- Email: [email protected]
Growth, cytopathogenicity and morphology of Mycoplasma gallisepticum and M. gallinarum in tracheal explants
J. COMP.PATH.
GROWTH,
455
l976.C:0~.86.
CYTOPATHOGENICITY AND 1IORPHOLOGY OF MKOPLASMA GALLISEPTICUhf AND Xi. GALLI.KARUM IN TRACHEAL EXPLANTS BY p\I. Fanar
Poultry
N.
ABU-ZAHR
Research Laboratories,
Lebanon Republic
and &I.
BUTLER*
INTRODUCTION
Several species of mycoplasmas grow in tracheal explants and those associated with disease are usually cytopathogenic, for example M. pneumoniue (Collier, Clyde and Denny, 1971; Collier, 1972; Cherry and Taylor-Robinson, 1973), &f. mycoides var. capri and A4. gallisepticum (Butler and Ellaway, 1971; Cherry and Taylor-Robinson, 1971, 1973). The mechanism of pathogenicity is not clear but the fact that the organisms are generally extracellular parasites implies that exotoxic substances are involved, although, apart from a neurotoxin associated with M. neuroiyticum (Tully, 1964; Thomas, 1967) there is little other evidence for toxin production (Kaklamanis and Thomas, 1970). It has been suggested that hydrogen peroxide, produced by some mycoplasma species, is responsible for cellular damage, but because both pathogenic and nonpathogenic mycoplasmas produce hydrogen peroxide (Whittlestone, 1972) its role in disease is uncertain. However, an important factor in pathogenicity may be the capacity of mycoplasmas to adsorb to cells (Sobeslavsky, Prescott and Chanock, 1968; Sethi and Muller, 1972) and the intimate nature of this condition in tracheal explants has been explored by transmission electron miscroscopy (Collier, 1972 ; Chu and Uppal, 1975). Butler, Abu-Zahr and Aghakhan (1973) used both transmission and scanning electron microscopy to study the parasitic mode of M. gallisepticum in explants and later extended the study to include L%l. gallinarum (Abu-Zahr and Butler, 1974). This work is now presented in detail with some additional data. MATERIALS
Organisms. ,1f. gallinarunz
AND
METHODS
(strain 10120, National Collection of Type Cultures, Colindalej and the S6 strain of hf. gallisepticum(Central Veterinary Laboratory. Weybridgej were used. Stock cultures of both mycoplasmas were prepared in growth medium; PPLO broth (Difco) enriched with 20 per cent. unheated horse including penicillin (1000 u./ml.j and serum and 10 per cent. yeast autolysate, thallium acetate (0.05 per cent. w/v) cultures were stored at -20 “C. until required for the preparation of fresh 24 h. cultures to inoculate tracheal explants. IZq?lants.These were prepared by cutting 1 or 3 mm. thick slices from the trachea * To whom correspondence and requests for reprints should be addressed.
456
M.
N.
ABU-ZAHR
AND
M.
BUTLER
from 20-day specific pathogen free chicken embryos. The explants were incubated at 37 “C. in 1 ml. medium in tubes (16 x 125 mm.) covered with loose caps in a humidified incubator containing 5 per cent. CC+ in air. The medium was Eagle’s MEM adjusted to pH 7.0 with 0.01 per cent. sodmm bicarbonate. Each explant was re-fed 1 ml. of fresh medium 24 h. after preparation, ready for inoculation, Growth. Explants were inoculated with 0.1 ml. of a 1: 1000 dilution of fresh 24 h. mycoplasma culture. The 1 mm. slices were examined daily for evidence of ciliostasis and mycoplasma growth. The colony counts were made on 0.1 ml. samples removed and pooled from 3 tubes selected at random from a set of 20 similar explants. At the same time, inoculated 3 mm. explants were removed for electron microscopy. Uninoculated explants of both sizes were appropriately examined and sampled as controls. The growth and survival of the mycoplasmas in 1 ml. volumes of explant medium and mycoplasma growth medium were determined by the colony count method. Transmission electron microscopy. Explants were washed in several changes of phosphate buffered saline (pH, 7.2; PBS), fixed in 5 per cent. buffered glutaraldehyde overnight at 4 “C. and post-fixed with 1 per cent. buffered osmium tetroxide for 1 h. at room temperature. Dehydration was in graded concentrations of ethyl alcohol, replaced by propylene oxide before each specimen was embedded in epoxyresin (Epon 812)*. Th in sections (100 nm.), cut on an LKB ultratome using glass knives, were stained with lead citrate followed by uranyl acetate and examined in a JEOL 1OOB EM operated at 60 kV. Scanning electron microscopy. Explants were cut in half, longitudinally, and rinsed with several changes of PBS to free the surface of mucilage. The cultures were fixed,
