Immunology 1976 30 611
Effects of active and passive immunization on Mycoplasma pulmonis-induced pneumonia in mice
GERALDINE TAYLOR* & D. TAYLOR-ROBINSON Division of Communicable Diseases, MRC Clinical Research Centre, Watford Road, Harrow, Middlesex
Received 9 October 1975; acceptedfor publication 30 October 1975
Summary. Parenteral immunization of C3H mice with viable Mycoplasma pulmonis organisms protected them from pneumonia induced by intranasal inoculation of these organisms. Spleen cells obtained from immunized mice were ineffective in preventing syngeneic recipients from developing respiratory disease. In contrast, convalescent-phase serum enhanced the clearance of mycoplasmas from the respiratory tract of mice challenged with a small number of organisms. Further, although 'immune' serum had no detectable effect on the number of mycoplasmas in the respiratory tract of mice challenged with a large number of organisms, such animals did not develop pneumonia. Since the pneumonia appears to be the result of the host's immune response to the mycoplasma, it is suggested that the transferred 'immune' serum may act by suppressing the immune response so that mice develop less severe lung lesions. This suggestion is supported by the observation that the complement-fixing antibody response of passively immunized mice was suppressed. * Present address: Agricultural Research Council, Institute
INTRODUCTION
Although mycoplasmas are an important cause of respiratory disease in a number of animal species (Taylor-Robinson, 1975), there are no fully effective vaccines to control these infections. In order to develop suitable vaccines it is necessary to understand the mechanisms involved in resistance to reinfection. Little is known of the parts played by serum antibody, cell-mediated immunity and local secretory antibody in resistance. However, examination of the ability of passively transferred antibody and of sensitized cells to confer immunity on syngeneic recipient animals may help to determine their relative importance. Investigations of this kind have been undertaken. Thus, Nocard and coworkers in 1899 (quoted by Nocard and Leclainche, 1903) and Gourlay and Shifrine (1966) demonstrated that transfer of convalescent-phase serum to cattle protected them against the effects of subcutaneous challenge with a virulent strain of Mycoplasma mycoides var. mycoides. On the other hand, Lloyd (1967) found that serum from cattle that were resistant to challenge with M. mycoides var. mycoides, did not protect recipient animals when given in amounts much greater than used by Nocard and co-workers. Despite these conflicting results, other
for Research on Animal Diseases, Compton, near Newbury, Berkshire. Correspondence: Dr D. Taylor-Robinson, Division of Communicable Diseases, MRC Clinical Research Centre, Watford Road, Harrow, Middlesex HAl 3UJ.
611
612
Geraldine Taylor & D. Taylor-Robinson
reports of the successful transfer of immunity to various mycoplasma respiratory diseases by administration of serum (Lam and Switzer, 1971; Goig, Kuksa and Franz, 1974), suggest that resistance to these diseases is mediated by serum antibody. However, inactivated mycoplasma vaccines, which stimulate high levels of serum antibody, do not always provide protection against intranasal challenge with live organisms (Goodwin, Hodgson, Whittlestone and Woodhams, 1969; Fernald and Clyde, 1970) and there is further evidence that there is often a poor correlation between serum antibodies and resistance to reinfection (Davies and Hudson, 1968; Goodwin et al., 1969; Lam and Switzer, 1971). These apparent discrepancies prompted investigations into the role of cell-mediated immune mechanisms in resistance to mycoplasma infections. There is evidence that thymus-dependent lymphocytes become sensitized during a mycoplasma respiratory infection (see Taylor and TaylorRobinson, 1975) and these cells appear to be involved in the development of the pneumonic lesions (Denny, Taylor-Robinson and Allison, 1972; Taylor, Taylor-Robinson and Fernald, 1974). However, there have been few reports of attempts to transfer immunity to mycoplasmal pneumonia by administration of cells. Lloyd (quoted by Lloyd and Trethewie, 1970) took lymph node cells from a calf resistant to M. mycoides var. mycoides and transferred them to its fully susceptible identical twin. The only indication that there had been a transfer of immunity to the recipient calf was a delay in its serological response to the mycoplasma after challenge compared with that of untreated, infected animals. In an attempt to understand more fully the relative importance of serum antibody and of cells in resistance to mycoplasma respiratory disease we have examined the effects of passive immunization on M. pulmonis-induced pneumonia in mice. M. pulmonis infection of mice appears to be a good model for many of the other respiratory mycoplasma infections (Lindsey and Cassell, 1973) and use of an inbred strain of mouse permits the transfer of cells between animals.
Ltd, and by the Animal Division of the Clinical Research Centre, Harrow. All mice were of the M.R.C. Laboratory Animal Centre's category 4(****) standard and were maintained in isolation throughout the experimental period. Mice were anaesthetized by intraperitoneal (i.p.) inoculation of 0-06 mg/g body weight of sodium pentobarbitone and were infected by intranasal (i.n.) inoculation of 0 05 ml of an appropriate dilution of an M. pulmonis stock culture. Uninfected control mice were inoculated i.n. in the same way with 0*05 ml of mycoplasma medium.
MATERIALS AND METHODS
Vaccination procedures Mice were actively immunized by intravenous (i.v.) inoculation of either a live or a heat-inactivated (560 for 1 h) suspension of M. pulmonis which contained 2 x 101 c.c.u. in 0-2 ml. As controls, mice
Mice Male, C3H mice weighing 20-30 g were supplied by Scientific Agribusiness Consultants (International)
Mycoplasma medium and strains Liquid medium for the isolation and growth of mycoplasmas has been described previously (Denny et al., 1972). Mice were infected with the 'JB' strain of M. pulmonis which was obtained from Dr J. Tully (National Institutes of Health, Bethesda); the strain was grown in medium free of thallium acetate and a fifth broth passage from that originally received was used for inoculation. The strain of M. mycoides var. capri (hereafter referred to as M. capri) was obtained from Dr G. R. Smith (Nuffield Institute, Regent's Park, London). Mycoplasma isolation, titration and identification These procedures were the same as those described previously by Denny et al. (1972). Numbers of organisms are expressed as colour-changing units
(c.c.u.). Complement-fixation (CF) test The M. pulmonis antibody titre of mouse sera was determined by a micro-technique. A washed, 300 mg pellet of M. pulmonis 'JB', resuspended in 6 ml of phosphate-buffered saline (PBS), was heated at 560 for 1 h and stored at - 700. The optimum dilution of this antigen in the CF test was 1/50. Serial dilutions of heat-inactivated (560 for 30 min) mouse sera were mixed with 3 HC5o of complement (Wellcome Reagents Ltd) and fixation was carried out at 40 overnight. The antibody titre was recorded as the dilution of serum at which there was approximately 50 per cent lysis of red cells.
