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Isolation of Mycoplasma moatsii from the intestine of wild Norway rats (Rattus norvegicus)
Veterinary Microbiology, 22 (1990) 23-29 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
23
I s o l a t i o n of Mycoplasma moatsii f r o m the I n t e s t i n e of Wild N o r w a y R a t s (Rattus norvegicus) J. GIEBEL, A. BINDER and H. KIRCHHOFF*
Institut f~r Mikrobiologie und Tierseuchen, Tieri~rztlicheHochschule, D-3000 Hannover (F.R.G.) (Accepted 12 September 1989)
ABSTRACT Giebel, J., Binder, A. and Kirchhoff, H., 1990. Isolation of Mycoplasma moatsii from the intestine of wild Norway rats (Rattus norvegicus). Vet. Microbiol., 22: 23-29. The intestinal tracts of twenty inbred SPF rats (LEW, BN, WKY, DA) and six wild Norway rats (Rattus norvegicus Berkenhout 1769) were investigated for mycoplasmas. Cultivation was in three different media. Mycoplasmas were not isolated from the intestine of the inbred SPF rats but were found in the epithelia and contents of caecum, colon and jejunum as well as in fecal samples of all of the wild Norway rats investigated. The mycoplasmas isolated all belonged to the same species, and were identified as Mycoplasma moatsii, originally isolated from grivit monkeys (Cercopithecus aethiops) and thought to be specific for this host.
INTRODUCTION In i n v e s t i g a t i o n s on t h e o c c u r r e n c e of mollicutes i n h a b i t i n g t h e gastrointest i n a l t r a c t a n a e r o b i c m y c o p l a s m a s were f o u n d in t h e r u m e n of c a t t l e a n d sheep ( R o b i n s o n a n d H u n g a t e , 1973; R o b i n s o n a n d Allison, 1975; R o b i n s o n et al., 1975; R o b i n s o n a n d R h o a d e s , 1977; R o b i n s o n , 1979) a n d in t h e i n t e s t i n e of swine ( B i n d e r a n d K i r c h h o f f , 1988). F a c u l t a t i v e l y a n a e r o b i c m y c o p l a s m a s , e.g. Mycoplasma (M.) alvi a n d M. sualvi h a v e b e e n isolated f r o m t h e i n t e s t i n e o f cattle ( G o u r l a y et al., 1977) a n d swine ( G o u r l a y et al., 1978). M y c o p l a s m a s r e l a t e d to M. alvi h a v e b e e n f o u n d in t h e i n t e s t i n e of voles ( G o u r l a y a n d Wyld, 1976 ). A c h o l e p l a s m a s w h i c h are k n o w n to grow u n d e r aerobic c o n d i t i o n s have b e e n d e t e c t e d in fecal samples of h o r s e s ( H e i t m a n n et al., 1982 ) a n d in t h e gut c o n t e n t s of various insects ( T u l l y et al., 1988). In our studies on t h e i n t e s t i n a l flora of l a b o r a t o r y r a t s a n d wild N o r w a y rats m y c o p l a s m a s were d e t e c t e d in t h e i n t e s t i n e a n d feces o f t h e wild N o r w a y rats. *To whom correspondence should be addressed.
0378-1135/90/$03.50
© 1990 Elsevier Science Publishers B.V.
24
J. GIEBEL ET AL.
MATERIALS AND METHODS
Animals Twenty inbred SPF rats, 12 LEW-rats, three BN-rats, three WKY-rats and two DA-rats (Zentralinstitut ffir Versuchstierzucht), and six wild Norway rats (Rattus norvegicus Berkenhout 1769) (Zoologisches Institut, Tier~irztliche Hochschule Hannover) were examined. The wild Norway rats investigated were the progeny of rats (F2 generation) which were caught by traps in the north of Germany. Rats were fed by conventional food and had body weights between 130 g and 160 g. The animals did not show any signs of clinical disease.
