GROWTH OF TREPONEMA PALLIDUM IN RABBITS

GILBERT SMOLIN, M.D., ROBERT A. Νοζικ, M.D., AND MASAO OKUMOTO, M.S. San Francisco, California

Treponematosis has been discussed with more than former frequency in the ophthalmic literature since the repeated recovery of spiral forms from the aqueous humor of human and animal eyes.1"3 Attempts have been made to identify these forms as Treponema pal­lidum by fluorescein-labeling techniques4 and animal inoculations,5 but positive identifica­tion by these means has not yet been possible.8

The following experiments were under­taken in an effort to enhance the infectivity of T. pallidum in the rabbit with antilympho-cyte serum (ALS) or steroids, or both. In this way we hoped to develop a model for the identification of small numbers of treponema-like organisms in the eye.

MATERIALS AND METHODS

The experimental animals were 48 New Zealand White male rabbits weighing four to five pounds and reacting negatively to the venereal disease research laboratory (VDRL) test.

The ALS, prepared as previously de­scribed,7 had a cytotoxicity titer of 1:256.

The normal horse serum (NHS) was ob­tained from a commercial source.

The strain of T. pallidum was a rabbit-adapted Nichols strain obtained from exper­imentally inoculated rabbit testes. The purif­ication of the organism8 and the numerical counts9 were done by the California State Public Health Department's Microbial Dis­ease Laboratory in Berkeley, California. The organisms were maintained in an atmo-

From the Francis I. Proctor Foundation for Re­search in Ophthalmology and the Department of Ophthalmology, University of California, San Francisco Medical Center. This investigation was supported in part by USPHS Research Grant EY-00310.

Reprint requests to Gilbert Smolin, M.D., Fran­cis I. Proctor Foundation, University of California, San Francisco Medical Center, San Francisco, Cali­fornia 94122.

sphere containing 10% C 0 2 and TPI basal media.

Four dilutions were used for four 0.1-ml intradermal injections into the shaven backs of each of the 48 rabbits. The dilutions con­tained, respectively, 5 X 104, 5 X 103, 5 X 102, and 5 X 101 organisms per 0.1-ml in­jection. There was a one-hour delay between when the organisms were harvested and puri­fied and when the injections begun. In addition, it required approximately 75 min­utes to make the dilutions and inoculate the animals. During these inoculations, the tré­ponèmes were not in the 10% C0 2 atmo­sphere. At the conclusion of the inoculations, over 80% of the tréponèmes retained their motility when observed by dark field micro­scopy.

EXPERIMENTAL DESIGN

The inoculated animals were divided into six groups of eight animals each, as follows:

Group I received intravenous ALS only.

Group II received intravenous ALS and intramuscular steroid.

Group III received intravenous NHS only.

Group IV received intravenous NHS and intramuscular steroid.

Group V received intramuscular steroid only.

Group VI received intravenous saline only.

The ALS, NHS, and saline were given intravenously in 2-ml doses. The steroid, cortisone acetate (Cortone, 25 mg/ml), was given intramuscularly in doses of 5 mg/kg. All medications were given on days -2, 0, 3, 5,7, 10, 12,14, 17, 19, and 21.

Clinical evaluation—The skin lesions were read twice a week for four weeks (day 13 to

273

274 AMERICAN JOURNAL OF OPHTHALMOLOGY ■ AUGUST, 1970

Group

ALS ALS + Steroid NHS NHS + Steroid Steroid Saline

13

0/8*

0/8 0/8

0/8 0/8 0/8

17

2/8

0/8 0/8

2/8 1/8 0/8

NUMBER(

TABLE 1 3F ANIMALS WITH SKIN LESIONS

(by days postinoculation)

20

4/7

3/8 2/8

2/7 2/8 1/7

Days After Inoculation

24

5/7

3/8 2/8

2/7 2/8 0/7

27

5/7

5/8 2/6

2/5 3/8 0/7

31

4/7

5/8 2/6

2/3 3/8 0/7

34

2/7

5/8 2/6

1/3 3/8 1/7

38

0/7

5/8 2/6

0/3 3/8 1/7

41

0/7

5/8 0/6

0/3 2/8 0/7

* The numerator represents the number of animals with skin lesions on the specific day. The denominator represents the number of animals alive in the given group on the same day.

day 41 ) by two observers who did not know what medication any animal had received. They recorded the coloration consistency, and presence or absence of induration, eleva­tion, and ulcération. The weight, appearance, activity, and appetite of the rabbits were also carefully noted.

