THE INEFFICIENCY OF BACTERIAL VIRUSES IN THE EXTERMINATION OF RATS.

By M. J. Rosenau,

Surgeon, U. S. Public Health and Marine-Hospital Service, Washington, D. C., now Professor of Preventive Medicine, Harvard University Medical School.

INTRODUCTION.

Rats are notoriously resistant to bacterial infections. About the only exception is the plague bacillus. Plague among rats occurs both in endemic and epidemic form. When we recall that this virulent disease, which spreads readily from rat to rat, neither eradicates these rodents nor, as a rule, makes any appreciable inroads on the number of rats, we can hardly expect that an artificially induced bacterial disease would be successful. No other known bacterial infection has such a virulence for rats as the plague bacillus has maintained. Epizootics of bacterial nature, therefore, can not be classed among the natural enemies of the rat. Despite this fact persistent efforts have been made to create artificial epizootics to combat these dangerous and destructive rodents, but with little success.

The bacterial viruses that have been used for the destruction of rats and mice belong to the colon-typhoid group^[AL] and excite enteritis of different characters and a septicemia.

In 1889 Loeffler discovered and described a bacillus which he called the bacillus of mouse typhoid (B. typhi murium). He isolated this organism from a spontaneous epidemic which occurred among white mice in the Hygienic Institute at Griefswald.^[AM] He determined that this bacillus not only caused the death of his mice in the laboratory, but also that the infection was taken into the system of the mouse by ingestion. He found the cultures to be especially virulent for field mice (Arvicola arvalis). Loeffler gives a complete description 180of the bacillus which, from a biologic standpoint, is the parent stock of almost all subsequent work along this line. The bacillus used by Danysz and other workers is either identical with or very closely allied to Loeffler’s bacillus of mouse typhoid.

In 1892 Loeffler^[AN] personally undertook a campaign against the field mice in Thessaly and reported satisfactory results. The depredations carried on by the mice were checked within eight to nine days.

The English commission^[AO] threw doubt upon the Thessaly operations and concluded that the bacillus as a means of destruction of mice has no value. They found the method to be expensive, affecting only one species of mice; further, that the epidemic-like spread of the disease in the fields was not sufficiently investigated, and that the infected material retains its virulence only for eight days and does not permit of being used in continued bad weather.

Loeffler’s optimistic report, however, stimulated many similar trials with varying success. Practically all these efforts were directed against mice, until 1900, when Danysz took up the subject from the standpoint of the rat and plague.

Danysz found that Loeffler’s Bacillus typhi murium proved to be pathogenic for ordinary mice (Mus musculus) and for field or harvest mice (Mus arvicolis), but not for rats.

The culture isolated by Laser^[AP] in 1892 was pathogenic for field mice (Mus agrarius); this organism killed 70 of the 76 mice which were used as experiment animals at the Hygienic Institute at Königsberg.

Mereshkowsky^[AQ] in June, 1893, isolated an organism belonging to this group from a ground squirrel known as the Zisel (Spermophilus musicus). This culture killed domestic and field mice when placed in their food, but was not pathogenic for rats.

The Japanese investigator Issatchenko,^[AR] in 1898, briefly described a bacillus obtained by him from gray [white?] rats, which proved virulent for rats and mice.

Each of these various bacilli is of such variable virulence that it could not be used for the destruction of all species of these rodents.

181Danysz^[AS] therefore conceived the notion that it would be of great interest first to extend the field of action of one of these organisms by increasing its virulence so that it would attack other species of rodents, and then maintain this increased virulence at its highest point.

In 1900 Danysz isolated a bacillus from a spontaneous epidemic among harvest mice. This organism was a cocco-bacillus presenting the general characteristics of the colon-typhoid group and resembling the bacillus of Loeffler—B. typhi murium. From the first this bacillus showed a slight pathogenicity for gray rats (M. decumanus). Out of 10 animals fed with a culture of this microbe 2 or 3 would die; several others would sicken and recover; others appeared completely refractory. The fact that a certain number of the rats fed with these cultures always succumbed led to the hope that it would be possible to increase the virulence of this particular microbe by the generally accepted methods—that is to say, by a certain number of passages from rat to rat.

Danysz first tried to increase the virulence of the organism by this means, but he found that successive passages from rat to rat, whether by feeding or by subcutaneous injection, ended by enfeebling rather than increasing the virulence of the microbe. He found that it was rarely possible to go beyond 10 to 12 passages. Sometimes the series was stopped at the fifth passage, or even sooner, by the survival of all the animals undergoing experiment. The result was exactly the same if, instead of alternating each passage through the animal by a culture in bouillon or agar, the bodies of animals dead of a preceding passage were fed to others.

It was therefore plain that in the evolution of an epidemic caused by this microbe it was necessary to take account of the indisputable diminution of the virulence of the microbe, as well as the natural resistance of the survivors.

Passage of cultures in collodion sacs inclosed in the peritoneal cavities of rats was tried, both in interrupted series and by alternating each sac culture with a culture in bouillon or on agar, but the end was invariably a notable diminution of virulence when administered by the digestive tract.

Finally, after long and painstaking procedures, Danysz obtained a very virulent culture that, contained in flasks and kept from the influence of light and air, preserved its virulence for several months. Planted on agar it preserved its virulence without appreciable diminution for two months under laboratory conditions. In bouillon, in flasks, or in tubes stoppered with cotton it altered very rapidly.

182

DESCRIPTION OF THE ORGANISM.

A culture of Danysz’s virus obtained from the Pasteur Institute had the following characteristics:

The organism is a cocco-bacillus showing distinct motility. Stains well by the ordinary stains, but does not stain by Gram’s method.

It grows well at ordinary room temperature, also in the incubator, and on all the ordinary media. In bouillon it produces a uniform cloudiness in twenty-four hours. A slight scum forms after several days’ growth, which falls to the bottom when shaken. In Dunham’s solution it grows well, but produces no indol in twenty-four hours’ growth.

It does not coagulate milk.

It grows the whole length of the stab in gelatin, forming small whitish colonies in the deeper portions of the tube. It does not grow over the entire surface of the gelatin tube; does not liquefy gelatin.

It grows under anærobic conditions.

It ferments glucose bouillon, but not lactose bouillon. In glucose bouillon it produces 1-CO₂, 5-H. It also produces H₂S.