Isolation procedure Epithelia as well as contents of caecum, colon and jejunum were investigated for mollicutes using modified Mycoplasma alvi medium, modified Friis medium and modified Hayflick medium as described by Binder and Kirchhoff (1988). Small samples (about 0.5 g) were taken from various parts of the intestinal tract and three times washed in phosphate buffered saline (PBS, consisting of NaC1, 8.0 g; KHzPO4, 0.2 g; Na2HPO4" 12 H20, 2.9 g; KC1, 0.2 g; distilled water to 1000 ml; pH 7.2). After that mucus and epithelia were scraped off with a scalpel, suspended in 4 ml M. alvi medium, filtered through membrane filters (Millipore Corp. ) of 0.45/tm pore size and transferred to plates as well as to tubes with modified M. alvi medium, modified Friis medium or modified Hayflick medium. Intestinal contents and fecal samples (about 0.3 g) were suspended in 6 ml modified M. alvi medium and treated in the same way as the epithelia. The incubation was carried out under aerobic conditions (5% CO2 in air) and in an anaerobic glove box (Forma Scientific, Marietta, OH) with an atmosphere of 85% N2, 10% H2 and 5% C02 at 37°C. Examination of the plates for growth and subcultures from the tubes on solid media were performed after 7 days.
Morphological studies The colonies of the mollicutes isolates were observed with a stereomicroscope (Leitz). Cellular morphology was examined by darkfield microscopy and by transmission electron microscopy of negatively stained and ultrathin sectioned cells. For negative staining cells were picked up directly from agar plates with formvar coated grids. The organisms were stained with 2% aqueous ammonium molybdate for 20 s to 60 s without any preceeding washing or fixation procedures. Thin sectioning was carried out as described by Kirchhoff et al. (1987).
Reversion experiments The mycoplasma strains isolated were five times subcultured in broth medium without any antimicrobial agents, inoculated onto antimicrobial agent-
ISOLATION OF MYCOPLASMA MOATSH FROM WILD NORWAY RATS
25
free solid medium, and observed for the morphological appearance of the colonies.
Sterol requirement The sensitivity to 5, 10, and 20% sodium-polyanethol-sulfonate (SPS) and 1.5% digitonin was tested by the disc method (Freundt et al., 1973). The test for cholesterol requirement was performed according to the method described by Tully (1983). Biochemical investigations The following reactions were tested as described by Aluotto et al. (1970): fermentation of glucose, hydrolysis of arginine, hydrolysis of urea, reduction of tetrazolium and tellurite, phosphatase activity, serolysis of horse and swine serum, hemolysis of sheep red blood cells. The test for hemadsorption of different erythrocytes (sheep, guinea pig, rat) was performed according to Clyde (1963). Serological tests Antiserum was prepared against one of our isolates, strain KE2, in rabbits according to the method described by Morton and Roberts (1967). All strains isolated were examined serologically by the growth inhibition test (Clyde, 1964 ) with antisera against mycoplasmas sharing the biochemical properties with the isolates and by the indirect immunofluorescence technique (Del Giudice et al., 1967) using antisera against all mycoplasma and acholeplasma species described so far. For the antisera tested and their origin see Kirchhoff et al. (1987). In addition to that antisera against M. anseris (from Dr. Stipkovits) and Acholeplasma entomophilum (from Dr. Tully) and M. moatsii (prepared in our laboratory) were tested. RESULTS
Isolation of mollicutes Mollicutes were not detected in the inbred rat strains investigated. They were isolated, however, in high numbers from all of the six wild Norway rats. Mollicutes were found in the epithelia as well as in the contents of jejunum, colon and caecum and in the fecal samples tested. Altogether 40 mycoplasma strains were obtained. Growth occurred in strictly anaerobic atmosphere as well as under aerobic conditions and in each of the media tested. Strain KE2, isolated from the colon was selected as reference strain. Morphology All strains isolated developed the typical fried-egg colony form (Fig. 1A). By dark-field microscopy pleomorphic, non motile cells were observed. Elec-
26
J. GIEBELET AL.
|
Fig. 1. Morphology of strain KE2. (A) Colonies revealing typical fried-egg morphology. Bar represents 200 #m. (B) Negatively stained organisms demonstrating cell pleomorphism. Bar represents 150 nm. (C) Ultrathin sectioned organisms showing a trilaminar cytoplasmic membrane and absence of a cell wall. Bar represents 250 nm.
tron micrographs of negatively stained preparations of strain KE2 showed a variety of pleomorphic cells, some of them with budding structures (Fig. 1B ). Formation of filaments was not observed. Ultrathin sections of strain KE2 demonstrated the absence of a cell wall and the presence of a triple layer membrane (Fig. 1C).