post-fixed and dehydrated as described above and, after being attached to aluminium mounting stubs, were coated with evaporated gold-palladium; care was taken to avoid over-heating the specimen and to ensure that the metal film was no thicker than 20 nm. Sections were examined in a Cambridge Stereoscan EM operated at 20 kV. RESULTS
Growth The growth of both organisms in explant cultures was similar (Fig. 1, curves A and B) but this growth was evidently dependent on conditions provided by viable explants because neither organism was able to multiply in the explant medium alone (Fig. 2, curves C and D), although M. gallinarum survived better than M. gallisepticum. It was noteworthy that M. gallinarum grew better than M. gallisepticum in mycoplasma growth medium (Fig. 2, curves A and B). Another difference between the behaviour of the two organisms was reflected by the relationship between growth and the development of ciliostasis; with M. gallisepticum ciliostasis occurred as soon as the maximum colony count was reached (Fig. 1, curves B and C), whereas with M. gallinarum ciliostasis was a relatively late development occurring after the colony count had fallen to low values (Fig. 1, curves A and D). The cilia of uninoculated cultures also eventually ceased to beat (Fig. 1, curve E). MorpholoD In infections with M. gallisepticum individual organisms and small clusters were first seen, after about 2 days, lying close to the cilia and microvilli of the * TAAB
Laboratories,
Reading,
England.
MYCOPLASMA
IN
TRACHEAL
$57
EXPLANTS
epithelial cells (Figs 3 and 4). Within a further day or two the epithelium sloughed into the lumen, where the cells degenerated and became surrounded by dense aggregates of organisms (Figs 5 and 6). In section the organisms were often pear-shaped with the narrow end adsorbed to and even pushed into the epithelial cells (Fig. 7) but under the stereoscan electron microscope the bodies were more elongated and there was evidence that one end protruded into the I
I
I
/
I
I
I
I
.-
100
1, 90
I30
70
-t
$0
.LD
-5 io
h .-;: ._ 0
-1
s
io
-2
I
-C ,,
II
II
,,
II
II
//
II
2
3
4
5
6
7
LL 88
1
Days
Fig.
1. Growth of’mycoplasmas in tracheal explants and the development (----) : growth (B) ; ciliostasis (C). M. gallinarum (---) : growth in uninoculated explants (. . .). (E).
of ciliastasis. (A) ; ciliostasis
M. gallise~ticunz (II). Ciliostasis
cell surface (Fig. 8). At a later stage some organisms were buried in the basement membrane (Fig. 9) and after this many organisms appeared in the lamina propria where they accumulated in the intercellular spaces and between the collagen fibres (Fig. 10) to which they were often intimately attached (Fig. 10, inset). The first visual evidence of infection with M. gallinarum was after about 3 days when dense aggregates of organisms appeared within the lamina propria (Fig. 11). After a further 2 or 3 days the mycoplasmas practically filled the whole of this tissue which became generally disorganized (Fig. 12), but in
458
M.
N.
ABU-ZAHR
AND
M.
BUTLER
contrast to M. gallisepticum the M. gallinarum bodies attachment to the tissue cells (Fig. 12, inset). At about cells started to slough and degenerate and mycoplasma or in clusters amongst them (Fig. 13) but did not form
did not form a specific this time the epithelial bodies appeared singly an intimate association
8-
?-
\ 6 -\,
, 5-
: : :
(I 4-
: /
, 3Days
Fig. 2. Growth of mycoplasmas in mycoplasma M. galliscpticum (---) : growth (B) ; survival
growth medium and survival in explant medium. (D). M. gallinarum (---) : growth (A) ; survival (C).
