INFECTION AND IMMUNITY, Oct. 1989, p. 3136-31410019-9567/89/103136-06$02.00/0Copyright C 1989, American Society for Microbiology

Functional Homology of Virulence Plasmids in Salmonellagallinarum, S. pullorum, and S. typhimurium

PAUL A. BARROW* AND MARGARET A. LOVELL

AFRC Institute for Animal Health, Houghton Laboratory, Houghton, Huntingdon,Cambridgeshire PE] 7 2DA, United Kingdom

Received 22 March 1989/Accepted 21 June 1989

The virulence-associated plasmids of strains of Salmonella gallinarum and S. pullorum were transferredseparately by mobilization with the F plasmid into virulence plasmid-cured derivatives of S. gallinarum, S.pullorum, and S. typhimurium and into a prototrophic Escherichia coli K-12 strain. The transconjugants weretested for virulence in chickens of different ages and in mice. The S. gallinarum and S. pullorum plasmids wereable to restore full virulence in the three Salmonella strains, thus demonstrating functional homology invirulence plasmids from these Salmonella serotypes and biotypes. The virulence phenotypes of the transcon-jugants remained the same as that of the parent strain of the recipient. This, together with the fact that E. coliK-12 containing either of the virulence plasmids was avirulent for chickens, suggested that in addition toplasmid genes, chromosomal genes are important in determining virulence, particularly in determining theability to survive and multiply in the cells of the reticuloendothelial system. The virulence plasmids were notself-transmissible and could not be transduced by temperate bacteriophages lysogenizing field strains of S.gallinarum. They were not in the same incompatibility group as F but were fi+.

There is a great deal of evidence that plasmid-linked genescontribute to the virulence of several Salmonella serotypeswhich produce systemic disease in animals and humans.Such serotypes include Salmonella typhimurium, S. dublin,S. enteritidis, S. cholerae suis, S. paratyphi C, and S.abortus ovis (1, 16, 17). The importance of an 85-kilobase-pair (kbp) plasmid to the virulence of S. typhimurium wasfirst demonstrated by Jones et al. (16), who showed thatcuring the plasmid produced strains which were less virulentfor mice and that reintroduction of the plasmid restoredvirulence. Similar results with S. dublin and S. enteritidis inmice have been produced by curing and reintroducing theirlarge plasmids (6, 15).By using plasmid curing and reintroduction, two recent

studies (2, 4) have shown that 85-kbp plasmids contribute tothe virulence of S. gallinarum and S. pullorum. These areSalmonella biotypes within the same serotype and are re-sponsible for fowl typhoid and pullorum disease, respec-tively, both of which are economically important diseases ofpoultry in many countries (11, 21). S. gallinarum producessevere disease with considerable mortality rates in chickensof all ages, whereas S. pullorum characteristically producessignificant mortality rates only in young chickens (25).Elimination of the large plasmids from S. gallinarum 9 and S.pullorum 3 produced strains with significantly reduced viru-lence for chickens. Reintroduction of the plasmids restoredfull virulence (2, 4). The plasmids were responsible for theability of the strains to survive and multiply in the cells of thereticuloendothelial system. It was also found that the plas-mids contributed to the ability of the strains to invade via thealimentary tract following oral inoculation.

Williamson et al. (28) recently demonstrated that an 8-kbpSalI-XhoI fragment derived from the virulence plasmid of astrain of S. typhimurium hybridized with similar fragmentsfrom 10 other serotypes, including S. gallinarum and S.pullorum. This indicated a considerable degree of genetichomology between virulence plasmids of serotypes with

* Corresponding author.

very different virulence characteristics and host ranges.Molecular similarities between virulence plasmids from dif-ferent serotypes were also demonstrated by Popoff et al.(19). Williamson et al. have also demonstrated functionalhomology between the virulence plasmids in S. dublin and S.typhimurium (28, 29). In the present study, we have at-tempted, by plasmid transfer, to determine whether func-tional homology exists between the virulence plasmids of S.gallinarum, S. pullorum, and S. typhimurium. The results ofsuch a study should also increase our understanding of thenature of the differences between S. gallinarum and S.pullorum.

