Louise Pearce

Experimental Transmission of Rabbit-Pox by a Filterable Virus

Experimental transmission of the spontaneous rabbit-pox discussed in the preceding communication 1 was attempted early in the course of the pandemic. Since the result of bacteriological examinations together with the clinical picture of the condition made it appear unlikely that we were dealing with any of the usual bacterial agents, the initial experiments were carried out with Berkefeld “V” filtrates of various organs and tissues from rabbits showing well marked symptoms of the disease. In other experiments, unfiltered suspensions were used. The organs employed included: testicle, liver, spleen, lung, popliteal lymph nodes, brain, spinal cord, blood, and skin. Injections were made into the testicles of rabbits procured from outside sources. The results of the first experiments were positive in that an orchitis and scrotal edema were pronounced within 48 hours, fever developed (105°-107°), the animals appeared ill, frequently a diarrhea was present, and death occurred in from 4 to 8 days. This condition has been produced successively from rabbit to rabbit by means of filtered material (usually testicle) and the agent is now in the fifteenth serial passage. Other routes of inoculation have also been successfully employed, namely, intracutaneous, subcutaneous, intravenous, intraperitoneal, intramuscular, intracerebral, and nasal and conjunctival instillation. By using small doses and certain routes of inoculation, it has been possible to reproduce all the clinical and pathological features of the spontaneous disease including the characteristic pock eruption of the skin and mucous membranes. In addition, complete recovery has occurred in these circumstances. The agent is active in tissue stored in 50% glycerol at ice box temperature for at least 94 days. Unfiltered material is much more potent than that filtered through a Berkefeld “V” candle but an “N” filtrate is also active.

Animal resistance and the endocrine system of the rabbit in experimental syphilis

In the course of the work that has been carried out in this laboratory during the past 8 years, it has become more and more apparent that of the several factors concerned in determining the general course and severity of syphilitic infections, the spirochete is of minor importance as compared with the number of other influences, especially the factor of animal resistance. This is strikingly illustrated by the experimental infection in the rabbit which, as a rule, is comparatively mild but in exceptional instances may assume a more malignant form or persist in an active state with severe constitutional symptoms for months or even years and in the end cause the death of the animal. Little has been known as to the nature or source of the forces employed by the animal in combating this disease but there is a great deal of evidence to indicate that resistance to this infection is a function of animal economy and is subject to the same influences as are concerned in the regulation of growth and development, and the maintenance of general metabolic activities. Moreover, there have been specific indications that resistance was influenced to some extent by the endocrine system. This possibility was first suggested to us by the occurrence of marked seasonal changes in the severity of the disease but was most forcibly impressed upon us by the abnormal resistance displayed at times by pregnant and lactating females.

Neoplasia in experimental syphilis

Neoplasia as a sequel to syphilitic infection is not uncommon in man but has never been recorded in an experimental animal. Recently, we have observed an atypical growth arising from the scar of an old syphilitic lesion in the scrotum of a rabbit which may prove to be a neoplasm. The animal was inoculated in the scrotum, June 16, 1916. Small chancres developed and then underwent spontaneous regression. Several months later, there was a recurrence and the lesion in the left scrotum persisted for some time. In October, 1920, there was a slight diffuse infiltration of the left scrotum and a small nodule appeared at the site of the old chancre. Although it was known that the animal still harbored spirochetes, 1 none could be demonstrated by dark-field examination of material from the nodule and it was excised for histological examination. There was a prompt recurrence and with the growth of the second cutaneous lesion, the left inguinal glands became markedly enlarged and indurated. Again no spirochetes could be demonstrated and the lesion with one of the adjacent glands was excised under ether anesthesia. Histological examination of the cutaneous nodules and gland showed a growth which presented more the appearance of a neoplasm than of a syphilitic lesion. It was composed for the most part of atypical epithelioid cells undergoing active proliferation and exhibiting marked invasive tendencies. Meantime there was a second recurrence and extension of the skin lesion over the mid line at the pubis with enlargement and induration of the right inguinal nodes. A deterioration in the physical condition of the animal was then apparent and progressed very rapidly, culminating in emaciation, weakness, severe anemia, loss of sphincter control with some spasticity of the hind legs and the formation of trophic ulcers about the anus and sheath.

Positive Wassermann Reaction Induced in Rabbits by Injection of Hamster Tissues.