Fig. 3. Transmission electron micrograph (TEM) of a section of an explant infected with M. gallisepticurn. Individual organisms (arrows) lie amongst the cilia and microvilli of an epithelial cell. x 9600. Fig. 4. Stereoscan electron micrograph (SEM) of the surface of an explant infected with M. gallisepticurn. Groups of organisms (arrows) are associated with the cilia and microvilli of the ciliated and non-ciliated areas of the epithelium. x 9600. Fig. 5. TEM of a section of sloughed epithelial cells from an explant infected with M. gallisepticum. The organisms (arrows) are closely associated with the degenerating epithelial cells. x 3200. Fig. 6. SEM of the surface of an explant infected with M. gaZliseptimm. The organisms have formed dense microcolonies around cells of the epithelium, some of which are protruding. x 3200. Fig. 7. TEM of a section of an epithelial cell of an explant infected with M. gallisepticum. The organisms (arrows) are closely associated with and even partly buried in the cell surface. x 56 000. Fig. 8. SEM of the surface of an explant infected with M. gallisepticum. The organisms lie intimately associated with the epithelial surface (arrow). x 9600.
MYCOPLASMA
IN TRACHEAL
EXPLANTS
459
460
M. N. ABU-ZAHR
AND M. Bw-LER
with them. Evidence for the presence of mycoplasma bodies on the epithelial surface wa.s not obtained by stereoscan electron microscopy. Cytopathologv
After inoculation with M. gallisepticum the epithelial cells shed their cilia and became swollen. The interdigitations or desmosomes disappeared so that the cells became detached from each other and the basement membrane. The cytoplasm became disorganized by the development of numerous vacuoles which replaced the mitochondria, endoplasmic reticulum and Golgi complex. The nuclei enlarged and the chromatin material became peripherally orientated and mixed with nucleolar material; the double nuclear membrane, although remaining distinguishable for some time, became loose and disintegrated so that the cell became an amorphous mass. Although the cells of explants inoculated with M. gallinarum also eventually degenerated, the process was unlike that induced by M. gallir@‘cum. Small densely staining amorphous inclusions appeared in the cytoplasm of the cells adjacent to the dense aggregates of mycoplasmas. The tissue cells became swollen and the inclusions tended to become more numerous and enlarge, displacing the nucleus and other cell organelles. The epjthelial cells in these regions eventually lost their cilia, became detached from the basement membrane and ultimately started to disintegrate, although some organelles like the nucleus and mitochondria, remained recognizable for some time as did the peculiar densely staining inclusions. The integrity of the control explants was retained for up to 2 weeks but then degenerative changes associated with ageing slowly developed; for instance, the cilia were shed from the epithelial cells and the cytoplasm became somewhat granular and vacuolated. DISCUSSION
Our observations provide visual evidence for significant differences in the mode of parasitism of M. gallisepticum and M. gallinarum and particularly draw attention to the close association between cytoadsorption by M. gallisepticum and its cytopathogenicity. With M. gallinarum, adsorption to cells did not occur and, although cytopathic effects eventually developed, they were qualitatively Fig. 9. TEM of a section of an explant infected with M. galliscptkn. Epithelial cells have sloughed and organisms (arrows) are intimately associated with the basement membrane (Bm). See also inset. (Lp = lamina propria). x 2560: inset x 28 000. Fig. 10. TEM of a section of the lamina propria of an explant infected with M. gallirepticum. The organisms (arrows) have accumulated in the spaces between the connective tissue and collagen fibres (Cf) with which they are intimately associated. See also inset. x 4800; inset 29 000. Fig. 11. TEM of a section of an explant infected with M. gallinarum. Organisms (arrow; Mg) are massed between the epithelial cells (Ep) and the lamina propria (Lp). (Cf = collagen fibres). x 7200. Fig. 12. TEM of a section of the lamina propria of an explant infected with M. gallinarum. The tissue is somewhat disorganized with mycoplasma bodies (arrows) arranged randomly but showing no signs of any specific attachment to cells or collagen fibres (Cf). See also inset. x 7200; inset x 29 000. Fig. 13. TEM of a section of an explant infected with M. gallinarum. The epithelial cells have sloughed and are partially degenerated. Mycoplasma bodies (arrows) are arranged randomly and show no signs of attachment to cells or debris. (Lp = lamina propria). x 2800.
MYCOPLASMA
IN TRACHEAL
EXPLANTS
46
1
462
M.
N.
ABU-ZAHR
AND
M.
