REVIEWS OF INFECTIOUS DISEASES • VOL. 12, NO.2. MARCH-APRIL 1990 e 1990 by The University of Chicago. All rights reserved. 0162-0886/90/1202-0018$02.00

HISTORICAL ARTICLE Eaton Agent - Science and Scientific Acceptance: A Historical Commentary B. P. Marmion Truth is the child of time, not of authority. - Bertold Brecht, Life of Gallileo

From the early 1960s, the discovery by Monroe Eaton of the etiology of primary atypical pneumonia (PAP) [1-4] had an electrifying and catalytic effect on the mycoplasmology of human disease and provided justification for the perseverance of those lonely pioneers - E. Klieneberger-Nobel, L. Dienes, D.G. Edward, E.A. Freundt, and others - who defined the cell biology of the pleuropneumonia-like organisms (PPLO) or mycoplasmas and developed appropriate methods for culture, antigenic comparison, and serodiagnosis in their efforts to relate them to disease. Yetfor two decades after its appearance, Eaton's work was either dismissed as a misinterpretation of data, or, further from the scene, outside the United States, accepted but with the lingering reservations that can subvert assessment of experimental data on their own merits. Apart from the intrinsic scientific merit of the work, the sequence of events and attitudes from Eaton's initial discovery in the 1940sto its vindication and acceptance in the early 1960s is a fascinating study for those interested in the "anthropology" of

the scientific life and the process of acceptance of scientific discovery, particularly in relation to the question of why some scientific discoveries are accepted more or less immediately, while othersapparently as well documented - are the subject of prolonged and sometimes unhappy controversy before they finally win through. It is also of interest to note how such rites of passage are affected by the hierarchical status of the person or institution where the work is done or by the array of competing interests in the field. The story has been told, in part, on a number of occasions (for example see [5]). The context of the problem of PAP in the 1940sand the impetus for the studies by Eaton and colleagues are summarized in table 1. After its clinical delineation in the 1930s, PAP was reported in sporadic cases among adolescents and young adults in schools and military installations. In general, the syndrome was characterized by relatively mild symptoms and signs that were disproportionate to the degree of infiltration visible on chest roentgenograms. Consequently, a common terminology was "walking pneumonia" in military medical circles. The condition attracted the attention of many eminent infectious diseases physicians and microbiologists in the United States and Europe. PAP became a particularly pressing problem in the early 1940s, when, as America moved to join World War II, large numbers of young susceptibles weregathered in military camps and other institutions - PAP occurred with case numbers in the thousands and an average period of absence of patients from duties

This article is based on a presidential address given to the Australian Society for Microbiology in May 1986. The author gratefully acknowledges discussions with, the recollections of, and comments from Drs. Gordon Meiklejohn, Wallace Clyde, Floyd Denny, Robert Chanock, Edwin Lennette, Hans Hers, Ruth Lemke, and Philip Plackett. Unfortunately, the state of Dr. Monroe Eaton's health precluded a reviewof the commentary with its main figure (Dr. Eaton died in 1989). Please address requests for reprints to Professor B. P. Marmion, Box 14, Rundle Mall PO, Adelaide, South Australia 5000.

338

Downloaded from http://cid.oxfordjournals.org/ at UB Giessen on May 25, 2015

The three classical papers published in 1944and 1945by Monroe A. Eaton and colleagues deal with the etiology of primary atypical pneumonia (PAP) and with the properties of a filterable agent (subsequently and for a number of years known as Eaton agent) from the sputum or lung of patients with PAP using cotton rats, hamsters, and chick embryos as laboratory hosts. The present review is first and foremost a tribute to Monroe Eaton and his colleagues for their trail-blazing discovery of a major cause of the atypical pneumonia syndrome and their steadfast vision of its importance. The organism was finally identified and designated Mycoplasma pneumoniae some 20 years after their papers first appeared in the Journal of Experimental Medicine.

Eaton Agent

339

Table 1. Primary atypical pneumonia-the problem. During the 1930s a form of pneumonia differing from pneumococcal pneumonia in its resistance to sulphonamides (hence "atypical") was recognized by Gallagher, Kneeland, Longscope, Reiman, Stokes, Horsfall, Dingle, and other workers in the United States and elsewhere A proportion of patients developed cold hemagglutinins [6-8] In the early 1940s there were many cases in young adults in colleges and in military camps

Figure 1. Dr. Monroe Eaton, circa 1960.

demic Diseases in the Army. Eaton had worked at the Rockefeller Institute on malaria and influenza and had contact with Horsfall and Sabin but had moved out to start the Virus Research Laboratory at Berkeley. In those days, the Berkeley laboratory was small scale and more isolated than it is today. Its development as a major virus and rickettsial diagnostic facility, which followed Lennette's arrival in 1944,was yet to come, along with the start of commercial air flights that decreased isolation and linked the laboratory with other virologic and medical centers in the eastern United States and elsewhere. Initial Observations on PAP in Berkeley Eaton tackled the problem of PAP in a fairly straightforward fashion, probably conditioned by his previous experience with the isolation of influenza virus by the intranasal inoculation of animals such as ferrets and mice. Suspensions of sputum - and of lung from the occasional patient with a fatal case of PAP - were inoculated into a wide variety of animals by the intranasal route; control animals were

Downloaded from http://cid.oxfordjournals.org/ at UB Giessen on May 25, 2015

of 30 days. A further delineation of this form of PAP from a syndrome caused by other agents was made possible by the discovery [6, 7] in 1943 that the sera of patients recovering from the disease agglutinated erythrocytes at 4°C-the so-called cold hemagglutinin reaction. The reaction was exhaustively investigated by Maxwell Finland and colleagues, who defined many of the clinical and laboratory parameters in a series of papers published in the JournalojClinicalInvestigation, of which the first is referenced [8]. For the purpose of the present commentary, it may be noted that the Finland group found that 68.5% of 200 PAP patients developed cold hemagglutinins. During the prevalence of PAP in the 1940s, there were three contending groups for the prize of discovering the etiology of the condition. The Commission on Acute Respiratory Diseases was based at Fort Bragg, North Carolina, directed by John Dingle, and well supported by numerous workers who either were already well known or were to become so in American infectious diseases medicine. The experienced and prestigious group at the Hospital of the Rockefeller Institute for Medical Research was led by Horsfall and comprised such workers as Lewis Thomas, Mirick, Ziegler, Curnen, and others. Finally, by comparison a numerically meager group, the Virus Research Laboratory of the California State Public Health Department was headed by Dr. Monroe Eaton and had a clinical research fellow, first Dr. John Talbot, then Dr. Gordon Meiklejohn (subsequently to become Professor of Medicine at the University of Colorado), a science graduate, Mr. van Herick, and three technicians. Figure 1 shows a photograph of Dr. Eaton taken in the early 1960s, at the time when his work on the PAP agent was at or near acceptance. The Virus Research Laboratory had some support from the International Health Division of the Rockefeller Foundation and from the Board for the Investigation and Control of Influenza and other Epi-

Marmion

340

Table 2. Frequency and extent of pneumonic lesions produced by PAP sputum and lung specimens after intranasal inoculation into cotton rats under anesthetic.

Specimens PAP sputum PAP lung Sterile inocula NOTE.

