INTERNATIONAL JOURNAL of SYSTEMATIC BACTERIOLOGY Vol

INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY Vol. 16, No. 2 April 1966 pp. 223-252

REVISION OF THE FAMILY CHLAMYDIACEAE RAKE (RICICETTSIALES): UNIFICATION OF THE PSITTACOSIS- LYMPHOGRANULOMA VENEREUM-TRACHOMA GROUP OF ORGANISMS IN THE GENUS CHLAMYDIA JONES, RAKE AND STEARNS, 1945'

L.A. Page

U. S. Department of Agriculture, Agricultural Research Service, Animal Disease and Parasite Research Division, National Animal Disease Laboratory, Ames, Iowa

ABSTRACT. Since 1945, taxonomists have made no less than seven attempts to define the bacteria of the family Chlamydiaceae Rake which includes the etiologic agents of psittacosis, lymphogranuloma venereum, and trachoma. These bacteria are often designated by the vernacular term, PLT group of agents. Clas- sifications were proposed by Moshovsky (1945), Rake (1 948, 1957), Krasil'nikov (1 949), Zhdan- ov and Korenblit (1949), Rhyzkov (l950), Zhdanov (1953) and Levaditi, Roger and Des- tombes (1964). The result was to increase the number of genera for the PLT group cul- minating in the Levaditi Et al. proposal of four genera for this bacterial group: Chlam- ydia Rake, Miyagawanella Brumpt, Bedsonia Meyer, and Rakeia Levaditi, Roger and Des- tombes. Evidence for continued recognition of these genera was examined in the light of recent knowledge of PLT organisms, and the validity of each name according to the rules of the International Code of Nomenclature of Bacteria and Viruses was ascertained. On the grounds that the taxonomic significance of properties common to bacteria of the PLT

In addition to the authpr, the following members of the Subcommittee on the Chlamydiaceae of the American Society for Microbiology's Taxonomy Committee have reviewed the manuscript and support the proposal: F. B. Gordon, J. W. Moulder, E. Jawetz, and M. Pollard. 224 I N 'T Ra A. T I 0 N A L J OU R N A L group (morphology, developmental cycle and group antigen) outweighed the value of the characteristics that separated the various species or strains (host preferences and specific antigenicity of lipoproteins of the cell wall), it was concluded that all of the species of bacteria of the PLT group properly be- longed in a single genus. Therefore, their unification in the genus Chlamydia Jones, Rake and Stearns, 1945, is proposed. The validly published generic name Chlamydia re- places Miyagawanella Brumpt 1938 which was not validly published and has priority over Bedsonia Meyer (1953) and Rakeia Levaditi, Roger and Destombes (1964). Furthermore, the term "virus" is incorrect and misleading when applied to these bacteria.

The development of a generally acceptable nomenclature for organisms of the psittacosislymphogranuloma venereum- trachoma (PLT) group has been hindered by serious disagreement among microbiologists as to the correct name for these bacteria. In the last two decades, no less than seven attempts, including three repetitive ones , have been made to define and name the members of the PLT group within the families Chlamydozoaceae and Ehrlichiaceae (Moshkovsky, 1945) or Chlamydiaceae (Rake, 1957) while the number of genera proposed by various authors has in- creased from Moshkovsky's original two to ten, some of which represent proposals of synonyms (Table 1). For the most part, the new generic names appeared, along with additional species names, in each new emendation of descriptions (Chlamydozoon, Ehrlichia Moshkovsky, 1945; Rickettsiaformis Zhdanov, 1953; Chlamydia Rake, 1957; Rakeia Levaditi, Roger and Destombes , 1964; Colesiota Rake, 1948, Ricolesia Rake, 1957, and Colettsia Rake, 1957, the latter three genera being for Coles' conjunctivitis organisms. Three distinct proposals for generic names were made apart from any attempt to circumscribe the group as a whole as inMiyagawanella Brumpt, 1938, Chlamydia Jones, Rake and Stearns, 1945, and Bedsonia Meyer, 1953). This proliferation of names most of which are synonyms has produced understandable restraint, if not confusion, among microbiologists in referring to organisms of the PLT SYSTEMATIC BACTERIOLOGY 225

Table 1. Condensed classification schema of various authors for the family Chlamydiaceae (Rake). (Names are listed as the author listed them without regard to error)

- .- -

3) conjunctivae Cobs nom. nov. 3 conjunctivae Coles Hosh. 2) conjunctivae-gai Coke nom. nova 4) conjunctivet-b&a C. n~m.

brlichiaceae

.. EbrUchia bsh. . Miyag-ellaBrruapt 1) lymphogranulomatosis Brumpt '1) IymphograuulOnatis. Br.

2) paittacii Lillie 2) psittaci Lillie 3) ornithosis sp. UOV.

3) broachopneurmniae Mosh. bronchopneumoniae %ah.

other species) 4) cds (Don. & Lesto.) 5) ovina hat. & Don.) 6) bods )Don. & Lesto.) 7) kurlovi (Mosh.) 226 INTERNATIONAL JOURNAL

Table 1 (continued)

--.- Cnlamydla Rake 1 tracEo~EF&p-- ><&enitale Mosh.

_lo, I 32- 12. I 3) conjunctivae Coles Moah. 13) conjunctivae 3) pecoris manov & bren. 13. 14. b) conjunctivae-galli Colts 4) pullorum 7&K 15* a) conjunctivae-bovis bsh. kerato-conjunctivae' 16. 16) 17. 18. 19. 20 * 2L hrlichiaceac 4:22; -.2. . Ehrllchia 25. lymphogranulometoeis Mosh. 10) lymJhophilus* 26. a) 27. 28. 29. 30 * 3. d) pnewniae Bergey et ~l. 32. 33.* 35. %. 11 opposum nm. MY. 37. ovia sp. nov. 14)151 bods ZBdc 38.39. other species) :other species ) other species) 40.41. 5 nrningophilus* 42. 9 mnocytaris* 43. 8 osteophilus* 4) columbarum ~p.nov. 44. 6 cricetia* 6) pneumolncningitidie sp.mv. 45. bpparentI SP. nov., 9) roaentium nov. EP. 46. lorn. nov., etc. thou& 47. lot so designated by 48. Luther. 492 SYSTEMATIC BACTERIOLOGY 227

