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A General Description of Mycoplasmas
Br. vet. J. (1969), 125,344
A GENERAL DESCRIPTION OF MYCOPLASMAS By
JENNIFER POLAND
Department of Pathology, The Royal Veterinary College, University of London
INTRODUCTION
Much interest has been taken in the mycoplasma group of organisms during the past few years, concurrent with improvements in techniques that have resulted in the more frequent isolation of these organisms. The number of known species now exceeds fifty; they have been isolated from a wide variety of domesticated mammals and birds as well as from man. Mycoplasmas have been found as contaminants of continuous cell line tissue cultures (Tully, 1966; Leach & Butler, 1966) and since some species produce cytopathic effects, inhibit the growth of viruses, cause haem adsorption and haemagglutination and affect chromosome numbers (Betts, 1967), confusion and difficulties can arise in virological investigations. A number of species of mycoplasmas have been isolated from cases of human leukaemia, although these are of doubtful significance (Fallon, 1966). The possibility that mycoplasmas are in some way concerned with human rheumatoid arthritis is a field of current research. Properties oj mycoplasmas (pleuropneumonia-like organisms) According to Hayflick & Chanock (1965) the properties of this group of organisms are as follows: (a) They can replicate in a cell-free environment. (b ) On agar they exhibit a characteristic colonial morphology with the centre or the whole colony embedded beneath the surface of the agar. (c) The smallest reproductive elements have a size range of 125- 150 mf),. Thus, they are the smallest free-living organisms of a size comparable to that of myxoviruses. (d ) They lack a rigid cell wall, and consist of protoplasm surrounded by a triple-layered cell membrane. (e) With the exception of saprophytic species, most mycoplasmas require sterols or protein for growth. (f ) All species exhibit an absolute resistance to penicillin, whereas many strains are sensitive to low concentrations of tetracyclines. (g) The growth of mycoplasmas is inhibited by specific antibody. (h ) Mycoplasmas do not revert to or from bacterial parent forms. Mycoplasmas were originally confused with viruses because of their small size and their inability to grow in the usual bacteriological media. Mycoplasmas have also been confused with "L" forms of bacteria; this confusion was natural since "L" forms are variants of bacteria which have lost their cell walls and so
DESCRIPTION OF MYCOPLASMAS
345
resemble mycoplasmas both in morphology and in colonial appearance (Morton, 1965). The lack of cell wall is the reason for mycoplasmas being more sensitive than bacteria to physical and chemical agents; parasitic species survive only for short periods outside the body of the host, whilst in the laboratory the fragility of mycoplasmas is illustrated by the fact that lyophilization may kill more than 90 p er cent of the organisms (Adler, 1965) and that even faint traces of detergents are lethal. Mycoplasmas lack the capsules' and flagella found in many bacteria, and so far only two species of mycoplasma have been found to produce exotoxins: M, neurolyticum and M. gallisepticum (Thomas, 1967). A few species, e.g. M. gallisepticum, haemagglutinate a variety of erythrocytes, and colonies of this species also absorb erythrocytes.
Morphology M ycoplasmas are highly pleomorphic (Turner, 1935) ; branched filaments have been observed in liquid cultures, hence the derivation of the term " mycoplasma" . On solid m edia, mycoplasm a colonies form central downgrowths and many species form characteristic mamillated colonies. However, considerable variation in size and appearance can occur even within the colonies of a single species, possibly due to variations in environment (Razin & Oliver, 196 I ). Cultivation Mycoplasmas have been cultivated in embryonated hens' eggs but there are considerable differences between species in the ability to grow in eggs (Dinter, D anielsson & Bakos, 1965). They have been cultivated in tissue cultures and in acellular liquid and solid media but there are considerable variations in their nutritional requirements and cultural properties. Some species have such fastidious nutritional r equirements that they are difficult to cultivate in any system. Most acellular media that support the growth of mycoplasmas contain 20 per cent serum and 10 per cent of a yeast extract, in addition to a broth. Most mycoplasmas ferment glucose and produce lactic acid. Thus growth can conveniently be det ected by adding glucose and phenol red to liquid media and observing a change in pH. Those sp ecies that do not ferment glucose usually degrad e arginine and so render the medium more alkaline. A few species split urea. Thallium acet ate and penicillin can be added to media to keep down bacterial contamination. However, p enicillin could induce the formation of "L" forms of bacteria and thallium acetate concentrations greater than I in 4000 may inhibit the growth of mycoplasmas particularly when they are present in low numbers. Epidemiology and pathogenicity The list of known species contains frank pathogens, organisms of low pathogenicity, benign parasites and a saprophyte, M. laidlawii.
