VISUALIZATION AND MORPHOLOGY OF PLEUROPNEUMONIALIKEORGANISMS IN CLINICAL MATERIAL'

MAURICE C. SHEPARD

Naval Medical Field Research Laboratory, Camp Lejeune, North Carolina

Received for publication July 2. 1956

In a previous communication (Shepard, 1956)it was reported that pleuropneumonialike or-ganisms (PPLO) which produce distinctive, veryminute colonies on solid medium, were recoveredfrom a high proportion of cases of nongonococcalurethritis (NGU) in human males. These char-acteristic, tiny PPLO colonies comprised ap-proximately 86 per cent of the recoveries ofpleuropneumonialike organisms (all types) fromnongonococcal urethritis patients. They weredesignated T strains of pleuropneumonialikeorganisms. In view of the relatively high pro-portion of cultures growing out only T straincolonies, it was felt desirable to determine whetherdiscrete, characteristic, intracellular PPLO in-clusions could be demonstrated in urethralscrapings from nongonococcal urethritis patientsyielding only T strain PPLO colonies in culture.The demonstration of tissue-phase PPLO in suchclinical material would provide additional in-formation supporting the concept that T strainPPLO are newly recognized members of thepleuropneumonia group. The methods employedfor their demonstration in clinical material bydirect microscopic methods, together with adescription of their morphology and characteristicappearance, comprise the substance of this report.

METHODS

Collection of material. The method of obtainingclinical material by urethral epithelial scrapingwas that previously described (Shepard, 1954).Material thus collected was immediately spreadin a long U-shaped manner on cleaned slides.A stiff, 22-gauge platinum wire loop was em-ployed. The material was streaked out in astraight line for a distance of approximately2 in, a "U" turn made with the loop, and suc-ceeding passes made back and forth in thismanner until no further material was deposited

' This work is not to be construed as necessarilyreflecting the views of the Department of theNavy.

on the slide. The loop was not lifted from theslide during the procedure. A sinall oIr medium-sized, blunt, Buck ear curette was also used toadvantage for taking epithelial scrapings. Thecollection of exudate alone is unsatisfactory andwill generally yield negative results, as discussedlater. The clinical material of choice is a scrapingfrom the region of urethral epithelium posteriorto the fossa navicularis. This portion of theanterior (cavernous) urethra is lined by stratifiedor pseudostratified columnar epithelium. How-ever, patches of stratified squamous epitheliumare common in this region (Maximow and Bloom,1952). Smears thus prepared were immediatelyplaced (without drying) in a jar containingabsolute methanol for fixation for 3 to 5 min.Prompt fixation of still wet smears yielded moresatisfactory preparations than delayed fixationfollowing variable periods of air drying. Fixationin mixtures containing equal parts of 95 per centethanol and diethyl ether (Papanicolaou, 1954)for 15 min gave similar results.

Staining procedures. Methanol-fixed epithelialscrapings were stained with Giemsa stain (azureB type, certified, Cat. No. 2281, Hartman-Leddon Company, Philadelphia, Pa.) for a periodof 3 to 6 hr in a Coplin jar, differentiated in pH7.0 buffer solution (Harkness, 1950) and air-dried ol- dried by gentle blotting between ab-sorbent filter papers. Stock Giemsa stain wasdiluted 1:50 in pH 7.0 phosphate buffer solutionimmediately before use.

Additional scrapings were stained by thebuffered azure-eosin technique of Lillie (1947,1948). MIethanol-fixed epithelial scrapings werestaine(l 3 to 6 hr in a Coplin jar employing thefollowing st aining solution: (listilled water, 32ml; pH 7.0 buffer solution, 2 ml; acetone, 5 ml;eosin Y (1 per cent aqueous solution), 0.5 ml;azure B (1 per cent aqueous solution), 0.5 ml.The staining solution was formulated in theorder shown. The distilled water used was doublvdistilled in an all-pyrex glass still. Buffer solutions