Table 1. (continued)

iri Rake (Bergey's No. 7th)~_- 1956 Leviditi, Roger, Destombes 1$4* 1. MICROTATOBIOI%S Philip SCEIZCMYCFPES von Baegeli 2. RIMBTTSIALES Buck&Buch. I ~CKE!ITSIALES Buch.-&.J&ch. - 3. __-____a-IEr~~&bb;-oov- - lanprdidae L. R- and D. 4. ' 2. Chlaydioideae (new subfamily) 5. 1. Chlmieae (new tribe) 6. r;. ~~-=a 1. Chlwdia Rake 1 trachomatfs Busacca 7. 2. oculogenitale Mosh. a. - 2 oculogenit&e Moeh. 9. 1. Colesiotoideae fnew subfamily) 10. - __ ___ ----- 1. Colesioteae (nev tribe) __ ll. 2. Colesiota Rake - 1. mi-- RIILI. 12. 1)co~Ju"ctivac~!L~------Ricolesia gen. nov. 2. RicouRake 13% 3. conjunctivae Colee nov. conjunctivae &lee Rake i4. 21) bovis Cole8 comb. nov.comb. 1) 15 - 16. 31 caprae coles comb. nov. 17 * 4 lestoquardii Don. & Gayvt ____ - -_I_ -__ 1s. . 2. Colettsieae (new tribe) - 19. . 20. 1) pecoris ~akc 21. 22. 23. 24. 25 * 26. 27. 28. 29. 30 - 31. 32. 33. 2) fells Rake 34. 35. * 9) opossumi Ryzhkov 36 10) Oy18 ZManOV 37. 38. 11) bods York & Baker 39. 12) pecoris Rake 1) pecoris Rake 40. 41. * Ihe authors' original names for 42. subfamilies and trike utilized endings 43. derived from the Botanical or Zoological 44. Nomenclature Codes. Tnese endings were 45. corrected to proper form according to 46. the Bacteriological Code. 47. 48. 49. .... --.-- 228 INTERNATIONAL JOURNAL group, and many authors, even in systematic studies, have resorted to the use of hyphenated monstrosities abbreviated as PLV (psittacosis- lymphog ranuloma vene reum) or even POMP (Psittacosis - ornithosis - mammalian - pneumonitis) (Terzin, 1958,). The names applied to the numerous diseases in birds and mammals caused by these organisms, has been further complicated: Meyer (1940) proposed the name "ornithosis'' for the diseases of non-psittacine birds caused by members of the PLT group, hence the etiologic agents were ornithosis agents. On the presumption that uniform coding was desirable for the grouping of new isolates of organisms causing trachoma and inclusion conjunctivitis, a group of microbiologists working with these organisms have agreed to a "Nomenclature of isolates of virus from trachoma and inclusionblenorrhea" (Gear $ &., 1963). This terminology involved the use of the abbreviation TRIC for TRachoma-inclusion _Conjunctivitis with additional coding for immunological grouping, country of origin, isolation number, tissue of origin, and ultimate diagnosis. Additional disagreement also exists on whether to desig- nate PLT organisms as viruses, rickettsiae or something neutral as agents or organisms. A summary of the most recent terminology used to describe organisms of the PLT group in titles of research papers published in the last 5 years illustrates the current range of terms (Table 2). Among the systematists, the taxonomic position of the whole group has remained unchallenged; that is, the PLT organisms, with minor variation, have been placed in the order Rickettsiales, separated by family from the true rickettsiae and clearly distinct from viruses as defined by Lwoff (1957). Recent studies by many workers on the morphology, cytology, chemical nature and independent metabolism of PLT organisms as summarized by Moulder (1964, 1966) has produced sufficient evidence to warrant recognition of organisms of the*PLT group as bacteria and not as viruses. Furthermore, those committed in recent years to the study of the taxonomy of viruses have not included organisms of the PLT group in that category (Andrewes, 1964; Huck, 1964; Wilner, 1964). Thus, the term virus has become incorrect and misleading for the etiologic agents of psittacosis, lymphogranuloma venereum, trachoma and the otherdiseases of man or animals caused by members of the PLT group. In the light of recent well authenticated research clarify- ing the relationship of the PLT group to other organisms, SYSTEMATIC BACTERIOLOGY 22 9

Table 2. Designation in titles of journal articles for PLT organisms or complexes thereof (1960-May 1965)

- Indexes* for , Terminology 1965 1964 1563 1962 1961 1960 Total Psittacosis (virus, group, agent)* 16 10 8 12 15 14 75 Ornithosis (virus, group, 18 agent 1 12 12 5 4 51 Psittacosis-Ornithosis (or reverse) virus, group 111 58

Psittacosis-lymphogranuloma 51 or PLV 3 9 1322

Psittacosis-lymphogranuloma- trachoma or PLT 2 3 32 3 114 Ornithosis-Lymphogranuloma 1

Ornithosis bedsoniae 1 1

Bedsonia 132 17 Miyegawanella 211 228

Trachoma-Inc lusion Con junctivities 58 13

Neorickettsia or Para- rickettsia 2 28 3 15 Bacteria 11 2

* Titles checked in: Index Veterinarius, 1961-1964; Index Medicus, 1960, 1963, 1965; Biological Abstracts, 1964-1965. ++ The term virus was the prevalent uswe. 230 INTERNATIONAL JOURNAL it was important that a proposal be developed that was both taxonomically satisfactory and nomenclaturally correct. The present paper is such an attempt. The task of preparing a proposal which blended the de- sires of microbiologists for convenient terminology with the requirements for correct nomenclature consisted of (1) re- viewing all past proposals of names for the taxa of the PLT group for their validity of publication, legitimacy and cor- rectness in accordance with the rules of bacteriological nomenclature expressed in the International Code of Nomen- clature of Bacteria and Viruses (1958); (2) avoiding proposals of new names for the taxa of the PLT group on the grounds that a terminology based upon names previously proposed was preferable. Selection of generic names and names of type species and strains for PLT taxa was made with due consideration of past proposals, current nomenclatural needs, and current research on the PLT group; (3) the conclusions and recommendations of this study were reviewed by competent authority consisting of persons mentioned in a footnote to the front page. Yet to be accomplished is the submission of the conclusions of this paper to the Judicial Commission of the Inter- national Committee on Bacteriological Nomenclature for consideration and any action that might be required or ap- p ropriate .