BRITISH VETERINARY JOURNAL, 125, 7
I
Almost all domesticated animals harbour benign parasites in the upper respiratory and/or urogenital tracts; in addition some animals may harbour potentially pathogenic mycoplasmas in the same sites. Although many of the pathogenic mycoplasmas cause respiratory disease, some cause inflammation of the pleura, pericardium and peritoneum, or of the joints. For example, M. mycoides causes pleuropneumonia in adult cattle whilst in young calves it also causes pericarditis and arthritis and M. arthritidis causes arthritis alone in rats and mice. Close contact is required for transmission of respiratory diseases caused by mycoplasmas since inhalation is the route of infection and the organisms do not survive for more than a few minutes outside the animal. Diagnosis is based on one or more of the following methods: Isolation and identification oj a pathogenic mycoplasma. Isolation is facilitated by passing material through a filter having an APD of 450 mfl since this removes contaminating bacteria. However, filtration may be impracticable when only a small quantity of infected material is available. Identification is based on: I. Biochemical tests to · detect glucose fermentation, arginine degradation, or the splitting of urea. II . Serological tests: i. Metabolism inhibition (Taylor-Robinson, Purcell, Wong & Chanock, 1966). ii. Growth inhibition (Edward & Fitzgerald, 1954; Clyde, 1964). iii. Agar gel double diffusion (Taylor-Robinson, Somerson, Turner & Chanock, 1963). iv. Fluorescent antibody techniques (Tully, 1965; Guidiche, Robillard & Carski, 1967). Detection oj antigens of pathogenic mycoplasmas within diseased tissue, usually jrom cadavers. Antigens have been detected by means of fluorescent antibody tech-
niques and agar del double diffusion tests. Both these tests have been widely used in the diagnosis of M. mycoides infection. Detection oj significant levels of circulating antibody in living infected animals. Tests that have been used to detect antibodies include: i. Metabolism inhibition (Taylor-Robinson et al., 1966 ). ii. Growth neutralization (Gourlay & Domermuth, 1967). iii. Indirect haemagglutination (Dowdle & Robinson, 1964). iv. Haemagglutination inhibition (Fahey & Crawley, 1954). v. Agglutination (Adler, 1958; Beharsefat & Adler, 1965). vi. Precipitation (Turner, 1962). vii. Complement fixation (Campbell & Turner, 1953). viii. Fluorescent antibody techniques (Chanock, Hayflick & Barile, 1962). These tests have been widely used in the diagnosis of avian mycoplasmosis, human atypical pneumonia, and in contagious bovine pleuropneumonia, but they have their limitations depending upon the species of animal and of mycoplasma involved. Thus, not all of these tests would be suitable for anyone mycoplasmal disease, even if high antibody titres were present in infected animals.