ISOLATIONOFMYCOPLASMAMOATSHFROMWILDNORWAYRATS
27
TABLE 1 Results of biochemical investigations on six aerobic mollicute strains from the intestinal tracts of wild Norway rats and M. moatsii MK 405 from the urogenitary tracts of grivit monkeys
Sensitivity to 1.5% digitonin Sensitivity to SPS Fermentation of glucose Hydrolysis of arginine Hydrolysisof urea Reduction of tetrazolium Reduction of tellurite Lysis of erythrocytes Hemadsorption
Mycoplasma strains from wild Norway rats (KE2, Rk2,367, Rk3, KE7, KE9)
M. moatsii (MK 405)
+ + + + -
+ + +
+ -
Biochemical investigations Strain KE2 was resistant to SPS but sensitive to digitonin (inhibition zone: 4-5 m m ) . Gr o wth could not be observed in sterol-free basal medium containing bovine serum albumin and palmitic acid b ut occurred in medium supplem e n t e d with 5, 10 or 20% cholesterol. T h e results of furt her biochemical tests on strain KE2 and five selected ot he r strains are summarized in Table 1. Serological investigations All of the 40 strains isolated from the wild Norway rats reacted in the indirect immunofluorescence test with the a nt i s erum against strain K E 2 indicating t h a t t h e y all belonged to the same species. In the growth inhibition test (inhibition zone 4-5 m m ) as well as in the immunofluorescence test strain K E 2 showed a strong positive reaction with the self-prepared antiserum against M. moatsii as well as with the M. moatsii antiserum obtained from Dr. F r e u n d t ( F A O / W H O International Reference Centre for Animal Mycoplasmas, Aarhus, Denmark). DISCUSSION T h e mycoplasmas isolated from the intestinal tracts of wild Norway rats have been identified as M. moatsii. Originally M. moatsii was isolated from grivit monkeys which were i m por t e d from Ethiopia to the U.S.A. (Madden et al., 1974). T h e authors isolated six strains from five animals. M. moatsii was t h o u g h t to belong to the normal flora of t he urogenitary and respiratory tracts and to be specific for grivit monkeys. T h e r e is no description of a furt her isolation of this species. T h e pr e s ent results show t h a t M. moatsii also occurs in
28
J. G I E B E L E T AL.
rats. I t is t h e r e f o r e p o s s i b l e t h a t r a t s are t h e n a t u r a l h o s t s a n d t h a t t h e m o n k e y s w e r e i n f e c t e d w i t h M. moatsii b y rats. M. moatsii w a s n o t f o u n d in t h e S P F rats. T h i s m a y be t h e c o n s e q u e n c e of originally d e r i v i n g S P F r a t s for b r e e d i n g b y c e s a r e a n s e c t i o n a n d b y feeding w i t h i n d u s t r i a l diets. N o t h i n g is k n o w n a b o u t t h e s i g n i f i c a n c e of t h e m y c o p l a s m a s f o u n d in t h e gut of t h e wild N o r w a y rats. It is v e r y u n l i k e l y t h a t t h e y h a v e p a t h o g e n i c p r o p e r t i e s b e c a u s e t h e y h a v e b e e n i s o l a t e d in high n u m b e r s f r o m quite h e a l t h y rats. W e s u p p o s e t h a t t h e y b e l o n g to t h e i n d i g e n o u s m i c r o b i a l flora of t h e wild N o r w a y rats. ACKNOWLEDGEMENTS W e t h a n k Drs. M.F. Barile, E.A. F r e u n d t , N.F. Friis, R . N . G o u r l a y , A. Hill, R. L e m c k e , M. O g a t a , R.F. R o s s , L. S t i p k o v i t s a n d J.G. T u l l y for p r o v i d i n g mycoplasma cultures and antisera.