History of the Development of Nomenclature of Organisms of the PLT group

The background to the initial discoveries and early descriptions of the first named taxa of the PLT group are ably provided by Thygeson (1934,1962) and Jawetz (1964), Meyer (1953) and Bedson (1959). The reader is referred to these articles for details; however, a summary of the past nomenclatural proposals related to this group and their rationale follow s : Nocard (1893) proposed the name Bacillus psittacosis for the purported etiologic agent of the disease psittacosis (Greek psittakos - parrot). The organism he described was probably Salmonella typhimurium which was often isolated from parrots concurrently infected with psittacosis, and oc- casionally from man; hence, he considered it to be the cause of psittacosis. This opinion prevailed until 1930 when at least five physicians and their colleagues, separately and concurrently , de s c r i be d the f ilt e r able, obligate int r acellula r SYSTEMATIC BACTERIOLOGY 231

parasite associated with psittacosis in parrots and man (Bedson, Western and Simpson, 1930; Lillie, 1930; Coles, 1930; Levinthal, 1930; Krumwiede ct 51. 1930). In two articles published in Lancet in February, 1930, Bedson, Western and Simpson pointed out the filterability of an infectious principle in tissues from parrots and from a man with clinical psittacosis and their failure to isolate any organism able to grow on bacteriological media. These authors were able to reproduce the disease in budgerigars and chickens but proposed no name for their filterable agents. Two months later, in an April 11, 1930, issue of the U. S. Public Health Reports, R. D. Lillie compared the intracellular organisms encountered in focal lesions in parrots and a human and concluded:

"Such minute intracellular coccoid and rod-shaped Gram-negative organisms which have not been cultivated are usually designated as Rickettsia, in which genus it would appear that they should provisionally be included. The specific name psittaci was chosen as indicating a connection with parrots. The name Rickettsia psittaci is herewith proposed and the species is defined as minute Gram- ne gative int racellula r co c coid and bipolar bacilliform bodies of about 0. 2p to 0. 3 p in diameter, found in reticulo-endothelial cells, mesothelial cells, and large mononuclear cells of the parrot (probably Amazon sp.) and in large mononuclear cells in man, associated with, but without established relationship to, psittacosis.I'

Today, members of the present genus Rickettsia are readily distinguished from PLT organisms on the basis of their antigenicity, pathogenicity, and metabolic patterns. While there are some similarities in size and affinity for dyes , the rickettsiae form a natural group metabolically distinct from PLT organisms (Bovarnick, 1956; Weiss, 1964, 1965). Thus, Lillie placed the etiologic agent of psittacosis in the previously described and named genus Rickettsia. The name was validly published, but the organism was later transferred to another genus. Coles reportedinthe May 20, 1930 issue of Lancet the finding of minute bodies in tissue smears from a chickenand two mice which had been inoculated with infectious material from cases of human psittacosis. He observed paired cocci 0. 6-0. 7 p in length and 0. 3-0. 4 p in diameter and opined: 232 INTERNATIONAL JOURNAL

"To my mind, they very closely resemble some of the rickettsia bodies. I compared them with a slide of Rickettsia prowazeki of typhus fever in the louse and they bore a striking similarity to the coccoid and paired coccoid stage of these bodies.. . The coccoid bodies are small enough to pass the Seitz filters and have been very definitely found in the filtrate, and these may prove to be the causal organisms of this obscure disease. They might provisionally be termed the coccoid X bodies of psittacosis."

Coles did give an English vernacular name which was quite proper. Although his description was validly published, he did not choose to propose a scientific name. Similar independent observations of the agent of psittacosis were made by W. Levinthal who reported his findings at the 1st International Congress of Microbiology in Paris in 1930 (published in 1932). He suggested the name Micro- bacterium multiforme psittacosis based on his clinical and micros copis obs e rvations . Levinthal' s trinomial is ille gi- timate for two reasons: Code rule 24d making later homo- nyms unacceptable and Code rule 6 which rules out trino- mials. In this case, Levinthal, perhaps unintentionally, used a generic name previously validly published by Orla Jensen in 1919 for the lactic acid bacteria. Throughout the period between 1930 and 1938, Bedson and his colleagues continued to study the relationship between the intracellular particles seen in infectious tissues from psittacosis cases and the disease in laboratory animals. He was the first to describe the unique developmental cycle, immunity, antigenic composition, and use of complement fixation tests for diagnosis of psittacosis. Bedson summarized his classical studies of psittacosis in the Harben Lec- tures of 1958 (published in 1959). Throughout all this work, Bedson maintained an extraordinary restraint in connection with the nomenclature of these organisms, preferring ap- parently, to await accumulation of sufficient evidence to warrant a definitive nomenclature. It was unfortunate that he gave no names to the organisms he described. No such hesitancy was exhibited by those persons working with trachoma. In the early thirties, the intracellular bodies originally described by Halberstaedter and von Prow- azek in 1907 were observed again by a new generation of workers and given names. In 1907, Halberstaedter and von Prowazek had proposed the name Chlamydozoa (from Gr. SYSTEMATIC BACTERIOLOGY 233

