SPIROCHAETES AND THEIR GRANULE PHASE.
By
H.B. FANTHAM, M.A.CANTAB., D.SC.LOND.,
Lecturer on Parasitology, Liverpool School of Tropical Medicine

[Transcriptet from: The British Medical Journal, March 18, 1916: 409-411]
[Sorry for any transcript errors, but the photocopy was very small and thus hard to read ]

SPIROCHAETES are thin, delicate, undulatory organisms which are widely distributed in Nature. Some of them are free-living and occur in stagnant water or in sea-water. Others live in the digestive tracts of animals, both vertebrate and invertebrate. In man Spirochaeta buccalis and S. dentium may be found in the mouth and S. bronchialis in the respiratory tract, while S. curygyrata and S. stenogyrata may occur in human faeces. Also, S. balbianii is a well-known species found in the intestinal caecum and crystalline style of oysters and allied Lamellibranchs. Other spirochaetes occur in the tissues of vertebrates, and are often pathogenic. The causal agents of relapsing fever, such as S. recurrentis and S. duttoni, occur in the blood during thie pyrexial periods. Another of these organisms - S. schaudinui - is found in tropical ulcers. Yet other spirochaetes, often placed in a separate genus Treponema, are the causal agents of syphilis and of yaws, and are known respectively as Treponema pallidum and T. pertenue.

Probably the longest spirochaetes are among those found free-living. Such an one is S. plicatilis, which may reach 200 in length. On the other hand, S. laveraui, found in the blood of mice, is a very short form, and may be only 3 long. Spirochaetes may wary from 0.25 to 2 in width. The outline of the body of one of these Pretista is corkscrew-like and varies in appearance owing to its great flexibility. Quickly moving spirochaetes show many waves of small amplitude, while slowly moving forms present fewer, larger waves of greater amplitude. Hence it will be seen that it is hardly accurate to take as specific characters the number of undulations or coils found in fixed and stained specimens of spirochaetes. The number of turns or waves is, then, more an index of rate of motion - as well as of thickness, which is also a factor - than one of differentiation between various species. Further, owing to the processes of growth and division the latter occurring by binary fission, there is much morphological variation among spirochaetes. This polymorphism results in differences in length and breadth within the same species, variations which tend to be overlooked unless a large number of specimens from a series of cases is examined and measured.
Internally, spirochaetes possess chromatin bars, rodlets, or granules distributed evenly along their body length. These chromatin granules are seen only with great difficulty in the smaller forms.

Spirochaetes of vertebrates may be transmitted from host to host directly by the contaminative method, or by the intermediation of an arthropod vector. Examples of the former are the causal organisms of syphilis and of yaws, while examples of the latter are the parasitcs of relapsing and African tick fevers. A consideration of the life cycles of spirochaetes leads to a discussion of their granule phase, or capacity for ''granule chedding'', as it has been termed, a most interesting phenomenon around which a certain amount of controversy has centred. There is no doubt that spirochaetes produce such granules; it is only their significance, whether cyclical or degenerative, that is in question.

One of the best investigated with respect to granule formation is S. duttoni which is transmitted from man to man by the tick Ornithodorus moubata. The life-cycle of this spirochaete was first outlined by Dutton and Todd in 1907, and was studied in detail by Leishman in 1909-10. Leishman's results essentially were that spirochaetes gave rise by multiple fission to granules or coccoid bodies inside the vertebrate host, and that these granules or coccoid bodies found their way move especialIy to the Malphigian tubules, gonads, and other organs of the tick. The granules themselves multiplied. The eggs of the female tick became infected with granules, and the progeny of infected females might be born infected. The observations of Leishman have been confirmed and extended by Balfour (1911), Fantham (1911), and Hindle (1911). The organisms investigated were S. duttoni, S. recurrentis, and S. gallinarum, or varieties of the last. The formation of granules in Treponema pallidum and in T. pertenue has been recorded by Balfour, by Ranken, and by Hoffman. It has also been observed in Spirochaeta bronchialis by Fantham in 1914-15. Similar multiple transverse fission has been seen in the larger molluscan spirochaetes-for example, S. balbianii and S. anodontae, by Besanquet (1911), Fantham (1911) and Gross (1912). It may be mentioned that, even as early as 1882, Zopf recorded and figured the formation of micrococcoid and bacillary forms from spirochaetes of stagnant water and of the teeth, and stated that these forms stand in genetic relationship to one another.