No. of specimens causing pneumonia/total no. of specimens (%)

Percentage of group with pneumonia (mean [range])

38/128 (30) 3/15 (20) 7/136 (4.2)

(48 [12-100]) (46 [38-63]) NA

Table is adapted from [1]. NA = data not available.

hamsters ranging from 2 to 4 x 102 : Once a reliable suspension of the PAP agent was available from chick embryo, it was possible to do neutralization tests with acute- and convalescent-phase sera from patients and to prepare antisera to the agent in chick embryo lung in rabbits. A remarkable amount of meticulous work was executed with this laborious and cumbersome system and established [1-3] the following main points. (1) An agent present in 20070-30070 of sputum or lung samples from PAP patients produced focal areas of pneumonia but rarely complete consolidation when inoculated intranasally into cotton rats or hamsters. The lesion-producing agent in these samples could be neutralized by the addition of convalescent-phase serum from the PAP patients to the inoculum given to the animals. The agent could not be serially passaged in cotton rats or hamsters because of the activation of and overgrowth by endogenous viruses. (2) The agent could be isolated from clinical specimens by direct inoculation of the chick embryo amnion and could be passaged serially in the chick embryo amnion and lung, with subinoculation of suspensions into hamsters by the intranasal route as a detection system. Titers of 102-103 hamster infective doses or 104 chick embryo infective doses were obtained in the chick embryo lung, and titrations showed that a 25- to lOO-fold greater concentration of infected chick embryo lung was required to produce lesions in hamsters than was required to infect other chick embryos. The infectivity of chick embryo lung suspension for hamsters could be neutralized by convalescent-phase but not by acute-phase sera from PAP patients. Five isolates from PAP patients were shown to be antigenically similar or identical in hamster immunization/cross-challenge experiments. Rabbit antisera to various endogenous viruses of hamsters and cotton rats - including a virus related to pneumonia virus of mice - did not neutralize the agent, but antisera raised to the PAP agent in chick embryo lung neutralized the infectivity of chick embryo lung-amnion suspensions for hamsters or cotton rats. (3) The PAP agent was stable for long periods at -70°C and survived for a few hours in serum broth at 20°C. Filtration experiments with infected chick embryo suspension and collodion membranes showed that infectivity was retained by a membrane of an average pore diameter of 300 um, but passed at 366 and 400 urn, suggestinga particle size of 180-250

Downloaded from http://cid.oxfordjournals.org/ at UB Giessen on May 25, 2015

given sterile broth. As Gordon Meiklejohn recalls, "Everything that had fur and moved was pressed into service." Of the wide range of rodents used, western cotton rats and hamsters proved to be of most value. A proportion (f\J45 070) of the cotton rats developed small focal pneumonic lesions; examples of the general pattern with clinical specimens and some controls are given in table 11 of Eaton et al. [1]. An important point was that the degree of lung consolidation, although reproducible, was rarely complete and involving all lobes as with, for example, a well-adapted influenza virus and may have been a difficulty with the isolation system that prejudiced its acceptance. Table 2 (adapted from Eaton et al. [1]) shows that 30070 of PAP sputa and 20070 of lung suspensions produced pneumonia in experimental animals, compared with 4.2070 of inoculations of sterile broth (X2 2 = 28; P < .0001). Specimens collected within 9 days of onset weremore often positive than those collected in convalescence. It was not possible, however, to passage isolates serially in hamster or cotton rat lung as potency declined or there was overgrowth by endogenous viruses or bacteria. Faced with this difficulty, Eaton and his colleagues turned to the chick embryo as a laboratory host [2]. Sterile tissue suspensions or filtrates of PAP sputa or lung were inoculated by the amniotic route into Il-day-old chick embryos - fortunately without the addition of antibiotic, which later was to become routine for the processing of virus specimens. Suspensions of lung, trachea, and amniotic membranes from groups of chick embryos were harvested after 5-8 days of further incubation (optimally, 7-8 days) and subinoculated intranasally into hamster or cotton rats to detect the presence of the agent. Serial passage in chick embryo proved possible, with infectious titers of chick embryo lung suspensions for

Eaton Agent

mission experiments with volunteers, the so-called Pinehurst trials. Detailed descriptions werepublished in 1946in the Bulletin ofthe Johns Hopkins Hospital [12]. Volunteers inhaled a spray of a bacteria-free filtrate from a pool of throat washings and sputa from PAP cases. After some initial problems with cross-infection or contamination of spraying equipment in an early transmission experiment, which led to cases of PAP and minor respiratory illness in those given autoclaved inoculum, clear results were obtained. Dingle and Jordan [13] give a good summary of the results: "Approximately 75070 of recipients developed respiratory illnessand 25% pneumonia, with incubation periods around 12days. Of 16volunteers developing pneumonia, 13 (80%) developed cold haemagglutinins and 2 (12%) Streptococcus MG agglutinins," figures compatible with those just described for the survey by Eaton et al. [3]. Dingle and Jordan further stated that "the presence or absence of Streptococcus MG in the throat before or after inoculation bore no relation to the development of pneumonia nor werethere significant changes in the bacterial flora of the respiratory tract before, during or after the illness." The Commission's conclusion was that PAP of the variety associated with cold hemagglutinins was due to a filter-passing agent, presumably a virus. A backward look at the findings of the two groups up to this stage suggests to this commentator that an important and possibly definitive opportunity was missed for a collaborative linking of the findings of Eaton and his colleagues on the one hand with those of the Commission on Acute Respiratory Diseases on the other. Both were working with the same clinical condition, and both had produced evidence of a filterable agent in respiratory secretions. Eaton [10] wrote: "The agent inoculated into human volunteers was unfortunately not studied for its ability to grow in chicken embryos and no inoculations of human volunteers have yet been done with the virus propagated in chick embryos." Certainly administration of chick embryo lung suspensions infected with Eaton agent to volunteers at Fort Bragg might have enabled a direct test of a major Koch's postulate, i.e., the reproduction of disease (in the host species) with laboratory cultures of a proposed etiologic agent. Eaton and his colleagues could also have tested preinoculation and postinoculation (sometimes convalescent-phase) sera from the Pinehurst volunteers in the chick embryo lung-hamster neutralizing antibody assay. And finally, volunteers who had re-

Downloaded from http://cid.oxfordjournals.org/ at UB Giessen on May 25, 2015

J.1m. There were no obvious lesions in infected chick embryos, but a certain underdevelopment was noted at the time of harvest, 5-7 days after inoculation. Attempts to neutralize the PAP agent in the chick embryo amniotic cavity gave variable but not decisive results. (4) Systematic surveys of some 69 PAP patients showed that 42 (61070) developed a fourfold or greater increase in titer of neutralizing antibody to the PAP agent in chick embryo culture. A subgroup of 28 patients from the 42 with neutralizing antibody was tested for agglutinins to Streptococcus 344 (Streptococcus MG), and 11 (39%) were positive. Only 17 of the subgroup could be tested for cold hemagglutinins but 11 (64070) were positive (compare the proportion obtained by Finland et al. [8]). On the other hand sera from patients with other forms of PAP (e.g., influenza virus, psittacosis) did not develop neutralizing antibody to the PAP agent. There was thus a reasonable correlation with the commonly used, if limited, laboratory markers for PAP, but some individuals infected with the agent did not develop Streptococcus MG or cold hemagglutinins. Of the 27 patients remaining from the total group of 69, seven with specimens collected later in the disease had raised but unchanging neutralizing antibody titers, and 20 (29070) were considered to be negative. From all of these results - short of the actual inoculation of volunteers - a substantial case for regarding the PAP agent as a cause of a significant segment of the PAP syndrome had been built up. In the immediate aftermath of the publications in 1944 and 1945, two aspects were to prove troublesome in the acceptance of the findings. The first was the failure to get reproducible neutralization of the PAP agent in the chick embryo amnion, although Eaton et al. [3] had pointed out that Burnet [9] had had similar problems in attempts to neutralize influenza virus in the chick embryo amnion. The second was the occurrence of heterogenetic antibodies in the sera of PAP patients - cold hemagglutinins, Streptococcus MG agglutinins, and complementfixing antibody to normal and virus-infected hamster or mouse lung, together with reactions such as a false-positive Wassermann reaction [1, 10, 11]. Meanwhile, the other two groups working on the PAP problem had produced results, some of which were ultimately to mesh precisely with those of Eaton and his colleagues. The larger of the two groupsthe Commission on Acute Respiratory Diseasesconducted extensiveand very welldocumented trans-

341

Marmion

342

from the patient. In earlier attempts Weir and Horsfall [16] produced lesions in the lung of the Jamaican mongoose with PAP sputa, a finding not confirmed in the Puerto Rican mongoose by the Commission on Acute Respiratory Disease in collaboration with Dammin and Weller [17]. Moreover, chick embryo lung infected with Eaton's PAP agent did not produce pneumonia in the mongoose [10]. Around 1943 Dr. Harold Johnson obtained lesions in cotton rat lungs with specimens from an outbreak of PAP in Alabama (cited in [1]). These limited animal transmission experiments mirrored to some extent the experience of Eaton and his colleagues, but neither they nor many other isolated attempts by other workers using a wide variety of experimental animals approached the body of systematic information gathered in Berkeley. Attempts were made by Eaton and his colleagues to have their findings confirmed by the Rockefeller Institute group, but chick embryo cultures of the PAP agent failed - rather surprisingly (see below)to give positive results in New York. Thus, acceptance of the findings of Eaton and his colleagues remained in limbo. The next significant development in the story came from the Department of Bacteriology at the Harvard Medical School after Monroe Eaton moved there from Berkeley.