BUTLER
from those induced by M. gallisepticum.Indeed, M. gallinarum is thought to be saprophytic, utilizing growth factors released from normal cultures (Reed, 1972) and the organisms had no effect on explants in which the medium was frequently changed (Taylor-Robinson and Cherry, 1972). The cytopathogenicity associated with M. gallisepticum developed rapidly and the organism evidently had a strong cell tropism which presumably not only ensured active colonization of the tissue, but was also an effective intimate system for the action of cytotoxic substances. It could also provide the mechanism for access to essential nutrients as suggested by Zucker-Franklin, Davidson and Thomas, 1966; Stanbridge, 1971 and Collier, 1972. Thus the differences demonstrated between the behaviour of M. gallisepticum and M. gallinarum can be regarded as support for the widely held view that Ad. gallisepticum, unlike M. gallinarum, has a pathogenic role for the avian host (Jordan, 1975). distinct
SUMMARY
Transmission and scanning electron microscopy was used to study the parasitic mode of M. gallisepticum and M. gallinarum in chicken embryo tracheal explants. Both mycoplasmas grew equally well but M. gallisepticum rapidly induced ciliostasis, whereas with M. gallinarum it was a much later development. M. gallisepticum first colonized the epithelium and later the lamina propria, usually forming a close association with the cells. This was in contrast to the behaviour of M. gallinarum which appeared first in the lamina propria and did not develop an intimate association with the cells. The cytopathology associated with M. gallinarum infections may be mainly due to competitive depletion of nutrients essential for the healthy maintenance of the explant, whereas with Ad. gallisepticum the cell tropism probably plays a significant role by providing an intimate system for the action of cytotoxic substances. ACKNOWLEDGMENTS
One of us (M.N. A.-Z.) is indebted to the Lebanese Research Council for a maintenance grant and to the Central Veterinary Laboratory, Weybridge, for providing specific pathogen-free eggs and laboratory facilities for part of the study. REFERENCES
Abu-Zahr, M. N., and Butler, M. (1974). A comparison of ultrastructural detail=f M. gallisepticum and M. gallinarum in infected tracheal explants. International Congress on Mycoplasmas of Man, Animals, Plants and Insects. September 11 th-17th, Bordeaux, France. Butler, M., Abu-Zahr, M. N., and Aghakhan, M. (1973). Study of a mixed infection with mycoplasma and adenovirus. 1st International Congress for Bacteriology. September 2nd-7th, Jerusalem, Israel. Butler, M., and Ellaway, W. J. (1971). Growth and cytopathogenicity of mycoplasma in human and chicken tracheal explants. Journal of Comparative Pathology, 81, 359-364. Cherry, J. D., and Taylor-Robinson, D. (1971). Growth and pathogenicity studies of Mycoplasma gallisepticum in chicken tracheal organ culture. Journal of Medical Microbiology, 4, 441-449.
MYCOPLASMA
IN
TRACHEAL
463
EXPLANTS
D. (1973). Mycoplasma pathogenicity studies Cherry, J. D., and Taylor-Robinson, in organ cultures. Annals of the New York Academy of Sciences, 225, 290-303. Chu, H. P., and Uppal, P. K. (1975). S in gl e and mixed infections of avian infectious bronchitis virus and Myco$usma gallisepticum. Developments in Biological S’tandardization, 28, 101-l 14. Clollier, A. M. (1972). Pathogenesis of Mycoplasma pneumoniae infection as studied in human foetal trachea in organ culture. In Pathogenic L2Qxoplasmas. (:iba Foundation Symposium, pp. 307-320. Elsevier, Amsterdam. C:ollier, A. M., Clyde, W. A., and Denny, F. W. (1971 j. L2
206-227. Taylor-Robinson, D., and Cherry, J. D. (1972). Non-pathogenic mycoplasmas inhibiting the effect of a pathogenic mycoplasma in organ culture. Jounlal of nedical Microbiology, 5, 291-298. Thomas, L. (I 967). The neurotoxins of M. neurolyticum and ,21. gallisepticum. :lnnol.s oj‘ fhe New York Academy of Sciences, 143, 2 18-224. Tully, J. C. (1964). Production and biological characteristics of an extraceilular neurotoxin from r\Iycoplasma neurolyticum. Journal oj‘ Bacteriology, 88, 381-388. \Vhittlestone, P. (1972). Pathogenicity of mycoplasmas in animals. In AGcrobial Pathogeni& in Man and Animals, H. Smith and J. H. Pearce, Eds. The Socict!, for General Microbiology, Symposium, 22, 2 17-250. Zucker-Franklin, D., Davidson, M., and Thomas, L. rl1966j. The interaction 1J1 mycoplasmas with mammalian cells. I. HeLa cells, neutrophils and eosinophils. Journal of IGperimental ;2ledicine, 124, 521-532. [Received for publication,
December I3th,
19751
GROWTH,
455
l976.C:0~.86.