The formation of coccoid bodies, as observed by me in various spirochaetes in fresh preparations, with or without the use of dark-ground illumination, is as follows:
The cytoplasm at first is very finely granalar, in fact, almost homogeneous. The chromatin bars appear as minute refractile masses. A concentration of some of the cytoplasm occurs around each chromatin rodlet. These small concentrations gradually become oval, the outer cytoplasmic layer differentiates as a thin coat, and ultimately a series of coccoid bodies or granules is formed, lying usually transversely or slightly obliquely within the periplast sheath. Sometimes the coccoid bodies are set at liberty by a rupture appearing at one end of the spirochaete; at other times, several ruptures, or disintegration of the sheath, can be observed. Stained specimens show a series of darker, lozenenge-like coccoid bodies alternating with relatively clear pale-staining areas. Within the Malpighian or genital cells of a transmitting tick and in certain mononuclear sputal cel1s penetrated by S. bronchialis, the coccoid bodies often seem to be liberated by the disintegration of the periplast. Groups of coccoid bodies still retaining the outline of the spirochaete from which they originated are of fairly frequent occurrence.
When the coccoid bodies are released by a terminal rupture of the parent they tend to form irregular clumps. The progressive elongation of the granules, the assumption of the sinuous form, and the emergence of very small Spirochaetes from the groups of granules have been observed in life. It is very probable that there is a definite period in the life of a spirochaete at which there is a marked differentiation of coccoid bodies. It must also be borne in mind that coccoid bodies may be present when spirochaetes as such cannot be detected. At the same time, the finding of chromatinic granules alone is not sufficient to justify the inference that spirochaetosis is indicated, as all such granules are not necessarily spirochaetal in origin. It is essential that motile spirochaetes should be found at some period of the disease, and the devolopmental stages leading to granules or coccoid bodies observed in vivo.

The coccoid bodies have been otherwise interpreted by Marchoux and Couvy (1912-13), Blanc (1911) and Wolbach (1914), who seem to consider these bodies either to be degenerative or to be unconnected with the life-cycle of spirochaetes. According to the first-named investigators, who worked on S. gallinarum in Argas persicus, the spirochaetes within an infected tick retain their spirochaetal facies, becoming at times so attenuated that they may cease to be visible, some having been observed already on the limits of visibility. Naturally, the question forces itself as to how it it possible to determine the spirochaetal facies if the organism has become invisible. Further, they state that granules occur in the Malpighian tubules, ova, and genital ducts of normal ticks and other arachnids, and that these have been mistaken by Leishman and his supporters for spirochaetal granules. However, it has not been denied that granules may occur in normal tick cells, nor was it asserted that all intracellular granules in infected tick's were spirochaetal of origen, for the granules seen in arachnid cells are not all of the same nature.

Regarding the development of S. recurrentis or its varieties in the louse, Pediculus vestimenti, Sergent and Foley (1914) state that the spirochaete in the louse assumes a very small form which is as virulent as the spirochaetiform stage. During eigth days following a meal of infected blood the body of the louse does not contain any spirochaetes as such, though the spiral organisms reappear later, as was first observed by Nicolle, Blaizot, and Conseil in 1912. Very probably this minute form will be found to be of the nature of a Leishman granule or coccoid body. Also, Nicolle and Blanc (1914) find that the causal agents of relapsing fever are virulent or infective in the louse just before they reappear as spirochaetes. They think that there is an invisible stage in the life-cycle, though they do not appear to have examined for a granule stage, which might easily be overlooked. Further, it should be noted that in experiments with the invertebrate transmitters of such spirochaetes as S. duttoni, S. recurrentis, and S. gallinarum, careful attention should be paid to the temperature, humidity, and other climatic conditions under which the investigations are conducted, since these factors undoubtedly influence the development of spirochaetes therein.

Granule stages have also been recorded from time to time in spirochaetes in the blood of vertebrates. Thus, Balfour, in 1908, found such bodies in the red blood corpuscles of Sudanese Fowls suffering from spirochetosis. Prowazek, in 1906, recorded intracorpuscular stages of S. gallinarum. Breinl (1907) and others have observed encysted forms of S. duttoni in the spleen, which forms broke into granular bodies that gave rise to new generations of spirochaetes. Sergent and Foley (1914) also found that a minute but infective form occurred in the blood of patients suffering from relapsing fever during apyretic intervals when spirochaetes were absent. Personally, I have seen a very few of these spirochaetes on rare occasions breaking up into coccoid bodics in the blood of the vertebrate host. These minute bodies may appear to occur in or on the red blood corpuscles. They may be found at the crisis and may possibly explain the ''after-phase,'' and may be connected with relapses in the vertebrate host. It is also interesting to note that Sir Patrick Manson, in his well-known book on tropical diseases, writes as folows regarding the etiology of relapsing fever: "Obermeier and von Jaksch describe certain refractile bodies present in the blood during the fever intermissions. The latter author says that he has observed the developmont of these bodies into short rods, from which the typical spirochaetes are eventually evolved."

Again the passage of S. bronchialis from man to man is most probably by means of the coccoid bodies that leave the human host in the spray with expired air and by way of nasal secretions, as was shown by the present writer in 1914 and 1915. Linen soiled by such secretions, and indiscriminately packed with other soiled clothing, may also aid. Owing to the fragility and short life of S. bronchialis extracorporally, the resistant coccoid bodies in air, dries sputum and dust, and possibly also on the bodies of flies and other insects, are probably instrumental in inducing attacks of bronchial spirochaetosis in human beings, especially those having a lowered bodily resistance, such as after a chill.