REFERENCES Aluotto, B.B., Wittler, R.G., Williams, C.O. and Faber, J.E., 1970. Standardized bacteriolytic techniques for the characterization of mycoplasma species. Int. J. Syst. Bacteriol., 20: 35-58. Binder, A. and Kirchhoff, H., 1988. Isolation of anaerobic mollicutes from the intestine of swine. Vet. Microbiol., 17: 151-158. Clyde, W.A., 1963. Hemolysins in identifying Eaton's pleuro-pneumonia-like organism. Science, 139: 55. Clyde, W.A., 1964. Mycoplasma species identification based upon growth inhibition by specific antisera. J. Immunol., 92: 958-965. Del Giudice, R.A., Robillard, F. and Carski, T.R., 1967. Immunofluorescence identification of mycoplasma on agar by use of incident illumination. J. Bacteriol., 93:1205-1209. Freundt, E.A., Andrews, H., Erno, H., Kunze, M. and Black, F.T., 1973. The sensitivity of Mycoplasmatales to sodium-polyanethol-sulfonate and digitonin. Zentralbl. Bakteriol., I. Abt. Orig. A, 225: 104-112. Gourlay, R.N. and Wyld, S.G., 1976. Ilsley-type and other mycoplasmas from the alimentary tracts of cattle, pigs and rodents. Proc. Soc. Gen. Microbiol., 3 (4): 142. Gourlay, R.N., Wyld, S.G. and Leach, R.H., 1977. Mycoplasma alvi, a new species from the bovine intestinal and urogenital tract. Int. J. Syst. Bacteriol., 27: 86-96. Gourlay, R.N., Wyld, S.G. and Leach, R.H., 1978. Mycoplasma sualvi, a new species from the intestinal and urogenital tracts of pigs. Int. J. Syst. Bacteriol., 28: 289-292. Heitmann, J., Kirchhoff, H., Chercheletzi, Ch., Jonas, E. and Deegen, E., 1982. Isolation of acholeplasmas from horse feces. Vet. Microbiol., 7: 273-276. Kirchhoff, H., Beyene, P., Fischer, M., Flossdorf, J., Heitmann, J., Khattab, B., Lopatta, D., Rosengarten, R., Seidel, G. and Yousef, C., 1987. Mycoplasma mobile sp. nov., a new species from fish. Int. J. Syst. Bacteriol., 37: 192-197. Madden, D.L., Moats, K.E., London, W.T., Matthew, E.B. and Sever, J.L., 1974. Mycoplasma moatsii, a new species isolated from recently imported Grivit monkeys {Cercopithecus ae thiops). Int. J. Syst. Bacteriol., 24: 459-464. Morton, H.E. and Roberts, R.J., 1967. Production of antimycoplasma antibodies (PPLO) in rabbits. Proc. Soc. Exp. Biol. Med., 125: 538-542.
ISOLATION OF MYCOPLASMA MOATSH FROM WILD NORWAY RATS
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Robinson, I.M., 1979. Special features of anaeroplasmas. In: M.F. Barile and S. Razin (Editors), The Mycoplasmas, Vol. I. Academic Press, New York, pp. 515-528. Robinson, I.M. and Allison, M.J., 1975. Transfer of Acholeplasma bactoclasticum Robinson and Hungate to the genus Anaeroplasma (Anaeroplasma bactoclasticum Robinson and Hungate comb. nov. ): Emended description of the species. Int. J. Syst. Bacteriol., 25: 182-186. Robinson, J.P. and Hungate, R.E., 1973. Acholeplasma bactoclasticum sp. n., an anaerobic mycoplasma from the bovine rumen. Int. J. Syst. Bacteriol., 23: 171-181. Robinson, I.M. and Rhoades, K.R., 1977. Serological relationships between strains of anaerobic mycoplasmas. Int. J. Syst. Bacteriol., 27: 200-203. Robinson, I.M., Allison, M.J. and Hartman, P.A., 1975. Anaeroplasma abactoclasticum gen. nov., sp. nov.: an obligately anaerobic mycoplasma from the rumen. Int. J. Syst. Bacteriol., 25:173181. Tully, J., 1983. Tests for digitonin sensitivity and sterol requirement. In: S. Razin and J.G. Tully (Editors): Methods in Mycoplasmology, Vol. I, Mycoplasma characterization. Academic Press, New York, pp. 355-362. Tully, J.G., Rose, D.L., Carle, P., Bov6, J.M., Hackett, K.J. and Whitcomb, R.F., 1988. Acholeplasma entomophilum sp. nov. from gut contents of a wide range of host insects. Int. J. Syst. Bacteriol., 38: 164-167.