Observations on PAP in Boston The move was fortunate as it brought Eaton into contact with Albert Coons and Melvin Kaplan, who had just developed the fluorescent antibody technique for detection of antigen and antibody. Eaton, with the help of a young research fellow, Chien Liu, applied the immunofluorescence (IF) technique to a study of the agent in the chick embryo lung [18]. Frozen sections of chick embryo lung were cut and stained by the IF method with use of either acuteand convalescent-phase sera from PAP cases or rabbit sera made against strains of Eaton agent previously isolated in the chick embryo. The results were quite striking (figure 2). Here at last was a more sensitiveand lesslabour-intensive method for measuring antibody to the organism and for demonstrating its presence and distribution in chick embryo inoculated with either prototype strains or clinical samples. Eight more strains of Eaton agent were isolated, one (SiL) from a fatal case of pneumonia at the Boston City Hospital.

Downloaded from http://cid.oxfordjournals.org/ at UB Giessen on May 25, 2015

covered from disease induced by filtrates from PAP patients in the Pinehurst trial might have been challenged with Eaton agent to demonstrate cross immunity, or the inocula from the successful Pinehurst trials might have been neutralized by pretreatment with rabbit antisera to Eaton's PAP agent in further volunteer experiments. It appears [10] that the nearest the two lines of investigation came at that time was in 1943-1944 when sera from 13 cases of PAP at Fort Bragg - not in the transmission trialwere tested by Eaton's group for neutralizing antibody to the PAP agent and seven were found to be positive. It seems that such a direct approach to the problem was less attractive than the dialectic about the epiphenomena of cold hemagglutinins and the possible artifactual properties of antitissue and neutralizing antibody. It was not until some 16years later that the two lines of investigationwerefinally brought together. Meanwhile, the Navy Research Unit at the Hospital of the Rockefeller Institute for Medical Research had fared less well. Thomas et al. [14] had isolated an indifferent Streptococcus, apparently of a single serotype (Streptococcus MG or Streptococcus 344), from the lungs or sputa of PAP patients and found that the sera of 35%-50070 of patients agglutinated the organism. There was a short-lived suggestion that it might have an etiologic role. However, the organism could be found in "-'25070 of healthy persons or those with other respiratory infections (see also comments on Pinehurst transmission experiments above). It seemed more likely that the Streptococcus shared a common antigen with the PAP agent, as already suggested by Eaton et al. [3]. The team also identified [11] the tendency of PAP patients to develop heterogenetic antibodies; 14 of 35 PAP patients developed a fourfold. or greater increase of complement-fixing antibodies to suspensions of mouse lung infected with pneumonia virus of mice, and sera from five of the patients reacted with preparations with influenza virus or psittacosis group antigens. These were valid observations; it was their interpretation that was to give rise to problems. Horsfall et al. [15] experienced substantial and misleading problems with activation of pneumonia virus of mice in their experimental animals but recorded the production of pneumonic lesions in cotton rats with one sample of sputum from a PAP case and neutralization with convalescent-phase serum

Eaton Agent

343

New Directions at Western Reserve and Bethesda Figure 2. Section of chick embryo mesobronchus infected with Hetter strain of Eaton agent and stained by immunofluorescence with convalescent-phaseserum from a patient with atypical pneumonia. Area A is that used to define the coincident location of coccobacillary bodies and antigen in figure 3.

A representative (FH) strain of the new isolates was shown by cross-neutralization in cotton rats and cross-IF tests with infected chick embryo lung to be antigenically closely similar, if not identical, to the Mac strain isolated in California in 1944. Titers of the PAP agent in the chick embryo lung, when titrated in chick embryos and detected by IF, ranged from 4.7 to 5.4 EID so; the value for the Mac strain, 4.8, was close to that, 4.0, obtained by Eaton et al. [2] in the original studies. A paper by Liu et al. [19] published in 1959 examined the kinetics of the development of IF antibody to the PAP agent (now termed a virus) in chick embryo lung sections. IF antibody developed between 2 and 3 weeks after onset of illness, and there was a good correlation between IF antibody and neutralizing antibody measured (as before) in cotton rats. The proportion of IF antibody-positive patients developing cold hemagglutinins or Streptococcus MG agglutinins was variable, depending on the selection of the patients in different groups, but was ""58070 and 30070, respectively, again in line with earlier experience. IF antibody was still detectable, at lower titer, 12-18 months after illness, whereas cold hemagglutinins had become negative by that time.

At this point two new investigators entered the field. Dr. Wallace Clyde, an NIH (National Institutes of Health) postdoctoral fellow, joined Drs. J. H . Dingle and Floyd Denny at the School of Medicine, Western Reserve (Cleveland , Oh.), and Dr. Robert Chanock joined Dr. Robert Huebner at the Laboratory of Infectious Diseases at the NIH (Bethesda, Md.), Clyde was given the task of examining, by Liu's technique, the stored preinoculation and postinoculation sera from the Pinehurst transmission experiments conducted by the Commission on Acute Respiratory Diseases some 18 years before. The results were remarkable; at last the observations of Eaton's group and the Commission came together. Volunteers who had been exposed to untreated or filtered inocula (sputum or throat washings) and who had developed pneumonia or minor respiratory illness showed substantial increases of IF antibody to the PAP agent by Liu's method. On the other hand volunteers given autoclaved material, with the exception of one who had broken quarantine by visiting another inoculated group, remained well and infrequently developed IF antibody (table 3). Despite a detailed and scrupulous analysis of the new evidence presented in the Journal of Clinical Investigation [21], and a carefully balanced discussion of the fmdings, the etiologic significance of Eaton agent for PAP was not finally conceded. The summary concludes, "These results cannot be interpreted as proof of the etiological role of the Eaton agent in primary atypical pneumonia until the exact specificity of the reaction between the agent and fluorescent stainable antibodies has been established." It

Downloaded from http://cid.oxfordjournals.org/ at UB Giessen on May 25, 2015

Of particular significance for this commentary is the fact that Liu et al. [19] showed that removal of cold hemagglutinins and Streptococcus MG agglutinins by absorption of PAP sera with erythrocytes or streptococcal suspensions did not remove the IF antibody, although titers werelower after the absorption with Streptococcus MG. The latter effect turned out to be caused by the removal of a complementlike factor from the sera, and addition of fresh antibody-negative human serum or guinea pig serum restored the IF antibody titers to the values before absorption [20]. Equally important, it was shown that chick embryo lung powder or mouse liver powder did not lower the IF antibody titer.

Marmion

344

Table 3. Immunofluorescence tests on sera from second Pinehurst transmission experiment.

Table 4. Inoculation of volunteers with Eaton agent in monkey kidney cell culture [24, 25].