From a general biological standpoint it is plain that this bacillus belongs to the para-typhoid group, and is very similar to the bacillus of hog cholera as well as the Bacillus icteroides, also B. enteritidis, so far as its morphological and cultural characteristics are concerned.

In the following table the fermentations produced by various members of this group upon certain carbohydrates are shown:

Organism. Lactose. Saccharose. Maltose. Mannete. Glucose. Levulose.

B. typhosus − − − − − −

B. dysenteriæ Shiga − − − − − ^[AT]

B. dysenteriæ Kruse − − − − − ^[AT]

B. para-typhosus A − − + + + +

B. para-typhosus B − − + + + +

B. para colon − − + + + +

B. acidi lactici + − + + + +

B. hog cholera − − + + + +

B. typhi murium Danysz − − + + + +

B. icteroides − − + + + +

B. enteritidis − − + + + +

B. coli communis + + + + + +

183

EXPERIMENTS UPON RAT VIRUS IN THE HYGIENIC LABORATORY.

In 1901 I made an investigation of this pathogenic microbe (B. typhi murium) applied to the destruction of rats under laboratory conditions.^[AU] One hundred and fifteen rats were fed with the cultures in various ways during the course of these experiments with the virus. Of these, 46 died—less than half.

Most of the rats used were the gray rat (M. decumanus or norvegicus) and the tame white rat. A few (8) of the wild black or house rats (M. rattus) were used.

The virus is in reality pathogenic for these three kinds of rats when ingested. No special difference was noted in its effects upon the various species.

As the work progressed it soon became evident to me that the result depended largely upon the amount of the culture ingested. By starving rats for a day or two and then giving them all they could be induced to eat and drink of the cultures, a very positive result was obtained. In one instance of 27 rats so fed all died within a week. If the rats are given a small amount the effect is uncertain—only a few die. In one instance I fed 70 rats with 4 agar tubes and only 7 died. Feeding them a second time with very large quantities 9 more died. The survivors were then fed with all they could be induced to eat every day for a week without effect.

It seems plain, therefore, that a large primary dose proves fatal, and a small dose is not only uncertain, but produces an immunity. This is a very important factor, for it is likely that in the wild state rats would often partake of an amount too small to cause death. Such rats may then subsequently eat large amounts of the culture with impunity.

It would seem then that, after all, the virus is not so different from the laying of a chemical poison, depending as it does for its effect upon the amount ingested. A chemical poison, however, does not possess the disadvantage of producing an immunity. Another disadvantage possessed by the virus is the rapid deterioration in virulence which occurs when it is exposed to the action of air and light, or when it becomes dry, as is very apt to happen when laid out for rats in the wild state.

Since these early experiments, tests have been made of various rat viruses in the Hygienic Laboratory, and the results are given in the following pages:

184

AZOA.

Series 1. Single feeding of azoa in oatmeal. Rats starved twenty-four hours before feeding. Three out of eight animals died in four, five, and seven days, respectively. Micro-organisms resembling the predominant one of azoa could not be isolated from their hearts’ blood. The organs of these dead rats were fed to fresh rats with the result that one of the three died. It must be mentioned that the mortality among our fresh rats was nearly as high as that in the experimental animals from a disease probably due to infection with an animal parasite.

Series 2. Daily feedings with azoa in oatmeal. Five white rats fed with the mixture, a constant supply being kept in the cage. These animals were picked rats freshly obtained from the dealer. One rat died after seven days. It was heavily infested with lice. The azoa organism could not be found in the blood. The rest remained well after twenty-five days.

Series 3. Black tame mice, daily feedings. One of the five died after seven days. The rest remained well after fourteen days.

These experiments indicate that azoa is not pathogenic for white rats and black tame mice to a degree rendering it applicable for vermin extermination on a practical scale, provided that its action is no more pathogenic for the wild than for the tame species.

RATITE.

The manufacturers recommend a single feeding of this substance rather than a continued exhibition of the virus.

Series 1. Five white rats starved twenty-four hours and then fed with a mixture of ratite and oatmeal. Subsequent daily feedings with plain oatmeal. Picked animals fresh from the dealer. One animal died after twelve days; too much putrefied for further examination; had been heavily infested with lice. The remaining rats are well after twenty-five days.

Series 2. Five black tame mice fed as above. Three were found dead after eighteen days and the other two after nineteen days. Further experiments were not made, as putrefaction was too far advanced when they were found. The room in which they were kept had been unusually cold just before their death owing to a sudden and unexpected drop in the external temperature.

Ratite does not appear to be very pathogenic for white rats. All the five mice fed with ratite died in eighteen to nineteen days (much longer than the advertised incubation period of the infection), but their death could be reasonably attributed to unusual cold. This part of the test is therefore invalidated except that the animals lived considerably longer than would be expected from the literature 185furnished by the manufacturers, which says that the effects of laying the virus will be apparent in eight to ten days.

DANYSZ VIRUS.

Twelve tubes of Danysz virus were sent to the laboratory for examination April 7 by the Independent Chemical Company, agent for Danysz Virus Company (Limited). The label stated “Keep in a cool place and at above temperature; use before May 15, 1909.”

The virus was kept at 15° C. until April 13, when it was turned over to Passed Asst. Surg. W. H. Frost, who made the following tests:

One tube opened. Cultures made on two agar tubes were found to correspond in cultural characteristics with the bacillus of Danysz virus as generally described. The remainder of the tube prepared according to directions in accompanying circular, using stale dry bread 2 ounces and suspension of the culture in normal salt solution.

Series 1. Approximately equal parts, then fed to six white rats in individual cages, they having not been fed for twenty-four hours previously. Rats all ate greater part of infected food.

Five rats of series 1 died within five to seven days and were partly eaten before being removed from the cage. The pathological changes in all cases were chiefly enlargement and congestion of spleen and liver, and in some cases inflammation of small intestine. In each case an organism was obtained, usually in pure culture, from one or more organs, corresponding culturally and morphologically with cultures taken from original tube.

Series 2. April 14: Rats all ate greater part of infected food. Transferred to large cage containing nine other white rats. Nine other white rats exposed to infection by being placed in a large cage with the rats of series 1. Four of the nine rats of this series died in four to seven days after eating infected rats.

Pathological changes and results of cultures from internal organs the same as with series 1. Autopsy and cultures impossible in one case, where body was almost completely devoured.

