Vol. 59, No. 11

Association between a Large Plasmid and 15- to 17-KilodaltonAntigens in Virulent Rhodococcus equi

SHINJI TAKAI,1* TSUTOMU SEKIZAKI,2 TOSHISUKE OZAWA,l TORU SUGAWARA,1YUKARI WATANABE,' AND SHIRO TSUBAKI'

Department ofAnimal Hygiene, School of Veterinary Medicine and Animal Sciences, Kitasato University,Towada, Aomori 034,' and National Institute ofAnimal Health, Tsukuba, Ibaraki 305,2 Japan

Received 2 May 1991/Accepted 26 August 1991

Rhodococcus equi strains showing 15- to 17-kDa antigens in immunoblots were found to be virulent in mice.To study the genes specific to these antigens in virulent R. equi, we compared plasmid profiles, immunoblotprofiles, and murine pathogenicity proffles of 10 strains of R. equi. All the strains showing 15- to 17-kDaantigens contained a large plasmid of approximately 85 kbp and were virulent in mice; however, the remainingstrains lacked both the antigens and the large plasmid and were avirulent in mice. Mutants of virulent strainsATCC 33701 and Li, which were cured of the large plasmid by repeated passage at 38C, lacked the 15- to17-kDa antigens and showed a dramatic decrease in lethality in mice. These results suggested that the presenceof an 85-kbp plasmid may be essential for virulence and expression of 15- to 17-kDa antigens of R. equi andoffered support for earlier observations that freshly isolated strains of R. equi killed mice, whereaslaboratory-adapted strains did not.

Rhodococcus equi is a pathogen causing severe, highlyfatal pneumonia or enteritis or both in foals 1 to 3 months old(1, 14). The disease is usually sporadic and rarely involvesmore than one animal on a farm; however, certain farms mayexperience a high rate of infection and mortality every year(17). R. equi is widespread in the environment of horse-breeding farms. It has been isolated from the soil of pad-docks in farms with and without a history ofR. equi infection(15, 23, 25) and from the feces of adult horses and of foalswith and without the disease (24, 25). These observationssuggest that there may be differences among horse popula-tions in their ability to resist infection or differences amongstrains of R. equi in their ability to initiate disease (26).Several attempts have been made to elucidate the virulenceof R. equi (3, 12, 22), and it has long been recognized thatvariations in the outcome of experimental infections of foalsare related to differences in bacterial virulence (7, 11, 20).However, little is known of the markers or factors associatedwith virulence of R. equi.

Recently, we identified by using immunoblotting a diffusegroup of proteins in clinical isolates of R. equi that band atthe 15- to 17-kDa region of an sodium dodecyl sulfate (SDS)gel (21). These proteins have also been found in environmen-tal isolates virulent in mice. It is well established thatextrachromosomal genetic elements are responsible for vir-ulence factors (19). We therefore examined several strains ofR. equi for plasmid DNA'and attempted to correlate thepresence of these elements with the production of 15- to17-kDa antigens and with murine pathogenicity. In thispaper, we report the presence of an 85-kbp plasmid invirulent R. equi that was associated with the 15- to 17-kDaantigens.

MATERIALS AND METHODSBacterial strains. The bacterial strains used in this study

were R. equi ATCC 33701, ATCC 33702, ATCC 33703, ATCC33704, ATCC 33705, ATCC 33706, ATCC 33707, ATCC 6939

* Corresponding author.

and two clinical isolates (strains CE220 and Li), from thelungs of two foals naturally infected with R. equi (Table 1).Strains ATCC 33701 to ATCC 33707 were obtained from J.F. Prescott, University of Guelph, Ontario, Canada. StrainCE220 was obtained from M. Nakazawa, National Instituteof Animal Health, Tsukuba, Japan. Strains were storedfrozen with 20% glycerol in small aliquots at -80C.

