Streptococcic Antibody Antigen Reactions of the Serum and Skin of Monkeys during Attacks of Experimental Poliomyelitis

In studies previously reported,^1,2,3 it was found that an interphase clouding occurred in precipitation tests using the serums of patients suffering from diseases caused by, or associated with, certain alpha streptococci and antiserums prepared with the respective freshly isolated streptococci and that an immediate erythematous and edematous reaction occurred in the skin of patients at the site of intracutaneous injection of the respective antiserums or antibody euglobulin. These antibody-antigen reactions⁴ indicate that a person whose serum and skin react selectively to one or more antistreptococcic serums and not to control horse and and anti-pneumococcic serums is suffering from an infection caused by streptococci antigenically related to those used in preparing the antiserum. The principle involved, including the effects of therapeutic injection of the poliomyelitis antistreptococcic serum, has been applied to epidemic poliomyelitis with striking results.^5,6,7 It occurred to me that, if similar reactions could be demonstrated in experimental poliomyelitis in monkeys, the result might throw light on the true nature of the infectious process which occurs after inoculation of virus.⁸ This problem has been under study as opportunity was afforded since February 22, 1934. The results have been so consistent in a large number of experiments that a report is thought advisable at this time.

Methods

Interphase precipitation tests were made in small tubes by superimposing over various whole antistreptococcic serums the serum obtained from the centrifuged clotted blood of normal monkeys or of monkeys during the acute stage of, and during and after recovery from, poliomyelitis and after injection of the poliomyelitis antistreptococcic serum. Readings were made under a strong shaded light in a dark room against a nonreflecting background of black velvet after incubation at 35° C. for one and a half hours and refrigeration overnight.

For the cutaneous tests the skin over the lower part of the back, ilium and outer aspect of the thighs of Macacus rhesus monkeys, from which the hair was closely clipped and shaved, was found most suitable. I used 10 per cent solutions of the centrifuged euglobulin fraction, obtained by 1:10 dilution in slightly acidulated water, of the serum of horses immunized with streptococci obtained in studies of poliomyelitis, and control euglobulins from the serums of horses immunized with streptococci obtained in studies of encephalitis, arthritis, influenza and ulcerative colitis, from normal horse serum, or merely normal horse serum diluted 1:10. The test consisted of the intracutaneous injection of 0.03 c.c. of the various euglobulins, as superficially as possible, into normal monkeys, into monkeys during the acute stage of experimental poliomyelitis, during and after recovery and immediately before and after therapeutic injections of the antistreptococcic serum. An edematous reaction with an average diameter of 6 mm. or greater was considered a positive reaction and absence of measurable increase in the blanched regions attributable to the injection of the different euglobulins was considered a negative reaction.

The poliomyelitis and control antiserums used in treatment and in precipitation tests and from which the euglobulin fraction was obtained for cutaneous tests were prepared in horses in a comparable manner. Weekly intravenous and subcutaneous injections of increasing doses of the respective freshly isolated streptococci were given. The antigenic specificity of these streptococci was maintained in dense suspensions of glycerol (2 parts) and 25 per cent solution of sodium chloride (1 part) throughout the period of immunization.

The poliomyelitis antistreptococcic serum used for treatment was injected intravenously or intramuscularly once or twice daily for several days in amounts of 0.5 c.c. per kilogram of body weight, after onset of unmistakable symptoms of poliomyelitis. The antiserum consisted of a solution of 1 part of the euglobulin and 2 parts of the pseudoglobulin fraction of the antiserum in concentrations of about that of the whole serum from which it was obtained. Control intramuscular injections of the arthritis antistreptococcic serum, of normal horse serum and of human poliomyelitis convalescent serum were made.

Two groups of monkeys were studied. Only the precipitation reaction of the serum of the monkeys before and after inoculation of virus and after therapeutic injection of the poliomyelitis antistreptococcic serum was determined in the first group; in the second, the cutaneous reaction was studied also. In the first group seven different batches of antiserums which were prepared in horses by me with suspensions of streptococci isolated respectively from nasopharynx, cerebrospinal fluid or tissue from brain and spinal cord in epidemic and experimental poliomyelitis were used. In the second group antiserums commercially prepared with similar suspensions furnished by me were used. Besides these two groups of monkeys another group of animals which consisted of both monkeys and rabbits was studied in order to determine the value of the precipitation and cutaneous reactions as measures of streptococcal antigen.