Series 3. May 5: Three of the six surviving rats (one from series 1, two from series 2) were placed in individual cages, deprived of food for twenty-four hours, then fed each with one-third agar tube cultures of Danysz virus (the same used in the original feeding). All three ate practically all the virus given.

All three of these rats remain alive and well after two months.

Summary.—Series 1. Six rats each fed one-twelfth to one-sixteenth agar culture Danysz virus. Five died within five to seven days.

Series 2. Nine rats exposed to infection by being placed in cage with series 1. Four died within eight to twelve days after death of first rat of series 1.

186Series 3. Three of the surviving rats (1 from series 1 and 2 from series 2) fed each with one-third agar tube of original Danysz virus as used in series 1. None died.

TRANSATLANTIC RATIN.

A can of this substance labeled “Transatlantic ratin” was furnished by the American agents representing the Bacteriological Laboratory, Copenhagen, Denmark. The can bore the date January 26, 1909, and was stated to be “effective for six months from date of production.”

On April 13, 1909, this sample was given to Passed Asst. Surg. W. H. Frost for examination, and he obtained the following results:

April 13, 1909: Can of ratin opened with aseptic precautions. Contents mixed with about equal bulk of clean fresh lard.

A portion of this about equal to one tablespoonful fed to each of six white rats previously deprived of food for twenty-four hours. All the rats ate some of the bait at once. Feeding at 2 p. m.

April 14: Five rats very sick, having convulsions; partially paralyzed. One dead.

April 15: Three more rats found dead. Remaining 2 recovering.

The pathological change in all the above cases consisted chiefly of intense congestion of intestines, both large and small.

Cultures from the original case of ratin, on agar, bouillon, and in fermentation tubes, negative except staphylococcus in one tube.

Cultures taken from heart’s blood and other organs of the 4 dead rats all negative, except in one case a growth of a staphylococcus resembling S. pyogenes citreus.

April 20: Two rats fed on half agar slant culture of the staphylococcus obtained from heart’s blood of rat No. 1. Result of feeding negative after several weeks.

Note. The absence of a colon-like organism in this virus and the rapid death of the animals with convulsions suggested a chemical poison, which it is believed this can contained.—M. J. R.

EXPERIMENTS WITH MICRO-ORGANISMS FOR DESTROYING RATS, CONDUCTED BY THE UNITED STATES BIOLOGICAL SURVEY.^[AV]

RATIN.

The Biological Survey, Department of Agriculture, in cooperation with the Bureau of Animal Industry, has experimented with “ratin.” The material (ratin No. 2—labeled “Transatlantic ratin”) was furnished by the American agents in New York. Although the agent claimed that this was a bacterial preparation and that “it would kill 187for six generations,” it proved to be a glucoside poison (probably squills) and contained no bacteria of any kind. A number of experiments were made with it, and it proved to be an effective rat poison. In some instances the animals died within two hours after eating it, and in two experiments all the animals fed died within twelve hours. In other experiments, however, a considerable percentage of the affected rats recovered, and subsequent attempts to kill them with ratin No. 2 failed. Some were immune to its effects and others too wise to eat it a second time. More than a hundred rats were used in the experiments; but the main object—to test the communicability of the disease caused by ratin bacteria in healthy rats—failed, of course, since, as above stated, the preparation experimented with contained no bacteria, but was merely a vegetable poison. Before its character was fully determined, 15 rats killed in the experiments were eaten by 5 healthy rats; the latter were unaffected.

It should be noted that the labels on the tins containing transatlantic ratin were misleading. The user was warned to open the packages in dim light and to allow no moisture to come in contact with the contents, as the bacteria were very sensitive to light and moisture. The contents of the can were to be used at once. As a matter of fact the contents of one can were exposed to severe drying in heat and sunlight for four days and then soaked in water for two days. Afterwards the preparation was fed to different rats for a further period of four days, and its virulence was retained to the last.

The transatlantic ratin is in a solid medium, apparently bread and molasses. Its keeping qualities are excellent, and it is an effective poison for rats, but far too expensive for extensive use. A can costing $1.50 is enough for only 15 baits.

Its harmfulness to domestic animals was not fully tested. Dogs and cats refused to eat it and vomited it when it was forced upon them. Several animals, including a dog, were killed by injections of the poison in concentrated form.

A shipment of ratin No. 1 (the solid bacterial ratin, said to retain its virulence for two months) was received June 4, 1909. This preparation was dated May 8 and should have been still virulent. The contents of a can mixed with milk was fed to 8 adult rats on June 7. All of the baits were eaten, but no result followed. Cultures of the bacteria showed strong growths of new colonies.

On July 6 the contents of another can were fed to 1 adult and 16 young rats. One of the young was found dead on the morning of July 14. Cultures were made from the dead rat, but the bacillus was not recovered. Up to July 28 none of the other rats have been affected.

188

AZOA.

Several trials of azoa for the destruction of rats have come under the observation of members of the Biological Survey. Experiments made in the building occupied by the Interstate Commerce Commission were at first promising, but from a second invoice of the virus no results were obtained. In the buildings of the National Zoological Park 72 bottles of azoa were used, but the results were for the most part negative. In a store in south Washington where this preparation had been used the stench of dead rats was very strong, showing a measure of success.

DANYSZ BACILLUS.

Some three years ago the Biological Survey, assisted by the Bureau of Animal Industry, tested the efficiency of Danysz virus. In the laboratory from 10 to 50 per cent of rats fed on the virus died. In the field, however, results obtained were unsatisfactory. Only 1 dead rat was found from which the bacillus was recovered. Experiments with field mice gave better results. All the mice fed in confinement died, and field experiments resulted in many dead mice from which the bacillus was recovered.

EXPERIMENTS DURING THE SAN FRANCISCO PLAGUE OUTBREAK.^[AW]

Several proprietary biological products sold as rat exterminators were made the subject of seven experiments on wild San Francisco rats, for the purpose of ascertaining whether they were efficient for the purpose for which they were sold. Seventy-six rats were used in these experiments. About 10 per cent died within a month and there was considerable doubt as to whether all of the deaths that occurred were due to the agent used.

The following is a brief statement of the work done with these agents. In each case the rats used were wild Mus norvegicus, caught in San Francisco.

RATIN NO. 1.

Made by the Bakteriologik Laboratorium, Copenhagen, marked: “Effective two months from April 28, 1908.” The preparation comes ready for use in the form of a moist, mealy mass. On May 28, about 6 ounces of the material was fed to 12 rats. They all remained well until thirty days after feeding them when the experiment was regarded as terminated.

