THE MICROBIAL FLORA OF THE ROCKY MOUNTAIN WOOD TICK, DERMACENTOR ANDERSONI STILES EDWARD A. STEINHAUS U. S. Public Health Service, Rocky Mountain Laboratory, Hamilton, Montana Received for publication, January 8, 194 This report is concerned with the results of a study of the microbial flora of 5 adult specimens of the Rocky Mountain wood tick, Dermacentor andersoni, obtained from various sources. The accumulated data suggest that the bacterial flora of this tick is largely fortuitous, consisting chiefly of adventitious organisms acquired while feeding on its host. MATERIALS AND METHODS Part of the ticks tested were from a laboratory strain initiated in 1934 and reared subsequently through several generations, and part were collected from nature in western Montana. There were 16 unfed specimens and 486 that had recently received a blood meal. One hundred and forty-five lots were tested. A few of the lots contained numerous ticks, some only two; the remainder averaged 1 ticks each. In addition, a considerable number of ticks were dissected and the various organs examined separately. Each lot was prepared for examination by first immersing the ticks in tincture of merthiolate for 4 to 5 hours, then rinsing them thoroughly with 5 changes of sterile saline (.5 ml. of the last wash was cultured), and finally by triturating the ticks (except those dissected) in sterile saline. The resulting suspension was then examined for bacteria, yeasts, molds, rickettsiae, and motile protozoa by the techniques outlined below. BACTERIA Techniques The triturated tick suspension was streaked on glucose beef-infusion agar, North's gelatin chocolate blood agar, and cystine heart agar plates. Tubes of Noguchi's leptospira medium were also frequently inoculated. As a routine test for anaerobes, deep tubes of sodium-thioglycollate broth containing minced pork brain were inoculated and the surface of the broth covered with a layer of vaseline. Occasionally pyrogallic-acid sodiuim-hydroxide as well as vacuum methods were also used. The cystine heart agar and one North's gelatin chocolate blood agar plate were incubated at 37.5 C. while another North's gelatin chocolate agar plate, one glucose plate, the leptospira medium, and the sodium-thioglycollate broth were incubated at room temperature (5 to 8C.). In addition to the culture work, slides of the triturated tick suspensions were made and stained with Ziehl-Neelsen's carbol fuchsin for the detection of acidfast organisms. Each strain of bacterium isolated was subjected to a complete cultural, 397
398 EDWARD A. STEINHAUS morphologic, physiologic and pathogenic examination. In determining physiologic properties the cocci were cultured in 1 differential media and all other forms were cultured in 3. For the pathogenicity tests each strain was injected into guinea pigs (intraperitoneally and subcutaneously), rabbits (intraperitoneally and intravenously), and mice (intraperitoneally) in varying dosages. Results Seventy-seven strains of bacteria were isolated and studied. These included 18 strains of gram-negative rods, 5 gram-positive rods, 47 gram-positive cocci, and 7 gram-positive spore-forming rods. No acid-fast bacteria were observed. Table 1 gives the number and percentage of bacteria in each morphologic group according to the sources of the ticks and to the condition of the ticks with respect to their blood meal. When male and female unfed ticks were examined separately no essential difference was noted in the kinds of bacteria isolated. Most of the "fed" ticks tested were partially to fully engorged females. In the case of those ticks which were dissected and the various organs or parts examined separately, cultivable bacteria were isolated from only the alimentary tract. It is known, however, that the bacterium causing tularemia may occur also in the Malpighian tubes and coelomic fluid of this tick (Francis, 197). Percentage of ticks harboring bacteria. In general, percentages of ticks harboring bacteria indicate that unfed ticks from vegetation contained the fewest bacteria (1. per cent) and the ticks from the domestic sheep harbored the most (17 per cent). If, however, one considers only the engorged female ticks from, for example, the domestic sheep, the percentage rises to almost. Since the latter ticks were examined in pairs, the percentage might have been higher if each tick had been examined individually. The failure to recover bacteria from ticks from nature subsequently fed on domestic rabbits apparently does not present a true picture since well over one per cent of those reared on rabbits for use in production of Rocky Mountain spotted fever vaccine harbor bacteria. Considering the data in general, it is convenient to suppose that each bacterial strain arose from one tick. Analyzed on this basis, the data give the following results: Of the 16 unfed ticks, 1.6 per cent harbored bacteria, while of the 486 recently fed ticks, 9.1 per cent yielded bacteria. In other words, on the average, over 5 times as many fed ticks harbored bacteria as unfed ticks. There was no essential difference between laboratory-reared ticks and those collected in nature so far as the percentage of lots from which bacterial strains were recovered. Kinds of bacteria. The 77 strains of bacteria were tentatively classified as to genus as follows: one of the genus Achromobacter, 4 Alcaligenes, 7 Bacillus, Corynebacterium, 4 of the coliform group (probably Escherichia), Flavobacterium, 47 Micrococcus, 4 Pasteurella, Pseudomonas, and gram-positive and gram-negative rods were unidentified. The 4 strains of Pasteurella were all
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4 EDWARD A. STEINHAUS P. tularensis isolated from the engorged female ticks obtained from domestic sheep. As far as the writer is aware, this is the first instance of P. tularensis GROUP 1. Pigment yellow to orange; gelatin not liquefied.... Pigment yellow to orange;f gelatin liquefied.... 3. Pigment red; gelatin notj liquefied... 4. No pigment, (white); gelatin not liquefied... 5. No pigment, (white); gelatinf liquefied... TABLE Reactions of micrococci in differential media STRAINS PRODUCING ACI IN Glucose Lactose Sucrose Mannitol 1 1 1 17 1 17 94.4 66.6 94.4 3 4.9 3 8.6 4.9 18 13 18 94.7 68.4 94. 7 1 14 88.8 STRAINS PRODUCING HS 1 1 14.3 57.1 1 63.1 NO, from NOs 17 94.4 8.6 5 16 6.3 9. NO. STRAINS TESTED All Groups... f 4 9 4 9 9 35 47 l 85.1 61.7 85.1 61.7 19.1 74.4 TABLE 3 Bacterial strains, according to genus, tested for pathogenicity in guinea pigs, rabbits and mice GENUS NO. OF STRAINS PATHOGENIC FOR NO. OF STRAINS TESTED Guinea Rabbits Mice pigs Rbis Mc Achromobacter... 1 1 Alcaligenes... 4 3 Bacillus... 7 1 3 Coliforms (probably Escherichia)...... 4 3 1 4 Corynebacterium....... Flavobacterium... Micrococcus... 47 4 4 Pasteurella... 4 4 4 4 Pseudomonas... 1 Unidentified... 4 1 1 Totals... 77 1 1 1 18 1 7 19 being isolated from Dermacentor andersoni by cultural methods. Parker and Spencer (194) detected the tularemia organism in ticks by animal inoculation.
