B. anthracis, S. faecalis and B. subtilis were found to survive, the first two indefinitely, in the alimentary canal of Argas persicus, and to be passed in its faeces. The stomach contents, in vitro, were bactericidal to B. anthracis, but in some of the tests a few individuals survived, and these may represent more resistant organisms, or spores, to which the infection of the tick by the relatively massive doses used in the feeding experiments was due. In smears of the stomach contents and faeces of the tick, B. anthracis appeared as a short thick bacillus in chains of about three to eight individuals provided with a very well-developed capsule. This capsule, not present in the agar cultures used for the bactericidal tests, may have afforded the bacillus protection within the tick. If this be true, then a very small dose of the capsulated bacilli from the blood of an infected animal might succeed in infecting the tick. A culture of B. anthracis recovered after one hundred days in the tick was found to be fully virulent and pathogenic.
B. subtilis seemed to behave like B. anthracis in the tick, but the investigation of this micro-organism was much less complete than that of B. anthracis. There is an apparent discrepancy between the results of the feeding experiments and the inhibitory tests in vitro. This might be explained by assuming that the two ticks which harboured the bacillus to the end were deficient in the inhibitory principle, which, as shown above, is not equally active in all ticks, or by suggesting, on analogy to the bacteriophage, that the inhibitory element was completely absorbed by the large bacterial dose used in feeding, leaving an excess of bacilli to establish the infection. This might also be put forward as an alternative explanation in the case of B. anthracis.
All the experiments with Streptococcus faecalis were consistent, and showed it to be an organism wholly resistant to the bactericidal and inhibitory elements in the tick's stomach. B. mycoides neither survived long in the tick nor appeared in its faeces. Only four ticks however lived, and of these only two were available for examination; a number perhaps too small to justify any definite conclusion being drawn from purely negative results. Why this bacillus should have behaved differently to the other sporing bacilli, B. anthracis and B. subtilis, is not apparent. It was not more susceptible to the lytic action of stomach contents than was B. anthracis, and, unlike B. subtilis, it was wholly resistant to the inhibitory principle.
Tests with B. pestis and B. melitensis were inconclusive, as both of these micro-organisms died out soon in the inactivated horse serum used in feeding; B. pestis in two weeks and B. melitensis in about three weeks. In the case of B. melitensis the organism apparently survived as long in the tick as it did in the serum control. In order to obtain more conclusive results with these two organisms, it is necessary to find a medium which, while providing a suitable food for the tick, will not prove inimical to the life of the bacillus. It is noteworthy, however, that the growth of these organisms was not inhibited, and B. melitensis was not destroyed, in vitro by ticks' stomach contents.
B. typhosus presented a curious problem in that, while it survived in the stomach for at least twenty-two days, it was never recovered from the faeces; this applies also to the case of B. melitensis. Whether or not this is due to some factor existing in the rectum or malpighian secretion, has not been shown. The cause of the eventual disappearance of B. typhosus from the tick is obscure. It may be that it is killed off by the products of its own life activities in a medium particularly favourable to its growth. No lysin or inhibitory principle affecting B. typhosus was found in the tick.
Staphylococcus aureus furnished very conclusive results. It died out quickly after ingestion by the tick, and the evidence of the feeding experiments was fully supported by the results of the tests in vitro which showed the stomach contents to be powerfully bactericidal and inhibitory to the coccus.
The source and nature of the lytic and inhibitory principles require further investigation which will be made the subject of a later communication by one of us (J.T.D.). For the present, the results of the control cultures on horse serum make it clear that this medium is not the source. Other tests carried out with the inactivated blood of the fowls used for the normal feeding of the ticks gave no appreciable evidence of bactericidal or inhibitory power.Therefore it may reasonably be assumed that the source of these elements is within the tick, but whether they are derived from the salivary glands or the stomach has not been shown. With regard to their nature, their action is not dependent upon the presence of alexin, and the lysin may be of the nature of an enzyme, or perhaps something similar to the “lysozyme” present in certain animal secretions, described by Fleming (1922). Both the lytic and inhibitory principles vary in amount in individual ticks, and neither is inactivated by exposure to a temperature of 58° C. for thirty minutes.
In all cases of ticks fed on bacterial cultures the micro-organisms remained within the confines of the alimentary canal, and no evidence of invasion of the haemocoele was found. Defaecation was the only process by which bacteria were found to leave the tick, and as only a few types were passed in this way, and defaecation occurs only at long intervals, and not as a rule during feeding, it seems very improbable that Argas persicus could be an efficient vector of any of the bacterial viruses employed in this work.