Plasmid-curing method. Two strains, ATCC 33701 and Li,were cultured and passaged 25 times consecutively at 38C inyeast extract-casein-cysteine (YCC) broth medium. At eachpassage, a 0.05-ml culture portion was inoculated into 3.0 mlof fresh medium and incubated further for 48 h. Every fivepassages, individual colonies from these broth cultures wereisolated on nutrient agar, and 10 colonies per culture wereinoculated into 3.0 ml of fresh broth medium and incubatedfor 48 h at 38C. Bacterial cells were screened for thepresence of 15- to 17-kDa antigens and a plasmid.

Serotyping of the strains lacking 15- to 17-kDa antigens.Serotyping of the derivatives from strains ATCC 33701 andLi was done by the method described previously by Prescott(13).Horse serum. Serum from a foal naturally infected with R.

equi was used for the immunoblotting procedures (21).Preparation of antigens. Whole-cell antigens were pre-

pared by harvesting bacteria grown for 48 h at 38C from thebroth. Samples were solubilized in SDS reducing buffer(0.0625 M Tris hydrochloride [pH 6.8], 10% [vol/vol] glyc-erol, 2% SDS, 5% 2-mercaptoethanol, 0.02% bromphenolblue) and boiled for 10 min. The undissolved material wassedimented by centrifugation at 7,000 x g for 3 min. Whole-cell antigen preparations contained approximately 107 cellsper 10 ,ul of sample buffer per lane.

Gel electrophoresis and immunoblot analysis. SDS-poly-acrylamide gel electrophoresis (SDS-PAGE) was carried outby the discontinuous method of Laemmli (8) with a verticalslab gel of 15% polyacrylamide and a stacking gel of 4.5%polyacrylamide. Ten microliters of each sample was loadedonto each lane and electrophoresed at 150 V for 1.5 h. Uponcompletion of SDS-PAGE, protein-containing bands were

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transferred to nitrocellulose sheets (Toyo Roshi Inc., Tokyo,Japan) for subsequent immunologic analysis.The blotted nitrocellulose sheets were incubated in Block

Ace (a blocking agent made from milk; Yukijirushi NyugyoInc., Tokyo, Japan) for 60 min at 37C to block any sites thathad not bound on the membranes. The sheets were incu-bated for 2 h at 37C with infected-foal serum which hadbeen diluted 1:100 in Block Ace. The sheets were washed(three times for 10 min each time) in 0.05% Tween 20 inTris-buffered saline (pH 7.4). Horseradish peroxidase-con-jugated goat anti-horse immunoglobulin G (Cappel Labora-tories, Cochranville, Pa.) was diluted 1:1,000 in Block Ace,and the sheets were incubated for 1 h at 37C. After thesheets were washed, the substrate diaminobenzoic acid(Bio-Rad Laboratories, Richmond, Calif.) and hydrogenperoxide were added. Development was stopped by washingthe sheets in distilled water. Except for the use of horserad-ish peroxidase-conjugated goat anti-rabbit immunoglobulinG (Cappel Laboratories), the same procedure was used forthe immunoblot made from hyperimmune rabbit serum.

Isolation of plasmid DNA. Plasmid DNA was isolated fromR. equi by the alkaline lysis method (2) with the followingmodifications, which were found to be necessary for isola-tion of plasmid DNA from Rhodococcus spp. (18). Thebacteria were incubated at 37C for 2 h in a buffer containing0.05 M Tris hydrochloride, 0.01 M EDTA, 0.05 M NaCl and20% (wt/vol) sucrose (pH 8.0) plus 5 mg of lysozyme per ml.Cells were then lysed in 3.0% (wt/vol) SDS in 0.05 M Trishydrochloride buffer (pH 12.6) at 55C for 2 h. ChromosomalDNA was precipitated with 5 M potassium acetate-acetatebuffer (pH 4.8) and centrifuged at 10,000 x g for 15 min. Thepartially purified DNA was subjected to electrophoreticanalysis for detection of plasmids. For restriction enzymedigestions, the plasmid DNA prepared by large-scale isola-tion was purified by means of cesium chloride-ethidiumbromide density gradient centrifugation (18). All restrictionenzymes were purchased from Takara Shuzo Co. Ltd.(Kyoto, Japan) and used according to the recommendationsof the manufacturer.Mouse pathogenicity test. Virulence of the parent strains