CYTOPATHOGENICITY AND 1IORPHOLOGY OF MKOPLASMA GALLISEPTICUhf AND Xi. GALLI.KARUM IN TRACHEAL EXPLANTS BY p\I. Fanar
Poultry
N.
ABU-ZAHR
Research Laboratories,
Lebanon Republic
and &I.
BUTLER*
INTRODUCTION
Several species of mycoplasmas grow in tracheal explants and those associated with disease are usually cytopathogenic, for example M. pneumoniue (Collier, Clyde and Denny, 1971; Collier, 1972; Cherry and Taylor-Robinson, 1973), &f. mycoides var. capri and A4. gallisepticum (Butler and Ellaway, 1971; Cherry and Taylor-Robinson, 1971, 1973). The mechanism of pathogenicity is not clear but the fact that the organisms are generally extracellular parasites implies that exotoxic substances are involved, although, apart from a neurotoxin associated with M. neuroiyticum (Tully, 1964; Thomas, 1967) there is little other evidence for toxin production (Kaklamanis and Thomas, 1970). It has been suggested that hydrogen peroxide, produced by some mycoplasma species, is responsible for cellular damage, but because both pathogenic and nonpathogenic mycoplasmas produce hydrogen peroxide (Whittlestone, 1972) its role in disease is uncertain. However, an important factor in pathogenicity may be the capacity of mycoplasmas to adsorb to cells (Sobeslavsky, Prescott and Chanock, 1968; Sethi and Muller, 1972) and the intimate nature of this condition in tracheal explants has been explored by transmission electron miscroscopy (Collier, 1972 ; Chu and Uppal, 1975). Butler, Abu-Zahr and Aghakhan (1973) used both transmission and scanning electron microscopy to study the parasitic mode of M. gallisepticum in explants and later extended the study to include L%l. gallinarum (Abu-Zahr and Butler, 1974). This work is now presented in detail with some additional data. MATERIALS
Organisms. ,1f. gallinarunz
AND
METHODS
(strain 10120, National Collection of Type Cultures, Colindalej and the S6 strain of hf. gallisepticum(Central Veterinary Laboratory. Weybridgej were used. Stock cultures of both mycoplasmas were prepared in growth medium; PPLO broth (Difco) enriched with 20 per cent. unheated horse including penicillin (1000 u./ml.j and serum and 10 per cent. yeast autolysate, thallium acetate (0.05 per cent. w/v) cultures were stored at -20 “C. until required for the preparation of fresh 24 h. cultures to inoculate tracheal explants. IZq?lants.These were prepared by cutting 1 or 3 mm. thick slices from the trachea * To whom correspondence and requests for reprints should be addressed.
456
M.
N.
ABU-ZAHR
AND
M.
BUTLER
from 20-day specific pathogen free chicken embryos. The explants were incubated at 37 “C. in 1 ml. medium in tubes (16 x 125 mm.) covered with loose caps in a humidified incubator containing 5 per cent. CC+ in air. The medium was Eagle’s MEM adjusted to pH 7.0 with 0.01 per cent. sodmm bicarbonate. Each explant was re-fed 1 ml. of fresh medium 24 h. after preparation, ready for inoculation, Growth. Explants were inoculated with 0.1 ml. of a 1: 1000 dilution of fresh 24 h. mycoplasma culture. The 1 mm. slices were examined daily for evidence of ciliostasis and mycoplasma growth. The colony counts were made on 0.1 ml. samples removed and pooled from 3 tubes selected at random from a set of 20 similar explants. At the same time, inoculated 3 mm. explants were removed for electron microscopy. Uninoculated explants of both sizes were appropriately examined and sampled as controls. The growth and survival of the mycoplasmas in 1 ml. volumes of explant medium and mycoplasma growth medium were determined by the colony count method. Transmission electron microscopy. Explants were washed in several changes of phosphate buffered saline (pH, 7.2; PBS), fixed in 5 per cent. buffered glutaraldehyde overnight at 4 “C. and post-fixed with 1 per cent. buffered osmium tetroxide for 1 h. at room temperature. Dehydration was in graded concentrations of ethyl alcohol, replaced by propylene oxide before each specimen was embedded in epoxyresin (Epon 812)*. Th in sections (100 nm.), cut on an LKB ultratome using glass knives, were stained with lead citrate followed by uranyl acetate and examined in a JEOL 1OOB EM operated at 60 kV. Scanning electron microscopy. Explants were cut in half, longitudinally, and rinsed with several changes of PBS to free the surface of mucilage. The cultures were fixed,