chlamys = cloak and zoa = animals) for a taxon coordinate with protozoa and bacteria. The purported etiologic agents of several diseases (including trachoma) were placed here but without species names other than vernacular names. Later (1907) von Prowazek proposed the generic name Chlamydozoon with a single species G. bombycis for the agent causing the "yellow disease" of the silkworm. This is the type species of the genus Chlamydozoon. He included the description of the etiologic agent of trachoma but used only a vernacular designation. The more recent conclusion that the etiologic agent of trachoma belongs with the bacteria and that the yellow disease is caused by a virus makes illegitimate the allocation of any species of bacteria to the genus Chlamydozoon. In 1933 and 1935, Busacca compared the trachoma organisms with Halberstaedter and von Prowazek's descriptions. On the basis of morphologic and tinctorial similarities of the trachoma organisms to the rickettsiae, Busacca proposed the name Rickettsia trachomae (sic). This. nomenclatural lapsus calami was corrected by Foley and Parrot (1937) to _R. trachomatis. These latter authors also regarded Rickettsia trachomatis as synonymous with Chlamydozoon trachomatis. The latter binomial was utilized in turn by Moshkovsky(l945) as the type species of the genus Chlamy- dozoon of his proposed family Chlamydozoaceae, and also by Rake in 1948 in Bergey's 6th Edition (Table 1). As previously pointed out by the Editorial Board of the Interna- tional Bulletin of Bacteriological Nomenclature ( 1955), the species name Chlamydozoon trachomatis was illegitimate as a name of a species of bacteria for the reason that CLa- mydozoon bombycis was validly published as a name for a virus. The family name Chlamydozoaceae was also illegitimate for use in bacteriology. Rake, in the 7th edition of Bergey's Manual proposed the family name Chlamydiaceae. He also placed the agents of trachoma in the genus Chlamy- -dia, a name proposed by Jones, Rake and Stearns (1945). The specific epithet for these organisms remained trachomatis. Later Zhdanov (1953) and Levaditi et 51. (1964) used the binary combination Chlamydia trachomatis. In 1935, Miyagawa and his colleagues published their four reports on the "Virus of lymphogranuloma inguinale Nicolas, Favre and Durand," in which he described the organism's morphology, filterability, transmission to laboratory animals and cultivatability in the chicken chorioallantoic nicni- brane. In 1935 the French parasitologist, E. Brumpt, \

Miyagawa's laboratory and was shown some slides of these organisms. In apparent appreciation for this courtesy Brumpt included a footnote in an article describing a new rickettsia1 species, ,R. culicis, a proposal for a new genus and type species Miyagawanella lymphogranulomatosis, in honor of Professor Miyagawa (Brumpt, 1938). His words in the March 1938 issue of Annales de Parasitologie in a footnote to the 5th paragraph, page 155 (in translation from the French) are:

"(2) The classification of the representatives of the family of rickettsiae is very difficult to establish; however, it appears that one can place, provisionally, alongside the genus Rickettsia of da Rocha-Lima (1916), [ a name] created for the parasites of world typhus and which is synonymous with the genus Dermacentroxenus Wolbach (1919), the genera Wolbachia Hertig (1936), Ehrlichia Mochkovski (1937), Ixodisymbiotes n. g. Brumpt, for the numerous polymorphic and specific symbiotes of the ixodides, and Miyagawanella n. g. Brumpt. I created [the name for] this last genus for the species ,M. lymphogranulomatosis n. sp., agent of climatic bubo which Professor Miyagawa showed and offered to me demonstrative preparations in Tokyo in November 1935. "It perhaps will be possible one day to include among the Rickettsia (sic) the undefined microbes of vaccinia (Cytoryctes vacciniae Guarnieri 1892), of rabies (Neuroryctes hydrophobiae Calkins), also those of the Borreliota Goodpasteur 1933, the human and animal variolas, of mollus cum contagiosum and various ultra- viruses in other diseases."

The validity of Brumpt's Miyagawanella is open to ques- tion. He employed the casualness of a footnote to propose a monotypic genus which he failed to describe. Rule 13 of the Code states:

"The name of a genus or of a subgenus is not validly published unless it is accompanied (a) by a description of the genus or subgenus; or.. . in the case of a monotypic new genus based on a new species, by a combined generic and specific description of one or more species included in the genus, SYSTEMATIC BACTERIOLOGY 235

(b) by a description of one or more species or by citation of a previous and effectively published description of one or more species included in the genus. I'

In actuality, Brumpt proposed a name for an organism whose taxonomic position was obscure to him for he could only place Miyagawanella alongside the genus Rickettsia "provisionally," and he suggested that various pox viruses and rabies virus be included among the rickettsiae. Rule 1.2 of the Code indicates that a name of a taxonomic group is validly published only if it has been definitely ac- cepted by the author who published it. In 1945, two attempts were made to recognize taxonomically the similarities among the organisms causing lymphogranuloma venereum, psittacosis, and trachoma. The first was by S. D. Moshkovsky of the Institute of Malaria and Medical Parasitology in Moscow. He had previously studied a rickettsia-like agent causing a leucocytosis of guinea pigs for which he established the monotypic genus Ehrlichia with the species canis in 1937. He also systemized a large group of obligateE. intracellular parasites under the name Chlamydozoa (Table 3). Moshkovsky applied this name to the group of organisms suggested by Brumpt (quoted above) as being generally related: that is, (1) rabies virus which Moshkovsky placed in the genus Neuroryctes (Calkins), (2) the Borreliota of Goodpasture, the Cytoryctes of Guar- nieri, and the Strongyloplasma of Lipschutz in a new family Cytoryctaceae, (3) the trachoma and inclusion conjunctivitis organisms and the animal conjunctivitis organisms of Coles in a new family Chlamydozoaceae, (4) Rickettsia and kr- macentroxenus genera in the family Rickettsiaceae (Pinker- ton), (5) the organisms of lymphogranuloma venereum, mouse pneumonia, and leucocytonosis of dogs, cattle, sheep and guinea pigs under the genusEhrlichia Moshkovsky all in a new family Ehrlichiaceae. Lastly, Moshkovsky placed the red blood cell parasites of the genera Bartonella, Haemo- bartone lla, G rahame lla, Epe r ythroz oon and Ae gypt ianella in a new family Bartonellaceae. As far as the inclusion of rabies among these organisms is concerned, Moshkovsky mistakenly considered the Negri bodies observed in brain tissue to be the rabies virus, and since the Negri body appeared to be similar morphologically to other "Chlamydo- -zoa," it was included. Moshkovsky separated these families on the basis of their 236 INTERNATIONAL JOURNAL

Table 3. Moshkovsky's 1945 separation of chlamydia1 organisms based on the origin of host cell attacked.