and derivatives was estimated by determining the 50% lethaldose by the probit method on groups of five mice. Theinoculum range used was 105 to 108 cells in 0.2 ml of thebacterial suspension for all of the strains. The number ofdeaths was recorded for 10 days after intravenous inocula-tion, and the 50% lethal dose was calculated by the methodof Reed and Muench (16).To study the course of infection, the parent and derivative

strains (approximately 2 x 106 to 4 x 106 viable bacteria per0.2 ml of inoculum) were injected intravenously into a tailvein. Mice were killed on days 0, 1, and 2 after infection, andtheir livers, spleens, and lungs were removed and placed inseparate sterile glass tissue grinders that contained 2.0 ml ofphosphate-buffered saline (PBS). The organs were homoge-nized, the homogenates were serially diluted in sterile PBS,and samples of these dilutions were plated on nutrient agar.The plates were incubated at 37C for 48 h, and then thecolonies were counted. Results were expressed as the value(mean + standard error of the mean) of log1o R. equi per g oforgan.

RESULTSAssociation of a large plasmid with 15- to 17-kDa antigens of

virulent R. equi. The presence of plasmids in a number ofRhodococcus strains has been described previously (6, 9).

TABLE 1. Bacterial strains used in these experiments

Strain Source Prescott's 509o lethal Referenceserotype dose

ATCC 6939 Horse lung 1 >1.0 X 108 10ATCC 33701 Horse lung 1 1.2 x 106 13ATCC 33702 Dog skin 2 >1.0 x 108 13ATCC 33703 Pig lymph nodes 3 >1.0 x 108 13ATCC 33704 Pig lymph nodes 4 7.9 x 106 13ATCC 33705 Pig lymph nodes 5 2.5 x 106 13ATCC 33706 Horse lung 6 >1.0 x 108 13ATCC 33707 Human abscess 7 >1.0 x 108 13

CE220 Horse lung 3 2.1 x 106 12Ll Horse lung 3 1.7 x 106 21

The plasmid content of 10 R. equi strains was examined toassess the association of the presence of plasmids withvirulence or with the presence of 15- to 17-kDa antigens in R.equi.Table 1 shows the pathogenicity of the 10 strains tested in

mice, and Fig. 1 shows immunoblot profiles of the 10 strains.R. equi ATCC 33701, ATCC 33704, ATCC 33705, CE220,and Li, showing 15- to 17-kDa antigens in immunoblots,were found to be virulent in mice. Figure 2 shows theplasmid profiles of the 10 strains. All five strains showing theantigens contained the large plasmid. The remaining fivestrains lacked both the large plasmid and the antigens andwere avirulent for mice. In addition, avirulent strain ATCC33702, which lacked the antigens, contained one smallplasmid of 4.8 kbp.Cesium chloride gradient-purified plasmid preparation of

strain ATCC 33701 was analyzed by restriction enzymedigestion to estimate the plasmid size (Fig. 3). The molecularsize of the plasmid was estimated to be 85 kbp.Mutants lacking both the plasmid and 15- to 17-kDa anti-

gens by repeated passages. To study the association ofplasmid to the virulence of R. equi, mutants lacking theplasmid were obtained by repeated passages at 38C. Therehave been some reports on the loss of 17.5- and 15-kDa

1 2 3 4 5 6 7 8 9 10

+ . _I~ -

FIG. 1. Immunoblot profiles of R. equi strains. Whole-cell prep-arations were analyzed by immunoblotting with serum from anaturally infected foal. Lanes: 1, strain ATCC 6939; 2, strain ATCC33701; 3, strain ATCC 33702; 4, strain ATCC 33703; 5, strain ATCC33704; 6, strain ATCC 33705; 7, strain ATCC 33706; 8, strain ATCC33707; 9, strain CE220; 10, strain Li. The molecular weight markers(with Mr in parentheses), phosphorylase b (106,000), bovine serumalbumin (80,000), ovalbumin (49,500), carbonic anhydrase (32,500),soybean trypsin inhibitor (27,500), and lysozyme (18,500) are indi-cated by bars on the right. The arrow on the left indicates the bandfor the 15- to 17-kDa antigens.