Mycoplasma-induced pneumonia in mice inoculated medium.
were
i.v.
613
RESULTS
with 02 ml of mycoplasma
M. pulmonis infection in actively immunized mice Passive immunization 'Immune' serum was obtained from mice which had been inoculated i.v. 3 weeks previously with 2 x 106 c.c.u. of M. pulmonis. Spleen cell suspensions were prepared from these animals by gently teasing chopped pieces of spleen through a 100-gauge stainless steel sieve into 199 medium (Gibco Biocult). Cell aggregates were allowed to sediment for 5 min and the supernatant cell suspension was gently forced through a series of needles of graded size so that a single cell suspension was produced. The cells were sedimented at 300 g for 10 min, washed twice with 199 medium and resuspended to give approximately 108 viable nucleated cells/ml. The ratio of donor to recipient animals was 2: 1. Recipient mice were inoculated i.v. with either 108 cells or with 0 4 ml of serum and challenged 24 h later with 5 x 105 c.c.u. of M. pulmonis inoculated i.n. Animals treated in a similar way with cells or serum obtained from mice that had been inoculated i.v. with mycoplasma medium served as controls. Mice were examined 14 days after i.n. challenge for mycoplasmas in the respiratory tract and for lung lesions.
Absorption of serum M. pulmonis or M. capri organisms used to absorb serum were grown in 600 ml of medium for 5 days, centrifuged at 27,000 g for 20 min and the pellets washed three times with PBS. Serum was absorbed twice by mixing 300 mg of the pellet with 4 ml of serum at 4° and occasionally shaking for 24 h. After absorption, the mycoplasmas were separated from the serum by centrifugation.
To investigate the ability of parenterally inoculated organisms to protect against respiratory disease, mice were inoculated i.v. with either a heat-killed or a live suspension of mycoplasmas estimated to contain 2 x 105 c.c.u. These animals were challenged i.n. 3 weeks later with 5 x 104 c.c.u. of viable M. pulmonis organisms and they were examined 14 days after challenge. As seen in Table 1, the numbers of mycoplasmas isolated from the respiratory tract, and the severity of lung lesions, after i.n. challenge, were no different in mice that had received dead organisms i.v. than in control mice that had been inoculated i.v. with mycoplasma medium. In contrast, the numbers of organisms isolated from the respiratory tract of mice that had received the live vaccine were significantly less than those isolated from control animals receiving medium (P0 2 0-1-02 0-02-0 05 0 001-001
* Probability that the lesion score of passively immunized mice is significantly different from that of controls (Student's t-test). t Lesion score+ s.d. $ Proportion of mice with lung lesions.
615
Mycoplasma-induced pneumonia in mice 10(D
Table 4. Effect of passively transferred serum on the isolation of mycoplasmas from the respiratory tract of mice inoculated intranasally with 5 x 103 c.c.u. of M. pulmonis 14 days previously
0
94
84
Treatment
c
0
6(
Number of organisms isolated from:
Pharynx
Lung
4-2+ 1-1* (5/5)t 50 (1/4)
5 8+ 2 3 (5/5) 2-0 (1/4)
or
0y
Untreated 'Immune' serum:
44
* Geometric mean of number of organisms isolated, expressed as log10 c.c.u./ml+s.d. t Proportion of mice from which M. pulmonis was isolated. t 'Immune' serum obtained from mice inoculated intravenously 21 days previously with M. pulmonis.
24 KI 10 20 30 40 Time (days) offer infection of donor mice
Figure 1. Effect of passively transferred 'imnnune' serum (0) and 'immune' cells (0) on the development c)f pneumonia in mice 14 days after intranasal inoculation of 5 x i05 c.c.u. of M. pulmonis.
isolated from control animals (P = 0-002-0 01; Student's f-test). The large challenge dose of 5 x 10O C.C.U. of M. pulmonis may have overwhelmed the ability of the transferred 'immune' serum to eliminEate the myco_ _r _ .1_ _ tne Tneretore, plasmas from the respiratory tract. 1r'l-_ of lion the isolat organ'immune' serum on effect of isms after infection of mice with a smalHer number of mycoplasmas was investigated. Mice w4ere inoculated i.v. with 21 day convalescent-phase serum. These and untreated animals were challenjged i.n. with
9-0r 8-0 7-0 0t O 7E 6-0 9
0
E Eo
= - =
5-0 4-0
I
W Wo
L
c 0 D0 0
3-01_ 2-0F_ 1-0 10
20 30 Time (days)
40
50
Figure 2. Effect of passively transferred 'imr the persistence of M. pulmonis in the lungs of mice inoculated intranasally with 5 x iO c.c.u. of M. pulmon is (control mice (0); mice treated with 'immune' serum (0)). B
5 x 103 c.c.u. of M. pulmonis and examined 14 days later. As seen in Table 4, mycoplasmas were isolated from the respiratory tract of all five of the untreated mice but from only one out of four mice that had been treated with 'immune' serum. This difference was statistically significant at the 5 per cent level (Fisher's exact test).