Serologie evaluation—All of the animals were bled before and at intervals during the course of the experiment. The blood was stored at +4°C for 24 hours and a VDRL test was then performed on the separated se­rum.

RESULTS

Clinical evaluation—The numbers of ani­mals with skin lesions in the various groups are recorded in Table 1. Skin lesions were observed in five of the Group II animals who received ALS and steroid over a 27-day period. Lesions appeared in five of the Group I animals treated with ALS alone, for a much shorter period (four days). Le­sions appeared in two or three of the animals in each of the groups receiving NHS, steroid, or both, for various lengths of time ; and in only one animal in the saline group for three brief periods.

Table 2 records the maximum number of lesions seen in each group during the 30-day

observation period. The appearance of the lesions did not vary significantly from one group to the next.

All of the animals receiving steroid had anorexia and a significant loss of weight. ALS, NHS, and saline did not have this ef­fect. One animal died in each of the ALS, saline, and steroid groups during the experi­ment. Two animals died in the NHS group, and five died in the group receiving NHS and steroid. The remaining animals in the latter group appeared to be seriously ill.

Serologie evaluation—Prior to the experi­ment, all of the animals were VDRL-nega-tive. Most of them became VDRL-positive between three and four weeks after the T. pallidum inoculation. All of the surviving animals in the groups receiving ALS ( seven

TABLE 2 NUMBER OF SKIN LESIONS PER GROUP

Group

I ALS II ALS+steroid III NHS IV NHS+steroid V Steroid VI Saline

Maximum No. Lesions per

Group

10 10 7 7 5 2

VOL. 70, NO. 2 TREPONEMA PALLIDUM GROWTH 275

TABLE 3 DEVELOPMENT OF A POSITIVE VDRL TEST BY DAY 35

No. Surviving Serology Group

I ALS II ALS+steroid III NHS IV NHS+steroid V Steroid VI Saline

Rabbits on Day 35*

7 8 6 3 8 7

Negative

0 0 2 0 4 2

Weakly Positive

1 3 2 0 3 3

Positive

6 5 2 3 1 2

* Of the 48 rabbits inoculated, nine had died by day 35

rabbits') and ALS + steroid (eight rabbits) became positive five weeks after inoculation. Only three rabbits in the NHS plus steroid group were living at five weeks, and all of these were VDRL-positive. In the NHS, steroid, and saline groups, two of six, four of eight, and two of seven animals, respectively, were still VDRL-negative (Table 3) .

DISCUSSION

The number of skin lesions produced in this experiment after the intradermal inocu­lation of T. pallidum was smaller than ex­pected. It may be that some of the organisms at the time of inoculation were not viable. Although the animals were fed meal without antibiotics while under our care, it is possi­ble that high doses of antibiotics were in their feed before we obtained them. Either of these factors would have altered the ani­mals' clinical responses.

The animals were inoculated in sequence from Group I through Group VI. If the via­bility of the organisms varied significantly during the 75 minutes needed for making the inoculations, the results would have been weighted accordingly. But in fact, there were more animals that developed lesions in Group II than in Group I, and more in Group V than in Groups III or IV.

Inspection of the number of animals that developed skin lesions suggests that ALS, and especially ALS plus steroid, increases the infectivity of T. pallidum in rabbits.

Among the affected animals, however, there was no significant difference from group to group in the number of lesions that devel­oped ; nor were there any intergroup differ­ences that could be considered significant so far as the sérologie conversion ratios were concerned.

NHS, and especially the NHS plus ste­roid combination, was tolerated very poorly by the animals; two died in the former group and five in the latter.

Since more data must be accumulated be­fore any conclusions can be drawn from this study, followup experiments are in progress in which some of the variables have been eliminated.

SUMMARY

An attempt was made to potentiate Tre-ponema pallidum infection in rabbits. The tréponèmes were inoculated into the shaven backs of the rabbits and the animals were then treated with antilymphocyte serum, normal horse serum and steroids in various combinations. The results suggest that ALS alone or in combination with steroids in­creases the infectivity of the organism.

ACKNOWLEDGMENTS

We thank Drs. Jerome Goldman and Leslie No­rms for their valuable advice in the planning of this study. We also thank Mrs. Peggy Yamada and Mr. Robert Cave for their technical assistance. Special thanks are extended to the staff of the Microbial Disease Laboratory of the California State Health

276 AMERICAN JOURNAL OF OPHTHALMOLOGY AUGUST, 1970

Department in Berkeley, both for help and advice and for providing and preparing the T. pallidum used in this study.