(Page is)* MESODERMOSIS Table 3

A. Leucocytomonosis B. Erythrocytonosis (Bartonellanosis). Characteristic localization of Characteristic localization of the the parasite in leucocytes agent in red blood cells

1. Mononuclear leucocytonosis. 1. Erythrocytonosis. Erythrocytes and oncoblasts are infected. Mononuclear and histocytic cells are infected.

(a) mononuclear are affected by Aroya fever Bartonella Ehrlichia bacilliformis

Basic habitat of the agent 2. True erythronosis. Only erythrocytes are infected.

Dog Ehrlichia cani Bartonellanosis Haembartonella Cattle Ehrlichia bovis anemia of rats muris Sheep Ehrlichia ovlna Guinea Pig Ehrlichia kurlovi

(b ) Mononuc lear histonosi s ; Melanosis of Orahamella besides mononuclear cells, Negroes t alpae fixed tissue elements are Eperythrozoon infected to some degree. coccoides

Inguinal E. (Miyagawanella) Aegyptiosis Aegyptianella Lymphoma- lymphogranumolatosis pullorum tosis Broncho- E. (Miyagawanella) pneumonia bronchopneumoniae of mice and rats Psittacosis E. (Miyagawanella) psittaci

2. Granulocytic leucocytonosis (granulocytonosis) Circulatory granulocytes are infected.

Field cytoecetes Cytoecetes microti

* Moshkovsky, S. D. "The cytoropic agents of infections and the positions of the rickettsiae in the system of Chlamydozoa" (in Russian). Advances in Modern

Biology (Uspekhi Sovremennoi Biologii) 19: 1-44 (1945). S YS T E MAT IC BA CTERIO LOG Y 237 apparent predilection for infecting cells only of a certain origin. According to him, organisms from some groups invaded cells derived only from ectoderm, and organisms from other groups invaded cells derived only from mesoderm; hence the terms ectodermosis and mesodermosis. Moshkovsky's rationale for this was contained in certain summary statements in his article, "The cytotropic agents of infections and the positions of the rickettsiae in the system of Chlamydozoa" (in Russian),

". . . 7. In principle, the cytotropic Chlamydozoa can be differentiated into two subgroups. The first subgroup consists of related parasites infecting cells which develop exclusively from the outer germinal layer (ectoderm) or sometimes derived from other layers. "The second subgroup consists of related parasites infecting cells originally derived from the mesoderm. "In the subgroup of the agents of ectodermoses in the sense mentioned above we have separated the following families : Neuroryctaceae - localization in nerve cells (genus Neuroryctes) Cytoryctaceae - agents of generalized epitheliosis (genera Cytoryctes, Borreliota) and also agents of epitheliosis of skin (genus Strongyloplasma) Chlamydozoaceae - agents of localized epithelj-osis of mucous membranes (genus Chlamydozoon) "In the subgroup of agents of mesodermosis we s eparate the following fami lie s : 1) Rickettsiaceae - characteristic localization in endothelium of vessels - (genera Rickettsia, C owdr ia, De rma cent r oxenu s ) 2) Ehrlichiaceae - characteristic localization in leucocytes circulating in blood (genera Ehrlichia, Miyagawanella, Cytoecetis) 3) Bartonellaceae - characteristic localization in e r ythrocytes (geneTa Bartone lla, Haemobartonella) .... I'll. The agents of trachoma like the Chlamydozoa and other agents that localize in the epithelium of the mucous membrane should not be ranked with the Rickettsia. Nonetheless, there is reason to suppose that they transfer by lice; therefore, the search for positive Weil-Felix reactions should be continued. The search for an intermediate stage between the trachoma 238 INTERNATIONAL JOURNAL

agents of Chlamydozoa and the bacterial type, bacillus Noguchi, or similar bacteria has been fruitless."

The validity of Moshkovsky's basis for separation of the families in his ''Chlarnydozoa system'' may be questioned, for cellularhabitat may be helpful in classification, but does not deserve dominance in any system. His classification represents, nevertheless, the first attempt to delineate the organisms in the order Rickettsiales beyond the previously existing Rickettaiaceae and Bartonellaceae of Gieszczykie- wicz (1939). It was unfortunate that his term Chlamydozoa and its subdivisions Chlamydozoaceae and Chlamydozoon had no nomenclatural standing because of Halbe rstaedter and von Prowazek's prior use of the genus to name a virus. Furthermore, Moshkovsky's inclusion of the lymphogranuloma venereum and mouse pneumonia organisms along with Donatien and Lestoquard's leucocyte parasites of dogs, cattle, and sheep in his previously described genus Ehrlichia was an unfortunate error. In this case, Ehrlichia canis was validly published in 1937 by Moshkovsky for the rickettsia-like, rod-shaped organisms he observed in the monocytes of infected guinea pigs. In 1945 he placed Donatien and Lestoquard's organisms as well as the agents of lymphogranuloma venereum, mouse pneumonia and psittacosis in the genus Ehrlichia while recognizing Miyagawanella as a subgenus for the latter three organisms. Rule 24d applies to his erroneous place- ment of the LGV-psittacosis organisms in Ehrlichia; for PLT organisms cannot be considered congeneric with the original Ehrlichia canis. The latter agent was readily transmitted by ticks and no group antigen nor developmental cycle similar to that observed in all of the PLT organisms has been demonstrated for ,E. canis. Three years prior to $he appearance of Moshkovsky's classification, the first nonmorphologic evidence that the trachoma and inclusion conjunctivitis organisms were similar to psittacosis-lymphogranuloma venereum organisms came fromthe work of Rake, Shaffer and Thygeson (1942) who demonstrated that all of these organisms contained a common group antigen. Then in 1945, Jones, Rake and Stearns proved the susceptibility of the agent of lymphogranuloma venereum to sulfonamide compounds and noted reports that other agents of the PLT group were also susceptible to these chemicals. The authors then stated: SYSTEMATIC BACTERIOLOGY 239

'I.. . In fact, the only so-called viruses whose susceptibility to the sulfonamide has been confirmed are members of the lymphogranuloma-trachoma- psittacosis group of agents (namely, lymphogranuloma venereum, trachoma, inclusion blenorrhea, and mouse pneumonitis). Evidence has accumulated serving to separate this group from the true viruses, analogous to the Rickettsiae, with the name Chlamydia. I'