MICROBIAL FLORA OF ROCKY MOUNTAIN WOOD TICK Excepting the four strains of P. tularensis, the only instances of multiple isolations were in the genus Micrococcus. Three species were each isolated twice, one 3 times, and one 5 times. Each isolation was from a different lot of ticks. The largest number of bacteria of any morphologic group were of the genus Micrococcus. No attempt was made to classify them specifically, but following the general scheme of Bergey's Manual, they have been divided into 5 groups. Their characteristics are given in table. In the main they show the characteristics typical of saprophytic micrococci. Pathogenicity tests. As shown in table 3, 1 of the 77 strains were pathogenic to guinea pigs, 1 to rabbits, and 1 to mice. In each case the criterion of pathogenicity was the death of the animal. With the exception of the 4 strains of Pasteurella tularensis, only two strains were pathogenic to all three animals: a coliform (probably Escherichia) and a Bacillus (Bacillus megatherium). In all, 3 different strains were pathogenic for at least one of the 3 species of test animals. In view of the observations of many investigators that the fermentation of mannitol is a characteristic property of pathogenic staphylococci, it is interesting to note that while 9 of the cocci isolated produced acid in mannitol only 5 of these were pathogenic. The sixth pathogenic coccus did not ferment this carbohydrate. Immunity tests. Because the rickettsia of Rocky Mountain spotted fever occurs spontaneously in Dermacentor andersoni, immunity tests against this disease were given to the guinea pigs which had been inoculated with the various strains of bacteria, in order to determine if there was any cross-immunity. To do this, 1. ml. of guinea pig blood virus of Rocky Mountain spotted fever (a D. andersoni strain) was inoculated into each pig which had survived the inoculation of bacteria for at least days. In no case was any immunity shown. OTHER MICROORGANISMS Yeasts and molds. Examinations for these higher fungi yielded two strains of yeast but no molds. The two yeasts were not identified as to species but very probably belong to the tribe Saccharomyceteae. They were non-pathogenic for guinea pigs, rabbits, and mice. Pickettsiae. The Rocky Mountain spotted fever rickettsia, Dermacentroxenus rickettsii Wolbach; the rickettsia of American Q fever, Rickettsia diaporica Cox; and a non-pathogenic rickettsia-like organism which has been studied by the writer and is the subject of a separate report, may occur spontaneously in D. andersoni. Several strains of Rocky Mountain spotted fever were isolated in guinea pigs inoculated with recently fed ticks from nature. Because of the difficulty in differentiating microscropically between these three species in the tick, the presence or absence of rickettsiae was recorded for each lot of ticks examined without any regard as to the species of rickettisa concerned. Of the 145 lots examined, rickettsiae were observed in 94. The 41
4 EDWARD A. STEINHAUS number of Rocky Mountain spotted fever rickettsiae observed would, of course, be much smaller than this (see Parker and Spencer (196)). All examinations for rickettsiae were made using both Macchiavello's and Giemsa's staining methods. Spirochaetes. The suspension from each lot of ticks was stained with Giemsa's spirochaete stain. Of the 13 lots so examined, none showed spirochaetes. Motile protozoa. The triturated tick suspensions from 13 lots of ticks were observed by the "wet mount" and occasionally by the "hanging drop" method for motile protozoa. In no case were any observed. These examinations were more or less cursory and should not be considered final. On several occasions forms which may have been encysted protozoa were seen but were not identified as such with certainty. Bacteriophage. Though no routine examinations for bacteriophage were made, one strain was found associated with a strain of Micrococcus isolated from engorged female ticks obtained from domestic sheep. Apparently this is the first record of bacteriophage being isolated from ticks. About this same time, on two different occasions, a gram-negative short rod was isolated from another species of tick, Dermacentor albipictus. This bacterium was also heavily infected with a strain of bacteriophage. ACQUISITION OF BACTERIA BY TICKS During the course of the investigation, it became apparent that fed ticks harbored bacteria more frequently than did unfed ones. This fact suggested that ticks must acquire bacteria from their hosts while feeding. Since most of the strains isolated were'saprophytes rather than pathogens, it did not seem reasonable that these bacteria were acquired with ingested blood. Therefore, the possibility that bacteria may be acquired adventitiously from the skin of the host was tested. Tests were made with Serratia marcescens and an unidentified Micrococcus isolated from D. andersoni. Serratia marcescens was chosen because of its pigment and because our