MATERIALS AND METHODS

Bacterial strains. S. gallinarum 9 and S. pullorum 3 wereisolated from field cases of fowl typhoid and pullorumdisease, respectively. S. typhimurium F98 was also isolatedfrom diseased chickens. The virulence of these strains forchickens has been demonstrated (25). In addition to plasmid-cured derivatives of these strains, a prototrophic Esche-richia coli K-12 strain (proto) (23) was used as a recipient.The derivatives of these strains that were used in the presentstudy are shown in Table 1. All strains were stored inglycerol-nutrient broth at -20°C. Unless otherwise indi-cated, nutrient broth or agar (Oxoid, Basingstoke, UnitedKingdom) was used for bacterial culture. Broth cultureswere incubated for 24 h at 37°C in a shaking bath (100 strokesper min). They contained between 5 x 108 and 1 x 109 viableorganisms per ml.

Plasmid isolation and enzyme digestion. Plasmid DNA wasvisualized by the method of Hansen and Olsen (14), using0.7% agarose gels for electrophoresis. PstI digestion wascarried out under conditions recommended by the manufac-turer (Bethesda Research Laboratories, Inc., Gaithersburg,Md.).Transposon mutagenesis, plasmid curing, and bacterial

mating procedures. Tn3-tagged virulence plasmids from S.gallinarum 9 and S. pullorum 3 were mobilized with the Fplasmid into Nalr mutants of E. coli K-12 proto and virulence

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TABLE 1. Salmonella and E. coli K-12 strains used

Strain Large-plasmid Description and referencecontent

S. gallinarum 9 pSGO90 Parent strain (4)S. gallinarum 9 F+ pSGO90, F F transferred to Nalr mutant of S. gallinarum 9S. gallinarum 9 VP- None Virulence plasmid cured from S. gallinarum 9 (4)S. gallinarum 9 VP- F+ F F transferred to Nalr mutant of S. gallinarum 9 VP-S. gallinarum 9(pSP3) pBL001::Tn3 Tn3-labeled virulence plasmid (pBL001::Tn3) transferred by F from Spcr mutant

of S. pullorum 3 into Nalr mutant of S. gallinarum 9 VP-

S. pullorum 3 pBLOO1 Parent strain (2, 25)S. pullorum 3 F+ pBL0O1, F F transferred to Nalr mutantS. pullorum 3 VP- None Virulence plasmid cured from S. pullorum 3 (2)S. pullorum 3 VP- F+ F F transferred to Nalr mutant of S. pullorum 3 VP-S. pullorum 3(pSG9) pSGO90::Tn3, F Tn3-labeled virulence plasmid (pSG090::Tn3) transferred by F from Rifr mutant

of S. gallinarum into Nalr mutant of S. pullorum 3 VP-

S. typhimurium F98 pTM980 Nalr mutant of parent strain (3, 25)S. typhimurium F98 F+ pTM980, F F transferred into F98S. typhimurium F98 VP- None Virulence plasmid cured from S. typhimurium F98 (3)S. typhimurium F98 VP- F+ F F transferred into F98 VP-S. typhimurium F98(pSP3) pBL001::Tn3 pBL001::Tn3 transferred by F from S. pullorum 3 Spc' into S. typhimurium

F98 VP-S. typhimurium F98(pSG9) pSGO90::Tn3 pSG090::Tn3 transferred by F from S. gallinarum 9 Rifr into S. typhimurium

F98 VP-

E. coli K-12 proto None Parent strain (23)E. coli K-12 F+ F F transferred into Nalr mutant of E. coli K-12 protoE. coli K-12(pSP3) pBL001::Tn3 pBL001::Tn3 transferred by F from S. pullorum 3 Spcr into E. coli K-12 NalrE. coli K-12(pSG9) pSGO90::Tn3 pSG090::Tn3 transferred by F from S. gallinarum 9 Rif into E. coli K-12 Nalr

plasmid-cured strains of S. gallinarum 9, S. pullorum 3, andS. typhimurium F98 as described previously (2-4). Trans-conjugants were checked for the presence of large plasmidsby plasmid analysis and by PstI digestion of plasmid DNA.The F plasmid was itself transferred to Nalr mutants of the

parent and plasmid-cured forms of S. gallinarum 9, S.pullorum 3, and S. typhimurium F98 from E. coli K-12 protoF+. Transconjugants which were Lac- were tested for thepresence of F by measurement of their sensitivity to bacte-riophage MS2, by plasmid analysis, and by PstI digestion.