Jones, Rake and Stearns also made an appropriate reference to a previously published description of the agent of lymphogranuloma venereum (Rake, McKee, and Shaffer, 1940). The statement quoted above and reference to an appropriate description of the organisms represents a valid publication of the generic name Chlamydia. A footnote on page 958 of Bergey's Manual of Determinative Bacteriology, 7th edition, 1957, states that Chlamydia Jones, Rake and Stearns, 1945, was a nomen nudum without nomenclatural standing. Re-examination shows that the generic name Chlamydia was in reality validly published in 1945 and was legitimate when published by Rake in 1957 with the type species Chlamydia trachomatis (Busacca) Rake 1957. Later, Rake defined the organisms in the familychlamy- dozoaceae Moshkovsky for the 6th edition of Bergey's Manual of Determinative Bacteriology (1948). He recognized the now invalid generic name Chlamydozoon Halberstaedter and von Prowazek and the generic name Migagawanella Brumpt but did not accept the generic name Ehrlichia Moshkovsky. In the genus Chlamydozoon he included ,C. trachomatis and -C. oculogenitale. In the genus Miyagawanella he included eight species; ,M. lymphogranulomatis (sic) Brumpt, ,M. psittaci Lillie, 2. bronchopneumoniae Moshkovsky, ,M. ornithosis Rake, M. pneumoniae Rake, ,M. felis Rake, 2. louisianae Rake, and 2. illinii Rake. In regard to the additional new species, Rake commented in 1953: "It will be noted that eight species are listed. The right of some of ,these, particularly, perhaps, ,M. illinii, to be considered as distinct species may be questioned. Further investigation may well show that true species do not exist but that the described differences are only at the strain level. Such lack of complete clarity is, in rn,y opinion, no reason for refusing to undertake classification at this time. Such classification should be regarded as an active undertaking open to change at 240 INTERNATIONAL JOURNAL

any time that new evidence compels such a change. It should indeed serve to point to areas of uncertainty, and be an incentive to further investigation. I'

Rake further pointed out morphologic, antigenic, toxigenic and mode of multiplication differences between PLT organisms, rickettsiae, and pox viruses. This contrasted with Moshkovsky's separation of these groups of agents on the basis of cytotropism. Thus, the basis for the separation of the PLT group from the rickettsiae and from the viruses became clearer, although their final exclusion from the viruses on the basis of their metabolic capabilities independent of the host cell was not shown until recently. Organisms of the PLT group catabolize glucose anaerobically under highly specialized conditions which include the addition of adenosine triphosphate (ATP) (Weiss, 1965). The rickettsiae, on the other hand, do not appear to utilize glucose at all, but respire vigorously in the presence of glutamate and the reaction results in a net gain in ATP (Bovarnick and Snyder, 1949; Bovarnick, 1956). Furthermore, one of the rickettsia1 species, 5. quintana has been cultivated on a lifeless medium (Vinson and Fuller, 1961). That PLT organisms possess both types of nucleic acids (RNA and DNA), reproduce by division and metabolize independently of host cells is sufficient to exclude them from the viruses according to the criteria of Lwoff (1957). In the late forties and early fifties, three Russian taxonomists presented their views of the classification of PLT organisms. In a 1949 monograph entitled "Guide to the Bac- teria and Actinomycetes" (in Russian), N. A. Krasil'nikov repeated Moshkovsky's outline for the Chlamydozoa while adding several more species (Table 1). This work added nothing of significance to the classification within this family of organisms, for he merely included Coles' animal conjunctivities strains as species under the genus Chlamydozoon (while ignoring the genus Colesiota Rake) but included Rake's new specific epithets pneumoniae, felis, louisianae, and illinii (leaving out ornithosis) to Moshkovsky's genus Er- lichia. Furthermore, he placed Carpano's fowl leucocytonosis organism as Ehrlichia avium and Mohamed's fish leucocytonosis organisms as 5.Pisces alongside _E. kurlovi of Moshkovsky. The latter two organisms described in 1936 and 1949, respectively, are of questionable nature. SYSTEMATIC BACTERIOLOGY 241

In 1950, V. L. Ryzhkov of the USSR's Academy of Medi- cine, Virus Institute in Moscow published in the Academy's journal his "Study of the systematics of viral' (in Russian). In this brief and imaginative study, Ryzhkov devised four classes for all plant, insect and animal viruses: Gamaleyae, Crystallobiotae, Arthrodophilaes, and Chlamydozoa. He placed the organisms of the PLT group in the last class with the following justification:

"IV Class Chlamydozoa (Prowazek) "They are parasites of animals and man. They contain thymonucleic acid and lipoids. Sometimes one can find amounts of yeast nuclei acid (Virus group). Largest representatives reach 700 in diameter. Diameters are often higher than 200 mt~.but rarely less than 100 m+~.Characteristically, they are highly specialized. Individual viruses infect definite species; sometimes definite varieties. Properties of virus can be modified by adaptation and passage through a suitable host. Natural spreading is not associated with a carrier. Wild animal reservoirs are rarely encountered. When this does occur, then the virus may be transferred directly to the bearer (transmission of rabies by wolves and sanguinovorus animals). Virus is widely dis semi- nated in skin lesions and mucosa. Pantropic viruses are rarely encountered. This class is less monolithic than previous ones. Many families are concerned." (page 15) "In this class one should distinguish, more or less, natural orders. We have added to the numbers of this class by including previously intact groups of viruses which at present are not unified in this group, and we have given the name only to the order, family, and species.