23
I s o l a t i o n of Mycoplasma moatsii f r o m the I n t e s t i n e of Wild N o r w a y R a t s (Rattus norvegicus) J. GIEBEL, A. BINDER and H. KIRCHHOFF*
Institut f~r Mikrobiologie und Tierseuchen, Tieri~rztlicheHochschule, D-3000 Hannover (F.R.G.) (Accepted 12 September 1989)
ABSTRACT Giebel, J., Binder, A. and Kirchhoff, H., 1990. Isolation of Mycoplasma moatsii from the intestine of wild Norway rats (Rattus norvegicus). Vet. Microbiol., 22: 23-29. The intestinal tracts of twenty inbred SPF rats (LEW, BN, WKY, DA) and six wild Norway rats (Rattus norvegicus Berkenhout 1769) were investigated for mycoplasmas. Cultivation was in three different media. Mycoplasmas were not isolated from the intestine of the inbred SPF rats but were found in the epithelia and contents of caecum, colon and jejunum as well as in fecal samples of all of the wild Norway rats investigated. The mycoplasmas isolated all belonged to the same species, and were identified as Mycoplasma moatsii, originally isolated from grivit monkeys (Cercopithecus aethiops) and thought to be specific for this host.
INTRODUCTION In i n v e s t i g a t i o n s on t h e o c c u r r e n c e of mollicutes i n h a b i t i n g t h e gastrointest i n a l t r a c t a n a e r o b i c m y c o p l a s m a s were f o u n d in t h e r u m e n of c a t t l e a n d sheep ( R o b i n s o n a n d H u n g a t e , 1973; R o b i n s o n a n d Allison, 1975; R o b i n s o n et al., 1975; R o b i n s o n a n d R h o a d e s , 1977; R o b i n s o n , 1979) a n d in t h e i n t e s t i n e of swine ( B i n d e r a n d K i r c h h o f f , 1988). F a c u l t a t i v e l y a n a e r o b i c m y c o p l a s m a s , e.g. Mycoplasma (M.) alvi a n d M. sualvi h a v e b e e n isolated f r o m t h e i n t e s t i n e o f cattle ( G o u r l a y et al., 1977) a n d swine ( G o u r l a y et al., 1978). M y c o p l a s m a s r e l a t e d to M. alvi h a v e b e e n f o u n d in t h e i n t e s t i n e of voles ( G o u r l a y a n d Wyld, 1976 ). A c h o l e p l a s m a s w h i c h are k n o w n to grow u n d e r aerobic c o n d i t i o n s have b e e n d e t e c t e d in fecal samples of h o r s e s ( H e i t m a n n et al., 1982 ) a n d in t h e gut c o n t e n t s of various insects ( T u l l y et al., 1988). In our studies on t h e i n t e s t i n a l flora of l a b o r a t o r y r a t s a n d wild N o r w a y rats m y c o p l a s m a s were d e t e c t e d in t h e i n t e s t i n e a n d feces o f t h e wild N o r w a y rats. *To whom correspondence should be addressed.
0378-1135/90/$03.50
© 1990 Elsevier Science Publishers B.V.
24
J. GIEBEL ET AL.
MATERIALS AND METHODS
Animals Twenty inbred SPF rats, 12 LEW-rats, three BN-rats, three WKY-rats and two DA-rats (Zentralinstitut ffir Versuchstierzucht), and six wild Norway rats (Rattus norvegicus Berkenhout 1769) (Zoologisches Institut, Tier~irztliche Hochschule Hannover) were examined. The wild Norway rats investigated were the progeny of rats (F2 generation) which were caught by traps in the north of Germany. Rats were fed by conventional food and had body weights between 130 g and 160 g. The animals did not show any signs of clinical disease.