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Were prei)ared in 25 perl cent methanol solutionafter the metho(d of Lillie (1947, 1948), employing0.1 M citric acid and 0.2 M disodium phosphatemixtures. After removing slides from the stainingsolution they were washed approximately 5 secin distilled water and dried between sheets offilter paper, or preferably, in a stream of com-pressed air. Epithelial scrapings stained by thebuffered azure-eosin technique described showa low degree of stained background debris.Further, the cytoplasm of epithelial cells generallystains a pale lavender shade, providiing a lightlystained cytoplasmic background for ease indetection of pleuropneumonia group organisms.Staining solutions for each batch of slides weIrenade up fresh, used once and dliscarded.Although certain extracellular PPLO can be

recognized without great difficulty, there isnevertheless ample opportunity for the confusionof free PPLO with extracellulai aIrtifacts instained epithelial scrapings. In order to eliminatethis type of error, major emphasis has beenplacedl on searching for PPLO in intracellularp)osition within the cytoplasm of intact epithelialcells. Cells which showed evidence of degenerativechanges were avoided, in order to further reducemisinterpretation due to intracy7toplasmic arti-facts.The photographic technique used was that

reported previously (Shepard, 1956). The theo-retical object magnification at film emulsioniplane was 900 X. However, actual magnification,as determined by center of field image measure-ments of 10- and 20-A line spaces (center tocenter) projected from the ruling of a No. 31-16-90 Bausch and L9)mb stage micrometer, was780 x. All films, with the exception of figure 13,were enlarged photographically by 2 X, yieldinga final magnification of 1560 X. Final magnifica-tion in figure 13 is 3,200 X. A green No. 58 anda yellow No. 15 Wratten color filter combinationwas employed to increase particle definition an(dcontrast for photographic purposes.

RESULTS

T issue-phase p)leuropneumonia group or-ganisms were successfully demonstrated inurethral epithelial scrapings and weere char-acterized chiefly by their peculiar pleomorphism,minute size, basophilic staining reaction to stainsof the Romanowski type, and by their intra-cytoplasmic (levelopment within parasitize(l

epithelial cells. In many instances, the termpolymorphism more appropiiately (lescribed theappearance of these minute microorganisms asseen in stained epithelial scrapings. With theGiemsa and azure-eosin techniques employed,the pleuropneumonialike organisms stained a(lelicate light blue to bluish purple. Stains dilutedin buffer solutions of pH 7.2 rather than of pH7.0 yielded slightly (leeper staining reactions, butgreater care had to be exercised in differentiation.Deep staining, such as the intense stainingreaction exhibited by commonly recognize(dbacteria, was never observed in the intracyto-plasmic PPLO (or extiracellular foims). Theorganisms weire not observed in intranuclearposition, nor were they recognized within poly-morphonuclear leucocytes with any degree ofcertaintv. At no time have we observed intra-cytoplasmic filamentous structures in ouIr prepa-rations. Tissue-phase PPLO were generally ob-served to parasitize medium and large-sizedepithelial cells. The precise origin of these cells isuncertain. They appeared to originate from theregion of stratified or pseudostratified columnarepithelium, but resembled metaplastic, roundedsquamous, or transitional type cells. Followingsuccessful treatment, tissue-phase PPLO could nolonger be demonstrated in epithelial scrapings,nor could T strain pleuropneumonialike or-ganisms any longer be recovered in culture.To facilitate documentation of our observations

an arbitrary morphological classification of thepredominant tissue-phase PPLO observed inparasitized epithelial cells was adopted. Thefollowing morphological categories were estab-lished: (1) coccoid and coccobacillarv forms;(2) short and elongated bacillary forms; (3)annular forms; (4) bipolar forms. Further, twoadditional categories were provided to includethose PPLO less frequently encountered, butconsidered highly characteristic of the pleuro-pneumonia gI-oup, and for viiruslike aggregationsand inclusions interpreted to be of PPLO origin.In all instances where intracytoplasmic PPLOwere observed in stained uiethral epithelialscrapings, T strain PPLO colonies were recoveredin primary culture in large numbeirs and fre-quently in pure culture. The only exception wascase No. 202, which showed PPLO inclusionsbut yielded negative cultures for PPLO.