studies had not yielded any organisms of this genus. Cultures of these organisms were thinly smeared over the surface of the skin area on which the ticks were to feed. The test ticks were confined to this area by metal capsules and allowed to feed to engorgement. Their exteriors were then sterilized in the usual manner and their internal organs cultured on glucose beef-infusion agar. The three stages, larvae, nymphs, and adults were all treated in this manner. In every case it was shown that the test bacteria could be acquired from the skin surface of the host animal. _ DISCUSSION The literature contains several statements suggesting that the alimentary tract of ticks is invariably sterile. This general conclusion appears to be based on the work of Hindle and Duncan (195) and Duncan (196), who found the gut contents of Argas persicus to be devoid of bacteria and to contain a bacteriostatic principle. From the results of the present investigation, it is appar-
MICROBIAL FLORA OF ROCKY MOUNTAIN WOOD TICK ent that in the case of Dermacentor andersoni the bacterial flora, consisting largely of organisms contained in the alimentary tract, is not an established one in the sense that it is in the case of certain of the Hexopoda. Instead, it appears that most of the bacteria this tick may harbor are the result of the fortuitous acquisition of these organisms from its host. This is shown by a comparison of the flora in D. andersoni and that found in some insects (Steinhaus, 1941). In the latter there were found 53 per cent gram-negative rods,.5 per cent gram-positive cocci, 1 per cent gram-positive short rods, and 14.5 per cent gram-positive spore-formers, while the flora of the tick consists of 3.4 per cent gram-negative rods, 61. per cent gram-positive cocci, 6.5 per cent gram-positive short rods, and 9.1 per cent gram-positive spore-formers. Since it is generally accepted that the bacteria of the skin surfaces of most animals consists largely of cocci and since this appears to be the principal source of the D. andersoni flora, it is not surprising to find that over 6 per cent of the bacteria of this tick belongs to this group of microbes nor that most of them were non-pathogenic. Nevertheless, 3 different strains were pathogenic for one or more of the 3 kinds of test animals used. Though we used media similar to that employed by Noguchi (196) we were unable to recover, with certainty, the three organisms (Bacillus rickettsiformis, Bacillus pseudoxerosis, and Bacillus equidistans) isolated by him from D. andersoni. On several occasions we did isolate gram-negative short rods which were similar to those he described. Since apparently none of Noguchi's original cultures are available, it was not possible to make a close comparison. SUMMARY A study of the internal microbial flora of 5 specimens of the Rocky Mountain wood tick, Dermacentor andersoni, obtained from various sources, yielded 77 strains of bacteria, strains of yeasts, at least 1 rickettsia (that of Rocky Mountain spotted fever), 1 rickettsia-like organism, and 1 strain of bacteriophage. No acid-fast bacteria, molds, spirochaetes or motile protozoa were observed in any of the ticks examined. Each of the 77 strains of bacteriawere studied in detail with respect to their cultural, morphologic, physiologic, and pathogenic characteristics. Of 16 unfed adult ticks, only 1.6 per cent harbored bacteria, but of 486 recently fed ticks, bacteria were found in a minimum percentage of 9.1. One possible explanation of the greater number of bacteria in recently fed ticks is the experimental finding that feeding ticks can ingest bacteria from surface of skin to which they are attached. The evidence suggests that the bacterial flora of D. andersoni is a fortuitous one consisting chiefly of adventitious organisms apparently acquired from its hosts. REFERENCES DUNCAN, J. T. 196 On a bactericidal principle present in the alimentary canal of insects and arachnids. Parasitology, 18: 38-5. FRANCIS, E. 197 Microscopic changes of tularemia in the tick Dermacentor andersoni and the bedbug Cimex lectularius. U. S. Pub. Health Repts., 4: 763-77. 43
44 EDWARD A. STEINHAUS HINDLE, E., AND DUNCAN, J. T. 195 The viability of bacteria in Argas persicus. Parasitology, 17: 434-446. NOGUCHI, H. 196 Cultivation of rickettsia-like microorganisms from the Rocky Mountain spotted fever tick, Dermacentor andersoni. J. Exptl. Med., 43: 515-53. PARKER, R. R., AND SPENCER, R. R. 194 Tularaemia. XI. Tularaemia infection in ticks of the species Dermacentor andersoni Stiles in the Bitterroot Valley, Mont. U. S. Pub. Health Repts., 39: 157-173. PARKER, R. R., AND SPENCER, R. R. 196 A study of the relationship between the presence of rickettsia-like organisms in tick smears and the infectiveness of the same ticks. U. S. Pub. Health Repts., 41: 461-469. STEINHAUS, E. A. 1941 A study of the bacteria associated with thirty species of insects. J. Bact., 4: 757-79. Downloaded from http://jb.asm.org/ on March 17, 19 by guest