IF antibody to Eaton agent

Inoculum* Untreated Filtered Autoclaved

No. of volunteers with indicated No. of condition volunWell teers PAP MRI 12 11 19

3 3

5 5

4 3

0

(l)t

18

No. with fourfold or greater No. positive rise in titer (%) 8 (2 NT) 8 8 (l NT)

80 72 5

was the ghost of the antitissue antibody problem, not yet exorcised despite the absorption experiments by Liu et al. [19]. Chanock and his colleagues for their part had conducted a number of well-planned laboratory-field investigations of children [22] and adults [23] with respiratory infections, particularly at the Marine Corps Recruit Depot at Parris Island, North Carolina. In essence, these confirmed the findings of Liu et al. [19]. Apart from the serologic response with IF antibody and confirmatory correlations with cold hemagglutinins and Streptococcus MG agglutinins, 14 more strains of the PAP agent were isolated [24], which, as Liu had found, were antigenically similar if not identical to the 1944 isolates from Berkeley and those obtained in 1954-1957 in Boston. In addition, Chanock et ale [24] adapted the organism in chick embryo lung to grow in cell culture, particularly in monkey kidney tissue culture (MKTC). They were also able to use MKTC for the primary isolation of the agent from clinical specimens. In an important advance they set up some volunteer transmission experiments with an MKTC-adapted PAP agent - the PI 898 strain isolated at the Parris Island camp - with results shown in table 4. An important aspect of the planning of this transmission trial [25, 26] was the division of the volunteers into those with and those without IF antibody to Eaton agent before inoculation (table 4). Of 27 volunteers initially without IF antibody, 16 (59070) became ill with pneumonia, febrile upper respiratory illness, or myringitis. On the other hand, in the group of 25 volunteers with antibody before inoculation, there was no pneumonia or febrile illness and

Inoculum PI 898 (1()6·2 10 50)

Serologic state* Positive

Fever, Pneu- respiratory illness Myringitis monia 0

0

3

4

(n = 25)

Negative

9

(n = 27)

* Antibody tests by Liu's method on chick embryo lung with sera taken before inoculation. Positive ~10 IF; negative .s.;;10 IF.

only one person (4070) with myringitis; however, a proportion developed afebrile respiratory illness suggestive of reinfection. The organism persisted in the respiratory tract of some volunteers for at least 20 days. The findings from these transmission experiments and those from Clyde et ale [21] probably mark the turn of the tide in the acceptance of Eaton's work. At this point we may ask how far the evidence accumulated by all groups of workers fulfilled Koch's postulates establishing an organism as etiologically related to a disease or to part of a disease syndrome (see table 5). Cases of PAP had regularly yielded the agentin California, in Boston, and at Parris Island. The patients had developed neutralizing or IF antibody to the PAP agent propagated in chick embryo, and there was a reproducible but not absolute correlation between antibody development and the presence of cold hemagglutinins and Streptococcus MG agglutinins. . Cultures (in chick embryo and MKTC) had been established in the laboratory - not perhaps "pure" cultures in the sense of propagation at limit dilution but, in the chick embryo, apparently uncontaminated Table S. Koch-Henle Postulates (modified for viruses and nonbacterial agents) Cases of the disease regularly yield agent and develop immune markers to it Cultures established in laboratory system such as animals, cell culture, or chick embryo Disease reproduced with pure laboratory cultures together with reisolation of agent and development of immune markers in experimental animals or volunteers The presence of antibody (or cell-mediated immunity) to the putative agent, induced by previous infection or vaccination, confers resistance to disease on natural or artificial challenge

Downloaded from http://cid.oxfordjournals.org/ at UB Giessen on May 25, 2015

NOTE. Data are taken from [21]. NT = not tested; MRI = minor respiratory illness. * Sputum and throat washings from earlier volunteer inoculations. t Failure of segregation.

Results of inoculation

Eaton Agent

Burnet [9] had not managed to neutralize influenza virus in the chick embryo amnion (although pneumonia produced by mouse-adapted strains of influenza are readily neutralized in mice). In fact, in the light of present knowledge, it is clear that in some host systems infected with viruses or nonbacterial agents neutralization is not dependent solely on the antibody mixed with the inoculum but is partly dependent on the cooperation of the immune response of the host after inoculation. Thus, with another cell-dependent prokaryote - Coxiella burnetii- disease in mice can be substantially modified by addition of antibody to the phase 1 antigen of the Coxiella to the inoculum [27]. However, the same mixture in athymic mice [28], chick embryos or cell cultures produces an unmodified infection. As for (5), the notion that an antitissue antibody could neutralize the infectivity of an infective agent in an animal or serve to localize antigen by IF in an infectedchick embryo mesobronchus without staining the surrounding tissue remains somewhat perplexing in terms of standard immunologic conceptseven given that in certain specialized circumstances anticell antibody may modify virus-cell interactions. Alternatively, it might be supposed that the PAP agent, coated with tissue (chick embryo) antigens, might be aggregated by antitissue antibody with a consequent diminution of titer that might appear as neutralization, In fact some evidence against the possibility of the neutralization of the PAP agent by antitissue antibody had been obtained indirectly by Eaton and van Herick [29] as part of a study of the artificial immunization of hamsters and cotton rats. Intraperitoneal inoculation with infected chick embryo lung suspension provoked a protective immunity against later intranasal challenge with the agent, whereas intraperitoneal inoculation of uninfected chick embryo lung into control animals neither protected them against intranasal challenge nor generated neutralizing antibody. Liu et al. [19] had shown that absorption of PAP convalescent-phase sera with tissue suspensions did not remove IF antibody to the Eaton "virus." But perhaps the latter findings were too late to influence the Dingle and Jordan chapter that appeared in the same year [13]. The stalemate over the acceptance of the evidence was eventually overtaken by observations on the nature of Eaton agent or virus and its recognition as a mycoplasma. In strict logic this should have made no difference to the acceptance of existing evidence, which, as I have tried to show, fulfilled Koch's postu-

Downloaded from http://cid.oxfordjournals.org/ at UB Giessen on May 25, 2015

by other organisms and, in MKTC, in which contaminant SV40 virus had been neutralized with antiserum. The disease had been reproduced [25, 26] with the agent in MKlC, and furthermore the PAP agent had been linked with the Pinehurst transmission experiments [21]. Finally, the prior possession of immunity (antibody) to the putative agent did confer resistance to the more important aspects of the disease (pneumonia or febrile respiratory illness), although reinfection was not prevented [25, 26]. What objections could remain to scientific acceptance of Eaton agent as the cause of PAP commonly associated with cold hemagglutinins? Probably the most considered account of the objections is given in the comprehensive and scholarly review of PAP by Dingle and Jordan [13]. Points of objection appeared to be that (1) although lesions had been produced in cotton rat or hamster lung, serial passage was unsuccessful; (2) although serial passage in chick embryo had been achieved, there was no evidence of infection (presumably infection in this context was intended to mean overt pathologic changes); (3) different workers had isolated different agents; (4) "no data has yet been presented with respect to the ability of convalescent sera from cases of primary atypical pneumonia to neutralise Eaton's virus directly in embryonated eggs" [13]; and finally (5)there were unusual immunologic reactions (i.e., in addition to cold hemagglutinins and Streptococcus MG agglutinins, there were the falsepositive Wassermann reaction and antitissue antibodies [detected by complement fixation test]). Thus, the authors conclude, "it is entirely possible that all of these [presumably including the neutralizing/IF antibody] are non-specific readings dependent in some fashion on the breakdown of pulmonary tissue...." [13]. Of these reservations (4) and (5) are probably the most substantial. As for (4), it was correct that the Berkeley group had failed to get clear-cut neutralization of the PAP agent in ovo with human convalescent-phase serum. Similarly Clyde (W. A. Clyde, personal communication) could not neutralize with human convalescent-phase serum in ovo, although an effect was obtained with strong concentrations of a hyperimmune rabbit antiserum and small doses of the agent; fresh antibody-free human serum was added to the serum-agent mixtures as a complement source. Eaton et al. [2] had already pointed out that

345

346

lates. However, the new knowledge gave a tangibility to the findings and, of course, at once allowed the introduction of a different set of techniques and predictions about how the organism should behave. Information in this final phase came from several different directions.