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1 2 3 4 5 6 7 8 9 10

4--Chr

FIG. 2. Plasmid profiles of R. equi strains. Plasmid DNA wasextracted by the method of Singer and Finnerty (18), resolved in a0.7% (wt/vol) agarose gel, and stained with ethidium bromide.Lanes: 1, strain ATCC 6939; 2, strain ATCC 33701; 3, strain ATCC33702; 4, strain ATCC 33703; 5, strain ATCC 33704; 6, strain ATCC33705; 7, strain ATCC 33706; 8, strain ATCC 33707; 9, strain CE220;10, strain Li. chr, chromosomal DNA.

antigens by repeated passages and on the virulence oflaboratory-adapted strains (4, 5, 11, 20).Two of the strains, R. equi ATCC 33701 and Li, were

chosen for further study because some of the virulencecharacteristics of these strains have already been described(22). Strains ATCC 33701 and Li were cultured and pas-saged 25 consecutive times at 38C in YCC broth. Every fivepassages 10 individual colonies of each culture were isolatedand screened for the presence of plasmid DNA by agarosegel electrophoresis and for the presence of 15- to 17-kDaantigens by immunoblotting. As shown in Table 2, of the 10colonies from a culture after passages, 8 lacked 15- to 17-kDaantigens in the immunoblot profiles and were cured ofplasmid DNA, as evidenced by the plasmid profiles. Subse-quently, all of the isolated colonies lacked both the antigensand the plasmid after passage 10. Strain Li was a freshisolate that was subcultured no more than five times. Of the10 colonies from a culture after 15 passages, 8 lacked boththe antigens and the plasmid, and all of the isolated colonieslacked both the antigen and the plasmid after passage 20.The curing of a plasmid was coincidental to a loss ofdetectable 15- to 17-kDa antigens in the two strains tested.There was no difference in colony morphology among thecolonies with or without the antigens.

FIG. 3. Restriction analysis of the plasmid DNA from R. equiATCC 33701. Purified plasmid DNA was digested with the restric-tion endonucleases indicated below and subjected to electrophoresison 0.8% agarose. Lanes: 1, EcoRI; 2, BamHI; 3, HindIll; 4, MluI;5, Notl; 6, A HindIII.

TABLE 2. Incidence of mutants that lack the plasmid and 15- to17-kDa antigens by repeated subculture

No. of colonies (n = 10) lacking plasmid andStrain antigens after passage:

1 5 10 15 20 25

ATCC 33701 0 8 10 10 10 10Li 0 0 0 8 10 10

Representative colonies were selected after passages 1and 10 of strain ATCC 33701 at 38C and after passages 1 and20 of strain Li at 38C for further experiments. These weredesignated ATCC 33701(17kDa+), ATCC 33701(17kDa-),L1(17kDa+) and L1(17kDa-), respectively. Figure 4 showsthe immunoblot profiles of the four strains and ATCC 6939,the type strain of R. equi, using the infected-foal serum.There were no differences in the immunoblot profiles of eachstrain, except for the presence of 15- to 17-kDa antigens.Figure 5 shows the plasmid profiles of the five strains.Strains ATCC 33701(17kDa-) and L1(17kDa-) were curedof the plasmid, and ATCC 6939 did not possess the plasmid.Strains ATCC 33701(17kDa-) and L1(17kDa-) were sero-typed Prescott's serotypes 1 and 3, respectively, the same asthe parent strain. No reversion to the parent phenotype of15- to 17-kDa antigens' production was demonstrated inderivative strains grown at 38C for 10 more serial passages.

