and University of South Florida, Tampa, Florida Accepted November 15, 1973 KWAPINSKI, J. B. G., E. H. KWAPINSKI, and N. M. MCCLUNG. 1974. The growth of Mycobac- terium leprae in snakes. Can. J. Microbiol. 20: 420422. Mycobacterium leprae, obtained from a human case of lepromatous leprosy, was successfully grown in newborn snakes, within 5 to 6 weeks, and it was transferred from snake to snake, upon intramuscular injection. KWAPINSKI, J. B. G., E. H. KWAPINSKI et N. M. MCCLUNG. 1974. The growth of Mycobacteriutn leprae in snakes. Can. J. Microbiol. 20: 42W22. Une souche de Mycobacterium leprae, isolee d'un cas humain de lepre Itpromateuse, a CtC cultivCe avec succes en 5 a 6 semaines, chez des serpents nouveau-nCs. La culture est transferable de serpent a serpent par injection intramusculaire. [Traduit par le journal] In the light of our hypothesis, the pathogen of species. Accordingly, in our search for a species leprosy has arisen as a strict intracellular parasite to which this parasite might have been best at the reptilian era, but its pathobiological pro- adapted, we have chosen newborn snakes. perties have been modified during the long The species used was Elaphe quadrioitta; period of interactions with some of the evolving 3- to 4-day-old yellow rat snakes, hatched from eggs in this laboratory and carefully 'Received July 31, 1973. protected against any possible infection or Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by University of P.E.I. on 11/23/14 For personal use only.

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Page 1: The growth of               Mycobacterium leprae               in snakes

420 CAN. J. MICROBIOL. VOL. 20, 1974

tableau 1). Cette variation peut Ctre expliqute par un certain nombre de facteurs. Le mode de fixation des cellules, A l'alcool pour les coupes de foie de rat et a l'acetone pour les fibroblastes, les effets engendrts par la mise en culture des fibroblastes, l'origine des cellules, les dilutions diffkrentes des serums (8), sont la autant de variantes pouvant expliquer en partie les modi- fications morphologiques de la fluorescence.

Remerciements

Les auteurs dtsirent remercier les Docteurs B. Rose et M. Mandl, The Harry Webster Thorp Laboratories, H6pital Royal Victoria, Montrtal, QuC.

1. ARANJO, F. G., E. V. BARNETT, L. 0. GENTRY et J. S. REMINGTON. 1971. False-positive anti-Toxo- plasma fluorescent-antibody tests in patients with antinuclear antibodies. Appl. Microbiol. 22: 27&275.

2. HANSHAW, J. B. 1969. Congenital Cytomegalovirus infection: laboratory methods of detection. J. Pediatr. 75(6): 1179-1 185.

3. HANSHAW, J. B., H. J. STEINFELD et C. J. WHITE. 1968.

Fluorescent antibody test for Cytornegalovirlrs macroglobulin. N. Engl. J. Med. 279: 566-570.

4. JACK, I., et K. C. Mc AULIFFE. 1968. Sero-epidemio- logical study of Cytotnegalovirirs infection in Mel- bourne children and some adults. Med. J. Aust. 1: 206213.

5. KRAUS, S. J., J. R. HASERICH et M. A. LANTZ. 1970. Fluorescent treponemal antibody-absorption test reactions in lupus erythematosus. N. Engl. J. Med. 282: 1287-1290.

6. KURSTAK, E., S. BELLONCIK, P. A. ONJI, S. MONT- PLAISIR et B. MARTINEAU. 1972. Localisation par I'immunoperoxydase des antigenes du virus cytome- galique en culture cellulaire de fibroblastes humains. Arch. Gesamte Virusforsch. 38(1): 67-76.

7. LANGENHUYSEN, M. M. A. C., T . H. THE, H . 0. NIEWEG et J. G. KAPSENBERG. 1970. Demonstration of IgM Cytomegalooirus-antibodies as an aid to early diagnosis in adults. Clin. Exp. Immunol. 6(3): 387-393.

8. MONIER, J. C., et J. THIVOLET. 1972. Patterns of antinuclear antibody fluorescence in leucocytes and hepatocytes. Rev. Eur. Etud. Clin. Biol. 17: 582-591.

9. MONTPLAISIR, S., et B. MARTINEAU. 1972. Infection causee par le virus Cytornigaliqire (VCM) dans la region de Montreal: Ctude CpidCmiologique. Can. J. Public Health, 63: 333-341.

10. MONTPLAISIR, S., S. BELLONCIK, N. P. LEDUC, P. A. ONJI, B. MARTINEAU et E. KURSTAK. 1972. Electron microscopy in the rapid diagnosis of Cytornegalovirus: ultrastructural observation and comparison of methods of diagnosis. J. Infect. Dis. 125(5): 533-538.