Nature of the PAP Agent

lian Goodburn and I started work on Eaton agent in the Virus Laboratory of the Public Health Laboratory Service (Leeds, U.K.), wellaway from the main scenes of activity in Boston and Bethesda. We obtained the Hetter (FH) strain of the agent from Professor Stuart Harris, Department of Medicine, Sheffield University, and the Mac strain from Dr. Eaton, primarily for the serodiagnosis of cold hemagglutinin-Streptococcus MG-positive pneumonia. The FH strain was passed in the chick embryo amnion, and lung suspensions were subinoculated intranasally into hamsters. I was at once struck by the resemblance of the small, variable grey pneumonic lesions in hamster lung to those produced by Mycoplasma pulmonis in mouse lung, which I had seen in the late 1940s while working in Wilson Smith's department at University College Hospital (London) on grey lung virus of mice, another agent of uncertain nature [40-42]. The size of the PAP agent, its sensitivity to broad-spectrum antibiotics and to streptomycin, its resistance to penicillin, and the macroscopic and microscopic nature of the pneumonia were all in line with the properties of a respiratory, pleuropneumonia-like organism (PPW) or mycoplasma. It is interesting that Eaton had already raised the possibility of the agent as PPW in his paper [34] on the streptomycin sensitivity of the PAP agent, but standard cell-free cultures for PPW were negative at that time, We did two things in initial attempts to substantiate the PPW hypothesis. The first was to determine whether the PAP agent was sensitive to organic gold salts (e.g., sodium aurothiomalate), as these were known to inhibit M pulmonis and Mycoplasma arthritidis in mice [43,44]. They proved to be inhibitory to the PAP agent in hamsters and chick embryos, as were organic arsenicals. Poliomyelitis, influenza and vaccinia viruses, and a chlamydiaNigg and Eaton's pneumonitis "virus" of mice - are not inhibited. The second step was to use a special intensified Giesma technique to stain small coccobacillary bodies associated with the chick embryo mesobronchial cells in areas positive for PAP agent antigen (figure 3). The bodies were in fact in the thin layer of mucus (demonstrable by stains such as alcian blue at pH 3.0)on top of the cellsand so were predominantly extracellular. It is possible that the latter position, and perhaps their fragility in fixatives such as Palade's osmium tetroxide, prevented their earlier detection by electron microscopy [30]. These findings

Downloaded from http://cid.oxfordjournals.org/ at UB Giessen on May 25, 2015

Although Eaton and his colleagues [1-3] had initially described their PAP agent as an agent or filterable agent, later descriptions, e.g., by Liu [18], called it a virus. Its antigens had been located to the superficiallayer of cells in the chick embryo mesobronchus, but hematoxylin and eosin staining [18] showed intact mesobronchial epithelial cells and cilia. Donald and Liu [30] examined thin sections of infected chick embryo trachea in the electron microscope and found particles of 150-250 J.1m in diameter, with thin envelopes of 300 J.1m in diameter inside rvl00Jo of the nonciliated epithelial cells (or perhaps between them?). The notion that the PAP agent might be a virus was reinforced to some extent in an analysis by Chanock and associates of the growth pattern of the PAP agent in MKTC [24] and in chick embryo entodermal tissue culture [31]. Infectivity was not detectable in the cells or fluid phase of the cultures until 3-5 days after inoculation, a pattern that could be interpreted as an eclipse phase of viral replication. Curiously enough, in the light of later experiments by Clyde [32], attempts to detect antigen in the MKTC were not successful, although antisera giving good IF results on chick embryo lung sections were available. On the other hand, against the view that it was a virus, Eaton and various colleagues had already shown [33-36] that the PAP agent was sensitive to aureomycin, chloromycetin, carbomycin, and erythromycin and variably sensitive to streptomycin, although resistant to penicillin. It was also sensitive to nitrocompounds and aldehyde semicarbazones. Some of these sensitivities resembled those of the psittacosis-lymphogranuloma group of organisms, which at the time (1950) were also classified as viruses; stains for P-LGV (psittacosis-lymphogranuloma venereum) organisms in PAP agent-infected chick entoderm cultures were negative [31]. The sensitivity of the PAP virus to broad-spectrum antibiotics agreed well with the clinical reports of the response of PAP to these antibiotics determined by Finland, Meiklejohn, Lennette, Kingston, and others [37-39]. At about this time (late 1950s), Gil-

Marmion

Eaton Agent

347

were reported in Nature in 1961 [45] and also at a meeting [46] in Prague, in May 1961, on respiratory tract diseases of viral and rickettsial origin; the findings were discussed with Dr. Robert Chanock at the meeting and shortly afterwards in Leeds. Efforts were also made to grow the (presumptive) mycoplasma in England and at the NIH. Drs. Chanock, Hayflick, and Barile [47] were the first to succeed using an ingenious IF staining of colonies to identify the isolates . The results were shortly confirmed [48] in Leeds, by Dr. Hans Hers and his colleagues in Leiden, and by Dr. D. G. Edward at the Wellcome Laboratories in Beckenham; Dr. Ruth Lemke (Lister Institute, Chelsea, London) prepared a CF antigen from the Hetter strain of Mycoplasma pneumoniae that was shown to react with sera from local patients with respiratory illness and pneumonia [48]; similar patterns were obtained in Holland (for summary see [63]). Dr. Wallace Clyde confirmed the findings of coccobacillary bodies in the mesobronchi of chick embryo lung. He also used the special Giemsa technique to stain individual organisms and colonies of the mycoplasma on the surface of cells in MKTC and showed the coincidence of these structures with the location of PAP agent antigen demonstrable by IF [32] (figure 4). In October 1962, a conference on newer respiratory disease viruses, mycoplasmas, and PPLO was

held at the NIH [49]. The session on mycoplasmas included Hans Hers, myself, Wallace Clyde, and Robert Chanock. The session was chaired by John Dingle and reviewed the evidence on the etiologic importance of Eaton agent in PAP and the nature of the organism. During the ensuing discussion, the chairman was asked by Dr. Langmuir, who had been a member of the original Commission on Acute Respiratory Disease at Fort Bragg, whether he now accepted the validity of the claims by Eaton and his colleagues; later in the meeting Dr. Dingle replied that he did indeed accept the evidence (table 6). Thus, the end of the long road to the acceptance of Eaton's claims was finally reached - truly, "truth is the child of time." In fact, Dingle's reference to etiologic postulates having been satisfied, in the context of the identification of Eaton agent as a mycoplasma, was not completely pertinent; such postulates were already satisfied before the nature of the organism was established. Indeed it is a little ironic to reflect that, had M. pneumoniae been isolated first in cell-free media and then such cultures used to fulfill Koch's postulates, the results with an organism attenuated by passage on agar would have been much less impressive in terms of production of pneumonia [50] when compared with decisive volunteer trials with the yet-to-be-identified organism in MKTC by Chanock et al. [25, 26].

Downloaded from http://cid.oxfordjournals.org/ at UB Giessen on May 25, 2015

Figure 3. Left: Palisade arrays of coccobacillary bodies on the surface of the epithelial cells of chick embryo mesobronchus infected with Hetter strain of Mycoplasma pneumoniae. Right: Distribution of IF antigen in next serial section .

Marmion

348

The ambiguities of the process of scientific acceptance remain. Why did it take so long, and was the evidence against Eaton's findings really compelling? The ostensible reasons - heterogenetic serologic reactions and failure to neutralize the agent in chick embryos - hardly seem sufficient when weighed against the other evidence summarized above. Some generalizations on the anthropology of the acceptance process and the influence of several nonscientific factors are given in table 7. Probably the first of these is the most important. It is easy to say, from the standpoint of superior wisdom, that we do not believea particular set of results. Skepticism born of a long experience of analogous research is, of Table 6. Excerpt from proceedings of the Conference on Newer Respiratory Disease Viruses, Bethesda,October 1962. Session: The Mycoplasma (PPLO) Agents Dr. Langmuir (to chairm an, Dr. Dingle) ". . . I am wondering if we could calion the chairman who . . . has written at length on this subject .. . to give his judgment of the role of PPLO in the causation of PAP?" Dr. Dingle (later) "I shall try to answer Dr. Langmuir. The etiology of PAP has been a difficult problem for all of us . . . I have been rather reluctant (although some had other words for it) to accept the evidence . . . the chief reason was because of the peculiarities of the serologic evidence and the fact that until recently it was not possible to neutralize the so-called agent." [He then went on to say that he was now convinced and that etiolog ic postulates had been satisfied .]

course, of central importance in the scientific process and keeps it honest; however, it must not be persisted in harshly or unreasonably - it is easy to nail one's colors to the mast. If the stance is to be one of persistent and publically expressed skepticism, then there is an obligation to attempt to repeat the work using exactly the same methods as the original workers and-as important-to provide experimental verification of the basis of the (intuitive) skepticism. In the 1940s, with Eaton's results as argued above, it appears that no attempt was made by the critics to show, for instance, that antibody against pulmonary or other tissue did in fact neutralize the agent in cotton rats or hamsters. Further, it seems that flawed efforts were made (at the Rockefeller Institute) to confirm Eaton's original work with chick embryo-passaged material, a surprising failure considering that susceptible cotton rats were available (15) and that Clyde, Chanock and his colleagues, Hans Hers, Goodburn, and I had no problems in passaging and identifying prototype strains of the PAP agent in chick embryo lung. In general terms again, failures or distortions of Table 7. Nonscientific impediments to scientificacceptance Intuitive skepticism without experiment Group or institutional competitiveness and territoriality "Winner takes all" philo sophy

Downloaded from http://cid.oxfordjournals.org/ at UB Giessen on May 25, 2015

Figure 4. Colonies (C) of Mycoplasma pneumoniae growing on sheet of monkey kidney cells, stained by intensified Giemsa stain (left) or by immunofluorescence (right). By courtesy of Dr. W. Clyde (32).