Antibody response Passively transferred antibody to a particular antigen may suppress antibody production in recipient animals when they are challenged with the homologous antigen (Dixon, Jacot-Guillarmod and McConahey, 1967). To see if suppression of the antibody response occurred in passively immunized mice infected with M. pulmonis, two groups of mice were investigated. One group was inoculated i.v. with mouse serum which had a CF antibody titre of 1/64 and the other group with normal mouse serum which did not contain detectable CF antibody to M. pulmonis. The mice were inoculated i.n. 24 h later with 5 x 105 c.c.u. of M. pulmonis, killed at various intervals thereafter, and the titre of CF antibody to M. pulmonis in the sera determined. The log2 mean CF antibody titres for each group of mice were plotted as shown in Fig. 3. As there were only five mice in each group, the standard deviations of the mean CF antibody titres were usually quite large. Despite this, it is evident that the antibody response of the passively immunized mice was suppressed compared with that of mice treated with normal serum. The large standard deviation of the mean CF antibody titre of sera from the passively immunized group of mice examined 42 days after infection was
616
Geraldine Taylor & D. Taylor-Robinson
0
N4.0 40 -
|
._ u3-0
I
20
1-0 10
20 30 Time (days)
40
50
titre of 1/64, was absorbed twice with either M. pulmonis or M. capri. Absorption of the serum with M. pulmonis reduced the CF antibody titre to 1/8, whereas absorption with M. capri had no effect on the titre of the serum. Groups of mice were passively immunized with either 'normal' serum, 'immune' serum immune' serum absorbed with M. pulmonis or 'immune' serum absorbed with M. capri. These animals were challenged i.n. 24 h later with 5 x 105 c.c.u. of M. pulmonis and examined 14 days after infection. There were no differences in the numbers of mycoplasmas isolated from the respiratory tract of any of the groups of mice. However, all the animals that had been treated with 'normal' mouse serum developed lung lesions, whereas none of the mice that had received 'immune' serum or serum absorbed with M. pulmonis or M. capri developed lesions.
Figure 3. Effect of passively transferred 'immune' serum on the CF antibody response of mice inoculated intranasally with 5 x 10' c.c.u. of M. pulmonis (mice treated with 'normal' serum (0); mice treated with 'immune' serum (0)).
due mainly to one animal. This mouse had developed lung lesions and had a serum CF antibody titre of 1/64, whereas the serum CF antibody titres of the other four mice in the group, which did not have lung lesions, were 1/4 or 1/8. As seen in Table 5, suppression of the antibody response was also observed in those mice that were shown previously (Table 3 and Fig. 1) to be protected by 'immune' serum. Table 5. Complement-fixing (CF) antibody response of passively immunized mice examined 14 days after challenge with 5 x 105 c.c.u. of M. pulmonis
Donor sera*
CF antibody titre of recipient mouse sera (log2)
Normal 4 day convalescent 9 day convalescent 21 day convalescent 35 day convalescent *
4-0 40 10
1-0 15
Details of donor sera are presented in Table 3.
DISCUSSION
The ability of transferred cells and serum to confer resistance to M. pulmonis-induced respiratory disease in mice was examined in order to evaluate the factors involved in the resistance of these animals to reinfection by mycoplasmas. Mice that had been inoculated i.n. with M. pulmonis still had lung lesions 9 weeks later (G. Taylor, personal observation) so that it was impractical to use these animals to determine whether they were resistant to disease following a subsequent i.n. inoculation. Therefore, the ability of parenterally inoculated M. pulmonis organisms to confer resistance against i.n. challenge was examined. The failure of heat-inactivated organisms to induce significant protection may have been due to inoculation of insufficient numbers. However, i.v. inoculation of viable organisms not only prevented mice from developing lung lesions but also suppressed the growth of mycoplasmas in the lungs. These results are in agreement with those of Cassell, Lindsey, Overcash and Baker (1973) who reported that a single i.v. inoculation of viable organisms prevented mice from developing pneumonia. These animals were therefore used in our experiments as donors of 'immune' cells and
Absorption of 'immune' serum
serum.
Sera were obtained from mice 21 days after i.v. inoculation of 2 x 106 c.c.u. of M. pulmonis and pooled. The pooled serum, which had a CF antibody
The failure of spleen cells from immune mice to protect recipient animals against M. pulmonisinduced respiratory disease suggests that cellmediated immunity may not play an important part
Mycoplasma-induced pneumonia in mice in resistance to reinfection. However, passively transferred spleen cells may be unable to confer immunity to a respiratory disease. Thus, Truit and Mackaness (1971) were unable to protect mice against respiratory disease caused by i.n. inoculation of Listeria monocytogenes by transferring spleen cells from animals known to be resistant to the disease. On the other hand, such spleen cells protected mice from disease induced by i.v. inoculation of the Listeria. In contrast to the inability of cells to transfer resistance to mycoplasma respiratory disease, 'immune' serum prevented pneumonia induced by i.n. inoculation of M. pulmonis. In addition, the 'immune' serum enhanced the clearance of mycoplasmas from the respiratory tract of mice challenged with a small number of organisms. However, it did not decrease the number of mycoplasmas in the lungs after challenge with a large number of organisms, although it prevented the mice from developing pneumonia. The question is, how does passively transferred antibody prevent pneumonia despite the presence of large numbers of organisms in the respiratory tract? The pneumonia appears to be the result of the animal's immune response to the mycoplasma, since many of the lymphocytes in the peribronchiolar and perivascular areas of the lung are known to be producing immunoglobulin (Cassell, Lindsey and Baker, 1974). Furthermore, the immune response to an antigen can be suppressed by passively transferred specific antibody (Dixon et al., 1967). It is thought that this antibody competes with receptors on the lymphocytes for antigen, so that the cells are not stimulated to produce immunoglobulin. Gois et al. (1974) observed that the titre of metabolisminhibiting antibody in the sera of pigs treated with 'immune' sera prior to i.n. challenge with M. hyorhinis was suppressed in comparison with the response of control animals. If this suppression is mediated by failure of lymphocytes to be stimulated by the mycoplasma, then the transferred antibody may also be expected to prevent the accumulation of sensitized lymphocytes in the peribronchiolar and perivascular areas of the lung. In other words, pneumonia may be suppressed even in the presence of mycoplasmas. The failure of some workers to confer on animals immunity to mycoplasma respiratory infections by transferring 'immune' serum to them may be because they used serum containing low affinity antibody. The higher the affinity of the transferred antibody, the more effective it is in
617
suppressing the immune response (Walker and Siskind, 1968). The techniques that have been used to measure serum antibodies, such as CF, metabolism inhibition and indirect haemagglutination, may not detect the antibodies that promote phagocytosis of mycoplasmas or that prevent sensitization of lymphocytes. This may account for the apparent lack of correlation that has been noted to exist between serum antibodies and resistance to reinfection with respiratory mycoplasmas. Furthermore, the surprising observation that 'immune' serum still protected mice after CF antibodies had been absorbed from it by mycoplasma organisms, suggests that antibodies other than those measured by the CF technique are involved in resistance to disease. However, there are other explanations for this finding. It may be that the protective capacity of the absorbed 'immune' serum had been reduced and that it is necessary to test not just undiluted serum but serial dilutions of it in mice in order to detect the reduction. It is also possible that protective antibody is directed not against the mycoplasma itself but against some product of the mycoplasma that is involved in the development of pneumonia. Since serum antibody is unlikely to be present in the respiratory tract of animals that have recovered from a mycoplasma infection, other factors are probably involved in resistance to reinfection. Thus, local secretory antibody (IgA) may prevent the attachment of mycoplasmas to epithelial cells of the respiratory tract and this may be the first defence mechanism. In fact, Brunner, Greenberg, James, Horswood, Couch and Chanock (1973) found a correlation between the presence of specific IgA in human bronchial secretions and resistance to M. pneumoniae infection. If this initial defence of previously infected animals is overwhelmed by i.n. challenge, the mycoplasmas may stimulate a local inflammatory reaction with consequent transudation of serum immunoglobulins into the respiratory tract. There is evidence that clearance of mycoplasmas from the respiratory tract is mediated by immune phagocytosis (Clyde, 1971; Cassell et al., 1974). Since IgA does not appear to be opsonic (Knop and Rowley, 1974), the outpouring of serum antibodies may be important not only in suppressing the development of pneumonia, as previously described, but also in clearing mycoplasmas from the respiratory tract. All these observations suggest that humoral immune mechanisms are relatively more important
618
Geraldine Taylor & D. Taylor-Robinson
than cell-mediated ones in resistance to mycoplasma respiratory disease.