Precipitation Tests in the First Series of Monkeys

The results obtained in precipitation tests in the first series of experiments are summarized in table 1. Altogether 658 specimens of serum from 216 Macacus rhesus monkeys were tested in this series of experiments, with consistent results throughout. Nine different virus strains were used to produce the disease. Four of these were strains directly from patients that had succumbed to epidemic poliomyelitis or strains that had undergone one or two passages through monkeys; the rest were highly adapted strains which had undergone many passages through monkeys and which were supplied by various workers in the virus field.

Precipitation and Cutaneous Reactions in the Second Series of Monkeys

The results of the tests on the second series of monkeys were as strikingly consistent as those on the first series. A blanching, edematous reaction developed at the site of intracutaneous injection of the poliomyelitis euglobulin during the acute stage of poliomyelitis. In nonpigmented skin this sometimes was surrounded by an areola of erythema, but little or no reaction occurred at the sites of injection of the control euglobulins or normal horse serum (figs. 1 and 2).

Fig. 1. Edematous cutaneous reactions in three monkeys (protocols 1, 2 and 3) on the second, third and second day, respectively, of typical symptoms during attacks of experimental poliomyelitis, thirty minutes after intradermal injection (reading downward) of three batches of poliomyelitis euglobulin, and of one batch of encephalitis euglobulin, arthritis euglobulin and in a, normal horse serum diluted 1:10. The monkeys had been inoculated previously, with a, virus from a case of epidemic poliomyelitis, after one passage through a monkey, b, the Flexner standard mixed virus strain which has been passed through monkeys for many years,. and c, experimental virus derived from a neurotropic type of streptococcus in the fifth passage through monkeys (x 1¼).

Fig. 2. Results of intracutaneous injections into monkeys of poliomyelitis euglobulins (top two in each row for a, b, and c) and encephalitis, arthritis and ulcerative colitis euglobulins (bottom three in a, b, and c): a. no reactions, normal monkey; b and c, left rows, edematous reaction to two batches of poliomyelitis euglobulin and no reaction to the control euglobulins, in two monkeys on the second day of experimental poliomyelitis; b, right row, no reduction in reactivity to reinjection of the two poliomyelitis euglobulins four hours after intramuscular injection of arthritis antistreptococcic serum; c, right row, complete disappearance of reactivity to the two poliomyelitis euglobulins four hours after intramuscular injection of poliomyelitis antistreptococcic serum; d, left row, blanching edema to multiple intracutaneous injections of the poliomyelitis euglobulin, and right row, no reaction to multiple injections of the arthritis euglobulin in a monkey on the second day of experimental poliomyelitis.

The results of precipitation tests with the poliomyelitis antistreptococcic serum and the serum of monkeys and the cutaneous tests with the poliomyelitis euglobulin were uniformly negative before inoculation of virus, positive only in a few instances during the period of incubation, and positive in nearly all instances as symptoms of poliomyelitis developed and throughout the course of the disease in monkeys not treated with serum (figs. 3a and 4a). This reactivity of the blood serum (fig. 3b) and skin (figs. 2c and 4b) promptly disappeared after treatment with the poliomyelitis antistreptococcic serum.

Fig. 3. Precipitation reactions between the poliomyelitis antistreptococcic serum and the serum of monkeys obtained during attacks of experimental poliomyelitis.

Fig. 4. Cutaneous reactions in Macacus rhesus monkeys to intradermal injection of the poliomyelitis euglobulin during the acute stage of experimental poliomyelitis.

The substance in the serum of monkeys which caused the clouding in the interphase precipitation tests with the poliomyelitis antistreptococcic serum resembled antigen in its properties. It was filtrable through Berkefeld N and Seitz filters and was removed completely by absorbing agents such as animal charcoal, aluminum silicate and colloidal kaolin.

Control precipitation tests were made with the serums obtained from seventy-six monkeys during the acute stage of experimental poliomyelitis and the poliomyelitis antistreptococcic serum after absorption with the poliomyelitic or arthritic streptococci and the unabsorbed poliomyelitis antistreptococcic serum. Clouding at the interphase between the serums of the monkeys and the unabsorbed antiserum occurred in each of the seventy-six, in only eleven with the antiserum after absorption with poliomyelitic streptococci, and in sixty-two after absorption with arthritic streptococci.