REFERENCES

1. Smith, L. J., and Israel, C. W. : Spirochetes in the aqueous humor in seronegative ocular syphilis. Arch. Ophth. 77:474, 1967.

2. Smith, L. J., and Israel, C. W.: The presence of spirochetes in the late seronegative syphilis. JAMA 199:980, 1967.

3. Golden, B., Watzke, R. C, Lindell, S. S., and McKee, A. A.: Treponema-like organisms in the aqueous of nonsyphilitic patients. Arch. Ophth. 80 : 727, 1968.

4. Wells, J. A., and Smith, J. L. : The fluorescent

Ocular gnathostomiasis is a rarity. Gna-thostoma spinigerum was first found in man by Levinsen1 in 1890 in the breast abscess of a woman living in what is now Thailand. Subsequently, additional cases in humans were reported from Malay, Thailand, China, and Japan; the disease is prevalent in Southeastern Asia, Indonesia, the Philip­pines, China, and Japan.2 The natural hosts of the parasite are fish-eating mammals and fowl,3 which serve as intermediate hosts that may pass the parasite on to humans. In hu­mans, however, the worm does not reach ma­turity.

In 1945, Sen and Ghose4 reported a case of ocular gnathostomiasis, and in 1968, Khin5 reported additional cases. I believe the present case is the first to be reported from East Pakistan.

CASE REPORT

A stout, 35-year-old Muslim reported to the eye clinic of the Rajshahi Medical College hospital on

From the Department of Ophthalmology, Raj­shahi Medical College, Rajshahi, East Pakistan.

Reprint requests to Abdur Rahim Choudhury, M.D., M.B., B.S. (DAC), D.O. (London), Associ­ate Professor of Ophthalmology, Rajshahi Medical College, Rajshahi, East Pakistan.

antibody tissue stain for Treponema pallidum in the eye. Am. J. Ophth. 63:410, 1967.

5. Turner, T. B., and Hollander, D. H.: Biology of the Treponematoses. Geneva, World Health Or­ganization, 19S7, p. 31.

6. Montenegro, E. N. R., Nicol, W. G., and Smith, J. L.: Treponema-like forms of artifacts. Am. J. Ophth. 68:197,1969.

7. Smolin, G. : Suppression of corneal graft reac­tion antilymophocyte serum. Arch. Ophth. 79 :603, 1968.

8. Serologie Tests for Syphilis, 1959 Manual USPHS publication No. 411. U.S. Government Printing Office, Washington, 1959.

9. Chandler, F. W., Jr., and Norins, L. C. : Studies on the quantitation of intraocular Treponema palli­dum. An in vitro model. Brit. J. Vener. Dis. 45:2, 117, 1969.

September 27, 1967, complaining of pain in his right eye, swelling of the right eyelid and the right temporal region, and dimness of vision. The patient had experienced these symptoms at varying inter­vals over the preceding several months. This epi­sode had had its onset three days earlier. In July, 1967, while working in a jungle area, he received an insect bite on the right temporal region. Follow­ing this, he experienced itching and pain at the site, with swelling, and a few blebs appeared in the con­junctiva near both the medial and lateral canthi. The initial swelling subsided after a few days, but swelling continued to recur at different sites of the right temporal region. Ultimately, palpebral swell­ing appeared, accompanied by pain and dimness of vision in the right eye.

The patient's normal diet consisted of fish, meat, vegetables, rice, and wheat. He gave no history of having eaten raw or partially cooked fish. However, at the time symptoms first appeared, a river was his source of water supply, both for drinking and bath­ing.

The right temporal region was swollen and the palbebral conjunctiva slightly conjested. Vision in the right eye was 6/36. The cornea was edematous ; there were many large keratic precipitates; the aqueous humor was turbid ; the pupil was drawn to one side. Inflammatory cells and exudate were de­posited on the lens surface, and the fundus could not be visualized.

The patient was treated initially for anterior uveitis, but the response was not satisfactory. Symptoms disappeared temporarily, but shortly re­curred. On November 27, 1967, the pain in the right eye increased, vision decreased to counting fingers at 10 feet, and a curved, dark-brownish mass was

OCULAR GNATHOSTOMIASIS

ABDUR RAHIM CHOUDHURY, M.D.

Rajshahi. East Pakùtan