REFERENCES BRUNNER H., GREENBERG H.B., JAMES W.D., HORSWOOD R.L., COUCH R.B. & CHANOCK R.M. (1973) Antibody to Mycoplasma pneumoniae in nasal secretions and sputa of experimentally infected human volunteers. Infect. Immun. 8, 612. CASSELL G.H., LINDSEY J.R. & BAKER H.J. (1974) Immune response of pathogen-free mice inoculated intranasally with Mycoplasma pulmonis. J. Immunol. 112, 124. CASSELL G.H., LINDSEY J.R., OVERCASH R.G. & BAKER H.J. (1973) Murine mycoplasma respiratory disease. Ann. N. Y. Acad. Sci. 225, 395. CLYDE W.A. (1971) Immunopathology of experimental Mycoplasma pneumoniae disease. Infect. Immun. 4, 757. DAVIES G. & HUDSON J.R. (1968) The relationship between growth-inhibition and immunity in contagious bovine pleuropneumonia. Vet. Rec. 83, 256. DENNY F.W., TAYLOR-ROBINSON D. & ALLISON A.C. (1972) The r6le of thymus-dependent immunity in Mycoplasma pulmonis infections of mice. J. med. Microbiol. 5, 327. DIXON F.J., JACOT-GUILLARMOD H. & MCCONAHEY P.J. (1967) The effect of passively administered antibody on antibody synthesis. J. exp. Med. 125, 1119. FERNALD G.W. & CLYDE W.A. (1970) Protective effect of vaccines in experimental Mycoplasma pneumoniae disease. Infect. Immun. 1, 559. GOIA M., KUKSA F. & FRANZ J. (1974) Influence of intraperitoneal administration of hyperimmune pig serum, IgG and IgM on the development of infection in gnotobiotic piglets infected intranasally with Mycoplasma hyorhinis. Zbl. Vet. Med. 21, 176. GOODWIN R.F.W., HODGSON R.G., WHITTLESTONE P. & WOODHAMS R.L. (1969) Immunity in experimentally induced enzootic pneumonia of pigs. J. Hyg. (Camb.), 67, 193. GOURLAY R.N. & SHIFRINE M. (1966) Passive transfer of immunity and formation of lung lesions in cattle following intravenous inoculation of antibody and Mycoplasma mycoides. Bull. epizoot. Dis. Afr. 14, 369.
KNOP J.G. & ROWLEY D. (1974) The antibacterial efficiencies of ovine IgA, IgM and IgG. J. infect. Dis. 130, 368. LAM K.M. & SWITZER W.P. (1971) Mycoplasmal pneumonia of swine: active and passive immunization. Amer. J. vet. Res. 32, 1737. LINDSEY J.R. & CASSELL G.H. (1973) Experimental Mycoplasma pulmonis infection in pathogen-free mice. Amer. J. Path. 72, 63. LLOYD L.C. (1967) An attempt to transfer immunity to Mycoplasma mycoides infection with serum. Bull. epizoot. Dis. Afr. 15, 11. LLOYD L.C. & TRETHEWIE E.R. (1970) Contagious bovine pleuropneumonia. The role of mycoplasmas and L-forms of bacteria in disease (ed. by J. T. Sharp), p. 172. C. C. Thomas, Springfield, Illinois. MACKANESS G.B. (1969) The influence of immunologically committed lymphoid cells on macrophage activity in vivo. J. exp. Med. 129, 973. NOCARD E. & LECLAINCHE E. (1903) In: Les Maladies Microbiennes des Animaux, 3rd edn, volume 1. Masson et Cie, Paris. TAYLOR G. & TAYLOR-ROBINSON D. (1975) The part played by cell-mediated immunity in mycoplasma respiratory infections. In: International Symposium on Immunity to Infections of the Respiratory System in Man and Animals, London, 1974. Develop. biol. St., volume 28, p. 195. Karger, Basel. TAYLOR G., TAYLOR-ROBINSON D. & FERNALD G.W. (1974) Reduction in the severity of Mycoplasma pneumoniaeinduced pneumonia in hamsters by immunosuppressive treatment with anti-thymocyte serum. J. med. Microbiol. 7, 343. TAYLOR-ROBINSON D. (1975) The importance of mycoplasmas in respiratory infections. International Symposium on Immunity to Infections of the Respiratory System in Man and Animals, London, 1974. Develop. biol. St., volume 28, p. 86. Karger, Basel. TRUITT C.L. & MACKANESS G.B. (1971) Cell-mediated resistance to aerogenic infection of the lung. Amer. Rev. resp. Dis. 104, 829. WALKER J.G. & SISKIND G.W. (1968) Studies on the control of antibody synthesis. Effect of antibody affinity upon its ability to suppress antibody formation. Immunology, 14, 21.