Isolation procedure Epithelia as well as contents of caecum, colon and jejunum were investigated for mollicutes using modified Mycoplasma alvi medium, modified Friis medium and modified Hayflick medium as described by Binder and Kirchhoff (1988). Small samples (about 0.5 g) were taken from various parts of the intestinal tract and three times washed in phosphate buffered saline (PBS, consisting of NaC1, 8.0 g; KHzPO4, 0.2 g; Na2HPO4" 12 H20, 2.9 g; KC1, 0.2 g; distilled water to 1000 ml; pH 7.2). After that mucus and epithelia were scraped off with a scalpel, suspended in 4 ml M. alvi medium, filtered through membrane filters (Millipore Corp. ) of 0.45/tm pore size and transferred to plates as well as to tubes with modified M. alvi medium, modified Friis medium or modified Hayflick medium. Intestinal contents and fecal samples (about 0.3 g) were suspended in 6 ml modified M. alvi medium and treated in the same way as the epithelia. The incubation was carried out under aerobic conditions (5% CO2 in air) and in an anaerobic glove box (Forma Scientific, Marietta, OH) with an atmosphere of 85% N2, 10% H2 and 5% C02 at 37°C. Examination of the plates for growth and subcultures from the tubes on solid media were performed after 7 days.
Morphological studies The colonies of the mollicutes isolates were observed with a stereomicroscope (Leitz). Cellular morphology was examined by darkfield microscopy and by transmission electron microscopy of negatively stained and ultrathin sectioned cells. For negative staining cells were picked up directly from agar plates with formvar coated grids. The organisms were stained with 2% aqueous ammonium molybdate for 20 s to 60 s without any preceeding washing or fixation procedures. Thin sectioning was carried out as described by Kirchhoff et al. (1987).
Reversion experiments The mycoplasma strains isolated were five times subcultured in broth medium without any antimicrobial agents, inoculated onto antimicrobial agent-
ISOLATION OF MYCOPLASMA MOATSH FROM WILD NORWAY RATS
25
free solid medium, and observed for the morphological appearance of the colonies.
Sterol requirement The sensitivity to 5, 10, and 20% sodium-polyanethol-sulfonate (SPS) and 1.5% digitonin was tested by the disc method (Freundt et al., 1973). The test for cholesterol requirement was performed according to the method described by Tully (1983). Biochemical investigations The following reactions were tested as described by Aluotto et al. (1970): fermentation of glucose, hydrolysis of arginine, hydrolysis of urea, reduction of tetrazolium and tellurite, phosphatase activity, serolysis of horse and swine serum, hemolysis of sheep red blood cells. The test for hemadsorption of different erythrocytes (sheep, guinea pig, rat) was performed according to Clyde (1963). Serological tests Antiserum was prepared against one of our isolates, strain KE2, in rabbits according to the method described by Morton and Roberts (1967). All strains isolated were examined serologically by the growth inhibition test (Clyde, 1964 ) with antisera against mycoplasmas sharing the biochemical properties with the isolates and by the indirect immunofluorescence technique (Del Giudice et al., 1967) using antisera against all mycoplasma and acholeplasma species described so far. For the antisera tested and their origin see Kirchhoff et al. (1987). In addition to that antisera against M. anseris (from Dr. Stipkovits) and Acholeplasma entomophilum (from Dr. Tully) and M. moatsii (prepared in our laboratory) were tested. RESULTS
Isolation of mollicutes Mollicutes were not detected in the inbred rat strains investigated. They were isolated, however, in high numbers from all of the six wild Norway rats. Mollicutes were found in the epithelia as well as in the contents of jejunum, colon and caecum and in the fecal samples tested. Altogether 40 mycoplasma strains were obtained. Growth occurred in strictly anaerobic atmosphere as well as under aerobic conditions and in each of the media tested. Strain KE2, isolated from the colon was selected as reference strain. Morphology All strains isolated developed the typical fried-egg colony form (Fig. 1A). By dark-field microscopy pleomorphic, non motile cells were observed. Elec-
26
J. GIEBELET AL.
|
Fig. 1. Morphology of strain KE2. (A) Colonies revealing typical fried-egg morphology. Bar represents 200 #m. (B) Negatively stained organisms demonstrating cell pleomorphism. Bar represents 150 nm. (C) Ultrathin sectioned organisms showing a trilaminar cytoplasmic membrane and absence of a cell wall. Bar represents 250 nm.
tron micrographs of negatively stained preparations of strain KE2 showed a variety of pleomorphic cells, some of them with budding structures (Fig. 1B ). Formation of filaments was not observed. Ultrathin sections of strain KE2 demonstrated the absence of a cell wall and the presence of a triple layer membrane (Fig. 1C).