The growth of Mycobactevium lepvae in snakes1

J. B. G. KWAPINSKI, E. H. KWAPINSKI, AND N. M. MCCLUNG Utriversity of Manitoba, Winnipeg, Manitoba

and University of South Florida, Tampa, Florida

Accepted November 15, 1973

KWAPINSKI, J. B. G., E. H. KWAPINSKI, and N. M. MCCLUNG. 1974. The growth of Mycobac- terium leprae in snakes. Can. J. Microbiol. 20: 420422.

Mycobacterium leprae, obtained from a human case of lepromatous leprosy, was successfully grown in newborn snakes, within 5 to 6 weeks, and it was transferred from snake to snake, upon intramuscular injection.

KWAPINSKI, J. B. G., E. H. KWAPINSKI et N. M. MCCLUNG. 1974. The growth of Mycobacteriutn leprae in snakes. Can. J. Microbiol. 20: 42W22.

Une souche de Mycobacterium leprae, isolee d'un cas humain de lepre Itpromateuse, a CtC cultivCe avec succes en 5 a 6 semaines, chez des serpents nouveau-nCs. La culture est transferable de serpent a serpent par injection intramusculaire. [Traduit par le journal]

In the light of our hypothesis, the pathogen of species. Accordingly, in our search for a species leprosy has arisen as a strict intracellular parasite to which this parasite might have been best at the reptilian era, but its pathobiological pro- adapted, we have chosen newborn snakes. perties have been modified during the long The species used was Elaphe quadrioitta; period of interactions with some of the evolving 3- to 4-day-old yellow rat snakes, hatched

from eggs in this laboratory and carefully 'Received July 31, 1973. protected against any possible infection or

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Page 2: The growth of               Mycobacterium leprae               in snakes

NOTES 42 1

contamination, were injected intramuscularly injected with live M. leprae. Much smaller or with about lo3 solidly stained M. leprae hardly visible lesions were observed in the organisms which were homogenized in 0.01% tetracycline.HC1 solution made in sterile phos- phate-buffered saline (PBS), pH 7.2. A control group of eight, 3- to 4-day-old yellow rat snakes were injected with the same organisms heated for 30 min at 100' C. The number of bacteria present in suspension was determined by counting in a Petroff-Hausser chamber.

The source of microorganisms used was the same as that used for previous research (2) : name- ly, M. leprae recovered from the spleen and lepro- mas of a woman who suffered from lepromatous leprosy for 30 years and had been without treatment for 10 years before her death, because of non-responsiveness to sulfones. These micro- organisms did not grow on any culture media, including Loewenstein-Jellsen's medium and thioglycollate medium, and did not multiply in mice, guinea pigs, or pigeons.

The snakes were observed for 5 months unless death occurred earlier. At the autopsy, histo- logical sections or smears made from the spleen, liver, muscles, and subcutaneous tissue were stained by Fite's (1) or Ziehl-Neelsen's method, respectively, and examined at 600 and 900 x magnification. Extracts from snake organs, in which acid-fast bacilli were detected, and extracts from organs of control snakes were made as follows: the tissue suspended in PBS (5 mg wet weightlml) and kept on crushed ice was homo- genized for 1-2 min in a Sorvall omni-mixer and then ultrasonicated for 5 min at 30 kHz in a Biosonik 111, centrifuged at 5000 g for 10 min, and supernatant fluid collected.

The extracts were examined against two anti-

remaining snakes. Because of the relatively small size of the snakes, it was difficult to assess whether pathological changes did occur in lymphoid organs. No lesions at all were detected in the organs of the snakes injected with heated M. leprae.

On microscopic examination, numerous solidly red-stained cylindrical bacteria, arranged in bundles or in globi, were detected in smears and l~istological sections made from the liver and lymphatic tissues of snakes injected with live material but not in the snakes injected with heated material. Thus, between 20 and 200 acid- fast bacteria per microscopic field were found in smears made from visible liver lesions, or in spleen tissue, whereas in the organs showing no macroscopic lesions, the average numbet of bacteria was 10 times smaller. No bacteria were observed in organs obtained from snakes injected with the preheated M. leprae.

A spleen, removed aseptically from one of the snakes above, was homogenized in 1 ml of a sterile 0.85% sodium chloride solution; 0.1 ml of the homogenate was then injected intra- muscularly into six, 3- to 4-day-old snakes. All the snakes died in 5 to 6 weeks, showing the symptoms described above. On autopsy no organ alterations, aside from an apparent en- largement of the spleen, were observed. Micro- scopic examinations of the smears made from the spleen, liver, and lymph nodes revealed the presence of round or cylindrical acid-fast organ- isms, situated mainly intracellularly.

No growth occurred within 3 months from the inoculates of the snake organs placed onto

M . leprae rabbit antisera and against normal blood agar, dextrose agar, -dextrose broth, rabbit sera (as controls) using the cellulose- thioglycollate medium, and Loewenstein- acetate immunodiffi~sion procedures described Jensen's medium. White mice injected intra- elsewhere (3).