Effects of active and passive immunization on Mycoplasma pulmonis-induced pneumonia in mice
GERALDINE TAYLOR* & D. TAYLOR-ROBINSON Division of Communicable Diseases, MRC Clinical Research Centre, Watford Road, Harrow, Middlesex
Received 9 October 1975; acceptedfor publication 30 October 1975
Summary. Parenteral immunization of C3H mice with viable Mycoplasma pulmonis organisms protected them from pneumonia induced by intranasal inoculation of these organisms. Spleen cells obtained from immunized mice were ineffective in preventing syngeneic recipients from developing respiratory disease. In contrast, convalescent-phase serum enhanced the clearance of mycoplasmas from the respiratory tract of mice challenged with a small number of organisms. Further, although 'immune' serum had no detectable effect on the number of mycoplasmas in the respiratory tract of mice challenged with a large number of organisms, such animals did not develop pneumonia. Since the pneumonia appears to be the result of the host's immune response to the mycoplasma, it is suggested that the transferred 'immune' serum may act by suppressing the immune response so that mice develop less severe lung lesions. This suggestion is supported by the observation that the complement-fixing antibody response of passively immunized mice was suppressed. * Present address: Agricultural Research Council, Institute
INTRODUCTION
Although mycoplasmas are an important cause of respiratory disease in a number of animal species (Taylor-Robinson, 1975), there are no fully effective vaccines to control these infections. In order to develop suitable vaccines it is necessary to understand the mechanisms involved in resistance to reinfection. Little is known of the parts played by serum antibody, cell-mediated immunity and local secretory antibody in resistance. However, examination of the ability of passively transferred antibody and of sensitized cells to confer immunity on syngeneic recipient animals may help to determine their relative importance. Investigations of this kind have been undertaken. Thus, Nocard and coworkers in 1899 (quoted by Nocard and Leclainche, 1903) and Gourlay and Shifrine (1966) demonstrated that transfer of convalescent-phase serum to cattle protected them against the effects of subcutaneous challenge with a virulent strain of Mycoplasma mycoides var. mycoides. On the other hand, Lloyd (1967) found that serum from cattle that were resistant to challenge with M. mycoides var. mycoides, did not protect recipient animals when given in amounts much greater than used by Nocard and co-workers. Despite these conflicting results, other
for Research on Animal Diseases, Compton, near Newbury, Berkshire. Correspondence: Dr D. Taylor-Robinson, Division of Communicable Diseases, MRC Clinical Research Centre, Watford Road, Harrow, Middlesex HAl 3UJ.
611
612
Geraldine Taylor & D. Taylor-Robinson
reports of the successful transfer of immunity to various mycoplasma respiratory diseases by administration of serum (Lam and Switzer, 1971; Goig, Kuksa and Franz, 1974), suggest that resistance to these diseases is mediated by serum antibody. However, inactivated mycoplasma vaccines, which stimulate high levels of serum antibody, do not always provide protection against intranasal challenge with live organisms (Goodwin, Hodgson, Whittlestone and Woodhams, 1969; Fernald and Clyde, 1970) and there is further evidence that there is often a poor correlation between serum antibodies and resistance to reinfection (Davies and Hudson, 1968; Goodwin et al., 1969; Lam and Switzer, 1971). These apparent discrepancies prompted investigations into the role of cell-mediated immune mechanisms in resistance to mycoplasma infections. There is evidence that thymus-dependent lymphocytes become sensitized during a mycoplasma respiratory infection (see Taylor and TaylorRobinson, 1975) and these cells appear to be involved in the development of the pneumonic lesions (Denny, Taylor-Robinson and Allison, 1972; Taylor, Taylor-Robinson and Fernald, 1974). However, there have been few reports of attempts to transfer immunity to mycoplasmal pneumonia by administration of cells. Lloyd (quoted by Lloyd and Trethewie, 1970) took lymph node cells from a calf resistant to M. mycoides var. mycoides and transferred them to its fully susceptible identical twin. The only indication that there had been a transfer of immunity to the recipient calf was a delay in its serological response to the mycoplasma after challenge compared with that of untreated, infected animals. In an attempt to understand more fully the relative importance of serum antibody and of cells in resistance to mycoplasma respiratory disease we have examined the effects of passive immunization on M. pulmonis-induced pneumonia in mice. M. pulmonis infection of mice appears to be a good model for many of the other respiratory mycoplasma infections (Lindsey and Cassell, 1973) and use of an inbred strain of mouse permits the transfer of cells between animals.
Ltd, and by the Animal Division of the Clinical Research Centre, Harrow. All mice were of the M.R.C. Laboratory Animal Centre's category 4(****) standard and were maintained in isolation throughout the experimental period. Mice were anaesthetized by intraperitoneal (i.p.) inoculation of 0-06 mg/g body weight of sodium pentobarbitone and were infected by intranasal (i.n.) inoculation of 0 05 ml of an appropriate dilution of an M. pulmonis stock culture. Uninfected control mice were inoculated i.n. in the same way with 0*05 ml of mycoplasma medium.
MATERIALS AND METHODS
Vaccination procedures Mice were actively immunized by intravenous (i.v.) inoculation of either a live or a heat-inactivated (560 for 1 h) suspension of M. pulmonis which contained 2 x 101 c.c.u. in 0-2 ml. As controls, mice
Mice Male, C3H mice weighing 20-30 g were supplied by Scientific Agribusiness Consultants (International)
Mycoplasma medium and strains Liquid medium for the isolation and growth of mycoplasmas has been described previously (Denny et al., 1972). Mice were infected with the 'JB' strain of M. pulmonis which was obtained from Dr J. Tully (National Institutes of Health, Bethesda); the strain was grown in medium free of thallium acetate and a fifth broth passage from that originally received was used for inoculation. The strain of M. mycoides var. capri (hereafter referred to as M. capri) was obtained from Dr G. R. Smith (Nuffield Institute, Regent's Park, London). Mycoplasma isolation, titration and identification These procedures were the same as those described previously by Denny et al. (1972). Numbers of organisms are expressed as colour-changing units
(c.c.u.). Complement-fixation (CF) test The M. pulmonis antibody titre of mouse sera was determined by a micro-technique. A washed, 300 mg pellet of M. pulmonis 'JB', resuspended in 6 ml of phosphate-buffered saline (PBS), was heated at 560 for 1 h and stored at - 700. The optimum dilution of this antigen in the CF test was 1/50. Serial dilutions of heat-inactivated (560 for 30 min) mouse sera were mixed with 3 HC5o of complement (Wellcome Reagents Ltd) and fixation was carried out at 40 overnight. The antibody titre was recorded as the dilution of serum at which there was approximately 50 per cent lysis of red cells.