DESCRIPTION OF MYCOPLASMAS
347
D etection of the hypersensitivity state in living irifected animals by means oj allergic skin tests. So far allergic skin tests have only been used in contagious bovine
pleuropneumonia. These have involved the observation of immediate and of delayed type reactions (Shifrine & Gourlay, 1965; Gourlay & Shifrine, 1965). Immune responses to mycoplasmal diseases
In some diseases, such as contagious bovine pleuropneumonia, the r ecovered animal is solidly immune for a long period, colostrum from immune mothers can protect calves for up to 6 months and vaccination with modern vaccines has proved to be efficacious. In contrast to this, in some mycoplasmal diseases recovered animals are not resistant to re-infection, and for many mycoplasmal diseases no vaccines are available. Antibiotic sensitivity in vitro and in vivo In vitro sensitivity tests with chemotherapeutic agents and antibiotics have
shown that, in general, mycoplasmas are: (a) Resistant to penicillin, polymixin B, sulphonamides, and anti-fungal antibiotics, e.g. nystatin and amphotericin. (b) Slightly susceptible to streptomycin, kanamycin and the nitrofurans and rather more susceptible to chloramphenicol. (c) Very susceptible to the tetracyclines, the erythromycin group, tylosin and carbomycin. There are differences in sensitivity in vitro between species of mycoplasmas. For example, M. neurolyticum is not resistant to penicillin whilst all other mycoplasmas are resistant (Hottle & Wright, 1966) and M . gallisepticum is susceptible to erythromycin whilst the non-pathogenic chicken mycoplasmas are resistant. In addition, there are differences between antibiotic sensitivity in vitro a nd effectiveness in vivo . In many cases of est ablished disease due to a mycoplasma, tetracyclines are of little use therapeutically other than by suppression of secondary bacterial invasion. This contrasts with the strongly bacteriostatic effect of tetracyclines in vitro . Control of mycoplasmal diseases
Since treatment with antibiotics has little or no effect on established cases of disease, treatment cannot be relied upon to eliminate infection. Control must, therefore, d epend on: (a) Prophylaxis by means of vaccination. (b) Eradication by means of slaughter of all infected animals. (c) Establishment of closed populations free from potentially pathogenic mycoplasmas. To d ate this technique has only been applied to pigs and chickens. REF ERENCES
ADLER, H. E. (1958). Poult. Sci., 37, 1116. ADLER, H . E. (1965). Adv. vet. Sci., 1O, 21I. BEHARSEFAT, M. & ADLER, H . E. (1965) . Avian Dis., 9, 460.
BRITISH VETERI NARY JOURNAL, 125, 7 BETTS, A. 0. (1967). Viral and Rickettsial Infections of Animals (ed. by A. 0 . Betts & C. J . York), p. 94. New York: Academic Press. CAMPBELL, A. D. & T URNER, A. W. (1953). Aust. uet. ] ., 29, 154. CHANOCK, R. M., HAYFLICK, L. & BARILE, M. F. (1962) . Proc. Soc. Acad. Sci., 48, 41. CLYDE, W. A. (1964), ], Immunol. , 92, 958. DINTER, Z., DANIELSSON, D. & BAKOS, K. (1965) .]. gen. Microbiol., 4 1 , 77. DOWDLE, W. R. & ROBINSON, R . Q. (1964). Proc. Soc. expo Biol. Med., 116, 947 . EDWARD, D. G .ff. & FITZGERALD, W. A. (1954), ] , Path. Bact., 68, 23. FAHEY, J. E. & CRAWLEY, J. F. (1954), Can.]. compo Med., 18, 264. FALLON, R. J. (1966). Vet. R ec., 79, 700. GOURLAY, R. N. & DOMERMUTH, C. H. (1967). Ann. N.T. Acad. Sci., 143, 325. GOURLAY, R. N . & SHIFRINE, M. (1965). ]. compo Path. Ther., 75, 375. GUIDICHE, DEL, R. A., ROBILLARD, N. F. & CARSKI, T. R. ( 1967). ]. Bact., 93, 1205. HAYFLICK, L. & CHANOCK, R. M . (1965). Bact. Rev., 29,1 85. HOTTLE, G . A. & WRIGHT, D. N . (1966). ]. Bact., 91, 1834. LEACH, R. H . & BUTLER, M. (1966).]. Bact., 9 1 , 934. MORTON, H . E. (1965). Bacterial and Mycotic Infections in Man (ed. by R. J. Dubos & J . G. Hirsch), p. 787 . London: Pitman Medical Publications. RAZIN, S. & OLIVER, 0. (1961 ). ]. Gen. Microbiol., 24> 225. SHIFRINE, M. & GOURLAY, R. N. (1965). J . compo Path. Ther. , 75, 381. TAYLOR-ROBINSON, D., SOMERSON, N . L., TURNER, H. C . & CHANOCK, R. M. (1963). J. Bact.,
85, 1261. TAYLOR-ROBINSON, D., P URCELL, R. H ., WONG, D. C . & CHANOCK, R. M . (1966).]. Hyg.,
Camb ., 64, 91. THOMAS, L. (1967). Ann. N .T. Acad. Sci., 143, 218. T ULLY, J. G. (1965). J. infect. Dis., 115, 266. TULLY, J. G. (1966) . Proc. Soc. expo Biol. Med., 122, 565. TURNER, A. W . (1935).]. Path. Bact., 41, I. TURNER, A. W . (1962) . Aust. uet. J., 38, 335.