Control intramuscular injections of arthritis antistreptococcic serum (fig. 2b), normal horse serum and human convalescent poliomyelitis serum had little or no effect in preventing the edematous reaction to intracutaneous injection of the poliomyelitis euglobulin in monkeys during experimental poliomyelitis or the interphase precipitation reaction between the serum of monkeys and poliomyelitis antistreptococcic serum. Ten strains of “natural” poliomyelitis virus, two before and eight after passage through monkeys, and six strains of “experimental” poliomyelitis virus derived from neurotropic streptococci were inoculated into the second series of monkeys. Fifty-seven of ninety-two monkeys received “natural” virus and thirty-five “experimental” virus. Five per cent emulsion of brain and cord or corresponding Berkfeld N filtrates were used for inoculation. Twenty-one of the fifty-seven were inoculated intracerebrally and thirteen intranasally with emulsion, twenty-three intracerebrally with filtrate. Sixteen of the thirty-five that received “experimental” virus were inoculated intracerebrally with emulsion and nineteen with filtrate. The incidence and degree of cutaneous and precipitation reactions depending on whether “natural” or “experimental” virus was inoculated did not differ notably.

Serial dilution cultures⁹ alternately in dextrose brain broth and dextrose brain agar or dextrose brain broth cultures of the filtrates injected yielded the streptococcus in seven instances. Cultures in dextrose brain broth of emulsions of the different strains of virus injected revealed the streptococcus in nearly all instances, those in dextrose broth revealed the streptococcus in only a few instances, and those on blood agar never revealed streptococci. Cultures of filtrates in dextrose broth and on blood agar never yielded the streptococcus.

The Precipitation and Cutaneous Reactions as Measures of Streptococcal Antigen

If the precipitation reaction between the poliomyelitis antistreptococcic serum and the serum of monkeys during the acute stage of experimental poliomyelitis and the cutaneous reaction following intradermal injection of the poliomyelitis euglobulin really represent specific streptococcal antibody antigen reactions, similar reactions should be obtained between the poliomyelitis antistreptococcic serum and the serum of animals that have been inoculated with suitable numbers of the dead streptococci suspended in normal serum or solution of sodium chloride. Accordingly experiments were carried out: From the dense stock suspensions in glycerol and solution of sodium chloride 1,000,000; 10,000,000; 100,000,000; 1,000, 000,000 or 10,000,000,000 dead streptococci of poliomyelitis or of arthritis were added per cubic centimeter of solutions of sodium chloride and normal serums. These suspensions were placed in the refrigerator overnight and then cleared by centrifugation.

The cleared supernatant solutions or extracts containing 1,000,000; 10,000,000 and 100,000,000 dead streptococci of poliomyelitis gave precipitation reactions with the poliomyelitis antistreptococcic serum only. These reactions were directly proportional to the number of streptococci contained in the corresponding suspensions. The cleared suspensions of serums or solutions of sodium chloride of extracts containing 1,000,000,000 and 10,000,000,000 dead streptococci of poliomyelitis gave maximal reactions with the poliomyelitis antistreptococcic serum and slight cross reactions with the encephalitis and arthritis antistreptococcic serums. None gave reactions with normal horse serum.

Two series of monkeys and rabbits then were inoculated intracerebrally and intravenously or intracerebrally alone with graded amounts of the solutions of sodium chloride or serum containing the dead streptococci. From 10,000,000 to 30,000,000,000 dead poliomyelitis or arthritis streptococci were given to some of the monkeys and a tenth of these amounts to some of the rabbits; for other rabbits and monkeys approximately a third of these amounts were used. Ten monkeys and ten rabbits were given the poliomyelitic streptococci and four monkeys and ten rabbits received the arthritic streptococci. Five of the ten monkeys and seven of the ten rabbits that received the poliomyelitic streptococci, and two of the four monkeys and nine of the ten rabbits that received the arthritic streptococci in largest dosage, respectively, died in from twenty-four to forty-eight hours. Great weakness or flaccid paralysis without lesions of muscles or joints developed in the animals that received the dead poliomyelitic streptococci whereas evidence of pain and stiffness of muscles and joints and hemorrhage with exudation in muscles and joints developed in the animals that received the arthritic streptococci.

Results of precipitation and cutaneous tests in monkeys were as follows: Both tests gave negative results before injection and after injection also when only 10,000,000 dead organisms were given. Both gave specifically positive results to the poliomyelitis and arthritis antistreptococcic serums at four, twelve and twenty-four hours after injection and again negative or only slightly positive results forty-eight hours after injection of the larger numbers of dead streptococci. The cutaneous reactions of the rabbits were negative before and after injection, whereas the precipitation reactions were specifically positive after injection, to the poliomyelitis and arthritis antiserums. The degree of reaction was roughly proportional to the dosage injected.