Coccoid and coccobacillarlJ forms. In centratstto the gener-al )lec)rnorlphisall of the pleuiro-

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pneumonia group, we have on many occasionsobserved predominantly monomorphic forms inintracytoplasmic position in parasitized epithelialcells. The organisms appeared round or ovoid inshape, and occurred singly, in pairs and rarely,in short chains of from 3 to 5 elements (figure 3).The stained particles were discrete, generallyevenly stained, and estimated to be between300 and 450 m,u particle size by optical measure-ment methods. Coccoid and coccobacillary formsappeared more deeply stained than other mor-phological forms, possibly as a result of the uni-form distribution of protoplasm within the PPLOparticle. Representative illustrations of pre-dominantly coccoid and coccobacillary forms areshown in figures 1 and 2.

Short and elongated bacillary forms. The term"bacillary" is used in the general, descriptivesense, to denote elongated forms-not bacterial"bacilli" and not funguslike filaments. Theseforms have been regularly observed to exhibit ahigh degree of pleomorphism. The short pleomor-phic bacillary form was the PPLO tissue-phasemost frequently observed in our preparationsfrom the mucous membrane of the anteriorurethra from cases of NGU. The organismsoccurred as small, short bacillary bodies, straightor curved, frequently rickettsialike or comma-shaped, tapering in outline to a thin, almostinvisible point at one end, occurring singly andin pairs. An additional form was frequentlyobserved in the cytoplasm of parasitized cellsexhibiting the short bacillary form, and is bestdescribed as triangular in outline. Its solidstaining reaction and density was identical tothat of the short bacillary form with which itfrequently occurs (figures 4 and 5) and it isinterpreted to be a morphological variant of theshort pleomorphic bacillary form. Elongatedbacillary PPLO forms were less frequently seen.They were very pleomorphic and gave the im-

pression of being highly plastic structures (figures6 and 13). This interpretation may explain theoccurrence of bizarre, elongated structuressometimes encountered in this morphologicalcategory.

Annular forms. This term was adopted toinclude a variety of circular and elliptical PPLOwhich are characterized chiefly by the lack orthinness of stainable material in the centralregion of the organism. The protoplasm appearedto be concentrated at the periphery of the circularPPLO particle-evenly distributed in the formof a ring, or unevenly distributed, yieldinglopsided rings, "signet rings," and monopolarforms. Included in this group are forms whichappeared as two particles interconnected by astrand of stainable material in such a manner asto suggest earmuffs (figure 13). Due principallyto the manner in which the protoplasm wasdistributed or concentrated, the staining reactionof annular forms was generally very delicate(figures 7 and 13). They are nonetheless easilvrecognized by virtue of their highly characteristicmorphology.

Bipolar forms. This is the classic bipolar PPLObody. In stained epithelial scrapings it appearedas a small (400 by 700 m,u average) ovoid bodyshowing a stained circular particle at each endand, except for a trace of stainable materialwhich forms the ovoid periphery of the bipolarbody, devoid of stainable material between them.This is perhaps the most easily recognized formseen in clinical material, both in extracellularand in intracellular position (figures 8 and 13).

Multipolar and developmental forms. Therewere other forms of pleuropneumonialike or-ganisms seen in epithelial scrapings which wereoccasionally observed to occur along with theforms described above. Most notable of thesewas a triangular group of three stained l)articleswhich appeared to be interconnected by a thin

Figure 1. Parasitized epithelial cell showing coccoid and coccobacillary forms of pleuropneumonialikeorganisms. 1,560 X.

Figure 2. Two of three adjacent epithelial cells showing intracytoplasmic coccoid and coccobacillaryforms. 1,560 X.

Figure S. Coccoid forms and short, chainlike structures. Note also tendency toward short, bacillaryform. Also visible is a bipolar form exhibiting a germinating body from one of the two polar particles.1,560 X.

Figures 4 and 5. Pleomorphic, short bacillary forms of pleuropneumonialike organisms. The cellshown in figure 4 is heavily parasitized and is showing early signs of disintegration. 1,560 X.