Mycoplasma-induced pneumonia in mice inoculated medium.
were
i.v.
613
RESULTS
with 02 ml of mycoplasma
M. pulmonis infection in actively immunized mice Passive immunization 'Immune' serum was obtained from mice which had been inoculated i.v. 3 weeks previously with 2 x 106 c.c.u. of M. pulmonis. Spleen cell suspensions were prepared from these animals by gently teasing chopped pieces of spleen through a 100-gauge stainless steel sieve into 199 medium (Gibco Biocult). Cell aggregates were allowed to sediment for 5 min and the supernatant cell suspension was gently forced through a series of needles of graded size so that a single cell suspension was produced. The cells were sedimented at 300 g for 10 min, washed twice with 199 medium and resuspended to give approximately 108 viable nucleated cells/ml. The ratio of donor to recipient animals was 2: 1. Recipient mice were inoculated i.v. with either 108 cells or with 0 4 ml of serum and challenged 24 h later with 5 x 105 c.c.u. of M. pulmonis inoculated i.n. Animals treated in a similar way with cells or serum obtained from mice that had been inoculated i.v. with mycoplasma medium served as controls. Mice were examined 14 days after i.n. challenge for mycoplasmas in the respiratory tract and for lung lesions.
Absorption of serum M. pulmonis or M. capri organisms used to absorb serum were grown in 600 ml of medium for 5 days, centrifuged at 27,000 g for 20 min and the pellets washed three times with PBS. Serum was absorbed twice by mixing 300 mg of the pellet with 4 ml of serum at 4° and occasionally shaking for 24 h. After absorption, the mycoplasmas were separated from the serum by centrifugation.
To investigate the ability of parenterally inoculated organisms to protect against respiratory disease, mice were inoculated i.v. with either a heat-killed or a live suspension of mycoplasmas estimated to contain 2 x 105 c.c.u. These animals were challenged i.n. 3 weeks later with 5 x 104 c.c.u. of viable M. pulmonis organisms and they were examined 14 days after challenge. As seen in Table 1, the numbers of mycoplasmas isolated from the respiratory tract, and the severity of lung lesions, after i.n. challenge, were no different in mice that had received dead organisms i.v. than in control mice that had been inoculated i.v. with mycoplasma medium. In contrast, the numbers of organisms isolated from the respiratory tract of mice that had received the live vaccine were significantly less than those isolated from control animals receiving medium (P0 2 0-1-02 0-02-0 05 0 001-001
* Probability that the lesion score of passively immunized mice is significantly different from that of controls (Student's t-test). t Lesion score+ s.d. $ Proportion of mice with lung lesions.
615
Mycoplasma-induced pneumonia in mice 10(D
Table 4. Effect of passively transferred serum on the isolation of mycoplasmas from the respiratory tract of mice inoculated intranasally with 5 x 103 c.c.u. of M. pulmonis 14 days previously
0
94
84
Treatment
c
0
6(
Number of organisms isolated from:
Pharynx
Lung
4-2+ 1-1* (5/5)t 50 (1/4)
5 8+ 2 3 (5/5) 2-0 (1/4)
or
0y
Untreated 'Immune' serum:
44
* Geometric mean of number of organisms isolated, expressed as log10 c.c.u./ml+s.d. t Proportion of mice from which M. pulmonis was isolated. t 'Immune' serum obtained from mice inoculated intravenously 21 days previously with M. pulmonis.
24 KI 10 20 30 40 Time (days) offer infection of donor mice
Figure 1. Effect of passively transferred 'imnnune' serum (0) and 'immune' cells (0) on the development c)f pneumonia in mice 14 days after intranasal inoculation of 5 x i05 c.c.u. of M. pulmonis.
isolated from control animals (P = 0-002-0 01; Student's f-test). The large challenge dose of 5 x 10O C.C.U. of M. pulmonis may have overwhelmed the ability of the transferred 'immune' serum to eliminEate the myco_ _r _ .1_ _ tne Tneretore, plasmas from the respiratory tract. 1r'l-_ of lion the isolat organ'immune' serum on effect of isms after infection of mice with a smalHer number of mycoplasmas was investigated. Mice w4ere inoculated i.v. with 21 day convalescent-phase serum. These and untreated animals were challenjged i.n. with
9-0r 8-0 7-0 0t O 7E 6-0 9
0
E Eo
= - =
5-0 4-0
I
W Wo
L
c 0 D0 0
3-01_ 2-0F_ 1-0 10
20 30 Time (days)
40
50
Figure 2. Effect of passively transferred 'imr the persistence of M. pulmonis in the lungs of mice inoculated intranasally with 5 x iO c.c.u. of M. pulmon is (control mice (0); mice treated with 'immune' serum (0)). B
5 x 103 c.c.u. of M. pulmonis and examined 14 days later. As seen in Table 4, mycoplasmas were isolated from the respiratory tract of all five of the untreated mice but from only one out of four mice that had been treated with 'immune' serum. This difference was statistically significant at the 5 per cent level (Fisher's exact test).