Experimental animals. Unsexed Rhode Island Red chick-ens were obtained from a specified-pathogen-free flock atthis laboratory (2). Six-week-old female BALB/c mice(weighing approximately 20 g each) were obtained fromInterfauna, Huntingdon, United Kingdom. They were fedLabsure pellets (Labsure, Manea, United Kingdom) andwater ad libitum.

Virulence and pathogenicity studies. The virulence ofstrains for chickens was tested largely by the methodsdescribed by Barrow et al. (4), with oral and intramuscularinoculations. Newly hatched (less than 24-h-old) and 3-week-old chickens were used.The 50% lethal doses for mice were calculated following

intravenous inoculation (in the tail vein) with serially dilutedcultures. These doses were calculated by using the Maxi-mum Likelihood Program statistical package (RothamstedExperimental Station, Harpenden, United Kingdom), whichfollows conventional methods of probit analysis (9). Thiswas not possible for the data presented in Table 4. In thiscase, the method of Reed and Muench (20) was used to givean approximation, and the death rates were compared byusing a modification of the t test (26).

Bacterial viability in the reticuloendothelial system was

assessed following intravenous inoculation of groups of 24mice or 3-week-old chickens with approximately 105 organ-isms (for all Salmonella strains) or 108 organisms (for E. coli

K-12 strains) in 0.1 ml. At intervals thereafter, three animalsfrom each group were killed and viable counts were made onsamples of cardiac blood, breast muscle (chickens) or gas-trocnemius muscle (mice), spleen, myocardium, and liver. Inone experiment, newly hatched chickens were inoculated viaone of the gastrocnemius muscles with 108 organisms of theE. coli K-12 strains. In addition to the organs mentionedabove, inoculated gastrocnemius muscle from these chick-ens was sampled. In all cases, viable bacteria were countedon MacConkey agar (Oxoid).

RESULTS

Plasmid content and other characteristics of strains. Thebacterial strains used are listed in Table 1. Their plasmidprofiles are shown in Fig. 1. By using methods describedpreviously (2-4), it was possible to cure S. gallinarum 9, S.pullorum 3, and S. typhimurium F98 of their 85-kbp plas-mids. The plasmids were cured at frequencies of approxi-mately 10-4, 10-4 and 10-17, respectively.The Tn3-tagged virulence plasmid, pSG090::Tn3, in S.

gallinarum 9 Rif' was mobilized by F into S. pullorum 3 VP-Nalr, S. typhimurium F98 VP- Nalr, and E. coli K-12 protoNalr to produce strains designated S. pullorum 3(pSG9), S.typhimurium F98(pSG9), and E. coli K-12(pSG9), respec-tively. By PstI digestion of total plasmid DNA (Fig. 2), wefound that F was also present in each of the above threetransconjugants.

Similarly, pBL001::Tn3 in S. pullorum 3 Spc' was mobi-lized by F into S. gallinarum 9 VP- Nalr, S. typhimuriumF98 VP- Nalr, and E. coli K-12 Nalr to produce strainsdesignated S. gallinarum 9(pSP3), S. typhimurium F98(pSP3), and E. coli K-12(pSP3), respectively (Fig. 2). Inaddition to pBL001::Tn3, F had mobilized the small 2.6-kbpplasmid present in SP3 but not associated with virulence (4).F was not present in S. gallinarum 9(pSP3) or S. typhimu-

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a b c d e f g h j k m rn

LP

C

SP

FIG. 1. Plasmid profiles of S. gallinarum 9, S. pullorum 3, S.typhimurium F98, and E. coli K-12 proto. Strains are listed in Table1. Lanes: a, S. gallinarum 9; b, S. gallinarum 9 VP-; c, S. pullorum3; d, S. pullorum 3 VP-; e, S. gallinarum 9(pSP3); f, S. pullorum 3(pSG9); g, S. typhimurium F98; h, S. typhimurium F98 VP-; i, S.typhimurium F98(pSP3); j, S. typhimurium F98(pSG9); k, E. coliK-12 proto; 1, E. coli K-12 F+; m, E. coli K-12(pSP3); n, E. coli K-12(pSG9). Abbreviations: LP, large plasmids; C, chromosomal DNA;SP, small plasmids. The positions of molecular size markers are

indicated on the right.

rium F98(pSP3) but was present in E. coli K-12(pSP3) (Fig.2).