189

DANYSZ VIRUS.

The Danysz Virus Company (Limited), of London, furnished a preparation in the form of a culture on a slant of solid medium, said to be gelatine. The tube was marked: “To be used before June 1, 1908.” The contents of the tube, mixed with bread, according to directions, was fed to 6 rats. On the twenty-first day but 4 rats remained, 2 having died and been devoured by their companions. The 4 that remained were chloroformed, as the cage was needed for other purposes. Post-mortem examination showed them to be entirely normal.

RATITE.

Furnished by the Pasteur Vaccine Company, Chicago. This preparation is in the form of a culture in a liquid medium, presumably broth. The bottle was dated April 10, 1908, and the label stated that it should be used within twenty days from date of preparation. On April 29, 1908, 9 rats were fed with about 6 ounces of the preparation, mixed according to directions. The rats all remained alive and well, and when chloroformed on June 1, 1908, presented no abnormality on post-mortem examination. In another experiment the contents of a bottle of ratite was fed to 6 medium-sized Mus norvegicus. None of the animals died from the effects of the agent, and when they were killed on the fifty-fifth day after the feeding were found to present no lesions.

The remaining work was done with rat virus, sometimes called “Mouratus.” It is made by the same concern that makes the ratite. The rat virus comes in the form of a culture on a solid medium. The contents of three tubes was fed to 6 rats on May 26, 1908. Three of these rats died within thirty days. Only one was secured for examination before it had been mutilated beyond the possibility of making a satisfactory examination. This rat had a large yellow liver and a very large, dark, firm spleen. These appearances were probably due to the agent used and it is not unlikely that these 3 rats died from its effect. It will be observed that a very large dose was given. On another occasion four tubes of Mouratus were used for feeding 6 Mus norvegicus. One of the rats died on the fifteenth day, showing at autopsy an enlarged granular spleen and a granular liver. The other rats were alive and well at the end of thirty-four days when the experiment was discontinued.

Subcultures on broth were made from this preparation on three occasions, always well within the time limit on the label. The cultures were incubated in the dark, at room temperature, for forty hours on each occasion. Liberal amounts of the subculture were fed to a total of 31 rats. At the end of thirty days, it was found that 190only 2 of these rats had died. The others were alive and apparently well.

One objection to these agents which I have not seen stated is the following: The lesions caused by at least some of these members of the paracolon group may readily be mistaken for the lesions of plague, or it will perhaps be more accurate to say they give rise to lesions that create in one’s mind a suspicion of plague infection, and I have had to put many a rat to the guinea-pig test in order to make certain that a Danysz infection was not associated with the infection of plague, or that a Danysz rat was not a plague rat. Of course, this is of no consequence except in a community where antiplague measures are being taken, and an observer of limited experience who did not put a rat to a pretty rigid test would probably call some plague infected when in reality such is not the case.

In addition to the data set forth in this report, I have on several occasions fed the tissue of rats dead of Danysz infection to other rats, but have never succeeded in reproducing the disease. In other words, I have had no success whatever in raising the virulence by passage through animals.

OPINIONS OF OTHERS.

Kitasato,^[AX] 1906, states that the typhoid bacillus of the rat, which has been effectively used for killing field mice, has been found useless for house rats (Mus rattus) and therefore they no longer employ it.

Melvin,^[AY] 1908, reports that recently several new rat viruses were investigated in the Bureau of Animal Industry, with the result that the experiments clearly demonstrated the ineffectiveness and unreliability of the preparations tested.

Räbiger and Schwinning,^[AZ] 1906, tested the culture discovered by G. Neumann and prepared by the joint stock company “Ratin” at Copenhagen by applying it to rat destruction. Of house rats 90 per cent died; black rats 42.9 per cent; while horses, dogs, goats, sheep, fowls, and pigeons suffered no harm. Of seven experiments practically carried out, six showed very good results; in one favorable results were absent, which agrees with the experiments made in Denmark. There it was likewise found that in individual locally limited places the rats were able absolutely to withstand the infection of ratin.

191Räbiger,^[BA] 1905, states that experiments with Loeffler’s mouse typhus bacillus and the bacillus of Danysz virus have been carried to the conclusion that these bacterial preparations must be characterized as practically worthless.

In 1903 Neumann discovered in Denmark a rat-killing bacillus, which has been placed on the market by a society under scientific control under the name of “ratin.” Feeding experiments with this bacillus were tried under conditions as nearly natural as possible upon white mice, gray house mice, long-tailed field mice, and gray rats. The experiment animals were fed with cubes of white bread impregnated with virulent cultures. White mice show the least power of resistance, since they die within six days; house mice died in six to nine days; the greater part of the rats died from the sixth to the sixteenth day after feeding; a small percentage lived. The long-tailed field mice, which are shown to be insusceptible to Loeffler’s bacillus, also remained perfectly healthy after repeated feedings with bread infected with ratin.

Brooks,^[BB] 1908, reports the results of tests made with azoa on rats and mice, both in captivity and at large, but without any apparent discomfort to the animals. One of the tests is described as follows:

A supply of the azoa was obtained direct from the laboratories of the manufacturers. On July 27, 1907, while the material was yet fresh, three young Norway rats were caught and kept confined in a large wire rat trap. Beginning with the date given, and for a period of forty days thereafter, azoa was fed to the rats at intervals of a few days until ten 75-cent bottles had been consumed. The rats ate the cracked grain with which the virus was mixed very readily, and other food was denied them each time the azoa was given until every particle was eaten. At the end of the forty days the rats were still apparently in a healthy condition, and were removed from the trap and killed with a club.

Thompson,^[BC] 1906, states that three laboratory attempts have been made to destroy rats with imported strains of Danysz rat virus without success. Danysz having arrived at Sydney to study a similar method of destroying rabbits, the opportunity was taken of making a further attempt under his supervision with virus which had been imported and subsequently increased to the requisite degree of virulence, and had been placed at Thompson’s disposition. The grounds of the Gladesville Asylum, a large institute for the insane, were chosen for the tests, which were conducted by Dr. R. J. Millard.

192Millard summarized his result by stating that they can not be considered a satisfactory demonstration of the efficacy claimed for the Danysz virus. The results indicate a rapid loss of virulence, which must be obviated if this virus is to be of utility for rat destruction.