Evidence of specificity of the respective dead streptococci was not limited to the precipitation and cutaneous reactions and symptoms produced, but was shown also by their truly remarkable elective localization or tropism. All cultures of suspensions injected, and of cerebrospinal fluid and knee joint fluids of the monkeys before and after injection, proved sterile. Cultures from brain and blood after death of animals that succumbed were likewise sterile. No bacteria were found in stained smears of the cerebrospinal fluid and fluid from the knee joint before injection. The poliomyelitic streptococci remained in the cerebrospinal fluid (fig. 5a) for a much longer time than the arthritic streptococci. They were almost never found in smears of the fluid from the knee joints (fig. 5b), whereas the streptococci obtained in studies of arthritis disappeared promptly from the cerebrospinal fluid (fig. 5c) and were found in varying numbers in the articular fluid of animals that had received the arthritic streptococci, and often in large numbers four, twenty-four (with no leukocytes, fig. 5d) and forty-eight hours (often with many leukocytes) after intracerebral and intravenous injection or after intracerebral injection only.

Fig. 5. Streptococci in the cerebrospinal fluid, a, and none in joint fluid, b, of a monkey twenty-four hours after intracerebral inoculation of dead poliomyelitic streptococci; absence of streptococci in the cerebrospinal fluid, e, and large numbers of diplococci in the joint fluid, d, twenty-four hours after intracerebral injection of dead arthritic streptococci (Gram-safranine stain, x 1,000).

Sections of the brain and spinal cord, moreover, revealed perivascular infiltration and large numbers of streptococci in various stages of disintegration in the meninges, and dilatation of blood vessels, small hemorrhages and degeneration of ganglion cells in the anterior horns of the spinal cords of monkeys that had received the poliomyelitic streptococci whereas sections of brain and spinal cord of monkeys inoculated with the arthritic streptococci were free from lesions and streptococci.

The four monkeys that had received intracerebral, and one that had received intracerebral and intravenous inoculations of the dead streptococci obtained in studies of poliomyelitis, and two that had received the streptococci obtained in studies of arthritis, and a normal monkey were inoculated intranasally, according to the method of Sabin and Olitsky,¹⁰ with a highly virulent poliomyelitic virus ten and twelve days, respectively, after injection of the dead streptococci. Four of the five monkeys that had received the dead streptococci obtained in studies of poliomyelitis remained well and the precipitation and cutaneous reactions remained negative. The monkey that had received intracerebrally the suspension containing the smallest number of dead streptococci obtained in studies of poliomyelitis became paralyzed but recovered. The two monkeys that had received the dead streptococci obtained in studies of arthritis and the normal control monkey died of typical poliomyelitis. The precipitation and cutaneous reactions became positive in the four monkeys in which symptoms of poliomyelitis developed.

Therapeutic Effects in the Monkeys that Were Treated with Poliomyelitis Antistreptococcic Serum

Improvement in symptoms of monkeys often occurred concomitantly with the disappearance of reactivity of the skin and of precipitinogen from the serum after the therapeutic injection of the poliomyelitis antistreptococcic serum. Tremors, twitchings of muscles and excitability also disappeared or diminished and rapidly progressing paralysis sometimes was stayed or arrested. As has been indicated, therapeutic injections of the serum were not made until after unmistakable symptoms of poliomyelitis had developed. The beneficial effects in retarding progression of symptoms, however, usually were apparent only for two or three days in monkeys that were not yet severely paralyzed. There was usually no noticeable effect on the poliomyelitis after paralysis had become conspicious. Of forty-three monkeys that received serum twenty-four (56 per cent) died, whereas of sixty-six that did not receive serum and among which conditions were comparable, fifty-six (85 per cent) died. These results are in accord with those obtained previously in epidemic and experimental poliomyelitis.^{5-8, 11}