Order Chlamydozoales

Typical Family Chlamydozoaceae. Probably the large s t naturally occurring group. We 11 diff e ren- tiated elementary bodies. I'

In Ryzhkov's table of classification for the family Chla- mydozoaceae that follows on page 17, a footnote indicates that "the family Ehrlichiaceae Moshkovsky and Chlamydo- zoaceae Moshkovsky have been unified on the advice of R. M. 2 42 INTERNATIONAL JOURNAL

Schen." Thus, Ryzhkov placed the entire PLT group in the genus Chlamydozoon and lists the specific epithets without indicating whether they were newly proposed names or previously proposed names of other authors.

ps ittaci hominus t r achomiati c olumbi o s t e ophilu s c onj un ctiva e murinus monocytaris -bovi s -felis lymphophilus oculoge nit ale meningophilus opposumi ke rat o - conjunctivae cricetis

While Ryzhkov's taxonomic descriptions were extremely brief, and he used an illegitimate generic name Chlamydo- -zoon Prowazek, his unification of Moshkovsky's families Ehrlichiaceae and Chlamydozoaceae strengthened the original proposal of Jones, Rake and Stearns (1945) that organisms of the PLT group should be placed in a single genus. The same year (1950) that Ryzhkov's "Study on systematics of viruses'' appeared, V. M. Zhdanov and R. S. Koren- blit published a report given in 1949 at a scientific conference in Kharkov entitled "Systematics and nomenclature of viruses" (in Russian). In this preliminary description of their system, Zhdanov and Korenblit proposed a series of newnames for pre-existing genera and species in the order Rickettsiales; Philip (1956) subsequently pointed out the invalidity of the numerous synonyms proposed bv Zhdanov and Korenblit. In regard to the nomenclature of PLT organisms Zhdanov and Korenblit included the specific epithets trachomatis Busacca and oculogenitale Moshkovsky in the genus Chlamydozoon Halberstaedter and Prowazek 1907. They also rejected Rake's genus Colesiotawhose specific epithets conjunctivae (Coles) and conjunctivae p;alli (Coles) were replaced by pecoris (Zhdanov and Korenblit) and pul- -lorum (Zhdanov and Korenblit), respectively. Furthermore, they proposed the genus name Rickettsiaformis as a substi- tute for Miyagawanella Brumpt and Ehrlichia Moshkovsky. Sixteen species were placed in the genus Rickettsiaformis; nine of which were entirely new names for various well known strains; two names were changed forms of previously suggested names, and four epithets were changed forms of previously suggested names, and four epithets were the same as those proposed by Rake in Bergey's 6th Edition Manual (1948) (Table 1). The extensive synonymy found in Zhdanov and Korenblit's SYSTEMATIC BACTERIOLOGY 243 proposal was repeated and extended in Zhdanov's later monograph, "Guide to Human and Animal Viruses!' (in Rus- sian) published in 1953. It is obvious that these authors considered the organisms of the PLT group as viruses, a concept which is no longer valid. Many of the new species proposed are highly questionable, e. g. -R. pullorum Zhdanov and Korenblit for the coccoid forms from chickens studied by Nelson in 1936 which are now considered to have been Mycoplasma gallisepticum. Since the criteria for specia- tion within the PLT group have not been stabilized, Zhdanov's species proposals may prove to have been premature. In any case, the genus name Rickettsiaformis is illegitimate because it is a synonym of the previously validly published generic name Chlamydia Jones, Rake and Stearns (1945). In 1952, at a New York Academy of Sciences conference on "Nomenclature and Taxonomy of the Rickettsiales, I' Meyer (1953) outlined his reasons for commemorating S. P. Bedson in the genus name Bedsonia for psittacosis organisms and rejecting Brumpt's commemoration of Miyagawa in the name Mjyagawanella as inappropriate. While many of Meyer's reasons for suggesting the change had merit on historical grounds, the actual proposal disregarded nomenclatural rules of priority; for, the generic name Bedsonia Meyer (1953) is a junior synonym of Chlamydia Jones, Rake and Stearns (1945) and therefore is illegitimate. Some American microbiologists have sympathized with the reasons for Meyer's proposal without considering the Code rules and have used the generic name Bedsonia and its plural bedsoniae in describing various strains of PLT organisms. This has effected a drift away from legitimate bacteriological nomenclature. In the 7th edition of Bergey's Manual of determinative Bacteriology (1957), Rake included the genus Chlamydia for the etiologic agents of trachoma and inclusion conjunctivitis and the genus Miyagawanella for other PLT organisms, but did not recognize Bedsonia. Under the genus Miyagawanella he added four new binary combinations, M. opposumi (Ryzh- kov), ,M. &s (Zhdanov), ,M. bovis (Hork and Baker), and -M. pecoris (Rake). Thus Rake's 1957 list of the PLT organisms did not differ substantially from his 1948 list, other than recognizing four additional species. Meyer's Bedsonia was described in 1964, however, by Levaditi, Roger and Destombes in their publication entitled "Attempts at a classification of the Chlamydiaceae (Rake, 1955) taking into account their tissue affinities and their 244 INTERNATIONAL JOURNAL epidemiology" (from author's English summary). In this proposal, Levaditi and his colleagues devised

If.. . a simple key utilizing experimental, epidemiologic, and nosologic characters which.. . separates the family of Colesidae noncultivated in the yolk sac, from that of the Chlamydiae, cultivated in the yolk sac, and the endocellular parasitism of which is more marked;. . . '' (from authors' English summary). The authors further clarify their rationale by stating that: "The family of the Chlamydidae (epithelial parasites of man) differs from that of the Miyagawellidae (sic) which affect mesenchyme, and in which the new subfamilies of Miyagawellinae (sic) affecting essentially ganglia) and of Bedsoninae (sic) (essentially septicemic) may still be distinguished. The new genera Rakeia and Bedsonia have been created in order to explain the essential epidemiologic duality of the Bedsoninae and as an homage to two foreign authors whose contribution to the knowledge of these groups is very important." (authors English summary).

In effect, the above authors attempted to resolve the "didactic difficulties" (to use their term) by recognizing four genera for the PLT group, Chlamydia for trachoma and inclusion conjunctivitis organisms, Miyagawanella for lymphogranuloma venereum and related organisms, Bedsonia for avian strains of the PLT group, and a new genus Rakeia Levaditi, Roger and Destombes for the mammalian strains. While this proposal appears to soften the "didactic difficulties" it creates a divisive effect on nomenclature. It promotes unnecessary proliferation of generic names, for the taxonomic value of the differences that Levaditi, Roger and Destombes claim sep'arates these organisms into genera (host or cellular preferences, epidemiology) does not out- weigh the taxonomic importance of the characters that unite them (common morphology, developmental cycle, and group antigen). Furthermore, Levaditi ct 51. have created additional unnecessarynames of subfamilies and tribes. Most of these terms incorrectly contain endings utilized in the zoological code instead of endings required by the bacteriological code. In some cases, suffixes were manufactured by the authors with no known precedent. SYSTEMATIC BACTERIOLOGY 245

In light of the knowledge of the fundamental similarities between the organisms of the PLT group and the clarifica- tion of their distinction from other organisms (such as rickettsiae or viruses), it is taxonomically and nomenclaturally correct to place the organisms of the PLT group in a single genus, Chlamydia Jones, Rake and Stearns ( 1945). By reason of nomenclatural priority, the type species is ,C. trachomatis (Busacca) Rake 1957. Descriptions of this organism are found in publications previously cited, and are reviewed by Jawetz (1964).