Figure 6. Parasitized epithelial cell showing elongated, bacillary forms developing in two well-definiedareas of the cell cytoplasm. Also visible are two "earmuff" forms. 1,560 X.

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strand of stainable material. The particles wereroughly equidistant from each other by a lengthapproximately one to one and a half times theirparticle diameter (300 to 450 m,u). More rarelyobserved was an elongated diamond-shaped groupincluding four such particles.Another form of PPLO occasionally observed

in the cytoplasm of parasitized epithelial cellswas that of a straight or curved sprout or germi-nating body. These "germinating bodies" ap-peared to originate from either coccoid, bipolar,or annular forms, and are interpreted as develop-mental structures (figure 3 and bottom of figure13). The staining reaction of these developmentalforms was identical to that exhibited by thepleomorphic bacillary forms. They are mor-phologically similar to a developmental formshown in an electron micrograph of pus from aPPLO-infected rat (Klieneberger-Nobel andCuckow, 1955).

Viruslike intracytoplasmic aggregations. Virus-like aggregations (one or more) have been ob-served within the cytoplasm of certain cells.Their staining reaction was identical to that ofthe various forms of PPLO described above.Moreover, close examination revealed that theviruslike aggregates were composed of structureswhich, both in size and morphology, were char-acteristic of organisms of the pleuropneumoniagroup. Small to large colonylike intracytoplasmicaggregates were observed in certain cells (figures8 and 9), numbering from one to three suchaggregates per cell. They occupied a positionnear the cytoplasmic membrane or adjacent tothe nucleus. Viruslike "crescents" have beenobserved developing adjacent to and huggingthe nucleus of the cell (figure 10). Such manner

of development, in our experience, was a rareoccurrence. Nevertheless, the existence of intra-cytoplasmic "crescents" due to pleuropneumonia-like organisms should be noted. Large, basophilicintracytoplasmic inclusions have been observed(figure 11) which contained large numbers ofbasophilic particles of characteristic PPLOmorphology, size, and staining reaction. Thecultures from the individual from whom thisurethral scraping was made grew out largenumbers of T strain and TR strain (Shepard,1956) PPLO colonies in almost pure culture. Inaddition to the viruslike structures describedabove, smaller, cystic structures were observed inrare instances in intracytoplasmic position(figure 12) which showed internal particles ofcharacteristic PPLO morphology, size and stain-ing reaction. The cell illustrated in figure 12shows a typical bipolar PPLO body in additionto coccoid and coccobacillary forms containedwithin two of the eystic structures. Severalparticles were also developing in extracysticposition in the cell cytoplasm. With the exceptionof the latter instance (epithelial scraping fromcase No. 202 positive; single culture negative forPPLO of any type), large numbers of T strainPPLO colonies were recovered in primary culturefrom all individuals exhibiting the viruslikestructures described above.

DISCUSSION

The classic morphology of organisms of thepleuropneumonia group, as seen in appropriatelystained preparations from cultures, has beenshown to consist of minute, basophilic structuresin the form of granules, globules, larger roundbodies, ring forms and finely curved filaments

Figure 7. Parasitized epithelial cell showing an intracytoplasmic mass composed chiefly of annularforms. Pale staining reaction is characteristic. Also present, although not readily visible, are monopolarforms ("signet rings"), open triangles, and diamond-shaped structures. 1,560 X.

Figure 8. Three small, colonylike aggregates. Note the bipolar form in the lower group. 1,560 X.Figure 9. Three large, colonylike aggregates. The two adjacent cells (partially visible above and

right) also showed intracytoplasmic, large, colonylike aggregates of pleuropneumonialike organisms.1,560 X.

Figure 10. A single large, colonvlike aggregate developing adjacent to the cell nucleus in the mannerof a virus "crescent." 1,560 X.

Figure 11. A viruslike intracytoplasmic inclusion body is shown below the cell nucleus. The denseiportion of the inclusion body is packed with particles identical in morphology to those visible in thethinner area of the inclusion. 1,560 X.