Virulence in mice. To compare the virulence of R. equiATCC 33701(17kDa+) and L1(17kDa+) with their plasmid-cured derivatives ATCC 33701(17kDa-) and L1(17kDa-),respectively, groups of mice were given intravenous injec-tions of serial dilutions of each of the strains. The 50% lethaldoses of ATCC 33701(17kDa+) and L1(17kDa+) were 2.6 x106 and 2.2 x 106, respectively, whereas none of the miceinjected with a maximum of 108 plasmid-cured derivativestrain (17kDa-) died (Table 3).To examine more precisely the growth of the four strains

in murine organs, the mice were inoculated intravenouslywith 106 bacteria of each of the strains. As shown in Fig. 6,at 1 and 2 days postinfection, the number of bacteria ofstrains possessing the 17-kDa antigen present in livers andspleens increased to 106 to 107. The number of bacteria inlungs decreased at 1 day postinfection and then increased at2 days postinfection. On the other hand, the numbers ofbacteria in the liver, spleen, and lung were significantlydiminished in strains lacking the 17-kDa antigen. It was

1 2 34 5

~~1~~~- ~-

FIG. 4. Immunoblot profiles of R. equi ATCC 33701, Li, andtheir plasmid-cured derivatives. Whole-cell preparations were ana-lyzed by immunoblotting with serum from a naturally infected foal.Lanes: 1, strain ATCC 33701(17kDa'); 2, strain ATCC 33701(17kDa-); 3, strain L1(17kDa+); 4, strain L1(17kDa-); 5, strainATCC 6939. Molecular markers are indicated by bars on the right(see the legend to Fig. 1).

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-chr

FIG. 5. Plasmid profiles of R. equi ATCC 33701, Li, and theirplasmid-cured derivatives. Lanes: 1, strain ATCC 33701(17kDa+);2, strain ATCC 33701(17kDa-); 3, strain L1(17kDa+); 4, strainL1(17kDa-); 5, strain ATCC 6939. chr, chromosomal DNA.

apparent from the above results that the plasmid-curedderivatives were functionally avirulent in mice.ATCC 33701 isolates without and with the 17-kDa antigen

after passage 5 and a similar pair of Li isolates after passage15 were also tested, and the results confirmed that the loss ofvirulence in mice was associated with loss of the plasmid andantigen expression (data not shown).

DISCUSSIONThis study demonstrated that virulent R. equi contained a

large plasmid of approximately 85 kbp and that curing of theplasmid was coincident with a loss of detectable 15- to17-kDa antigens and a dramatic decrease in lethality formice. In the previous study, we revealed that 15- to 17-kDaantigens of R. equi were associated with virulence for miceand that these antigens were present in epidemiologicallyimportant strains, making them potentially useful as viru-lence markers in antigen detection assays (21). This evidencefor the presence of plasmids in virulent R. equi might be thefirst step in genetic studies on virulence-associated antigensand molecular pathogenesis of the disease.Nothing has been reported on the molecular mechanisms

involved in the virulence or attenuation of R. equi (14).However, it is well established that extrachromosomal ge-netic elements are responsible for many phenotypic charac-teristics of bacterial cells, including virulence factors (19).With this in mind, we examined the plasmid profiles of R.equi strains by the method of Singer and Finnerty (18), whodevised a method for isolation of plasmid DNA from Rhodo-coccus spp. In the study reported here, we demonstrated thepresence of a large plasmid of approximately 85 kbp invirulent R. equi strains showing 15- to 17-kDa antigens inimmunoblots and demonstrated the absence of plasmids inavirulent R. equi strains which lack the antigen. The expres-sion of 15- to 17-kDa antigens was clearly associated with thepresence of the plasmid. However, it must be shown that not

TABLE 3. Virulence of R. equi ATCC 33701, Li, andtheir derivatives

Strain Passage 85-kbp 15- to 17- 50o(parent) no. plasmid kDa antigens lethal dose