A GENERAL DESCRIPTION OF MYCOPLASMAS By
JENNIFER POLAND
Department of Pathology, The Royal Veterinary College, University of London
INTRODUCTION
Much interest has been taken in the mycoplasma group of organisms during the past few years, concurrent with improvements in techniques that have resulted in the more frequent isolation of these organisms. The number of known species now exceeds fifty; they have been isolated from a wide variety of domesticated mammals and birds as well as from man. Mycoplasmas have been found as contaminants of continuous cell line tissue cultures (Tully, 1966; Leach & Butler, 1966) and since some species produce cytopathic effects, inhibit the growth of viruses, cause haem adsorption and haemagglutination and affect chromosome numbers (Betts, 1967), confusion and difficulties can arise in virological investigations. A number of species of mycoplasmas have been isolated from cases of human leukaemia, although these are of doubtful significance (Fallon, 1966). The possibility that mycoplasmas are in some way concerned with human rheumatoid arthritis is a field of current research. Properties oj mycoplasmas (pleuropneumonia-like organisms) According to Hayflick & Chanock (1965) the properties of this group of organisms are as follows: (a) They can replicate in a cell-free environment. (b ) On agar they exhibit a characteristic colonial morphology with the centre or the whole colony embedded beneath the surface of the agar. (c) The smallest reproductive elements have a size range of 125- 150 mf),. Thus, they are the smallest free-living organisms of a size comparable to that of myxoviruses. (d ) They lack a rigid cell wall, and consist of protoplasm surrounded by a triple-layered cell membrane. (e) With the exception of saprophytic species, most mycoplasmas require sterols or protein for growth. (f ) All species exhibit an absolute resistance to penicillin, whereas many strains are sensitive to low concentrations of tetracyclines. (g) The growth of mycoplasmas is inhibited by specific antibody. (h ) Mycoplasmas do not revert to or from bacterial parent forms. Mycoplasmas were originally confused with viruses because of their small size and their inability to grow in the usual bacteriological media. Mycoplasmas have also been confused with "L" forms of bacteria; this confusion was natural since "L" forms are variants of bacteria which have lost their cell walls and so
DESCRIPTION OF MYCOPLASMAS
345
resemble mycoplasmas both in morphology and in colonial appearance (Morton, 1965). The lack of cell wall is the reason for mycoplasmas being more sensitive than bacteria to physical and chemical agents; parasitic species survive only for short periods outside the body of the host, whilst in the laboratory the fragility of mycoplasmas is illustrated by the fact that lyophilization may kill more than 90 p er cent of the organisms (Adler, 1965) and that even faint traces of detergents are lethal. Mycoplasmas lack the capsules' and flagella found in many bacteria, and so far only two species of mycoplasma have been found to produce exotoxins: M, neurolyticum and M. gallisepticum (Thomas, 1967). A few species, e.g. M. gallisepticum, haemagglutinate a variety of erythrocytes, and colonies of this species also absorb erythrocytes.
Morphology M ycoplasmas are highly pleomorphic (Turner, 1935) ; branched filaments have been observed in liquid cultures, hence the derivation of the term " mycoplasma" . On solid m edia, mycoplasm a colonies form central downgrowths and many species form characteristic mamillated colonies. However, considerable variation in size and appearance can occur even within the colonies of a single species, possibly due to variations in environment (Razin & Oliver, 196 I ). Cultivation Mycoplasmas have been cultivated in embryonated hens' eggs but there are considerable differences between species in the ability to grow in eggs (Dinter, D anielsson & Bakos, 1965). They have been cultivated in tissue cultures and in acellular liquid and solid media but there are considerable variations in their nutritional requirements and cultural properties. Some species have such fastidious nutritional r equirements that they are difficult to cultivate in any system. Most acellular media that support the growth of mycoplasmas contain 20 per cent serum and 10 per cent of a yeast extract, in addition to a broth. Most mycoplasmas ferment glucose and produce lactic acid. Thus growth can conveniently be det ected by adding glucose and phenol red to liquid media and observing a change in pH. Those sp ecies that do not ferment glucose usually degrad e arginine and so render the medium more alkaline. A few species split urea. Thallium acet ate and penicillin can be added to media to keep down bacterial contamination. However, p enicillin could induce the formation of "L" forms of bacteria and thallium acetate concentrations greater than I in 4000 may inhibit the growth of mycoplasmas particularly when they are present in low numbers. Epidemiology and pathogenicity The list of known species contains frank pathogens, organisms of low pathogenicity, benign parasites and a saprophyte, M. laidlawii.