During the observation period, it had been noted that the snakes injected with non-heated M. leprae showed considerable loss of weight, became emaciated beginning at the 3rd month after injection, and died in 11 to 13 weeks. The snakes injected with heated M. leprae survived, but were sacrificed and autopsied in the 5th month.

At autopsy, numerous pin-point, orange- yellow lesions, filled with a brittle material, were discovered in the liver of half the snakes

peritoneally or intravenously with- the infected liver homogenates survived the observation time of 2 months; no bacteria were detected in their spleens or livers when examined micro- scopically.

Extracts obtained from all six livers that on autopsy had visible lesions reacted with the anti-M. leprae antisera, but not with normal rabbit sera forming one or two precipitation bands. No bands were formed by the extracts from snakes injected with heated M. leprae.

The data obtained from the preliminary

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Page 3: The growth of               Mycobacterium leprae               in snakes

422 CAN. J. MICROBIOL. VOL. 20, 1974

studies indicate that M. leprae multiply vigo- rously in newborn snakes so that these micro- organisms can be easily detected in different organs. The infection has also been transmitted from snake to snake, with an apparent shortening of the incubation time and course of the disease. Compared with the similar experiments per- formed earlier in caymans, the newborn snakes seem to be much more susceptible and responsive to the infection with M. leprae. It is proposed to use newborn snakes (and possibly snake tissue

cultures) for experimental diagnosis of leprosy and for testing of anti-leprosy drugs.

1. FITE, G. L. 1940. The fuchsin formaldehyde method of staining acid-fast bacilli in paraffin sections. J. Lab. Clin. Med. 25: 743-744.

2. KWAPINSKJ, J. B. G., and E. H. KWAPINSKJ. 1973. Immunological reactions of Mycobacterium leprae and Mycobacterium lepraemurium grown in cayman. Can. J. Microbiol., 19: 764-766.

3. KWAPINSKI, 3. B. G., 3. 0. DE ALMEIDA, and E. H. KWAPINSKJ. 1972. Immunological analysis of Myco- bacteriunz leprae using reactions with cytoplasmic antigens of Actinomycetales. Can. J. Microbiol. 18: 391-396.

Phacidin, a fungal growth inhibitor from Potebniamyces balsamicola var. boyceil

A. FUNK AND E. E. MCMULLAN Department of the Environment, Canadian Forestry Seruice, Pacific Forest Research Centre, Victoria, British Coltrmbia

Accepted November 14, 1973

FUNK, A., and E. E. MCMULLAN. 1974. Phacidin, a fungal growth inhibitor from Potebtriatnyces balsamicola var. boycei. Can. J. Microbiol. 20: 422-425.

Potebniamyces balsamicola Smerlis var. boycei Funk produces a fungal growth inhibitor for which the name "phacidin" is proposed. It was extracted from cultures with benzene and purified as a yellow crystalline substance with the formula C16HZ2o5, which inhibited growth of a wide variety of fungi.

FUNK, A., et E. E. MCMULLAN. 1974. Phacidin, a fungal growth inhibitor from Potebnian~yces balsatrlicola var. boycei. Can. J. Microbiol. 20: 422-425.

Potebniamyces balsarnicola Smerlis var. boycei Funk produit un inhibiteur de la croissance fongique; le nom de phacidine est propose pour ce compose. I1 a kt6 extrait des cultures par le benzene; et une substance cristalline jaune ayant la formule C16H22O5 a ete obtenue a l'etat pur. Cette derniere inhibe la croissance de nombreuses moisissures. [Traduit par le journal]

Introduction

The canker fungus, Potebniamyces balsamicola Smerlis var. boycei Funk, produced visible quan- tities of a pale yellow substance in culture (Funk 1970). Crude extracts of this metabolite had inhibitory effects on the growth of yeast and molds. Subsequent experimentation has led to production of the antibiotic in liquid culture and to its purification as a crystalline substance, which has strong inhibitory effects on the growth of a wide variety of fungi.

Examination of its properties indicates that it is different from known antibiotics and the

' name phacidin is therefore proposed for this antibiotic (from Phacidiales, the order of asco-

mycetes to which the fungus belongs). However, the chemical structure will be presented in a later publication.

Materials and Methods Production of Phacidirz*

The fungus was maintained on 2% malt agar and grown at 15°C. Culture No. 357, used in production, was ob- tained from conidia in diseased Abies grandis (Doug].) Lindl. at Yahk, B.C., Canada. (Specimen deposited in Herb. DAVFP, 19112.)

Inoculumwas prepared by macerating petri platecultures on 2% malt agar in a micro Waring Blendor in sterile distilled water for 10-15 s. The macerated mycelium was also used to inoculate additional petri plate cultures, which were allowed to grow for 3 weeks at lS°C.

A solution of 5% malt extract (Difco) in distilled water

'Received July 23, 1973. *Patent applied for.

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