Treponemata

The genus Treponema (Schaudinn, 1905), includes minute, thread-like organisms, with spirally coiled bodies, the spirals being preformed or fixed. No membrane or crista is present, according to Schaudinn, though a slight one is said by Blanchard to be present in the organism of yaws. The ends of the organisms are tapering and pointed. Multiplication is by longitudinal and transverse division. The most important members of the genus are T. pallidum, the agent of syphilis, and T. pertenue, which is responsible for frambœsia or yaws.

Treponema pallidum, Schaudinn, 1905.

Syn.: Spirochæta pallida.

Treponema pallidum was first described by Schaudinn and Hoffmann in 1905 under the name of Spirochæta pallida. It has also been described under the names of Spironema pallida, Microspironema pallida and Trypanosoma luis. Siegel in 1905 described an organism which he called Cytorhyctes luis and considered to be the agent of syphilis. Schaudinn reinvestigated Siegel’s work and found T. pallidum, which he considered to be the causal agent of the disease, and pronounced against Cytorhyctes luis. It is probable now that both workers were correct, for Balfour (1911) has seen the emission of minute granules or “coccoid” bodies from T. pallidum and these granules probably correspond to the C. luis of Siegel. Recently E. H. Ross, having observed a spirochæte stage in the development of Kurloff bodies, thinks that T. pallidum is a stage in the life-history of a Lymphocytozoon. MacDonagh has also described a complicated and somewhat similar cycle, but these observations require further study and confirmation.

T. pallidum varies from 4 µ to 10 µ in length, its average length being 7 µ, while its width is usually about 0·25 µ. Longer individuals of 16 µ to 20 µ have been recorded. The body has from eight to ten spiral turns and forms a tapering process at each end (fig. 56). The organism is most difficult to stain, and its internal structure is little known. It is possibly like that of Spirochæta duttoni or S. balbianii, as the “granule shedding” observed by Balfour is strongly suggestive of the formation of resistant bodies by those spirochætes. Hoffmann (1912) has seen the formation of spores in T. pallidum.

The Treponemata occur in the primary and secondary sores, but are difficult to find in the tertiary eruptions of syphilis. Noguchi and Moore (1913) and Mott (1913) have demonstrated T. pallidum in the brain in cases of general paralysis of the insane. Marie and Levaditi (1914), however, consider that the treponeme found in the brain in such cases is different from T. pallidum.

Cultivation of T. pallidum.—This has been accomplished successfully by Noguchi, using a modification of his method for spirochæte cultivation, for T. pallidum is much more difficult to grow than spirochætes, being a strict anaerobe.

The apparatus consists of two glass tubes, the upper being connected to the lower by a narrower tube passing through a rubber cork (fig. 57). Both tubes are carefully sterilized.

A piece of fresh, sterile rabbit’s kidney is placed in the lower tube, which is filled with ascitic fluid, or ascitic fluid and bouillon mixture. The tube is inoculated with syphilitic material and corked by inserting the upper tube. In the bottom of the upper tube a piece of sterile rabbit’s kidney is placed and syphilitic material poured over it. A mixture of one part ascitic fluid and two parts of slightly alkaline agar is then poured over the tissue and allowed to solidify. When solid, a layer of sterile paraffin oil is poured on top of it, and the top plugged with cotton wool (fig. 57). The whole is then incubated at 37° C. for two or three weeks. The tissue removes traces of oxygen from the lower levels of the medium and also probably provides a special form of nourishment. At first T. pallidum grows in the solid medium, and then when the cultural conditions in the lower fluid portion become favourable, the organisms migrate thither and multiply abundantly. At first the culture is impure, but after several transferences a pure culture is obtained readily.

The syphilitic material for culture is prepared by cutting off pieces of tissue from the lesions, washing in sterile salt solution containing 1 per cent. sodium citrate, and then emulsifying the tissue in a mortar with sodium citrate.

Good cultures show rapid multiplication, which is invariably by longitudinal division.

In his various cultivation experiments Noguchi found morphological and pathogenic variations in T. pallidum. Three forms of the organism were found, namely, thicker, average and thinner types. The lesions caused in the testicle of the rabbit differ according to the variety inoculated, but more work is necessary on the subject.

Noguchi has cultivated a separate organism, T. calligyrum, from the surface of human genital or anal lesions, either syphilitic or non-syphilitic. It is apparently non-pathogenic, and is 6 µ to 14 µ long.

Hata (1913) has modified the Noguchi technique for the cultivation of spirochætes and treponemes, with a view to simplification and convenience. Hata substitutes normal horse serum for ascitic fluid and the “buffy coat” of the clot of horse blood in place of the small pieces of rabbit’s kidney. It is unnecessary to place sterile paraffin on the surface of the medium.