BRITISH VETERINARY JOURNAL, 125, 7
I
Almost all domesticated animals harbour benign parasites in the upper respiratory and/or urogenital tracts; in addition some animals may harbour potentially pathogenic mycoplasmas in the same sites. Although many of the pathogenic mycoplasmas cause respiratory disease, some cause inflammation of the pleura, pericardium and peritoneum, or of the joints. For example, M. mycoides causes pleuropneumonia in adult cattle whilst in young calves it also causes pericarditis and arthritis and M. arthritidis causes arthritis alone in rats and mice. Close contact is required for transmission of respiratory diseases caused by mycoplasmas since inhalation is the route of infection and the organisms do not survive for more than a few minutes outside the animal. Diagnosis is based on one or more of the following methods: Isolation and identification oj a pathogenic mycoplasma. Isolation is facilitated by passing material through a filter having an APD of 450 mfl since this removes contaminating bacteria. However, filtration may be impracticable when only a small quantity of infected material is available. Identification is based on: I. Biochemical tests to · detect glucose fermentation, arginine degradation, or the splitting of urea. II . Serological tests: i. Metabolism inhibition (Taylor-Robinson, Purcell, Wong & Chanock, 1966). ii. Growth inhibition (Edward & Fitzgerald, 1954; Clyde, 1964). iii. Agar gel double diffusion (Taylor-Robinson, Somerson, Turner & Chanock, 1963). iv. Fluorescent antibody techniques (Tully, 1965; Guidiche, Robillard & Carski, 1967). Detection oj antigens of pathogenic mycoplasmas within diseased tissue, usually jrom cadavers. Antigens have been detected by means of fluorescent antibody tech-
niques and agar del double diffusion tests. Both these tests have been widely used in the diagnosis of M. mycoides infection. Detection oj significant levels of circulating antibody in living infected animals. Tests that have been used to detect antibodies include: i. Metabolism inhibition (Taylor-Robinson et al., 1966 ). ii. Growth neutralization (Gourlay & Domermuth, 1967). iii. Indirect haemagglutination (Dowdle & Robinson, 1964). iv. Haemagglutination inhibition (Fahey & Crawley, 1954). v. Agglutination (Adler, 1958; Beharsefat & Adler, 1965). vi. Precipitation (Turner, 1962). vii. Complement fixation (Campbell & Turner, 1953). viii. Fluorescent antibody techniques (Chanock, Hayflick & Barile, 1962). These tests have been widely used in the diagnosis of avian mycoplasmosis, human atypical pneumonia, and in contagious bovine pleuropneumonia, but they have their limitations depending upon the species of animal and of mycoplasma involved. Thus, not all of these tests would be suitable for anyone mycoplasmal disease, even if high antibody titres were present in infected animals.