It was not possible to transfer the Tn3-tagged plasmidfrom S. typhimurium F98 into any of the recipients testedhere.The F plasmid was transferred into Nalr mutants of S.

gallinarum 9, S. gallinarum 9 VP-, S. pullorum 3, S.pullorum 3 VP-, S. typhimurium F98, and S. typhimurium

a b c d e f g h MW

: ~~~~~~~~~~~~~~~~~~.EFIG. 2. Pstl fragments from plasmids of the following strains: E.

cli K-12 F' (lane a) S gallinarum 9 (parent strain with large

plasmid tagged with Tn3) (lane b); S. pullorum 3 (parent strain with

large plasmid tagged with Tn3) (lane c); S. gallinarum 9(pSP3) (lane

d); S. pullorum 3(pSG9) (lane e); S. typhimurium F98(pSP3) (lane f0;S. typhimurium F98(pSG9) (lane g); E. ccli K-12(pSP3) (lane h); E.

ccli K-12(pSG9) (lane i). Lane MW contains HindIII and 4~X174HaeIII fragments.

a b c d e f g

FIG. 3. Large-plasmid content of F plasmid-containing strains ofS. gallinarum 9 and S. pullorum 3 listed in Table 1. Lanes: a, E. coliK-12 F+; b, S. gallinarum 9; c, S. pullorum 3; d, S. gallinarum 9VP- F+; e, S. gallinarum 9 F+; f, S. pullorum 3 VP- F+; g, S.pullorum 3 F+.

F98 VP-. These transfers were carried out because thepresence of F in S. pullorum 3(pSG9), S. typhimuriumF98(pSG9), E. coli K-12(pSG9), and E. coli K-12(pSP3)might conceivably have affected the expression of the plas-mid-mediated virulence determinant. S. gallinarum 9 VP-F+, S. pullorum 3 VP- F+, S. typhimurium F98F+, and S.typhimurium F98 VP- F+ were all sensitive to lysis bybacteriophage MS2, indicating the presence of F in a dere-pressed state. S. gallinarum 9 F+ and S. pullorum 3 F+ wereMS2 resistant. However, since F (166 kbp) is considerablylarger than either pSGO90 (ca. 85 kbp) or pBLOO1 (ca. 78kbp), its presence in these strains could be detected byextended electrophoresis (Fig. 3). The presence of F in allthese strains was confirmed by PstI digestion (data notshown). These results indicated that although the 85-kbpplasmids do not belong to incompatibility group FIA, thosefrom S. gallinarum 9 and S. pullorum 3, but not that from S.typhimurium F98, are fertility inhibiting (f+).As found previously (2, 4), the plasmids were found not to

be associated with serotype-specific lipopolysaccharide,metabolic activity (including dulcitol and maltose fermenta-tion, characteristics useful in distinguishing S. gallinarumfrom S. pullorum), or nutrient requirements.

Plasmids pSGO90::Tn3 and pBL001::Tn3 were not self-transmissible in broth or on agar plates by the methoddescribed by Bradley et al. (5). Ten field strains of S.gallinarum were found to harbor lysogenic bacteriophageswhich were lytic for a Nalr Spcr mutant of S. gallinarum 9VP-. The 85-kbp plasmids of these strains were tagged withTn3 and tested for the transducibility to S. gallinarum 9 VP-of ampicillin resistance (24). No transduction of ampicillinresistance was detected.

Virulence of S. gailinarum, S. pullorum, E. coli K-12, andtheir derivatives for chickens. The virulence of S. gallinarum9, S. pullorum 3, E. coli K-12, and their derivatives forchickens was assessed by oral and intramuscular inoculationof 3-week-old and newly hatched chickens (Table 2). Inpreliminary experiments the presence of F appeared to haveno effect on the virulence of S. gallinarum 9, S. pullorum 3,or E. coli K-12 proto, and the results are not given here.