Again, in 1907, during the seventh outbreak of plague in Sydney, Thompson^[BD] had Millard test the preparations known as azoa and ratin. The laboratory results with these preparations were similar to those made by other investigators. Experiments made upon the ship Hartfield with azoa produced no considerable epizootic. The fatality among such rats as were infected was small. The practical tests with azoa upon several areas along the harbor front also resulted in disappointment.

The tests made with ratin upon the bark Quilpe produced no epizootic among the rats, and of the rats caught none of them showed infection; and the field experiment at Gladesville also resulted negatively so far as dead or sick rats were concerned. Nevertheless, there was apparently considerable diminution in the rat population of this area.

Foster,^[BE] 1908, reports unfavorably upon the results of tests made of some of these rat viruses. Laboratories were opened for the use of different parties who wished to make tests. The tests were conducted under their own supervision. The rats which were not fed on anything but grain died as freely as those that had been fed on azoa. So far as this preparation is concerned Foster states that it is absolutely useless to depend upon it.

Several reports are found in print in which the rat virus was laid out in certain localities and shortly afterwards the rats disappeared—at least no more were noticed. Such observations are apt to be misleading, for rats are migratory. They come and go, especially when disturbed. Further, it is doubtful, as far as plague is concerned, whether it is desirable to drive the rats away, for they may thus scatter the infection.

S. S. Mereshkowsky and E. Sarin^[BF] have recently studied ratin II, put out by a Copenhagen firm—“Bakteriologisches Laboratorium Ratin.” The label upon the can of ratin II states that it is a bacterial culture, which produces in rats an infectious and fatal disease, killing them in two to eight days. The samples used by the authors were obtained as needed from the St. Petersburg representative of the firm. Feeding experiments carried out with gray rats (Mus decumanus) 193showed that the rapidity and severity of the symptoms was proportional to the amount ingested. No positive results were obtained from the bacteriological examination of the bodies.

The ratin itself was sometimes found to be sterile, sometimes found to contain several varieties of bacteria and fungi, but no one variety was constantly present.

The potency of the ratin was not altered by exposure to 100° C. for one hour or 120° C. for five minutes. It was destroyed, however, by burning to an ash.

Identical poisonous results were obtained upon rats by feeding them with “Scilla maritina cum bulbo rubro.”

Microscopical examination disclosed a small portion of a lamella, identified as belonging to the Liliaciæ, to which family squill belongs.

The authors conclude that ratin II is not a bacterial culture, but a poison rendered more dangerous to persons and domestic animals by the misleading statements of its makers.

PATHOGENICITY FOR MAN.

Loeffler^[BG] rather took it for granted at first that his Bacillus typhi murium was harmless for man. In order to remove the fears of the peasants in his campaign against the field mice in Thessaly he fed pieces of bread impregnated with the cultures to chickens, pigeons, dogs, hogs, horses, asses, sheep, and goats. No ill effects resulted. Further, some of the men who were distributing the prepared virus ate pieces of the infected bread in the presence of all and, it appears, suffered no ill effects.

Up to this time Loeffler had made no human experiments, but thought it improbable that his bacillus was harmful to man. He considered this view confirmed by the fact that he and his companions and still more so the peasants, handled large quantities of the virus without thorough disinfection of their hands and suffered no untoward effects.

Since that time, however, several mishaps have occurred. Instances of serious sickness and even death have been attributed to infection with the bacterial virus used for the destruction of rats.

Further, there is practically no difference between the Bacillus typhi murium and the para-typhoid bacillus which is the well-known cause of meat poisoning, and the Bacillus enteridion of Jarbues, which is associated with intestinal disorders.

It is true that persons have purposely partaken of the rat virus to prove that it is harmless to man; but it must be remembered that persons have partaken of cultures of cholera, typhoid, and other 194bacteria without apparent injury to themselves. The flora and condition of the gastro-intestinal tract, the amount and virulence of the infection, and other conditions (“Y” and “Z” of Pettenkofer) play an important rôle in the production of these diseases.

The following references from the literature give the instances in which the B. typhi murium, or similar rat viruses, have been held responsible for the disease in man:

Trommsdorff^[BH] carefully studied 13 suspected cases near Munich in early May, 1903. Nine of these came into direct contact with the virus, three ate and associated with these, and the remaining one only smelled of the virus. One died from vomiting and severe diarrhea. The illness, which set in usually two days after contact with the virus, was for the most part simple diarrhea of two to seven days’ duration (two to eight stools daily); in only three or four cases was there vomiting. The one fatal case seemed due to a confusing chain of circumstances, gross dietetic and alcoholic excesses in a weak, emaciated, presumably phthisical man whose three brothers had died of phthisis. One man, case No. 2 in the table, known to have eaten three pieces of infected bread, suffered only with a mild diarrhea.

In all cases errors of diet could be proven, and diarrhea was not uncommon at that season. The same physician attended during this period ten other cases of similar diarrhea in the vicinity having nothing to do with rat virus. The stools, however, did not have the same pathogenicity for mice, guinea pigs, or rabbits.

Trommsdorff specially points out the fact that the bacillus of mouse typhoid can multiply vigorously in the human intestine. It demands greater caution in the application of the cultures and more careful supervision over their use.

Finally, attention is invited to the fact that, contrary to the usual custom, the cultures of rat virus here used had been grown on milk, which might account for the increased virulence.

195The following table gives a brief summary of ten cases with the results of the agglutination tests:

Agglutination tests with serum of recovered cases, May 17, 1908.

Case. How infected; symptoms. Strains. Serum dilutions.

¹⁄₂₀ ¹⁄₄₀ ¹⁄₆₀ ¹⁄₁₀₀ ¹⁄₂₀₀

1 B. K. (From this case stool 2.) Laid rat poison 5–2,5–5; some days later diarrhea; later vomiting; convalescence and recovery May 10. A + + + ± 0

B + + + ± 0

C + + + ± 0

D + + + + ±

2 K. E. Ate three pieces of infected bread May 2; mild diarrhea several days. A + 0 0 0 0

B + ± 0 0 0

C 0 0 0 0 0

D + ± 0 0 0

3 H. B. Brought the virus April 28; perhaps touched it; next day diarrhea and vomiting; recovery after several days. A + 0

B + 0

C + 0

D − − − − −

4 G. S. Father of man that died; laid virus; two days later mild diarrhea for one or two days. A + + + + 0

B + + + + 0

C + + + 0 0

D + + + + ±

5 J. K. On April 27 laid virus; 2 days later diarrhea for several days. A ± 0 0 0 0

B ± 0 0 0 0

C ± 0 0 0 0

D − 0 0 0 0

6 J. N. Laid virus April 27; two days later had diarrhea for several days. A 0 0 0 0

B + + + ±

C 0 0 0 0

D + + + ±

7 G. I. “Held” the virus April 28. Next day diarrhea for four days. A + + + + ±

B + + + + ±

C + + + + ±

D − − − − −

8 H. R. Ate and associated with persons who handled virus; diarrhea several days. A + + 0

B + − 0

C + ± 0

D − − −

9 K. S. Associations as case 8; headache several days and loss of appetite. A + + + +

B + + + +

C + ± 0

D − − − − −

10 Associations as case 8; diarrhea for eight days; vomiting several days. A 0 0 0 0

B + + + ±

C 0 0 0 0

D + + + ±

A = Strain from Loeffler.