DESCRIPTION OF MYCOPLASMAS
347
D etection of the hypersensitivity state in living irifected animals by means oj allergic skin tests. So far allergic skin tests have only been used in contagious bovine
pleuropneumonia. These have involved the observation of immediate and of delayed type reactions (Shifrine & Gourlay, 1965; Gourlay & Shifrine, 1965). Immune responses to mycoplasmal diseases
In some diseases, such as contagious bovine pleuropneumonia, the r ecovered animal is solidly immune for a long period, colostrum from immune mothers can protect calves for up to 6 months and vaccination with modern vaccines has proved to be efficacious. In contrast to this, in some mycoplasmal diseases recovered animals are not resistant to re-infection, and for many mycoplasmal diseases no vaccines are available. Antibiotic sensitivity in vitro and in vivo In vitro sensitivity tests with chemotherapeutic agents and antibiotics have
shown that, in general, mycoplasmas are: (a) Resistant to penicillin, polymixin B, sulphonamides, and anti-fungal antibiotics, e.g. nystatin and amphotericin. (b) Slightly susceptible to streptomycin, kanamycin and the nitrofurans and rather more susceptible to chloramphenicol. (c) Very susceptible to the tetracyclines, the erythromycin group, tylosin and carbomycin. There are differences in sensitivity in vitro between species of mycoplasmas. For example, M. neurolyticum is not resistant to penicillin whilst all other mycoplasmas are resistant (Hottle & Wright, 1966) and M . gallisepticum is susceptible to erythromycin whilst the non-pathogenic chicken mycoplasmas are resistant. In addition, there are differences between antibiotic sensitivity in vitro a nd effectiveness in vivo . In many cases of est ablished disease due to a mycoplasma, tetracyclines are of little use therapeutically other than by suppression of secondary bacterial invasion. This contrasts with the strongly bacteriostatic effect of tetracyclines in vitro . Control of mycoplasmal diseases
Since treatment with antibiotics has little or no effect on established cases of disease, treatment cannot be relied upon to eliminate infection. Control must, therefore, d epend on: (a) Prophylaxis by means of vaccination. (b) Eradication by means of slaughter of all infected animals. (c) Establishment of closed populations free from potentially pathogenic mycoplasmas. To d ate this technique has only been applied to pigs and chickens. REF ERENCES
ADLER, H. E. (1958). Poult. Sci., 37, 1116. ADLER, H . E. (1965). Adv. vet. Sci., 1O, 21I. BEHARSEFAT, M. & ADLER, H . E. (1965) . Avian Dis., 9, 460.
BRITISH VETERI NARY JOURNAL, 125, 7 BETTS, A. 0. (1967). Viral and Rickettsial Infections of Animals (ed. by A. 0 . Betts & C. J . York), p. 94. New York: Academic Press. CAMPBELL, A. D. & T URNER, A. W. (1953). Aust. uet. ] ., 29, 154. CHANOCK, R. M., HAYFLICK, L. & BARILE, M. F. (1962) . Proc. Soc. Acad. Sci., 48, 41. CLYDE, W. A. (1964), ], Immunol. , 92, 958. DINTER, Z., DANIELSSON, D. & BAKOS, K. (1965) .]. gen. Microbiol., 4 1 , 77. DOWDLE, W. R. & ROBINSON, R . Q. (1964). Proc. Soc. expo Biol. Med., 116, 947 . EDWARD, D. G .ff. & FITZGERALD, W. A. (1954), ] , Path. Bact., 68, 23. FAHEY, J. E. & CRAWLEY, J. F. (1954), Can.]. compo Med., 18, 264. FALLON, R. J. (1966). Vet. R ec., 79, 700. GOURLAY, R. N. & DOMERMUTH, C. H. (1967). Ann. N.T. Acad. Sci., 143, 325. GOURLAY, R. N . & SHIFRINE, M. (1965). ]. compo Path. Ther., 75, 375. GUIDICHE, DEL, R. A., ROBILLARD, N. F. & CARSKI, T. R. ( 1967). ]. Bact., 93, 1205. HAYFLICK, L. & CHANOCK, R. M . (1965). Bact. Rev., 29,1 85. HOTTLE, G . A. & WRIGHT, D. N . (1966). ]. Bact., 91, 1834. LEACH, R. H . & BUTLER, M. (1966).]. Bact., 9 1 , 934. MORTON, H . E. (1965). Bacterial and Mycotic Infections in Man (ed. by R. J. Dubos & J . G. Hirsch), p. 787 . London: Pitman Medical Publications. RAZIN, S. & OLIVER, 0. (1961 ). ]. Gen. Microbiol., 24> 225. SHIFRINE, M. & GOURLAY, R. N. (1965). J . compo Path. Ther. , 75, 381. TAYLOR-ROBINSON, D., SOMERSON, N . L., TURNER, H. C . & CHANOCK, R. M. (1963). J. Bact.,
85, 1261. TAYLOR-ROBINSON, D., P URCELL, R. H ., WONG, D. C . & CHANOCK, R. M . (1966).]. Hyg.,
Camb ., 64, 91. THOMAS, L. (1967). Ann. N .T. Acad. Sci., 143, 218. T ULLY, J. G. (1965). J. infect. Dis., 115, 266. TULLY, J. G. (1966) . Proc. Soc. expo Biol. Med., 122, 565. TURNER, A. W . (1935).]. Path. Bact., 41, I. TURNER, A. W . (1962) . Aust. uet. J., 38, 335.