Figure 12. Cystic intracytoplasmic inclusions. Three of the cystic structures are showing typicalbasophilic elements. Coccoid and coccobacillary forms are also present in extracystic position. althoughnot in focus. 1,560 X.

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Figure 18. Parasitized epithelial cell showing intracytoplasmic tissue-phase inclusions of pleuro-pneumonialike organisms. A majority of the possible morphological forms of tissue-phase pleuropneu-monialike organisms is exhibited within the cytoplasm of this single cell at the same time. 3,200 X.

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(Bordet, 1910; Borrell et al., 1910; Sabin, 1941;Edward, 1954). Pleomorphism was considered tobe one of the distinguishing characteristics ofmembers of the pleuropneumonia group or-ganisms. Further, an elementary body stage wasfound to exist which yielded viable particlesestimated by filtration experiments to range from125 to 250 m,u in size. The excellent electronmiciographs of pleuropneumonia group organismspublished by Klieneberger-Nobel and Cuckow(1955) clearly illustrate the nature of some of thepolymorphic structures which characterize thegroup.

Relatively little is known, however, of theappearance and morphology of pleuropneumoniagroup organisms in clinical material from humansources. One of the earliest descriptions of pleuro-pneumonialike organisms in human clinicalmaterial is that of Johnston and MeEwin (1945),who reported finding small, intracytoplasmiccoccobacillary basophilic inclusions ("elementarybodies") in Giemsa and Castaneda staine(durethral epithelial scrapings from two cases ofnongonococcal urethritis. Pleuropneumonialikeorganisms weere recovered in culture from bothindividuals. Harkness (1945) noted the presenceof rings accompanying elementary bodies inGiemsa stained material from cases of kerato-dermia blennorrhagica, and suggested that ringswere developing from the granules and that thelatter may have been a granular stage in thedevelopment of PPLO.The presence of polymorphic structures, in-

cluding minute, discrete, ovoid or rod-shapedparticles and vibrio, rod, and coccoid forms,was reported by Williams (1946) in urethralscrapings from cases of nongonococcal urethritis.The most striking forms observed were minuterings with one or more thickenings at the poles.In some scrapings, only certain morphologicalcategories predominated (vibrios, rods, or coccoidforms, or ring forms). The organisms stainedbluish purple wvith Leishman's stain and wereoften arranged in small masses or clusters in thecytoplasm of large epithelial cells. The particlesize was estimated to be between 300 and 500m,u. The organisms were not found in leucocytesor in smaller epithelial cells, and intranuclearforms were not observed. The intracytoplasmicinclusions were considered to be an intracellular(tissue) phase of the pleuropneumonialike or-ganism. Our observations, based on examination

of urethral scrapings froim cases of NGU fromwhom T strain PPLO were recovered in culture,are in close agreement with those of Williams(1946). Unfortunately, his observations were notsupported by adequate PPLO culture studies onthe NGU cases from whom urethral scapingswere taken. Harkness and Henderson-Begg(1948) and Harkness (1950) described similarintracytoplasinic structures in Giemsa stainedepithelial scrapings from NGU cases includingsmall, bluish staining spherules, ovoids, rickettsia-like forms and elementarylike bodies, with a highproportion of ringlike formns. In rare instances onlyhave we observed predominantly either ring formsor bipolar forms. Illustrations (Harkness, 1950)depicting only these forms as representative of themorphology of pleuropneumonialike organisms instained epithelial scrapings, may be misleading. Inmany of the cases reported by Harkness and Hen-derson-Begg in which cultures were positive forPPLO, positive urethiral scrapings were obtained.A higher proportion of positive scrapings thanpositive cultures for PPLO was reported. Thisfinding is not in agreement with our observations.We feel that properly performed cultures, em-ploying an appropriate culture medium, providethe suipeirior method of demonstrating PPLOin clinical material from human sources. Thedirect microscopic examination of stained urethralscrapings, as herein described, is the more rapidmethod but is considered at present to be lessreliable than the culture method.