post-fixed and dehydrated as described above and, after being attached to aluminium mounting stubs, were coated with evaporated gold-palladium; care was taken to avoid over-heating the specimen and to ensure that the metal film was no thicker than 20 nm. Sections were examined in a Cambridge Stereoscan EM operated at 20 kV. RESULTS
Growth The growth of both organisms in explant cultures was similar (Fig. 1, curves A and B) but this growth was evidently dependent on conditions provided by viable explants because neither organism was able to multiply in the explant medium alone (Fig. 2, curves C and D), although M. gallinarum survived better than M. gallisepticum. It was noteworthy that M. gallinarum grew better than M. gallisepticum in mycoplasma growth medium (Fig. 2, curves A and B). Another difference between the behaviour of the two organisms was reflected by the relationship between growth and the development of ciliostasis; with M. gallisepticum ciliostasis occurred as soon as the maximum colony count was reached (Fig. 1, curves B and C), whereas with M. gallinarum ciliostasis was a relatively late development occurring after the colony count had fallen to low values (Fig. 1, curves A and D). The cilia of uninoculated cultures also eventually ceased to beat (Fig. 1, curve E). MorpholoD In infections with M. gallisepticum individual organisms and small clusters were first seen, after about 2 days, lying close to the cilia and microvilli of the * TAAB
Laboratories,
Reading,
England.
MYCOPLASMA
IN
TRACHEAL
$57
EXPLANTS
epithelial cells (Figs 3 and 4). Within a further day or two the epithelium sloughed into the lumen, where the cells degenerated and became surrounded by dense aggregates of organisms (Figs 5 and 6). In section the organisms were often pear-shaped with the narrow end adsorbed to and even pushed into the epithelial cells (Fig. 7) but under the stereoscan electron microscope the bodies were more elongated and there was evidence that one end protruded into the I
I
I
/
I
I
I
I
.-
100
1, 90
I30
70
-t
$0
.LD
-5 io
h .-;: ._ 0
-1
s
io
-2
I
-C ,,
II
II
,,
II
II
//
II
2
3
4
5
6
7
LL 88
1
Days
Fig.
1. Growth of’mycoplasmas in tracheal explants and the development (----) : growth (B) ; ciliostasis (C). M. gallinarum (---) : growth in uninoculated explants (. . .). (E).
of ciliastasis. (A) ; ciliostasis
M. gallise~ticunz (II). Ciliostasis
cell surface (Fig. 8). At a later stage some organisms were buried in the basement membrane (Fig. 9) and after this many organisms appeared in the lamina propria where they accumulated in the intercellular spaces and between the collagen fibres (Fig. 10) to which they were often intimately attached (Fig. 10, inset). The first visual evidence of infection with M. gallinarum was after about 3 days when dense aggregates of organisms appeared within the lamina propria (Fig. 11). After a further 2 or 3 days the mycoplasmas practically filled the whole of this tissue which became generally disorganized (Fig. 12), but in
458
M.
N.
ABU-ZAHR
AND
M.
BUTLER
contrast to M. gallisepticum the M. gallinarum bodies attachment to the tissue cells (Fig. 12, inset). At about cells started to slough and degenerate and mycoplasma or in clusters amongst them (Fig. 13) but did not form
did not form a specific this time the epithelial bodies appeared singly an intimate association
8-
?-
\ 6 -\,
, 5-
: : :
(I 4-
: /
, 3Days
Fig. 2. Growth of mycoplasmas in mycoplasma M. galliscpticum (---) : growth (B) ; survival
growth medium and survival in explant medium. (D). M. gallinarum (---) : growth (A) ; survival (C).