Eaton Agent

Epilogue: Eaton Agent into

Mycoplasma pneumoniae What happened after the Washington Conference and the events in 1961-1962? Dr. Eaton appeared in Time Magazine as one who had opened up a new area of medical research, as indeed he had. There were many other sequels. In general, there was a great expansion of interest in and support for mycoplasmology that not only extended knowledge of the cell and molecular biology of the organisms but also led to the identification of new mycoplasmas a?d some, but not many, new human mycoplasmal diseases, The activity at the time was well reviewed by Hayflick and Chanock [54]. The spectrum of disease associated with M. pneumoniae was expanded to infections involving the central nervous system occasionally the heart and pericardium, and the skin: The ability to culture M. pneumoniae on serum agar and to prepare antigens for complement fixation and other serologic tests simplified clinical-laboratory diagnosis and facilitated numerous laboratory-field studies of the clinical presentation and epidemiology of M. pneumoniae infection (e.g., the excellent stu~ies of Grayston and colleagues in Seattle [55]). Ultimately, agar culture of the organism as a method of routine laboratory confirmation proved to be suboptimal and slow. Recent approaches to direct diagnosis have employed detection of antigen or specific nucleotide sequences in respiratory secretions (for summary see [56-58]) as well as improved methods of serodiagnosis. In conclusion, two sequels may be explored in slightly more detail because they bear on the tale just told. The first relates to the coccobacillary bodies and to the difficulty in visualizing the organism in chick embryo lung experienced by Donald and Liu [30]. Wallace Clyde and his colleagues, particularly Collier, using organ cultures of tracheal rings from either human fetuses or hamsters and following ultrastructural observations by Biberfeld and Biberfeld [59], showed [60-62] that the M. pneumoniae cell absorbs to the host cell surface through a foot attachment process (figure 5). There is an impression of the organism sitting like a slug on the cell, perhaps injecting enzymes and thriving on the host breakdown products. The second sequel relates to the immunochemistry of M. pneumoniae antigens and to an explanation of the heterogenetic serologic reactions that proved such an obstacle to the acceptance of Eaton's finding. Liu and his colleagues [19] had established

Downloaded from http://cid.oxfordjournals.org/ at UB Giessen on May 25, 2015

skeptical assessment may interact with the second impediment to scientific acceptance - group or institutional competitiveness and territoriality. It would be idle to deny the existence of institutional hubris, which has both negative and positive, beneficial aspects. Max Charlesworth and his colleagues in a recent book entitled Life Among the Scientists [51] say, in a discussion of scientific motivation "Burnet's idea, that science is a game involving a cer: tain amount of boyish competitiveness, serves to obscure the fact science is very often concerned with power and struggles for power." It is perhaps true that the desire for power is a dominant human motivation, as pointed out years ago by Bertrand Russell [52]. There may, however, be more complex patterns for less-senior members of an institution - a highoctane mixture of intense curiosity, the desire for recognition, and intellectual impatience. Above all, there is the desire to be first with a discovery - what Charlesworth calls the "winner takes all" philosophy. He cites as an example the discovery of DNA with Rosalind Franklin and others involved in the DNA quest relegated to nonentity. In fact, the attitude is of respectable antiquity. Rene Dubos, in his book LouisPasteur, Freelance ofScience [53], recalls how Pasteur, with charming candor, said, "How disturbing to lose by hasty publication, the charm of following a fruitful idea with calm and prolonged meditation. And yet I would be evenmore disturbed if M. Marback should arrive first at the general idea which I follow!" All of these attitudinal strands come together in a mutually supportive sense of the superiority of the institution and those within its walls. Again, this esprit de corps plays a positive and important role in fueling the inexorable progress of the juggernaut of scientific research and refurbishing the institute with the brightest and best of the young workers. However, it may sometimes nourish the illusion that truth is not to be found extramurally. In the particular history we are considering, institutional or group competitiveness at least appears to have impeded rather than facilitated the process of discovery. The failure of collaboration in 1944 left the full significance of the Pinehurst transmission experiments unrealized for nearly 20 years. The overall problem is how to harness these ambivalent, very human, and powerful attributes to drive along the great juggernaut of science without the vehicle's straying from the road or crushing too many devotees beneath the wheels.

349

350

apparently attached to the surface of a ciliated epithelial cell in a tracheal organ culture. By courtesy of Dr. A. W. Collier (x 30,150).

from cross-absorption experiments with antisera from PAP patients that the cold hemagglutinin and the Streptococcus MG agglutinin appeared to be distinct from IF antibodies reacting with the PAP agent. Hers and I [63] found, however, that a rabbit antiserum to Streptococcus MG reacted weakly by IF on colonies of the Mac strain of M pneumoniae, a finding suggesting a minor shared antigen. After my move in 1963 to the Monash Medical School, Melbourne, I began collaborative work with Dr. Philip Packett (Animal Health Division, CSIRO) on cross-reactions between M. pneumoniae, Mycoplasmamycoides, and Streptococcus MG; in this we were joined by Dr. Ruth Lemcke as a World Health Organization Visiting Research Fellow and by Dr. Elizabeth Shaw. Plackett had had extensive experience with the fractionation and characterization of serologicallyreactive materials from M mycoides, which we found shared carbohydrate determinants with M pneumoniae. A major part ofthe AI. pneumoniae complementfixing antigen is extractable in choloroform-methanol (CM) 2:1 v/v [64, 65]. The CM extract reacted strongly in the complement fixation test with Streptococcus MG antiserum, and the determinant was sensitive to carbohydrase and sodium metaperiodate but resistant to heat, protease, and lipases [64]. Fractionation [66] of the CM extract showed the serologic activity to be associated with four or more neutral glycolipids- di- and trigalactosyldiglyceride and di- and trihexosyldiglyceride (glucose and galactose).

The main phosphatides were phosphatidyl glycerol and phosphatidyl monoglyceride. The glucosecontaining glycolipids from AI. pneumoniae reacted by complement fixation with Streptococcus MG antiserum. In reciprocal tests, a diglucosyldiglyceride from Streptococcus MG reacted strongly with M. pneumoniae antiserum as well as with the homologous antiserum. The phosphatides showed no mycoplasma-specific serologic activity, but the phosphatidyl monoglyceride reacted with Wassermann reaction-positive antiserum. The observations cast some light on the possible underlying mechanisms of the Streptococcus MG reaction and false-positive Wassermann reaction in PAP. It was also noted that lactosylceramide (cytolipin H) reacted with M pneumoniae antiserum; as the cerami de is one example of a mammalian cell surface marker, the reaction may point to a mechanism for the complement-fixing activity of PAP sera with tissue antigens [11]. Some insights into the mechanism of cold hemagglutinin formation in PAP followed later from the observations by Costea et aI. [67] that cold agglutinins with specificity for the human erythrocyte I antigen are formed in rabbits immunized with killed (or live) AI. pneumoniae and that their activity is inhibited by a- or /3-galactosides. Earlier work by the same group demonstrated that the I antigen of human erythrocytes is inactivated by a-galactosidase [68] and that cold agglutinins produced in the rabbit by immunization with killed Listeria monocytogenes are inhibited by galactose or various di- or trisaccharides containing galactose. Janney et aI. [69] report that CM extracts of erythrocyte ghosts containing a glycoprotein with I antigen reactivity will fix complement with rabbit antisera to AI. pneumoniae. These observations suggest that cross-reactions based on the galactose-containing glycolipids of the mycoplasma underlie the stimulation of cold agglutinins in PAP. The biological functions of antibodies to the neutral and acidic lipids of the organism have been partly defined [70]. Thus, antiglycolipid antiserum inhibits hemadsorption to M pneumoniae colonies, sensitizes the organism to complement lysis, and inhibits metabolism. However, the identity of the antigen provoking protective immunity and neutralizing antibody remains unclear. Major immunogens of the mycoplasma for human beings are the 169-kDa PI protein (adhesin) associated with the foot attachment process and a smaller 88-kDa protein [71]. It is tempt-