In connection with a previous study of the susceptibility of the Chinese hamster (Cricetulus griseus) to syphilitic infection, 1 it was noted that normal rabbits developed strongly positive Wassermann reactions after being injected with hamster tissues in which the syphilitic virus could not be demonstrated by rigid biological methods. The question arose as to whether the tissues of the normal hamster, when injected into the rabbit, are capable of inducing Wassermann reactive bodies in the circulating blood. Consequently, further studies were made on rabbits injected with various tissues of normal hamsters. The following is a summary of the results obtained with injections of various tissues by different routes. Unless stated otherwise the hamster tissues used were emulsified in sterile normal saline shortly before being injected into the rabbit. Wassermann tests were made on the blood serum according to the Kolmer technique. 2 This method proved to be quite reliable in our hands. Among 71 normal rabbits tested there was only one false positive reaction in an animal which was in poor physical condition. 1. Testis. Seventeen rabbits received 0.5–1.0 cc. of the emulsion intratesticularly, and all of these developed a positive Wassermann reaction. In one rabbit injected intraperitoneally the same positive result was obtained. Two other animals injected subcutaneously into the right flank, gave similar results. Two rabbits injected intravenously with the emulsion which had been filtered through sterile gauze, in doses of 0.3 and 0.5 cc, showed a weakly positive reaction in the blood. 2. Brain. All 7 rabbits injected intratesticularly with the emulsion developed a strongly positive Wassermann reaction, while 2 other animals injected intravenously gave negative results. 3. Whole blood (not treated with saline). Each of 5 intratesticularly injected rabbits developed a fairly strongly positive Wassermann reaction. In another animal, injected subcutaneously (0.2 cc.), the reaction was weakly positive.

Seasonal changes in organ weights and their relation to meteorological conditions.

It is well known that many of the endocrine glands of normal animals undergo rhythmic changes in weight per unit of body weight which conform, in general, with seasonal conditions. Still, the cause of these changes is not clearly understood nor is it generally known that other organs and tissues such as the heart, the liver, the kidneys, the spleen, the thymus, the lymphoid tissues, and even the brain, undergo changes of a similar character. In November, 1921, we began a series of investigations which included among other things a study of the ratio of the weights of various organs and tissues to the body weight of normal rabbits and the limits of variation that might be encountered, with especial reference to the causes of such variations. The factors studied included age, breed, length of caging, time of killing (with reference to the regular system of feeding), and meteorological conditions. All of the animals used were males, and an effort was made to minimize the influence of dietary factors by maintaining a uniform system of feeding and a uniform diet consisting of hay, oats, and cabbage, which is the regular diet given all rabbits in these laboratories. The plan followed was to kill and examine two groups of rabbits each month selected from stocks that were being used for experimental work. All animals were well nourished and free from any external evidence of disease. During the first few months the groups were small and intentionally of a mixed character, but beginning with June, 1922, the monthly groups consisted of from ten to twenty rabbits. The animals were killed prior to the daily feeding; they were anesthetized with ether and bled from the inferior vena cava while the heart was still beating. The organs were removed, freed of all adventitious material, and weighed on suitable balances.

Reciprocal Influence of Concomitant Infections: Syphilis and Vaccinia.

The influence which one infection may have upon the manifestations of another has been investigated in a number of conditions in connection with a general study of the factors concerned in determining the course and outcome of disease processes. In the case of syphilis and vaccinia of the rabbit, the result of simultaneous inoculations of Tr. pallidum and vaccine virus has been studied. Other experiments have dealt with the syphilitic reaction in rabbits immune to vaccine virus, and conversely, with the vaccine reaction in rabbits infected with Tr. pallidum. The Nichols strain of Tr. pallidum and the Noguchi strain of vaccine virus were used; groups of 5 or 10 male rabbits were employed for each series. The syphilitic tissue emulsion was injected in one testicle or intracutaneously on the sheath; the inoculation of vaccine virus was made on the shaved skin of the body by rubbing the infected tissue emulsion into scarified areas, and by intracutaneous injection. In other groups, the syphilitic and vaccine virus emulsions were both injected in the same testicle. Control series of rabbits were inoculated with each of these materials. The observation period varied from 3 to 5 months. An essential requirement of the work was the frequent examination of the rabbits in order that the syphilitic process in the several groups could be compared. The features of this infection which are especially important for such comparisons are illustrated by the following examples: the incubation period, character and duration of the primary and metastatic orchitis, the occurrence of scrota1 edema, the time of development of generalized lesions, their distribution, number and duration, and the initiation of latency, that is when all manifestations of the disease have healed.

Reaction of the Rabbit to Vaccine Virus

Little information is available regarding the clinical reaction of the rabbit to cutaneous inoculation of dermo-vaccine virus except as regards the local reaction at the site of injection. Furthermore, factors which presumably might affect the response as, for example, age, have not generally been considered. Our interest in the matter arose from the results obtained in the vaccination of the rabbits of a large breeding colony. The experiments are still in progress but enough has been learned to show that the clinical reaction of the rabbit to vaccination is extremely variable and that these variations are associated with a number of factors which concern the host. Approximately 1800 rabbits were vaccinated in December, 1933, and January, 1934, with culture dermo-vaccine virus obtained through the kindness of Dr. T. M. Rivers. The injections were made intracutaneously with dilutions ranging from 1:50 to 1:40,000. Previous tests had shown that the virus was active in a titre of 1:100,000 to 1:1,000,000 injected intradermally. Certain outstanding results of the experiments may be briefly summarized as follows. A typical local reaction developed in the adult non-immune stock of both sexes but it was much more severe in bucks than in does, and more pronounced in resting than in pregnant and nursing does. Generalized manifestations consisting in particular of a cutaneous maculo-papular eruption in areas remote from the injection site, as for example, the ears, were not infrequent. A lymph adenitis was also observed and an orchitis in male animals developed in many instances. There were a few cases of pronounced illness and prostration but only 3 fatalities. The results were quite different in the younger stock, all of which were presumably susceptible. In the first place, a local reaction did not develop in a large proportion of these rabbits and the incidence of negative results was inversely proportional to age.