Fig. 3. Transmission electron micrograph (TEM) of a section of an explant infected with M. gallisepticurn. Individual organisms (arrows) lie amongst the cilia and microvilli of an epithelial cell. x 9600. Fig. 4. Stereoscan electron micrograph (SEM) of the surface of an explant infected with M. gallisepticurn. Groups of organisms (arrows) are associated with the cilia and microvilli of the ciliated and non-ciliated areas of the epithelium. x 9600. Fig. 5. TEM of a section of sloughed epithelial cells from an explant infected with M. gallisepticum. The organisms (arrows) are closely associated with the degenerating epithelial cells. x 3200. Fig. 6. SEM of the surface of an explant infected with M. gaZliseptimm. The organisms have formed dense microcolonies around cells of the epithelium, some of which are protruding. x 3200. Fig. 7. TEM of a section of an epithelial cell of an explant infected with M. gallisepticum. The organisms (arrows) are closely associated with and even partly buried in the cell surface. x 56 000. Fig. 8. SEM of the surface of an explant infected with M. gallisepticum. The organisms lie intimately associated with the epithelial surface (arrow). x 9600.
MYCOPLASMA
IN TRACHEAL
EXPLANTS
459
460
M. N. ABU-ZAHR
AND M. Bw-LER
with them. Evidence for the presence of mycoplasma bodies on the epithelial surface wa.s not obtained by stereoscan electron microscopy. Cytopathologv
After inoculation with M. gallisepticum the epithelial cells shed their cilia and became swollen. The interdigitations or desmosomes disappeared so that the cells became detached from each other and the basement membrane. The cytoplasm became disorganized by the development of numerous vacuoles which replaced the mitochondria, endoplasmic reticulum and Golgi complex. The nuclei enlarged and the chromatin material became peripherally orientated and mixed with nucleolar material; the double nuclear membrane, although remaining distinguishable for some time, became loose and disintegrated so that the cell became an amorphous mass. Although the cells of explants inoculated with M. gallinarum also eventually degenerated, the process was unlike that induced by M. gallir@‘cum. Small densely staining amorphous inclusions appeared in the cytoplasm of the cells adjacent to the dense aggregates of mycoplasmas. The tissue cells became swollen and the inclusions tended to become more numerous and enlarge, displacing the nucleus and other cell organelles. The epjthelial cells in these regions eventually lost their cilia, became detached from the basement membrane and ultimately started to disintegrate, although some organelles like the nucleus and mitochondria, remained recognizable for some time as did the peculiar densely staining inclusions. The integrity of the control explants was retained for up to 2 weeks but then degenerative changes associated with ageing slowly developed; for instance, the cilia were shed from the epithelial cells and the cytoplasm became somewhat granular and vacuolated. DISCUSSION
Our observations provide visual evidence for significant differences in the mode of parasitism of M. gallisepticum and M. gallinarum and particularly draw attention to the close association between cytoadsorption by M. gallisepticum and its cytopathogenicity. With M. gallinarum, adsorption to cells did not occur and, although cytopathic effects eventually developed, they were qualitatively Fig. 9. TEM of a section of an explant infected with M. galliscptkn. Epithelial cells have sloughed and organisms (arrows) are intimately associated with the basement membrane (Bm). See also inset. (Lp = lamina propria). x 2560: inset x 28 000. Fig. 10. TEM of a section of the lamina propria of an explant infected with M. gallirepticum. The organisms (arrows) have accumulated in the spaces between the connective tissue and collagen fibres (Cf) with which they are intimately associated. See also inset. x 4800; inset 29 000. Fig. 11. TEM of a section of an explant infected with M. gallinarum. Organisms (arrow; Mg) are massed between the epithelial cells (Ep) and the lamina propria (Lp). (Cf = collagen fibres). x 7200. Fig. 12. TEM of a section of the lamina propria of an explant infected with M. gallinarum. The tissue is somewhat disorganized with mycoplasma bodies (arrows) arranged randomly but showing no signs of any specific attachment to cells or collagen fibres (Cf). See also inset. x 7200; inset x 29 000. Fig. 13. TEM of a section of an explant infected with M. gallinarum. The epithelial cells have sloughed and are partially degenerated. Mycoplasma bodies (arrows) are arranged randomly and show no signs of attachment to cells or debris. (Lp = lamina propria). x 2800.
MYCOPLASMA
IN TRACHEAL
EXPLANTS
46
1
462
M.
N.
ABU-ZAHR
AND
M.