Antibody response Passively transferred antibody to a particular antigen may suppress antibody production in recipient animals when they are challenged with the homologous antigen (Dixon, Jacot-Guillarmod and McConahey, 1967). To see if suppression of the antibody response occurred in passively immunized mice infected with M. pulmonis, two groups of mice were investigated. One group was inoculated i.v. with mouse serum which had a CF antibody titre of 1/64 and the other group with normal mouse serum which did not contain detectable CF antibody to M. pulmonis. The mice were inoculated i.n. 24 h later with 5 x 105 c.c.u. of M. pulmonis, killed at various intervals thereafter, and the titre of CF antibody to M. pulmonis in the sera determined. The log2 mean CF antibody titres for each group of mice were plotted as shown in Fig. 3. As there were only five mice in each group, the standard deviations of the mean CF antibody titres were usually quite large. Despite this, it is evident that the antibody response of the passively immunized mice was suppressed compared with that of mice treated with normal serum. The large standard deviation of the mean CF antibody titre of sera from the passively immunized group of mice examined 42 days after infection was
616
Geraldine Taylor & D. Taylor-Robinson
0
N4.0 40 -
|
._ u3-0
I
20
1-0 10
20 30 Time (days)
40
50
titre of 1/64, was absorbed twice with either M. pulmonis or M. capri. Absorption of the serum with M. pulmonis reduced the CF antibody titre to 1/8, whereas absorption with M. capri had no effect on the titre of the serum. Groups of mice were passively immunized with either 'normal' serum, 'immune' serum immune' serum absorbed with M. pulmonis or 'immune' serum absorbed with M. capri. These animals were challenged i.n. 24 h later with 5 x 105 c.c.u. of M. pulmonis and examined 14 days after infection. There were no differences in the numbers of mycoplasmas isolated from the respiratory tract of any of the groups of mice. However, all the animals that had been treated with 'normal' mouse serum developed lung lesions, whereas none of the mice that had received 'immune' serum or serum absorbed with M. pulmonis or M. capri developed lesions.
Figure 3. Effect of passively transferred 'immune' serum on the CF antibody response of mice inoculated intranasally with 5 x 10' c.c.u. of M. pulmonis (mice treated with 'normal' serum (0); mice treated with 'immune' serum (0)).
due mainly to one animal. This mouse had developed lung lesions and had a serum CF antibody titre of 1/64, whereas the serum CF antibody titres of the other four mice in the group, which did not have lung lesions, were 1/4 or 1/8. As seen in Table 5, suppression of the antibody response was also observed in those mice that were shown previously (Table 3 and Fig. 1) to be protected by 'immune' serum. Table 5. Complement-fixing (CF) antibody response of passively immunized mice examined 14 days after challenge with 5 x 105 c.c.u. of M. pulmonis
Donor sera*
CF antibody titre of recipient mouse sera (log2)
Normal 4 day convalescent 9 day convalescent 21 day convalescent 35 day convalescent *
4-0 40 10
1-0 15
Details of donor sera are presented in Table 3.
DISCUSSION
The ability of transferred cells and serum to confer resistance to M. pulmonis-induced respiratory disease in mice was examined in order to evaluate the factors involved in the resistance of these animals to reinfection by mycoplasmas. Mice that had been inoculated i.n. with M. pulmonis still had lung lesions 9 weeks later (G. Taylor, personal observation) so that it was impractical to use these animals to determine whether they were resistant to disease following a subsequent i.n. inoculation. Therefore, the ability of parenterally inoculated M. pulmonis organisms to confer resistance against i.n. challenge was examined. The failure of heat-inactivated organisms to induce significant protection may have been due to inoculation of insufficient numbers. However, i.v. inoculation of viable organisms not only prevented mice from developing lung lesions but also suppressed the growth of mycoplasmas in the lungs. These results are in agreement with those of Cassell, Lindsey, Overcash and Baker (1973) who reported that a single i.v. inoculation of viable organisms prevented mice from developing pneumonia. These animals were therefore used in our experiments as donors of 'immune' cells and
Absorption of 'immune' serum
serum.
Sera were obtained from mice 21 days after i.v. inoculation of 2 x 106 c.c.u. of M. pulmonis and pooled. The pooled serum, which had a CF antibody
The failure of spleen cells from immune mice to protect recipient animals against M. pulmonisinduced respiratory disease suggests that cellmediated immunity may not play an important part
Mycoplasma-induced pneumonia in mice in resistance to reinfection. However, passively transferred spleen cells may be unable to confer immunity to a respiratory disease. Thus, Truit and Mackaness (1971) were unable to protect mice against respiratory disease caused by i.n. inoculation of Listeria monocytogenes by transferring spleen cells from animals known to be resistant to the disease. On the other hand, such spleen cells protected mice from disease induced by i.v. inoculation of the Listeria. In contrast to the inability of cells to transfer resistance to mycoplasma respiratory disease, 'immune' serum prevented pneumonia induced by i.n. inoculation of M. pulmonis. In addition, the 'immune' serum enhanced the clearance of mycoplasmas from the respiratory tract of mice challenged with a small number of organisms. However, it did not decrease the number of mycoplasmas in the lungs after challenge with a large number of organisms, although it prevented the mice from developing pneumonia. The question is, how does passively transferred antibody prevent pneumonia despite the presence of large numbers of organisms in the respiratory tract? The pneumonia appears to be the result of the animal's immune response to the mycoplasma, since many of the lymphocytes in the peribronchiolar and perivascular areas of the lung are known to be producing immunoglobulin (Cassell, Lindsey and Baker, 1974). Furthermore, the immune response to an antigen can be suppressed by passively transferred specific antibody (Dixon et al., 1967). It is thought that this antibody competes with receptors on the lymphocytes for antigen, so that the cells are not stimulated to produce immunoglobulin. Gois et al. (1974) observed that the titre of metabolisminhibiting antibody in the sera of pigs treated with 'immune' sera prior to i.n. challenge with M. hyorhinis was suppressed in comparison with the response of control animals. If this suppression is mediated by failure of lymphocytes to be stimulated by the mycoplasma, then the transferred antibody may also be expected to prevent the accumulation of sensitized lymphocytes in the peribronchiolar and perivascular areas of the lung. In other words, pneumonia may be suppressed even in the presence of mycoplasmas. The failure of some workers to confer on animals immunity to mycoplasma respiratory infections by transferring 'immune' serum to them may be because they used serum containing low affinity antibody. The higher the affinity of the transferred antibody, the more effective it is in
617
suppressing the immune response (Walker and Siskind, 1968). The techniques that have been used to measure serum antibodies, such as CF, metabolism inhibition and indirect haemagglutination, may not detect the antibodies that promote phagocytosis of mycoplasmas or that prevent sensitization of lymphocytes. This may account for the apparent lack of correlation that has been noted to exist between serum antibodies and resistance to reinfection with respiratory mycoplasmas. Furthermore, the surprising observation that 'immune' serum still protected mice after CF antibodies had been absorbed from it by mycoplasma organisms, suggests that antibodies other than those measured by the CF technique are involved in resistance to disease. However, there are other explanations for this finding. It may be that the protective capacity of the absorbed 'immune' serum had been reduced and that it is necessary to test not just undiluted serum but serial dilutions of it in mice in order to detect the reduction. It is also possible that protective antibody is directed not against the mycoplasma itself but against some product of the mycoplasma that is involved in the development of pneumonia. Since serum antibody is unlikely to be present in the respiratory tract of animals that have recovered from a mycoplasma infection, other factors are probably involved in resistance to reinfection. Thus, local secretory antibody (IgA) may prevent the attachment of mycoplasmas to epithelial cells of the respiratory tract and this may be the first defence mechanism. In fact, Brunner, Greenberg, James, Horswood, Couch and Chanock (1973) found a correlation between the presence of specific IgA in human bronchial secretions and resistance to M. pneumoniae infection. If this initial defence of previously infected animals is overwhelmed by i.n. challenge, the mycoplasmas may stimulate a local inflammatory reaction with consequent transudation of serum immunoglobulins into the respiratory tract. There is evidence that clearance of mycoplasmas from the respiratory tract is mediated by immune phagocytosis (Clyde, 1971; Cassell et al., 1974). Since IgA does not appear to be opsonic (Knop and Rowley, 1974), the outpouring of serum antibodies may be important not only in suppressing the development of pneumonia, as previously described, but also in clearing mycoplasmas from the respiratory tract. All these observations suggest that humoral immune mechanisms are relatively more important
618
Geraldine Taylor & D. Taylor-Robinson
than cell-mediated ones in resistance to mycoplasma respiratory disease.