Emended Description of the Genus Chlamydia, Jones, Rake and Stearns (1945)

Spherical organisms, Gram-negative, parasites of the cytoplasm of host cells, ranging in size from 200 mp to 1000 mp, and which have a limiting membrane resembling in chemical composition the cell walls of Gram-negative bacteria. They have a unique developmental cycle following entry of the small (200-300 "y.) form of the infectious organism into the cytoplasm of a host cell. The architecture of the invading organism changes sequentially from large (up to 1000 mp) homogeneous forms, which multiply by division, to intermediate forms with dense central masses and small forms with dense central masses tightly sur- rounded with outer membranes. The organisms remain structurally intact through all stages with some degree of infectivity, although the infectivity of the large forms is very low. An antigenically similar carbohydrate lipoprotein complex is found in all organisms of the group and is resistant to heating at 100°C for 30 minutes. Cell envelopes may contain heat labile, strain- specific antigens. The organisms possess metabolic activities independent of those of the host cell. The organisms are found in tissues of numerous species of animals. Pathogenicity varies considerably. Some strains exhibit marked cytotropism. All of the organisms are influenced by tetracyclines, some are sensitive to penicillin or sulfa compounds.

Additional Characteristics of Members of the Genus Chlamydia

(1) Chemical constituents. Isolates examined thus far contain (a) both RNA and DNA; (b) muramic acid in their cell envelopes along with other compounds whose general 246 INTERNATIONAL JOURNAL composition resembles that found in the walls of Gram- negative bacteria, i. e. , relatively high proportion of lipid, carbohydrates, and a variety of amino acids; (c) a common group antigen made up of lipo-protein-carbohydrate complex; in some agents a carbohydrate is present within the intracellular microcolony which is differentially stained by iodine solution or by periodic acid-Schiff's reagent. (2) Host range. Numerous isolates, especially those of avian origin, have been shown experimentally to be able to grow, multiply and produce cellular and gross pathology in a wide range of avian and mammalian hosts. Natural inter- species transfers occur, but the range and extent of transfers are not known. Arthropods may be involved in the mechanical transmission of certain agents between hosts, but are not known to support the multiplication of any of the agents. Some agents, such as those that cause trachoma, inclusion conjun ctiviti s or 1ymphogr anuloma vene r eum have a marked, natural specificity for man (and for primates, experimentally) with a further marked specificity for epithelial cells of the conjunctiva or the genital tract. Cellular specificityas a basis for separation of various organisms of the group into genera is probably artificial. (3) Metabolism. At present, propagation of any of the chlamydiae is possible only by allowing the agents to infect living tissue cells. However, several isolates were shown to contain enzyme systems which are not of host origin. They can synthesize folic acid (Colon, 1960) or convert host folic acids to their own specific forms. They decarboxylate diaminopimelic acid to lysine (Moulder, 1963) and, possibly produce other cell wall components (Moulder, 1964), and degrade their own protein and ribonucleic acid. It was also shown that under certain conditions several strains can catabolize glucose (Weiss, 1964). This reaction requires added adenosine triphosphate (ATP) and MgSt and is en- hanced by added nicotinaqide adenine dinucleotide phosphate (NADP) and by high relative concentration of K'. The ATP requirement is eliminated by the substitution of glucose-6- phosphate for glucose. These effects are obtained with intact cells (Weiss, 1965). Also cell-free extracts of purified chlamydiae contain glucose- 6-phosphate and 6-phosphoglu- conate dehydrogenase and phosphoglucose isomerase (Moul- der, 1965). Synthesis of folic acid is also suggested on the basis of the susceptibility of the organisms to sulfonamides. On the other hand organisms resistant to sulfonamides have been shown to be uninhibited by folic acid analogues thereby SYSTEMATIC BACTERIOLOGY 247 suggesting that essential folic acid precursors are supplied by the host cell. (4)Susceptibility to antibiotics. Except for experimentally-derived mutants, all of the agents of the group tested this far have been shown to be inhibited in their multiplication by tetracyclines. Many are inhibited by sulfonamides, penicillin and other antibiotics. Many isolates are resistant to streptomycin, tyrothricin, polymixin and neomycin. (5) Antigens. All isolates contain a common antigen which is heat stable (lOO°C, 30 minutes or longer) and which is destroyed by treatment with periodate. Most isolates also have a specific antigen found in the cell envelope which is destroyed by heating to 60°C or treated with any of a large number of chemicals. Other antigenic components with varying heat sensitivities may be present. Many isolates possess in varying amounts a lipoprotein component which causes severe damage to the vascular endothelium of susceptible hosts resulting in a "toxic" death. The antigenic specificity of this component has been useful in grouping strains, but does not regularly permit the separation of species.

The genera Colesiota Rake 1948, Colettsia Rake 1957, and Ricolesia Rake 1957 for organisms found in the conjunctiva of animals are regarded as insufficiently characterized to determine their possible inclusion in the genus Chlamydia. In summary, the thesis is an argument for the desir- ability of recognition of Chlamydia Jones, Rake and Stearns, 1945, as the name of a genus under which all of the species of the PLT group of bacteria are included, with the type species being ,C. trachomatis (Busacca) Rake 1957.

ACKNOWLEDGMENT

The author expresses his gratitude to Dr. George Lam- bert of the National Animal Disease Laboratory, Ames, Iowa, for translating the Russian passages presented in this study. 248 INTERNATIONAL JOURNAL

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