B = Strain from market virus.

C = Strain from stool 1 (fatal case).

D = Strain from stool 2 (case 1 of table).

− = No test made.

0 = Negative.

± = Slight.

Controls = Serum of five normal persons tested as above; gave in no case agglutination higher than 1 : 20.

196The attending physician, noting that most of his patients had come into recent contact with rat virus (B. typhi murium) and suspecting that to be responsible, sent specimens of stools to the Hygienic Institute at Munich, where they were carefully examined with reference to this subject.

Organisms identical with Loeffler’s B. typhi murium were isolated from the two stools examined and these cultures were compared with and conformed with a culture of B. typhi murium obtained from Loeffler and also a culture from the virus on the local market.

Two guinea pigs injected with cultures from the two stools gave, after the second injection, serum which agglutinated all the above organisms 1:200. A mouse typhoid serum obtained from Loeffler agglutinated all the above strains distinctly in dilutions 1:640 and slightly in 1:1280.

In conclusion, the author considers three possibilities: 1. The mouse typhoid bacillus was the cause of the illness. 2. The bacillus was accidentally present, having no part in the production of the symptoms. 3. The bacillus was able to multiply only in case pre-existing intestinal trouble; then, however, causing the inflammation.

The case of Mayer, who became infected during the course of some laboratory experiments, is particularly instructive.

During an epidemic of mouse typhoid among his laboratory mice, evidently spread from some inoculated mice by ants, Mayer^[BI] who had personally handled the infected mice and their cages, became sick July 15, just seven days after the first appearance of the ants and after the observed rise in virulence of the mouse typhoid among the mice. His clinical history is as follows:

July 15: Weakness, epigastric pain, obstipation, temperature 37.7, pulse 90.

July 16: Slight diarrhea, increase of pain in region of trans-colon, temperature 38.3, pulse 98.

July 17: Diarrhea continued, pains increased—severe, chill in evening, temperature 39.1, pulse 102.

July 18: Obstipation, symptoms worse, chill again in evening, temperature 39.4, pulse 104.

July 19: Symptoms better, stools from purgative, evening temperature 36.9, pulse 68.

July 20: Left bed. Temperature and pulse normal, but weakness and slight epigastric pains continued till August 7.

197Patient’s serum agglutinated as follows:

Typhoid. Paratyphoid. Mouse-typhoid.

July 23 1–50+ 1–50+ 1–250+

August 7 1–50+ 1–50+ ^[BJ]1–100+

August 16 1–50+ 1–50+ 1–50 +

The bacillus of mouse typhoid was isolated from the patient’s stools July 21 and 23. Negative results thereafter. Examinations continued a month.

Same organism isolated from urine July 21. Negative thereafter.

The author concludes that the B. typhi murium is able to cause in man a rather severe acute illness of short duration.

Shibayama^[BK] gives the following report of outbreaks of human infection that have come to his knowledge in Japan, where mouse-typhoid virus has been used in considerable quantities.

Outbreak 1. In April, 1905, in a village of the Province of Saitama, 30 people became ill and 2 died with severe gastro-intestinal symptoms. Outbreak investigated by Dr. H. Sezuki, district medical officer, formerly of the Tokio Institute for Infectious Diseases.

It was found that all the 30 people had partaken of a dish of cooked vegetables served at a meeting of the town council, and that for application of sauce to these vegetables (after cooking) a wooden vessel had been used which two days before had been used for mixing mouse-typhoid virus with meal, without subsequent cleansing or sterilization.

The symptoms came on within twelve to forty-eight hours thereafter (usually twenty hours), chill or chilly sensations, rise of temperature to 38° or 39° C., or even to 40° C.; face flushed; pulse accelerated; great weakness; thirst, nausea, colicky pains in abdomen followed by severe diarrhea and vomiting. In general, the fever and diarrhea lasted two or three days; but malaise, anorexia, weakness, and mucous stools persisted for several days. The more severe cases showed choleraic symptoms of collapse. Two persons died in spite of medical treatment—a 6-year old boy and a man of 43, on the second and third day, respectively.

From the intestinal contents of these two cases, from the stools of several other cases, and from the remnants of the dish of vegetables in the wooden bowl an organism was isolated, which was demonstrated to be identical with the bacillus of mouse typhoid.

198These results were confirmed at the Tokio Institute for Infectious Diseases by Shibayama, by biological and immunizing tests.

Outbreak 2. On December 7, 1905, a peasant of a village in the Province of Miyaki brought home some mouse virus mixed with meal in cakes. This being mistaken for “mochi” was eaten about 2 p. m. the next day by two little girls, 3 and 8 years old, respectively, and their grandfather, 61 years old.

The man and the 8-year-old girl became sick at 9 p. m. the same day and the other child at about 3 p. m. on December 9. The symptoms in all cases were those of severe gastro-enteritis, as described under outbreak 1.

The man died December 12, the 8-year-old child died on the 10th; the 3-year-old child recovered after several days’ illness. These three alone ate of the virus and no other persons in the house became sick.

No bacteriological examination was practicable.

Outbreak 3.—In a village of the Province of Iskawa, on April 22, 1906, a lot of rat poison was prepared by mixing agar cultures of the mouse-typhoid bacillus with meal and water in a large wooden bowl.