ISOLATIONOFMYCOPLASMAMOATSHFROMWILDNORWAYRATS
27
TABLE 1 Results of biochemical investigations on six aerobic mollicute strains from the intestinal tracts of wild Norway rats and M. moatsii MK 405 from the urogenitary tracts of grivit monkeys
Sensitivity to 1.5% digitonin Sensitivity to SPS Fermentation of glucose Hydrolysis of arginine Hydrolysisof urea Reduction of tetrazolium Reduction of tellurite Lysis of erythrocytes Hemadsorption
Mycoplasma strains from wild Norway rats (KE2, Rk2,367, Rk3, KE7, KE9)
M. moatsii (MK 405)
+ + + + -
+ + +
+ -
Biochemical investigations Strain KE2 was resistant to SPS but sensitive to digitonin (inhibition zone: 4-5 m m ) . Gr o wth could not be observed in sterol-free basal medium containing bovine serum albumin and palmitic acid b ut occurred in medium supplem e n t e d with 5, 10 or 20% cholesterol. T h e results of furt her biochemical tests on strain KE2 and five selected ot he r strains are summarized in Table 1. Serological investigations All of the 40 strains isolated from the wild Norway rats reacted in the indirect immunofluorescence test with the a nt i s erum against strain K E 2 indicating t h a t t h e y all belonged to the same species. In the growth inhibition test (inhibition zone 4-5 m m ) as well as in the immunofluorescence test strain K E 2 showed a strong positive reaction with the self-prepared antiserum against M. moatsii as well as with the M. moatsii antiserum obtained from Dr. F r e u n d t ( F A O / W H O International Reference Centre for Animal Mycoplasmas, Aarhus, Denmark). DISCUSSION T h e mycoplasmas isolated from the intestinal tracts of wild Norway rats have been identified as M. moatsii. Originally M. moatsii was isolated from grivit monkeys which were i m por t e d from Ethiopia to the U.S.A. (Madden et al., 1974). T h e authors isolated six strains from five animals. M. moatsii was t h o u g h t to belong to the normal flora of t he urogenitary and respiratory tracts and to be specific for grivit monkeys. T h e r e is no description of a furt her isolation of this species. T h e pr e s ent results show t h a t M. moatsii also occurs in
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J. G I E B E L E T AL.
rats. I t is t h e r e f o r e p o s s i b l e t h a t r a t s are t h e n a t u r a l h o s t s a n d t h a t t h e m o n k e y s w e r e i n f e c t e d w i t h M. moatsii b y rats. M. moatsii w a s n o t f o u n d in t h e S P F rats. T h i s m a y be t h e c o n s e q u e n c e of originally d e r i v i n g S P F r a t s for b r e e d i n g b y c e s a r e a n s e c t i o n a n d b y feeding w i t h i n d u s t r i a l diets. N o t h i n g is k n o w n a b o u t t h e s i g n i f i c a n c e of t h e m y c o p l a s m a s f o u n d in t h e gut of t h e wild N o r w a y rats. It is v e r y u n l i k e l y t h a t t h e y h a v e p a t h o g e n i c p r o p e r t i e s b e c a u s e t h e y h a v e b e e n i s o l a t e d in high n u m b e r s f r o m quite h e a l t h y rats. W e s u p p o s e t h a t t h e y b e l o n g to t h e i n d i g e n o u s m i c r o b i a l flora of t h e wild N o r w a y rats. ACKNOWLEDGEMENTS W e t h a n k Drs. M.F. Barile, E.A. F r e u n d t , N.F. Friis, R . N . G o u r l a y , A. Hill, R. L e m c k e , M. O g a t a , R.F. R o s s , L. S t i p k o v i t s a n d J.G. T u l l y for p r o v i d i n g mycoplasma cultures and antisera.