BUTLER
from those induced by M. gallisepticum.Indeed, M. gallinarum is thought to be saprophytic, utilizing growth factors released from normal cultures (Reed, 1972) and the organisms had no effect on explants in which the medium was frequently changed (Taylor-Robinson and Cherry, 1972). The cytopathogenicity associated with M. gallisepticum developed rapidly and the organism evidently had a strong cell tropism which presumably not only ensured active colonization of the tissue, but was also an effective intimate system for the action of cytotoxic substances. It could also provide the mechanism for access to essential nutrients as suggested by Zucker-Franklin, Davidson and Thomas, 1966; Stanbridge, 1971 and Collier, 1972. Thus the differences demonstrated between the behaviour of M. gallisepticum and M. gallinarum can be regarded as support for the widely held view that Ad. gallisepticum, unlike M. gallinarum, has a pathogenic role for the avian host (Jordan, 1975). distinct
SUMMARY
Transmission and scanning electron microscopy was used to study the parasitic mode of M. gallisepticum and M. gallinarum in chicken embryo tracheal explants. Both mycoplasmas grew equally well but M. gallisepticum rapidly induced ciliostasis, whereas with M. gallinarum it was a much later development. M. gallisepticum first colonized the epithelium and later the lamina propria, usually forming a close association with the cells. This was in contrast to the behaviour of M. gallinarum which appeared first in the lamina propria and did not develop an intimate association with the cells. The cytopathology associated with M. gallinarum infections may be mainly due to competitive depletion of nutrients essential for the healthy maintenance of the explant, whereas with Ad. gallisepticum the cell tropism probably plays a significant role by providing an intimate system for the action of cytotoxic substances. ACKNOWLEDGMENTS
One of us (M.N. A.-Z.) is indebted to the Lebanese Research Council for a maintenance grant and to the Central Veterinary Laboratory, Weybridge, for providing specific pathogen-free eggs and laboratory facilities for part of the study. REFERENCES
Abu-Zahr, M. N., and Butler, M. (1974). A comparison of ultrastructural detail=f M. gallisepticum and M. gallinarum in infected tracheal explants. International Congress on Mycoplasmas of Man, Animals, Plants and Insects. September 11 th-17th, Bordeaux, France. Butler, M., Abu-Zahr, M. N., and Aghakhan, M. (1973). Study of a mixed infection with mycoplasma and adenovirus. 1st International Congress for Bacteriology. September 2nd-7th, Jerusalem, Israel. Butler, M., and Ellaway, W. J. (1971). Growth and cytopathogenicity of mycoplasma in human and chicken tracheal explants. Journal of Comparative Pathology, 81, 359-364. Cherry, J. D., and Taylor-Robinson, D. (1971). Growth and pathogenicity studies of Mycoplasma gallisepticum in chicken tracheal organ culture. Journal of Medical Microbiology, 4, 441-449.
MYCOPLASMA
IN
TRACHEAL
463
EXPLANTS
D. (1973). Mycoplasma pathogenicity studies Cherry, J. D., and Taylor-Robinson, in organ cultures. Annals of the New York Academy of Sciences, 225, 290-303. Chu, H. P., and Uppal, P. K. (1975). S in gl e and mixed infections of avian infectious bronchitis virus and Myco$usma gallisepticum. Developments in Biological S’tandardization, 28, 101-l 14. Clollier, A. M. (1972). Pathogenesis of Mycoplasma pneumoniae infection as studied in human foetal trachea in organ culture. In Pathogenic L2Qxoplasmas. (:iba Foundation Symposium, pp. 307-320. Elsevier, Amsterdam. C:ollier, A. M., Clyde, W. A., and Denny, F. W. (1971 j. L2
206-227. Taylor-Robinson, D., and Cherry, J. D. (1972). Non-pathogenic mycoplasmas inhibiting the effect of a pathogenic mycoplasma in organ culture. Jounlal of nedical Microbiology, 5, 291-298. Thomas, L. (I 967). The neurotoxins of M. neurolyticum and ,21. gallisepticum. :lnnol.s oj‘ fhe New York Academy of Sciences, 143, 2 18-224. Tully, J. C. (1964). Production and biological characteristics of an extraceilular neurotoxin from r\Iycoplasma neurolyticum. Journal oj‘ Bacteriology, 88, 381-388. \Vhittlestone, P. (1972). Pathogenicity of mycoplasmas in animals. In AGcrobial Pathogeni& in Man and Animals, H. Smith and J. H. Pearce, Eds. The Socict!, for General Microbiology, Symposium, 22, 2 17-250. Zucker-Franklin, D., Davidson, M., and Thomas, L. rl1966j. The interaction 1J1 mycoplasmas with mammalian cells. I. HeLa cells, neutrophils and eosinophils. Journal of IGperimental ;2ledicine, 124, 521-532. [Received for publication,
December I3th,
19751