Downloaded from http://cid.oxfordjournals.org/ at UB Giessen on May 25, 2015

Figure 5. Electron micrograph of filamentous cell of

Mycoplasma pneumoniae with a specialized foot process

Marmion

Eaton Agent

351

Table 8. Possible scheme relating Mycoplasma pneumoniae antigens or haptens to specific and heterogenetic serologic reactions in primary atypical pneumonia. M. pneumoniae

antigen or hapten Glycolipids Galactose-containing Glucose-containing Acid lipids Phosphatidyl monoglyceride

Cold agglutinins, antibrain antibody Streptococcus MG antibody False-positive Wassermann reaction Specific antibody, neutralizing antibody

ing to speculate that neutralizing antibody, as demonstrated in Eaton's hamster-cotton rat system, may be directed against these proteins, therebyexplaining the dichotomy between specific antibody and heterogenetic antibodies. Table 8 attempts to draw together the (incomplete) evidence in terms of the equivalence between M pneumoniae antigens or haptens on the one hand and heterogenetic or specific antibodies on the other. This includes reference to antibrain antibody, which may be a cross-reacting antibody directed against a galactose-containing determinant (see review in [72]). From this scheme it seems that Eaton's suggestion [3] that the unusual serologic reactions in PAP are akin to the Weil-Felix phenomenon, which depends on shared haptens between rickettsias and Proteus species, was not too wide of the mark. So in the end, the harvest from those early and difficult days in Berkeley was considerable. The tale is heartening in some ways, however frustrating for the participants. The scientific merit of the early work eventually prevailed; the process of scientific acceptance remained sound, albeit glacially slow. It is also encouraging that over the entire period from 1944 the publications of Eaton and his colleagues appeared in leading journals, particularly the Journal ofExperimental Medicine. Whatever the climate of skepticism may have been, the editors published and let the evidence speak for itself. And for that we must be exceedingly grateful. References 1. Eaton MD, Meiklejohn G, van Herick W. Studies on the etiology of primary atypical pneumonia: a filterable agent

Downloaded from http://cid.oxfordjournals.org/ at UB Giessen on May 25, 2015

Other antigens Membrane protein(s) (e.g., 169-kDa, 88-kDa)

Equivalent serologic response in human infection

transmissible to cotton rats, hamsters and chick embryos. J Exp Med 1944;79:649-68 2. Eaton MD, Meiklejohn G, van Herick W, Corey M. Studies on the etiology of primary atypical pneumonia. II. Properties of the virus isolated and propagated in chick embryos. J Exp Med 1945;82:317-28 3. Eaton MD, van Herick W, Meiklejohn G. Studies on the etiology of primary atypical pneumonia. III. Specific neutralisation of the virus by human serum. J Exp Med 1945;82:329-42 4. Chanock RM, Dienes L, Eaton MD, Edward DG, Freundt EA, Hayflick L, Hers JF, Jensen KE, Liu C, Marmion BP, Morton HE, Mufson MA, Smith PF, Somerson NS, Taylor Robinson D. Mycoplasma pneumoniae: proposed nomenclature for atypical pneumonia organism (Eaton Agent). Science 1963;140:662 5. Denny FW. Atypical pneumonia and the Armed Forces Epidemiological Board. J Infect Dis 1981;143:308-15 6. Peterson OL, Ham TH, Finland M. Cold agglutinins (autohaemagglutinins) in primary atypical pneumonias. Science 1943;97:167 7. Turner JC. Development of cold agglutinins in atypical pneumonia. Nature 1943;151:419 8. Finland M, Peterson OL, Allen HE, Samper BA, Barnes MW. Cold agglutinins. I. Occurrence of cold isohaemagglutinins in various conditions. J Clin Invest 1945;24:451-457 9. Burnet FM. Influenza virus infections of chick embryo by amniotic route: titration and serum neutralisation tests. Aust J Exp Bioi Med Sci 1941;151:39-44 10. Eaton MD. Virus pneumonia and pneumonitis viruses of man and animals. In: Doerr R, Hallauer C, eds. Handbuch d, Virusforschung. Vienna: Springer, 1950:87-126 11. Thomas L, Curnen EC, Mirick GS, Ziegler JE, Horsfall FL. Complement fixation with dissimilar antigens in primary atypical pneumonia. Proc Soc Exp BioI Moo 1943;52:121-5 12. Commission on Acute Respiratory Diseases. The transmission of primary atypical pneumonia to human volunteers. I. Experimental methods. II. Results of inoculation. III. Clinical features. IV. Laboratories studies. Bulletin of the Johns Hopkins Hospita11946;79:97-167 13. Dingle JH, Jordan WS. Primary atypical pneumonia. In: Rivers,Horsfall, eds. Viraland rickettsial infections of man, 3rd ed. Philadelphia: J. B. Lippincott, 1959:600-12 14. Thomas L, Mirick GS, Curnen EC, Zeigler JE, Horsfall FL. Studies on primary atypical pneumonia. II. Observations concerning the relationship of a nonhaemolytic streptococcus to the disease. J Clin Invest 1945;24:227-40 15. Horsfall FL, Curnen EC, Mirick GS, Thomas L, Zeigler JE. A virus recoveredfrom patients with primary atypical pneumonia. Science 1943;97:289-91 16. Weir JM, Horsfall FL. The recovery from patients with acute pneumonitis of a virus causing pneumonia in the mongoose. J Exp Med 1940;72:595-610 17. Commission on Acute Respiratory Diseases in collaboration with Dammin GJ, Weller JH. Attempts to transmit primary atypical pneumonia and other respiratory tract infections to the mongoose. J Immunol 1945;50:107-14 18. Liu C. Studies on primary atypical pneumonia. I. Localization, isolation and cultivation of a virus in chick embryos. J Exp Med 1957;106:455-67 19. Liu C, Eaton MD, Heyl JT. Studies on primary atypical pneu-

352

38. Meiklejohn G, Thalman G, Waligova OJ, Kempe CH, Lennette EH. Chemotherapy of primary atypical pneumonia. JAMA 1954;154:553-7 39. Kingston JR, Chanock RM, Mufson MA, Hellman LP, James WD, Fox HH, Manko MA, Boyers J. Eaton Agent pneumonia. JAMA 1961;176:118-23 40. Andrewes CH, Glover RE. Grey lung virus: an agent pathogenic for mice and other rodents. Br J Exp Pathol 1945; 26:379-83 41. Andrewes CH, Niven JSE Action of arsenicals on infection of mice with greylung virus. J Pathol BacterioI1953;66:565 42. Gay FW. Fine structure and location of mycoplasma-like grey lung and rat pneumonia agents in infected mouse lung. J Bacteriol 1967;94:2048-61 43. Sabin AB, Warren J. The therapeutic effectiveness of a practically nontoxic new compound (calcium aurothiomelate) in experimental, proliferative, chronic arthritis of mice. Science 1940;92:535 44. Edward DG. Catarrh of the upper respiratory tract in mice and its association with pleuropneumonia-like organisms. J Pat hoI Bacteriol 1947;59:209 45. Marmion BP, Goodburn GM. Effect of an organic gold salt on Eaton's primary atypical pneumonia organism and other observations. Nature 1961;1.89:247 46. Goodburn GM, Marmion BP. Investigations of the nature of Eaton's primary atypical pneumonia organism. Proc Cong on Respiratory Tract Diseases of Virus and Rickettsial Origin. Prague. May 1961 J Hyg Epidemiol Microbiol Immunoll962;6:176 47. Chanock RM, Hayflick L, Barile ME Growth on artificial medium of an agent associated with atypical pneumonia and its identification as a PPLO. Proc Natl Acad Sci USA 1961;48:41 48. Goodburn GM, Marmion BP, Kendall EJC. Infection with Eaton's primary atypical pneumonia agent in England. Br Med J 1963;1:1266-70 49. Conference on Newer Respiratory Disease Viruses USPHS. Bethesda, Md: National Institutes of Health, 1962:198-231 50. Couch RB, Cate TR, Chanock RM. Infection with artificially propagated Eaton Agent (Mycoplasma pneumoniaei. JAMA 1964;187:442-7 51. Charlesworth M, Farrall L, Stokes T, Turnbull D. Life among the scientists - an anthropological study of an Australian scientific community. Oxford, England: Oxford University Press, 1989 52. Russell B. Power. London: Unwin Books, 1938 [1960] 53. Dubos RJ. Louis Pasteur. Freelance of science. Boston: Little Brown, 1950 54. Hayflick L, Chanock RM. Mycoplasma species of man. Bacteriol Rev 1965;29:185-221 55. Grayston JT, Foy HM, Kenny GE. The epidemiology of mycoplasma infections of the human respiratory tract. In: Hayflick L, ed. The mycoplasmatales and the L phase of bacteria. Amsterdam: North Holland, 1969:651-82 56. Kok T-W, Varkanis G, Marmion BP, Martin J, Esterman A. Laboratory diagnosis of Mycoplasma pneumoniae infection: direct detection of antigen in respiratory exudates by enzyme immunoassay. Epidemiol Infect 1988;101:669-84 57. Harris R, Marmion BP, Varkanis G, Kok T, Lunn B, Martin J. Laboratory diagnosis of Mycoplasma pneumoniae infection: comparison of methods for direct detection of spe-