REFERENCES BRUNNER H., GREENBERG H.B., JAMES W.D., HORSWOOD R.L., COUCH R.B. & CHANOCK R.M. (1973) Antibody to Mycoplasma pneumoniae in nasal secretions and sputa of experimentally infected human volunteers. Infect. Immun. 8, 612. CASSELL G.H., LINDSEY J.R. & BAKER H.J. (1974) Immune response of pathogen-free mice inoculated intranasally with Mycoplasma pulmonis. J. Immunol. 112, 124. CASSELL G.H., LINDSEY J.R., OVERCASH R.G. & BAKER H.J. (1973) Murine mycoplasma respiratory disease. Ann. N. Y. Acad. Sci. 225, 395. CLYDE W.A. (1971) Immunopathology of experimental Mycoplasma pneumoniae disease. Infect. Immun. 4, 757. DAVIES G. & HUDSON J.R. (1968) The relationship between growth-inhibition and immunity in contagious bovine pleuropneumonia. Vet. Rec. 83, 256. DENNY F.W., TAYLOR-ROBINSON D. & ALLISON A.C. (1972) The r6le of thymus-dependent immunity in Mycoplasma pulmonis infections of mice. J. med. Microbiol. 5, 327. DIXON F.J., JACOT-GUILLARMOD H. & MCCONAHEY P.J. (1967) The effect of passively administered antibody on antibody synthesis. J. exp. Med. 125, 1119. FERNALD G.W. & CLYDE W.A. (1970) Protective effect of vaccines in experimental Mycoplasma pneumoniae disease. Infect. Immun. 1, 559. GOIA M., KUKSA F. & FRANZ J. (1974) Influence of intraperitoneal administration of hyperimmune pig serum, IgG and IgM on the development of infection in gnotobiotic piglets infected intranasally with Mycoplasma hyorhinis. Zbl. Vet. Med. 21, 176. GOODWIN R.F.W., HODGSON R.G., WHITTLESTONE P. & WOODHAMS R.L. (1969) Immunity in experimentally induced enzootic pneumonia of pigs. J. Hyg. (Camb.), 67, 193. GOURLAY R.N. & SHIFRINE M. (1966) Passive transfer of immunity and formation of lung lesions in cattle following intravenous inoculation of antibody and Mycoplasma mycoides. Bull. epizoot. Dis. Afr. 14, 369.
KNOP J.G. & ROWLEY D. (1974) The antibacterial efficiencies of ovine IgA, IgM and IgG. J. infect. Dis. 130, 368. LAM K.M. & SWITZER W.P. (1971) Mycoplasmal pneumonia of swine: active and passive immunization. Amer. J. vet. Res. 32, 1737. LINDSEY J.R. & CASSELL G.H. (1973) Experimental Mycoplasma pulmonis infection in pathogen-free mice. Amer. J. Path. 72, 63. LLOYD L.C. (1967) An attempt to transfer immunity to Mycoplasma mycoides infection with serum. Bull. epizoot. Dis. Afr. 15, 11. LLOYD L.C. & TRETHEWIE E.R. (1970) Contagious bovine pleuropneumonia. The role of mycoplasmas and L-forms of bacteria in disease (ed. by J. T. Sharp), p. 172. C. C. Thomas, Springfield, Illinois. MACKANESS G.B. (1969) The influence of immunologically committed lymphoid cells on macrophage activity in vivo. J. exp. Med. 129, 973. NOCARD E. & LECLAINCHE E. (1903) In: Les Maladies Microbiennes des Animaux, 3rd edn, volume 1. Masson et Cie, Paris. TAYLOR G. & TAYLOR-ROBINSON D. (1975) The part played by cell-mediated immunity in mycoplasma respiratory infections. In: International Symposium on Immunity to Infections of the Respiratory System in Man and Animals, London, 1974. Develop. biol. St., volume 28, p. 195. Karger, Basel. TAYLOR G., TAYLOR-ROBINSON D. & FERNALD G.W. (1974) Reduction in the severity of Mycoplasma pneumoniaeinduced pneumonia in hamsters by immunosuppressive treatment with anti-thymocyte serum. J. med. Microbiol. 7, 343. TAYLOR-ROBINSON D. (1975) The importance of mycoplasmas in respiratory infections. International Symposium on Immunity to Infections of the Respiratory System in Man and Animals, London, 1974. Develop. biol. St., volume 28, p. 86. Karger, Basel. TRUITT C.L. & MACKANESS G.B. (1971) Cell-mediated resistance to aerogenic infection of the lung. Amer. Rev. resp. Dis. 104, 829. WALKER J.G. & SISKIND G.W. (1968) Studies on the control of antibody synthesis. Effect of antibody affinity upon its ability to suppress antibody formation. Immunology, 14, 21.