The horse serum is mixed with twice its volume of physiological saline solution. The mixture is placed in tubes which are heated on a water-bath at 58° C., the temperature being raised gradually until it reaches 70° or 71° C. in three hours. The tubes are then heated at 71° C. for half an hour. After cooling, the contents will consist of an opaque semi-coagulated mass. This semi-coagulated serum and saline mixture may be substituted for Noguchi’s ascitic fluid.

The buff coagulum is cut into small pieces, about 1 c.c. in volume. They must be forced with a sterile glass rod to the bottom of the semi-coagulated serum and saline mixture. The medium is inoculated with a small quantity of infected blood and kept at 37° C. In the case of S. recurrentis, growth of spirochætes is observed on the second day, reaching a maximum in five to seven days. The growth of the organisms proceeds rather more slowly, they live for a longer period and maintain their virulence better than in Noguchi’s medium.

Treponema pertenue, Castellani, 1905.

Syn.: Spirochæta pertenuis; S. pallidula, Castellani, 1905.

Castellani discovered the organism in 1905, in scrapings of yaws pustules. He first described it under the name of Spirochæta pertenuis.

Treponema pertenue (fig. 58), though delicate and slender, shows great morphological variation both in length and thickness. It may be short, e.g., 7 µ, but can attain 18 µ to 20 µ in length and may be even larger. In cultures made by Noguchi, thick, medium and thin forms were found, each giving rise to a different type of frambœsial lesion when inoculated into the testicles of rabbits, thus suggesting the possibility of the occurrence of varieties of T. pertenue.

The organism is difficult to stain, but occasionally deeper staining granules are found along its body. They may represent a diffuse nucleus. Granule formation similar to that of T. pallidum has been observed by Ranken, using dark-ground illumination.

Many experiments have been made with a view to establishing the identity of the organism of yaws and also of differentiating between the causative agents of yaws and syphilis. Both monkeys and the human subject have been experimentally inoculated with yaws material and have developed the disease.

In an early experiment, negroes were inoculated with the secretion from lesions of yaws. All of them developed the disease, nodules appearing, chiefly at the seat of inoculation, in from twelve to twenty days, followed by the usual eruption. Similar results were obtained with thirty-two Chinese prisoners, who were inoculated with yaws, twenty-eight becoming infected.

A naturally infected yaws patient when inoculated with syphilis, contracted that infection, thus showing that yaws does not confer immunity to syphilis. This has also been observed naturally, when yaws patients have contracted syphilis.

Experiments with monkeys have been successfully performed. The incubation period varies from sixteen to ninety-two days. Lesions appear first at the seat of inoculation, and in some monkeys the eruption is localized to this spot, though the infection is general, T. pertenue occurring in the spleen, lymphatics, etc. Monkeys inoculated with splenic blood of a yaws patient, and also sometimes with blood from the general circulation, have become infected.

Castellani and others have shown that monkeys successfully inoculated with syphilis do not become immune to yaws, and vice-versâ.

Craig and Ashburn, using the monkey Cynomolgus philippinensis, found these animals susceptible to yaws but not to syphilis.

The ulcerated lesions of frambœsia are rapidly invaded by numerous bacteria as well as by different spirochætes, of which Castellani has described three distinct species. One is identical with Spirochæta refringens, Schaudinn, the other two are thin and delicate. One, S. obtusa, has blunt ends; the other S. acuminata, has pointed ends. T. pertenue is also present.

The reasons for considering T. pertenue to be the specific cause of frambœsia are:—

(1) T. pertenue is the only organism present in non-ulcerated papules, in the spleen and in the lymphatics of yaws patients, or of monkeys artificially infected with the disease. By no method has any other organism been obtained.

(2) Extract of frambœsia material, free from all organisms other than T. pertenue, reproduces the disease if inoculated.

(3) Extract of frambœsia material deprived by filtration of T. pertenue is no longer infective on inoculation.

The method of infection is contaminative, by direct contact. Women in Ceylon are frequently infected by their children. Any slight skin abrasion is sufficient to admit the parasite. In some cases, insects may carry the disease from person to person, and even in hospitals, when dressings are removed, it has been noticed that flies greedily suck the secretion from the ulcers. T. pertenue has been recovered from flies that have fed on yaws, and monkeys have contracted the disease when flies were placed and retained on them for a short time, after the insects had fed on yaws material.

Cultivation.—T. pertenue has been cultivated by Noguchi, who finds three types of parasites in his cultures, as before mentioned. Its multiplication is by longitudinal division.

Noguchi (1912), has cultivated species of Treponema from the human mouth, e.g., T. macrodentium, T. microdentium and T. mucosum, the latter from pyorrhea alveolaris. These parasites in the past may have been confused under the name Spirochæta dentium.