ATCC 33701 1 + + 2.6 x 10610 - - >1.4 x 108

Li 1 + + 2.2 x 10620 - - >1.0x108

Bi707(40

.t50cm

0)4

O 1 2 0 1 2Days post inoculation

FIG. 6. Mean viable counts of bacteria in different tissues. Thebacteria in the liver (0, 0), spleen (A, A), and lung (U, l) of miceafter intravenous inoculation with 2 x 106 to 4 x 106 R. equi ATCC33701(17kDa+) (solid symbol), ATCC 33701(17kDa-) (open symbol),L1(17kDa+) (solid symbol), and L1(17kDa-) (open symbol) werecounted. Each point represents the geometric mean standarddeviation for three mice.

only is curing the plasmid associated with loss of virulencebut that virulence can be restored by reintroduction of theplasmid. It is very likely that the plasmid encodes forunidentified virulence factors which are critical in R. equiinfection, since the molecular weight of the plasmid is solarge. Further studies are needed to clarify the genes en-coded by the large plasmid in virulent strains and by thesmall plasmid in the avirulent strain ATCC 33702.

Studies have shown that repeated passages of originallyvirulent clones of pathogenic bacteria promotes a populationshift in favor of variants lacking those characteristics (19).Dafaala et al. (5) reported that freshly isolated strains of R.equi killed mice, whereas laboratory-adapted strains did not.Loss of the antigens by repeated passages at 42C was alsoreported by Chirino-Trejo and Prescott (4). They reportedthat the 17.5- and 15-kDa bands were not produced by ATCC6939 and were lost on repeated passages in vitro. Theyspeculated that loss of the antigens in the type strain was dueto repeated passages. Unfortunately, they did not determinethe relationship between the antigens and virulence in R.equi. In the present study, a large plasmid in virulent R. equiwas successfully eliminated by repeated passages at anelevated temperature. Strain ATCC 6939, the type strain ofR. equi, which was isolated from a foal with fatal pneumoniain 1923 (10), has been passaged repeatedly since it wasisolated. ATCC 6939 has been used in experimental infec-tions in foals to reproduce the disease (11, 20); however, allthese experiments have resulted in failure. It is very likelythat the attenuation of strain ATCC 6939 occurred as a resultof curing the plasmid in the strain by repeated passage.

R. equi grows well over a wide range of temperatures (10to 40C), with the optimum temperature reported as 30C (1).In the preliminary experiment, plasmid-cured derivatives ofstrain ATCC 33701 were not found in the colonies isolatedfrom subculture at 30C after 25 passages, and the cultureafter 25 passages was virulent in mice (unpublished data).From the present data, the maintenance of the 85-kbpplasmid at 38C in vitro was unstable. Whether the replica-tion and inheritance characteristics of the 85-kbp plasmid istemperature sensitive is unclear, because an appropriate

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marker of the plasmid which permits the calculation of aprecise rate of plasmid segregation is not available. How-ever, we speculated that the replication of the plasmid is nottemperature sensitive, because the plasmid of freshly iso-lated strain Li was relatively stable. The plasmid-curedclone could overgrow the plasmid-carrying cells in a nonse-lective in vitro environment, i.e., without the selectivepressure of the host defense mechanisms.Most members of the genus Rhodococcus are saprophytic

soil organisms, although several pathogenic species exist,including Rhodococcus bronchialis (a human pathogen), R.equi (an animal pathogen), and Rhodococcus fascians (aplant pathogen). Previous work has shown that several R.fascians strains harbor large plasmids; the presence of a138-kbp plasmid correlated with resistance to cadmium in R.fascians D188 (6, 9). In this study, we first demonstrated alarge plasmid in R. equi strains correlated with virulence formice and the expression of 15- to 17-kDa antigens. It mightbe interesting to examine the plasmid content of virulent R.bronchialis.

Further studies will be made to define the plasmid genesinvolved in the pathogenesis of R. equi infection in foals bymolecular cloning and mutagenesis experiments.

ACKNOWLEDGMENTSThis study was supported by a grant-in-aid from the Equine

Research Institute, Japan Racing Association, and by a grant-in-aidfor scientific research (no. 03856078) from the Ministry of Educa-tion, Science and Culture, Japan.

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