Downloaded from http://cid.oxfordjournals.org/ at UB Giessen on May 25, 2015

monia. II. Observations concerning the development and immunological characteristics of antibody in patients. J Exp Med 1959;100:545-56 20. Liu C. Studies on primary atypical pneumonia. III. A factor in normal serum which enhances the reaction between PAP virus and convalescent serum. J Exp Med 1961;113:111-23 21. Clyde WA, Denny FW, Dingle JH. Fluorescent stainable antibodies to the Eaton Agent in human primary atypical pneumonia transmission studies. J Clin Invest 1961; 40:1638-47 22. Chanock RM, Cook MK, Fox HH, Parrott RH, Huebner RJ. Serologic evidence of infection with Eaton Agent in lower respiratory illness in childhood. N Eng J Med 1960;262:648-54 23. Chanock RM, Mufson MA, Bloom HH, James WD, Fox HH, Kingston JR. Eaton Agent Pneumonia. JAMA 1961; 175:213-20 24. Chanock RM, Fox HH, James WD, Bloom HH, Mufson MA. Growth of laboratory and naturally occurring strains of Eaton Agent in monkey kidney tissue culture. Proc Soc Exp BioI Med 1960;105:371-5 25. Chanock RM, Rifkind 0, Kravetz HM, Knight V, Johnson K. Respiratory disease in volunteers infected with Eaton Agent: a preliminary report. Proc Natl Acad Sci USA 1961;47:887 26. Rifkind 0, Chanock RM, Kravetz H, Johnson K, Knight V. Ear involvement (myringitis) and primary atypical pneumonia following inoculation of volunteers with Eaton Agent. Am Rev Respir Dis 1962;85:479-89 27. Abinanti FR, Marmion BP. Protective or neutralizing antibody in Q fever. Am J Hyg 1957;66:173-95 28. Kishimoto RA, Rozmiarek H, Larson EW. Experimental Q fever infection in congenitally athymic mice. Infect Immun 1978;22:69-71 29. Eaton MD, van Herick W. Experimental immunisation with the virus from primary atypical pneumonia. J Infect Dis 1947;81:116-21 30. Donald HB, Liu C. Cytological studies of chick embryo cells infected with the virus of primary atypical pneumonia. Virology 1959;9:20-9 31. Gordon FB, Quan AL, Cook MK, Chanock RM, Fox HH. Growth of Eaton Agent of primary atypical pneumonia in chick entodermal tissue culture. Proc Soc Exp BioI Med 1960;105:375-7 32. Clyde WA. Demonstration of Eaton's Agent in tissue culture. Proc Soc Exp BioI Med 1961;107:715 33. Eaton MD. Action of aureomycin and chloromycetin on the virus of atypical pneumonia. Proc Soc Exp BioI NY 1950;73:24 34. Eaton MD, Liu C. Studies on sensitivity to streptomycin of the atypical pneumonia agent. J Bacteriol 1957;74:784 35. Eaton MD, Perry ME, Grocke 1M.Effect of nitrocompounds and aldehyde semicarbazones on virus of primary atypical pneumonia. Proc Soc Exp BioI NY 1957;77:422 36. Eaton MD. Effect of some newer antibiotics on the agent of primary atypical pneumonia. In: Welch H, MartiIbanez, eds. Antibiotics annual. New York: Medical Encyclopaedia, 1954-55:1046 37. Finland M. Antimicrobial treatment for viral and related infections. 1.Antibiotic treatment of primary atypical pneumonia. N Engl J Med 1952;247:317-29

Marmion

Eaton Agent

67.

68. 69.

70.

71.

72.

munochemical analysis of Mycoplasma pneumoniae. 3. Separation and chemical identification of serologicallyactive lipids. Aust J Exp BioI Med Sci 1969;47:171-95 Costea N, Yakulis VJ, Heller P. The mechanism of induction of cold agglutinins by Mycoplasma pneumoniae. J Immunol 1971;106:598-604 Yakulis VJ, Costea N, Heller P. a Galactoside determinants of the I-Antigen. Proc Soc Exp Biol Med 1966;121:812-6 Janney FA, Lee LT, Howe C. Cold hemagglutinin crossreactivity with Mycoplasma pneumoniae. Infect Immun 1978;22:29-30 Razin S, Prescott B, Chanock RM. Immunogenicity of Mycoplasma pneumoniae glycolipids. A novel approach to the production of antisera to membrane lipids. Proc Nat! Acad Sci 1970;67:590-7 Hu PC, Huang C-H, Collier AM, Clyde WA. Demonstration of antibodies to Mycoplasma pneumoniae attachment protein in human sera and respiratory secretions. Infect Immun 1983;27:437-9 Rothstein TL. CNS disease. In: Mycoplasma as agents of human disease. Proceedings of an interdisciplinary conference. Waukegan, Ill: Windemere Communications, 1985: 109-17

Downloaded from http://cid.oxfordjournals.org/ at UB Giessen on May 25, 2015

cific antigen or nucleic acid sequences in respiratory exudates. Epidemiol Infect 1988;101:685-94 58. Bernet C, Garret M, De Barbeyrac B, Bebear C, Bonnet J. Detection of Mycoplasma pneumoniae using the polymerase chain reaction. J Clin Microbiol 1989;27:2492-6 59. Biberfeld G, BiberfeldP. Ultrastructuralfeaturesof Mycoplasma pneumoniae. J Bacteriol 1970;102:855-61 60. Collier AM, Clyde WA. Relationship between Mycoplasma pneumoniae and human respiratory epithelium. Infect Immun 1971;3:694-701 61. Wilson MH, Collier AM. Ultrastructural study of Mycoplasma pneumonia in organ culture. J Bacteriol 1976;125:332-9 62. Collier AM. Pathogenesis of Mycoplasma pneumoniae infection as studied in the human foetal trachea in organ culture. In: Pathogenic mycoplasmas. Amsterdam: Ciba Foundation Association Scientific Publishers, 1972:305-27 63. Marmion BP, Hers JF. Observations on Eaton primary atypical pneumonia agent and analogous problems in animals. Am Rev Respir Dis 1963;88:198-211 64. Lemke RM, Marmion BP, Placket P. Immunochemical analysisof M. pneumoniae. Ann NY Acad Sci 1967;143:691-702 65. Kenny GE. Heat-lability and organic solvent-solubility of mycoplasma antigens. Ann NY Acad Sci 1967;143:676-81 66. Plackett P, Marmion BP, Shaw EJ, Lemcke RM. Im-

353