Susceptibility of the Chinese Stripted Hamster (Cricetulus Grisens) to Treponema pallidum

Experiments to determine whether the Chinese hamster is susceptible to inoculation with Treponema pallidum have shown that organisms pathogenic for the rabbit may be recovered from certain tissues of this animal species. It would appear that there is dissemination of the organisms from the local focus of inoculation and presumably a multiplication of them occurs. Hamsters were injected in one testicle with a tissue (orchitis) emulsion rich in spirochetes obtained from rabbits which had been inoculated with either the Nichols strain or with a strain (P III) isolated from a patient in this hospital in 1926. At varying periods, the hamsters were killed by ether anesthesia and different tissues, emulsified with sterile normal saline were injected intratesticularly in normal rabbits. The rabbits were under observations approximately 3 months. In the case of those in which a clinical orchitis was not detected together with darkfield demonstration of spirochetes, the inoculated testicle and the popliteal lymph nodes were injected in a second set of rabbits; in a few instances, this procedure was repeated in a third set of rabbits. The available results on the first generation of rabbit transfers may be summarized as follows: (1) In rabbits injected with the inoculated hamster testicle, 37, 64, and 138 days previously, a syphilitic orchitis developed in 2 to 3 weeks. (2) The inguinal lymph nodes of a hamster which was inoculated 64 days previously, induced a syphilitic orchitis in 2 rabbits in 7 and 9 weeks respectively; nodes from a hamster inoculated 138 days previously gave negative results in one rabbit. (3) Brain tissue of a hamster injected 138 days previously, induced a syphilitic orchitis in a rabbit in 43 days; negative results followed the injection of brain tissue of 2 hamsters injected 37 to 64 days previously.

Comparison of the Blood Picture in Treated and Untreated Syphilis Patients

Blood examinations were made on a number of syphilitic patients 20 who were divided into 2 groups. One group consisted of 58 patients who had received no treatment up to the time of the blood examination. Thirty-two were seen in the stage of an active primary infection, 18 had active secondary lesions, and 8 had signs and symptoms of tertiary disease. The second group consisted of 55 patients who had received varying amounts of specific treatment, ranging from less than one full course of arsphenamine and mercury to several such courses. In 20 of these, treatment was instituted during the primary stage of the disease, in 16 treatment was begun during the secondary stage, and in 19 during the tertiary stage. Since the patients were ambulatory and repeated counts could be obtained only with great difficulty, one complete blood examination was made on each individual. Each examination included a total red and white cell count made with standardized pipettes, a hemoglobin determination by the Newcomer method, a platelet count by the Ringer-heparin method of Casey, 1 , 2 and a differential white cell count made with the supravital technique, 100 cells being counted on each of 2 smears. The blood findings in these 2 groups were then compared, and the mean and standard error of the mean was determined for each blood element. A difference was considered to be significant when the probability of its occurrence by chance was less than one in 100. In the treated group as compared with the untreated patients, the hemoglobin level and the absolute and relative numbers of lymphocytes were significantly higher, while the total white cells, the number of platelets, the absolute and relative numbers of neutrophils, and the absolute and relative numbers of monocytes were significantly lower.

Effect of controlled conditions of illumination upon malignancy of transplantable neoplasm.

Periodic variations in the malignancy of a transplantable neoplasm of the rabbit, which have been studied during the last four years, have been correlated with certain meteorological conditions and especially with the actual hours of sunshine. It has been found, that periods of maximum and minimum sunlight corresponding with summer and winter were periods of relatively low malignancy; while the periods of greatest malignancy occurred at times of abrupt and rapid changes in the hours of sunshine corresponding roughly with spring and autumn. 1 , 2 The influence of these factors has been interpreted as operating upon or affecting animal economy, while susceptibility or resistance to disease is considered as a function of the animal organism. This conception is supported by analogous variations in experimental syphilis of the rabbit, and the observation that in normal rabbits rhythmic changes in the mass relationship of practically all organs of the body have been found which have a definite relationship to seasonal changes and prevailing meteorological conditions. 3 While a variety of factors are probably involved in the production of these fluctuations, there appears to be a close relationship with sunlight and, in particular, first with the amount of sunlight as represented by the actual hours of sunshine and, second, with the rate and extent of change in the curve of sunshine. Furthermore, it is of considerable interest that these biological variations take place in the usual animal room with its illumination determined, in the first instance, by prevailing weather conditions and, in the second, by the interposition of window glass through which passes all or most of the sunlight. In order to test this conception of the relationship between sunlight, and the physical state and functional activities of the animal, experiments were undertaken in which conditions of illumination could be controlled.