W\e have experienced occasional failures indemonstrating pleuropneumonialike organisms inepithelial scrapings from cases of NGU whichhave yielded large numbers of T strain PPLOin culture. The explanation for these failures isnot too well understood. Klieneberger-Nobel(1954) stated that recognition of pleuropneu-monia group organisms from infective lesions wasvery difficult, and that she tried unsuccessfullyfor many years to identify these organisms inclinical material. Several factors undoubtedlyinfluence the success or failure of the directmicroscopic method. Williams (1946) foundPPLO inclusion bodies present in decreasingnumbers during the first week of nongonococcalinfection and usually absent in the second week,although pus cells and epithelia persisted. Ourexperience indicates that PPLO inclusions, whenpresent early in the course of NGU infection,may persist for longer periods, although de-

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creased in numbers. Chlonic NGU infections maNrequiie repeated scrapings to demonstrate tissue-phase PPLO. In addition, a stage of predomi-nantly elementary bodies at certain periods,consisting of particles 125 to 250 m,u in size,could easily escape detection. It is felt thatproper choice of clinical material and treatment ofsmears (scrapings) prior to staining play a con-siderable part in the outcome of the procedure.The collection of exudate alone has proved unsat-isfactory and will generally yield negative find-ings, due principally, we feel, to the scarcity anddegenerative character of epithelial cells in suchmaterial and the difficulty encountered in recog-nizing certain of the extracellular PPLO. Theclinical material of choice in NGU is a scrapingfrom the region of urethral epithelium posteriorto the fossa navicularis. Failure to immediatelyfix slides bearing still wet epithelial scrapingsmay, under certain conditions, result in alterationor destruction of the organisms before they canbe stained.The association of pleuropneumonialike or-

ganisms with nongonococcal urethritis in humanmales is complicated by the fact that the morecommon primary isolation media may fail tosupport development of T strain PPLO, or thatdevelopment of T strain colonies may occurunrecognized. In either instance (in the absenceof other PPLO types) a negative PPLO culturewould be reported. However, the demonstrationof tissue-phase PPLO in stained urethral epi-thelial scrapings, in the face of possible falsenegative culture findings would provide directmicroscopic evidence of pleuropneumonialikeorganisms in such clinical material.Under such circumstances, however, the

associative picture may be further complicatedby confusion of tissue-phase PPLO with virusinclusions in stained urethral scrapings. Thevirus inclusions of Chlamydozoon oculogenitalebear, in many cases, (Durel et al., 1950; Borel,1950; Brisou, 1951; Moustardier et al., 1954)striking resemblance to certain tissue-phasePPLO inclusions we have observed (figures 8-11).Further, our observations suggest that the useof such terms as "monomorphic" vs. "pleomor-phic" (Harkness, 1950) or "monomorphic" vs."polymorphic" (Moustardier et al., 1954) tomorphologically distinguish virus inclusions frominclusions due to PPLO, may be misleading. Wehave observe(d "monomorphic" inclusions re-

peate(lly in scrapings from NGU cases from whomonly T strain pleuropneumonialike organismswere r-ecovered in culture. In the absence ofappropriate culture technique for T strain PPLO,the virus term "inclusion urethiritis" coulderroneously be applied. The awareness of thesemorphological similarities in stained urethralscrapings should be emphasized and their signifi-cance interpreted with caution in the etiologicalassessment of nongonococcal urethritis.

SUMMARY

Intracytoplasmic inclusions observed withinepithelial cells from cases of nongonococcalurethritis in human males are interpreted to bethe tissue-phase of T strain pleuropneumonialikeorganisms (PPLO). They were found to exhibitstructures that are consistent in size, morphology,and staining reaction to those of the pleuro-pneumonia group as a whole. They are char-acterized by their minute size and exhibition ofcharacteristic polymorphic structures and bytheir staining ieaction as visualized in Giemsa oIrazure-eosinate stained epithelial scrapings. Theformation of characteristic particle aggiegationsand viruslike inclusions is discussed.The demonstration of intracytoplasmic PPLO

inclusions in stained epithelial scrapings fromindividuals yielding only T strain PPLO inculture, provides additional evidence that Tstrain PPLO are newly recognized members of thepleuropneumonia group organisms. Followingsuccessful treatment, PPLO inclusions could nolonger be demonstrated in stained epithelialscrapings, nor could T strain pleuropneumonia-like organisms again be recovered in culture.