On April 24 there was a festival in the village at which about 170 persons were served with 240 pounds of rice, which, after being cooked, was kneaded into cakes in a wooden bowl. About 80 pounds of this rice was so kneaded in the bowl previously used for preparing the rat poison. Twenty to twenty-four hours later 120 people who had eaten of the rice became ill with the already described symptoms of gastro-enteritis, of mild type among the strong but severe among the children and old people. Eighty-nine cases came under medical treatment. There were no deaths, but a number of cases were confined to bed for a week or more; mild cases recovered in one to three days.

No bacteriological examination was made, but the physicians and town officials were unanimously of the opinion that the rat virus was the cause of the outbreak.

Outbreak 4.—A peasant of the province of Niigata brought home on May 14, 1906, some rat virus (cultures of mouse-typhoid bacilli mixed with meal) which he laid away. Two of his grandchildren—a boy of 5 and a girl of 7—together with the 4-year-old daughter of a neighbor, found and ate the rat virus. The next day all three children became ill with severe gastro-enteritis, of which the 4-year-old child died on the third day. The others recovered after several days of medical treatment.

Outbreak 5.—On May 16, 1906, a peasant in the province of Jamagata brought home some rat virus (6 c. c. cultures of mouse-typhoid bacillus mixed with meal), which was accidentally mixed with the feed given to a healthy horse next morning. The same evening the 199horse showed loss of appetite and appearance of sickness. Within two days he developed a severe enteritis, of which he died on the seventh day. The body was buried, but was dug up in the night by a laborer who cut off the hind quarters, took them home, and distributed the meat among friends and neighbors.

Within three days 34 persons who had eaten of this meat became ill with symptoms of severe gastro-enteritis. A 72-year-old man died after five days; the others recovered in three to eleven days.

This outbreak was investigated by Dr. H. Segawa, a medical officer of the province and former member of the institute at Tokyo, who isolated from the remains of the horseflesh by plate cultures and animal inoculations, an organism identical with the bacillus of mouse typhoid. A culture was sent to Shibayama, who carefully verified it (details not given).

Shibayama concludes: In all cases the close relationship between the bacillus of mouse typhoid and the illness was established; and he thinks this organism must be accepted as the direct cause of the outbreaks.

Referring to Loeffler’s uniformly negative human experiments, he calls attention to known cases where men have taken virulent cultures of typhoid, diphtheria, etc., without infection. According to many bacteriological investigations, B. typhi murium is identical with the bacillus of enteritis. If it is proven that the latter is a cause of acute gastro-enteritis then the conclusion is likewise justified that the B. typhi murium is frequently pathogenic for man, causing an acute gastro-enteritis.

Fleischanderl^[BL] a reports six cases of illness—three severe and three mild—occurring in his practice in the latter part of April, 1908, presenting the following symptoms: Onset with rapidly increasing body pains, followed in a few hours by diarrhea, rise of temperature, and general prostration; in the next two or three days aggravation of the symptoms, fever (39° to 40° C.), copious diarrhea, vomiting (in one case), severe body pains, vertigo, and considerable prostration. Symptoms abated quickly in a few days, leaving considerable prostration, convalescence requiring two weeks in one case. In the less severe cases there was no fever, and the other symptoms were generally milder.

The simultaneous appearance of these and other similar rumored mild cases among the neighbors (about 20 in all) pointed to a common cause. It was found that three of the six cases were in people who had handled mouse-typhoid cultures the day before their illness, taking no precautions to avoid infection.

200The other three occurred in a family which, on the day before the onset of the illness, had drunk raw milk obtained from a house where the rat virus had been used shortly before, and only three members of the family who drank the milk became ill.

In order to prove the etiology of these cases Fleischanderl, who had never suffered any intestinal troubles, had had nothing to do with any case of typhoid fever for a year, and was in excellent health, took a culture of the mouse-typhoid bacillus as used in the neighborhood, rubbed a glass rod over the surface, washed it off in a glass of water, and drank this before breakfast on the morning of May 3.

In twenty-two hours he experienced mild, increasing body pains, followed within a few hours by diarrhea, and a few hours later by slight chill, rise of temperature to 38.2, pulse 106, severe pains in body, and feeling of great weakness.

May 4, 9 p. m.: Temperature 39.2° C., pulse 120. Height of symptoms.

May 5: Temperature 38.2° to 38.5° C., pulse 106 to 120. Other symptoms continued.

May 6: Temperature and pulse normal. All symptoms disappeared except weakness, which lasted two days.

Bacteriological investigations conducted by Herbert Berger in the K. K. Serotherapeutischen Institut and by Doctor Reichel, assistant in the Hygienic Institute of the University of Vienna, follow:

From the stools of one of the patients infected from milk an organism was isolated which, injected into mice (1 c. c. emulsion of forty-eight-hour culture), killed them in two to five hours. Mice infected by eating these dead mice died in thirty to forty-eight hours.

Control mice inoculated similarly with a culture of the market mouse-typhoid virus died in twenty to thirty hours, while the mice infected through eating these died after three to four days.

The following strains were used for cultural agglutination tests:

A. From stools of patient infected from milk.

B1. Market virus used in injecting mice.

B2. Market virus taken by author.

C25. From stools of author twenty-five hours after infection.

C55. From stools of author fifty-five hours after infection.

LL. Stock culture of Loeffler’s mouse-typhoid bacillus.

LP. Stock culture of para-typhoid bacillus.

All organisms (A-C55) were demonstrated as motile bacilli, not liquefying gelatine, not forming lactic acid, and forming gas from dextrose.

The serum of a rabbit after two injections of LL agglutinated LL and LP in dilution of 1:1280, did not agglutinate A, B1, B2, C25, and C55.

201Serum of rabbit after one injection of B2 agglutinated in 1:320 dilution A, B1, B2, C25, C55, and LL; did not agglutinate LP.

Serum of rabbit after one injection of C25 gave exactly similar results.

Doctor Reichel considers it proven that the organisms A to C55 are undoubtedly identical with Loeffler’s bacillus of mouse typhoid, and distinct from para-typhoid bacilli. The author considers it proven that this bacillus was the sole cause of the cases of enteritis observed.

Recently Mallory and Ordway^[BM], in a paper read before the American Association of Pathologists and Bacteriologists held in Boston, reported that lesions analogous to the early stages of typhoid lesions may be produced in rats by the use of Danysz virus.

In view of these facts the statements of some of the advertising matter of certain rat viruses call for revision.

REFERENCES TO THE LITERATURE.