A historical interest attaches to the Cytoryetes lais of Siegel. These structures are probably explained by the granules shed by Treponema pallidum (Spirochaeta pallida).
Further evidence for the transition and growth of granules or coccoid bodies into spirochaetiform organisms may be briefly mentioned. Thus, Nogushi (1911), in his paper on the cultivation of Treponema pallidum, records in the explanation of one of his figures that ''it is not rare to find a round body connected with one or two young pallida, as though the latter were hust sprouting from the former.'' Balfour (1913) thinks that he seems to have succeeded in growing spirochaetes in vitro from infected tick eggs in which granules only could be demonstrated. Sir William Leishman (1913) repeated many of his former experiments with altered technique, employing dark-ground illumination instead of fixation and staining. He finds the ''the granule clumps are not all alike, but that some of them show a much higher degree of refractility than others. Continuous observations of these refractile granules, with the microscope in a thermostat, has further shown me, in the case of two ticks, the definite extrusion of small and actively motile spirochaetes from these granule clumps . . . After about ten days at a temperature of 28 C. to 30 C., and with relatively high humidity, some of these clumps give rise to young spirochaetes, which become free, are actively motile, multiply by fission, and may persist in the tissues of the tick for many months.''

In conclusion, the value of the recognition of the granule phase in the Spirochaetacea has been strikingly set forth by Noguchi, who, in an address given before the Royal Society of Medicine in London on October 20th, 1913, stated that he ''was able to demonstrate . . . granules in the pure cultures of Treponema pallidum. This phenomenon, however insignificant it may appear in itself, was destined to furnish a key to one of the most disputed problems of the past fifty years-namely, the problem of so-called parasyphilis, since it was this very idea that prompted me to undertake to search for Treponema pallidum in one form or another in the brains of general paralytics and in the spinal cord from cases of tabes dorsalis.'' And again, ''I was led by the observation that Treponema pallidum sometimes assumes a granular form in cultures to re-study sections of paretic brains stained for the pallidum.''

REFERENCES.

Balfour, A. (1908, 1911): Spirochaetosis of Sudanese Fowls. Third Report Wellcome Labs., Khartoum. Pp. 35-58. Also Fourth Report, vol. A., pp. 76-107.
Balfour, A. (1913-14): Notes on the Life-Cycle of the Sudan Fowl Spirochaete. Trans. XVII Internat. Congress of Med., London, pt. ii, sect. xxi, pp. 275-278.
Dutton, J.E. and Todd J.L. (1907): A Note on the Morphology of Spirochaeta duttoni. Lancet, November 30th, 1907, pp. 1523-1525
Fantham, H.B. (1911): Some Researches on the Life-Cycle of Spirochaetes. Annals Trop. Med. and Parasitol., v, pp. 479-496.
Fantham, H.B. (1914): The Granule Phase of Spirochaetes. Annals Trop. Med. and Parasitol., viii, pp. 471-484.
Fantham, H.B. (1915): Spirochaeta bronchialis, Castellani. 1907, together with Remarks on the Spirochaetes of the Human Mouth, Annals Trop. Med. and Parasitol., ix, pp. 391-412.
Hindle, E. (1913): On the Life-Cycle of Treponema pallidum. Parasitology, iv, pp. 463-477
Leishman, W.B. (1910): An Address on the Mechanism of Infection in Tick Fever, and on Hereditary Transmission of Spirochaeta duttoni in the Tick. Lancet, January 1st, pp. 11-14. Also Trans. Soc. Trop. Med. and Hyg., iii, pp. 77-95
Leishman, W.B. (1913-14): Relapsing Fevers. Trans. XVII Internat. Congress of Med., London, pt. ii, sect. xxi, pp. 282.
Marchoux, F. et Couvy, L. (1913): Argus et Spirochaetes. Annales Inst. Pasteur, xxvii, pp. 450-480 and 620-643.
Nicolle, C. et Blanc, G. (1914): Les Spirilles de la fievre recurrente sont-ils virulent aux phases successives de leur evolution chez le pou? Demonstration de leur virulence un stade invisible. Compt. Rend. Acad. Sci., clviii, pp. 1815-1817.
Noguchi, H. (1913): On Some of the Recent Advances in the Field of Microbiology; with Demonstrations of the Pure Cultures of Various Spirochaetes, of the Viruses of babies and Poliomyelitis, and of Treponema pallidum in the Brains of General Paralytics [Occasional Lectures] Trop. Ren. Soc. Med., vii, pt. i, pp. 3-30
Sergent, E., et Foley, H. (1914): Des periodes de latence du Spirille chez la maladie atteint de fievre recurrente. Compt. Rend. Acad. Sci., clviii, pp. 1926-1928
Sergent, E., et Foley, H. (1914): De la periode de latence du Spirille chez le Pou infecte de fievre recurrente. Compt. Rend. Acad. Sci., clix, pp. 119-122

Further references will be found at the ends of some of the papers cited.