REFERENCES Aluotto, B.B., Wittler, R.G., Williams, C.O. and Faber, J.E., 1970. Standardized bacteriolytic techniques for the characterization of mycoplasma species. Int. J. Syst. Bacteriol., 20: 35-58. Binder, A. and Kirchhoff, H., 1988. Isolation of anaerobic mollicutes from the intestine of swine. Vet. Microbiol., 17: 151-158. Clyde, W.A., 1963. Hemolysins in identifying Eaton's pleuro-pneumonia-like organism. Science, 139: 55. Clyde, W.A., 1964. Mycoplasma species identification based upon growth inhibition by specific antisera. J. Immunol., 92: 958-965. Del Giudice, R.A., Robillard, F. and Carski, T.R., 1967. Immunofluorescence identification of mycoplasma on agar by use of incident illumination. J. Bacteriol., 93:1205-1209. Freundt, E.A., Andrews, H., Erno, H., Kunze, M. and Black, F.T., 1973. The sensitivity of Mycoplasmatales to sodium-polyanethol-sulfonate and digitonin. Zentralbl. Bakteriol., I. Abt. Orig. A, 225: 104-112. Gourlay, R.N. and Wyld, S.G., 1976. Ilsley-type and other mycoplasmas from the alimentary tracts of cattle, pigs and rodents. Proc. Soc. Gen. Microbiol., 3 (4): 142. Gourlay, R.N., Wyld, S.G. and Leach, R.H., 1977. Mycoplasma alvi, a new species from the bovine intestinal and urogenital tract. Int. J. Syst. Bacteriol., 27: 86-96. Gourlay, R.N., Wyld, S.G. and Leach, R.H., 1978. Mycoplasma sualvi, a new species from the intestinal and urogenital tracts of pigs. Int. J. Syst. Bacteriol., 28: 289-292. Heitmann, J., Kirchhoff, H., Chercheletzi, Ch., Jonas, E. and Deegen, E., 1982. Isolation of acholeplasmas from horse feces. Vet. Microbiol., 7: 273-276. Kirchhoff, H., Beyene, P., Fischer, M., Flossdorf, J., Heitmann, J., Khattab, B., Lopatta, D., Rosengarten, R., Seidel, G. and Yousef, C., 1987. Mycoplasma mobile sp. nov., a new species from fish. Int. J. Syst. Bacteriol., 37: 192-197. Madden, D.L., Moats, K.E., London, W.T., Matthew, E.B. and Sever, J.L., 1974. Mycoplasma moatsii, a new species isolated from recently imported Grivit monkeys {Cercopithecus ae thiops). Int. J. Syst. Bacteriol., 24: 459-464. Morton, H.E. and Roberts, R.J., 1967. Production of antimycoplasma antibodies (PPLO) in rabbits. Proc. Soc. Exp. Biol. Med., 125: 538-542.
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Robinson, I.M., 1979. Special features of anaeroplasmas. In: M.F. Barile and S. Razin (Editors), The Mycoplasmas, Vol. I. Academic Press, New York, pp. 515-528. Robinson, I.M. and Allison, M.J., 1975. Transfer of Acholeplasma bactoclasticum Robinson and Hungate to the genus Anaeroplasma (Anaeroplasma bactoclasticum Robinson and Hungate comb. nov. ): Emended description of the species. Int. J. Syst. Bacteriol., 25: 182-186. Robinson, J.P. and Hungate, R.E., 1973. Acholeplasma bactoclasticum sp. n., an anaerobic mycoplasma from the bovine rumen. Int. J. Syst. Bacteriol., 23: 171-181. Robinson, I.M. and Rhoades, K.R., 1977. Serological relationships between strains of anaerobic mycoplasmas. Int. J. Syst. Bacteriol., 27: 200-203. Robinson, I.M., Allison, M.J. and Hartman, P.A., 1975. Anaeroplasma abactoclasticum gen. nov., sp. nov.: an obligately anaerobic mycoplasma from the rumen. Int. J. Syst. Bacteriol., 25:173181. Tully, J., 1983. Tests for digitonin sensitivity and sterol requirement. In: S. Razin and J.G. Tully (Editors): Methods in Mycoplasmology, Vol. I, Mycoplasma characterization. Academic Press, New York, pp. 355-362. Tully, J.G., Rose, D.L., Carle, P., Bov6, J.M., Hackett, K.J. and Whitcomb, R.F., 1988. Acholeplasma entomophilum sp. nov. from gut contents of a wide range of host insects. Int. J. Syst. Bacteriol., 38: 164-167.