Loeffler,^[BN] 1889, gives an account of two spontaneous outbreaks among the mice kept at the Hygienic Institute at Griefswald. It was from these animals that he obtained and described the original B. typhi murium. He determined that the infection was by ingestion and that the organism was especially virulent for field mice. He described the organism in detail and also the lesions.

Laser,^[BO] 1892, reports that on the morning of February 6, 1892, 70 of the 76 field mice (Mus agrarius) used as experiment animals in the Hygienic Institut at Königsberg were found dead. A small bacillus twice as long as broad, displaying a very lively specific motility, was isolated from the spleen. It was tested upon animals and all the results compared with Eisenberg’s tables and found to be closely allied to the bacillus of ferret plague (Ebert-Schummelbusch), to the bacillus of American swine plague (Billings), and to that of French swine plague (Chantamesse and Cornil).

Mereshkowsky,^[BP] 1893, isolated an organism at the Royal Bacteriological Institute at St. Petersburg from a stock of Zisel (Spermophilus musicus) among which a spontaneous epizootic had occurred. The author found this culture to be virulent for domestic and field mice.

202Zupnik,^[BQ] 1897, states that Joseph, of the Agricultural Institute of Breslau, in 1882 originated the use of favus fungus for the destruction of mice. Zupnik tested B. typhi murium and Danysz virus upon mice. No experiments with rats.

Issatschenko,^[BR] in 1898, described briefly a bacillus obtained by him from gray rats. Recent investigation showed this bacillus to be very virulent for rats and mice, but harmless for the different species of domestic animals. Four hundred and forty-three experiments were made upon rats with pure cultures of the bacillus combined with dough and fed to the rats. He gives a table showing that the mortality occurred in 431 rats at an average of ten and one-half days. The greatest mortality occurred during the first fifteen days (84.2 per cent), with the greatest number on the seventh day (20.1 per cent).

Danysz,^[BS] 1900, isolated a cocco-bacillus during an outbreak of spontaneous disease amongst field mice which presented the general characteristics of the colon bacillus and to this extent resembled Loeffler’s bacillus (B. typhi murium), and which from the beginning exhibited some pathogenicity for gray rats (M. decumanus). Of ten such rats fed upon a culture of this organism, two or three died, while others that had fallen sick recovered and the same remained well. This small mortality offered some hope that it would be possible to increase the virulence of the bacillus by ordinary methods; that is, passing it from rat to rat. It was found, however, that the opposite was true; the virulence was always weakened by this process regardless of the method of administration. Thus in every series the first culture killed the animals in seven to twelve days; occasionally after one or two passages five to seven days; but subsequent passages decreased the virulence so that none died.

The general result is that it is difficult to maintain the virulence of the cocco-bacillus of the rat or to increase it when it is found to be small. It can only be effected by constantly making a large number of experiments and frequently testing the virulence of the culture. Danysz succeeded in keeping up a supply of cultures of sufficient strength for eight years. In 60 per cent of the operations where this culture has been used it has been successful in causing the absolute disappearance of the rats. In 15 per cent the result was entirely negative, and in the remaining 25 per cent there was a large diminution.

203Oettinger,^[BT] in 1903, increased the virulence of the Danysz bacillus by growth in an egg rendered alkaline after the method previously introduced by Wiener.

Pfreimbtner,^[BU] 1904, sees the reason for a partial failure in the application of Loeffler’s bacillus to the destruction of field mice. In the use of a solid medium (agar-agar), upon which the bacterial cultures only grow upon the surface, too few bacteria are transferred to the pieces of bread, and consequently too few virulent bacteria are consumed by the mice. An active infection depends not upon the existence of virulent bacteria, but rather upon the entrance of a definite number of virulent bacilli.

The author used skimmed milk as a nutrient medium and describes a series of 9 experiments and gives an estimate of the cost of the process with the use of milk instead of agar. The advantage of the use of milk is in the greater certainty of the results and cheapness as compared with other methods. The bread cubes impregnated with skimmed milk were well taken by the mice and desiccation of the cultures, which not infrequently occurs in the use of more solid media, is excluded. Notwithstanding the excessive thinning, the liquid still contains many virulent bacilli. The washing out of bacilli after rain is slightly less possible as in the use of the more solid media. The action of light, where the bread cubes contain many bacilli, is insignificant, and hence the carrying out of the process in the daytime is made possible. Milk is easily obtained and the thinning and application less bothersome. Finally the author expresses himself against the view that the B. typhi murium causes serious diseases in man.

Teichert,^[BV] 1905, speaks of Loeffler’s mice typhus bacillus and Pfreimbtner’s method of growing it upon sterilized skimmed milk. A number of experiments carried out at the bacteriological laboratory of the Vreschen experiment station shows the utility of Loeffler’s bacillus for the destruction of house and field mice. The long-tailed field mouse was, on the other hand, not harmed by it.

Bahr,^[BW] 1905, gives a complete and satisfactory summary of the literature upon the subject of the destruction of rats and mice with bacteria, including original work of his own.

204Zielander,^[BX] 1908, obtained fairly good results with Danysz virus in the laboratory, also with ratin. No fixed tests were recorded.

RÉSUMÉ.

Rats are notoriously resistant to bacterial infection. Even plague usually fails markedly to diminish their prevalence. An epizootic of bacterial nature, therefore, can not be classed with the natural enemies of the rat. We are not surprised, then, to learn that the bacterial viruses have signally failed to accomplish their mission.

These bacterial viruses belong to the colon-typhoid group of organisms. They are either identical with or closely related to the original bacillus of mouse typhoid discovered by Loeffler, or the para-typhoid bacillus type B, which is frequently the cause of meat poisoning, or the Bacillus enteritidis of Gærtner, which has been associated with gastro-intestinal disorders.

The claim that these rat viruses are harmless to man needs revision, in view of the instances of sickness and death reported by various observers. The pathogenicity for man depends upon the virulence of the culture, the amount ingested, the nature of the medium in which it grows, and many other factors.

Danysz virus is pathogenic for rats under laboratory conditions, but has feeble powers of propagating itself from rat to rat. It rapidly loses its virulence, especially when exposed to light and air. The result depends largely upon the amount ingested. The other viruses have proven even less satisfactory.

Under natural conditions these rat viruses may be likened to a chemical poison, with the great disadvantage that they rapidly lose their virulence and are comparatively expensive. They also have the further disadvantage that chemical poisons do not possess of rendering animals immune by the ingestion of amounts that are insufficient to kill or by the ingestion of cultures that have lost their virulence.

205