Illustrative Protocols

Protocol 1.—A Macacus rhesus monkey was inoculated intracerebrally on June 24, 1940, with 3 c.c. of an emulsion of glycerolated brain and cord of a monkey in which typical poliomyelitis had developed after inoculation of virus obtained in a case of epidemic poliomyelitis that had occurred in Tacoma, Washington, in the summer of 1940. The animal remained well until July 6, when undoubted beginning symptoms of poliomyelitis developed. The temperature was 103.2° F. The animal was hyperirritable, its face was flushed, it had a staccato voice and there were fine tremors of the body. On July 8 the animal was worse, there was marked weakness of all extremities, it was easily exhausted and could scarcely climb into its cage. Cutaneous tests were made; there were areas of blanching edema surrounding the sites of injection of three different batches of poliomyelitis euglobulin but there was no reaction to the encephalitis and arthritis euglobulins and normal horse serums diluted 1:10 (fig. 1a). On July 9 the animal dragged its right hind leg and weakness in all extremities was so severe that it could not climb to its perch. The cutaneous test was repeated with similar results. The animal was then given a therapeutic injection of the poliomyelitis antistreptococcic serum intramuscularly. On July 10 there was no extension of paralysis and another cutaneous test gave negative results. Therapeutic injection of the antiserum was repeated. On July 11 there was no extension of paralysis, cutaneous reactions were again negative and a third therapeutic injection of the serum was given. On July 12 there was decided improvement in strength of the left hind leg and of both fore extremities but the right hind leg was still flaccid. The cutaneous test was repeated, again with negative results. On July 13 the animal was killed. Aside from diffuse congestion of the brain, results of necropsy were negative.

Protocol 2.—A Macacus rhesus monkey was inoculated intracerebrally on March 3, 1942, with 2 c.c. of a filtrate of a 2.5 per cent emulsion of the spinal cord of a monkey that had died after inoculation of the Flexner mixed virus strain. The animal remained well until March 8 when typical symptoms of poliomyelitis developed. It was slightly tremulous, the face was flushed, the hair was rough and the animal was excitable. On March 9 there was almost complete flaccid paralysis of all extremities. On March 10 the animal was prostrate, respirations were shallow and the voice was weak but circulation was unimpaired; temperature was normal. Cutaneous tests made with three batches of poliomyelitis euglobulin, arthritis euglobulin, and normal horse serum diluted 1:10 produced blanching edema at the sites of injection of the three poliomyelitis euglobulins, which was maximal in thirty minutes, and no reaction to the arthritis euglobulin and normal horse serum (fig. 1b). On March 11 the animal was barely alive and was killed. Necropsy revealed moderate diffuse congestion of the brain but no lesions of the viscera.

Protocol 3.—A Macacus rhesus monkey, was inoculated intracerebrally on March 3, 1942, with 2 c.c. of filtrate of a 2.5 per cent emulsion of an “experimental” virus strain in the fifth monkey passage, which was derived from a neurotropic type of streptococcus. The animal remained well until March 9 when typical symptoms of poliomyelitis developed. Fine tremors of the head, congestion of the face, abnormal excitability, staccato voice and weakness of hind extremities were noted. On March 10 almost complete flaccid paralysis of all extremities and shallow respiration developed but circulation was unimpaired and temperature was normal. The cutaneous test was made with three different batches of poliomyelitis euglobulin, with the arthritis euglobulin and normal horse serum diluted 1:10. A blanching edema occurred at the sites of injection of the poliomyelitis euglobulins but there was no reaction to the arthritis euglobulin and normal horse serum (fig. 1c). On March 11 the animal was completely paralyzed, the voice was weak and respirations were shallow and wholly diaphragmatic. The animal was killed. Necropsy revealed only moderate diffuse congestion of the brain, without lesions of the viscera. Typical lesions of poliomyelitis were found in sections of the spinal cord.

Summary

Antibody-antigen reactions specifically related to the streptococcus isolated in studies of poliomyelitis were demonstrated in the skin and serum of Macacus rhesus monkeys during attacks of experimental poliomyelitis. Therapeutic injection of the poliomyelitis antistreptococcic serum apparently had beneficial action and caused the reactivity of skin and serum to disappear.

Reactivity of the skin and serum of monkeys to homologous streptococcal antibodies was demonstrated in four, twelve and twenty-four hours after intracerebral inoculations of the respective dead poliomyelitic and arthritic streptococci and disappeared after forty-eight hours. A corresponding specific affinity for, and toxic action on, tissues of the nervous system and joints was shown by the respective dead streptococci.

Monkeys that recovered from intracerebral inoculation of the dead poliomyelitic streptococci resisted intranasal inoculation of highly virulent virus to which all control monkeys, including those that had received the arthritic streptococci, succumbed.

The results of these experiments indicate that the streptococcus associated with poliomyelitis is antigenic and biologically related to what is now considered as virus.

References Cited:

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