REFERENCES

BORDET, J. 1910 La morphologie du microbede la p6ripneumonie des bovid6s. Ann. inst.Pasteur, 24, 161-167.

BOREL, L. J. 1950 Techniques de la recherchedu Chlamnydozoon-oculo-ggnitale et du "pleuro-pneumonia-like organism" en blennorrologie.La Prophylaxie Antiv6n6rienne, 22, 308-323.

BORRELL, DUJARDIN-BEAUMETZ, JEANTET, AND

JOUAN 1910 Le microbe de la p6ripneu-monie. Ann. inst. Pasteur, 24, 168-179.

BRISOU, M. J. 1951 Les urethrites a virus. J.Med. Bordeaux, 128, 1063-1067.

DUREL, P., ROIRON-RATNER, V., SIBOULET, A.,AND BOREL, L. J. 1950 Blennorragie A in-clusions cellulaires; interet de l'aur6omycine.Sem. Hop. Paris, 26, 3386-3398.

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EDWARD, D. G. 1954 The pleuropneumoniagroup of organisms: a review, together withsome new observations. J. Gen. Microbiol.,10, 27-64.

HARKNESS, A. H. 1945 The cutaneous manifes-tations of gonorrhoea. Brit. J. Veneral Dis-eases, 21, 93-113.

HARKNESS, A. H. 1950 Non-gonococcal ure-thriti.s. E. and S. Livingstone Ltd., Edin-burgh.

HARKNESS, A. H. ANI) HENDERSON-BEGG, A.1948 The significance of Pleuropneumonia-like or "L" organisms in non-gonococcalurethritis, Reiter's disease and abacterialpyuria. Brit. J. Venereal Diseases, 24, 50-58.

JOHNSTON, G. A. W. AND McEwIN, J. 1945 Non-gonococcal urethritis: considerations of aeti-ology; findings in two cases. Med. J. Aus-tralia, 1, 368-371.

KLIENEBERGER-NOBEL, E. 1954 Micro-organ-isms of the pleuropneumonia group. Biol.Revs., 29, 154-184.

KLIENEBERGER-NOBEL, E. AND CUCKOW, F. W.1955 A study of organiisms of the pleuropneu-monia group by electron microscopy. J. Gen.Microbiol., 12, 95-99.

LILLIE, R. D. 1947 In Staining Procedures, com-piled and edited by H. J. Conn and M. A.Darrow. 2nd ed. leaflet, Part III, Micro-

organisms; Section B, stains for microorgan-isms in sections, pp. 6-7. Biotech. P'ublica-tions, Geneva, N. Y.

LILLIE, R. D. 1948 Histopathologic technic. TheBlakiston Company, Philadelphia.

MAXIMOW, A. A. AND BLOOM, W. 1952 Y'extbookof histology, 6th ed. W. B. Saunders Com-pany, Philadelphia.

MOUSTARDIER, J., BRISOU, J., AND PERREY, M.1954 A propos des ur6trites A inclusions.Ann. dermatol. syphilig. 81, 521-527.

PAPANICOLAOU, G. N. 1954 Atlas of exfoliativecytology, pp. 5-6. Harvard University Press,Cambridge.

SABIN, A. B. 1941 The filtrable microorganismsof the pleuropneumonia group. Bacteriol.Revs., 5, 1-66.

SHEPARD, M. C. 1954 The recovery of pleur o-pneumonia-like organisms from Negro menwith and without nongonococcal urethritis.Am. J. Syphilis, Gonorrhea, Venereal Dis-eases, 38, 113-124.

SHEPARD, M. C. 1956 T-form colonies of pleuro-pneumonialike organisms. J. Bacteriol., 71,362-369.

WILLIAMS, S. 1946 Non-gonococcal urethritisin Australian troops stationed in Borneo.Med. J. Australia, 1, 693-695.

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