S. gallinarum 9 was highly virulent for chickens of bothages and by both routes of inoculation. S. gallinarum 9 VP-was completely avirulent for the older chickens and hadreduced virulence in the younger birds. Virulence wascompletely restored by reintroducing either pSG9O::Tn3from S. gallinarum 9 (4) or pBL001::Tn3 from S. pullorum 3,even though S. pullorum 3 was not virulent for chickens ofthis age (see below). Three-week-old chickens which diedfrom oral infection with S. gallinarum 9 or S. gallinarum9(pSP3) showed characteristic signs of acute fowl typhoid,particularly an enlarged and bronze-colored liver.

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TABLE 2. Virulence of S. gallinarum 9, S. pullorum 3,E. coli K-12 proto, and their derivatives for

21-day-old and newly hatched chickens

Virulencea of strain for following chickens:

21-day-old Newly hatchedStrain

.nOral Intramuscular ° Intramuscularlainocu- inoculationc 'no°cu- inoculationc

S. gallinarum 9 100 1.08 ± 0.27 100 0.05 ± 0.26S. gallinarum 9 VP- 0 >8.89 81 4.74 ± 0.29S. gallinarum 9(pSP3) 100 0.87 ± 0.25 100 0.00 ± 1.26

S. pullorum 3 5 >9.08 69 3.96 ± 0.54S. pullorum 3 VP- 5 >9.11 6 5.56 ± 0.39S. pullorum 3(pSG9) 5 >9.08 62 3.56 ± 0.27

E. coli K-12 proto 0 >9.53 0 9.03dE. coli K-12(pSP3) 0 >9.30 0 8.22dE. coli K-12(pSG9) 0 >9.54 0 8.32d

a Differences in virulence between pairs of strains (where calculated) are asfollows. Orally inoculated newly hatched chickens: S. gallinarum 9 - S.gallinarum 9 VP-, X2 = 3.31 (P = 0.08); S. pullorum 3 - S. pullorum 3 VP-,X2 = 13.3 (P < 0.01); S. pullorum 3 - S. pullorum 3(pSG9), x2 = 1.25 (P =0.25). Intramuscularly inoculated newly hatched chickens: S. gallinarum 9 -S. gallinarum 9 VP-, x2 = 43.1 (P = 0.01); S. gallinarum 9 - S. gallinarum9(pSP3), x2 = 2.63 (P = 0.1); S. pullorum 3 - S. pullorum 3 VP-, x2 = 6.23(P = 0.01); S. pullorum 3 - S. pullorum 3(pSG9), x2 = 2.29 (P = 0.15); E. coliK-12 proto - K-12(pSP3), not significant; E. coli K-12 proto - E. coliK-12(pSG9), not significant. Significant differences were calculated by themethod of Walters (26).

b Percent mortality observed by 3 weeks after inoculation of chickens with108 organisms.

c Log1o 50%o lethal dose ± standard error.d Not possible to calculate standard error.

S. pullorum 3 did not kill many 3-week-old chickens, andsubstitution of its virulence plasmid with pSGO90::Tn3 didnot increase its virulence. It was considerably more virulentin newly hatched chickens. S. pullorum 3 VP- was statisti-cally less virulent by both routes of inoculation than was S.pullorum 3, and virulence was restored by reintroduction ofeither pBLOO1::Tn3 from S. pullorum 3 (2) or pSGO90::Tn3from S. gallinarum 9. Newly hatched chickens which diedafter oral inoculation of S. pullorum 3 or S. pullorum3(pSG9) generally showed signs characteristic of either acutepullorum disease, including caked vents (diarrhea), or morechronic disease, such as large areas of necrosis in themyocardium and smaller necrotic areas in the liver andspleen. Older chickens inoculated with the same strainsremained healthy, but when they were killed they werefound to possess similar lesions.

E. coli K-12 proto, E. coli K-12 F+, E. coli K-12(pSP3),and E. coli K-12(pSG9) were all avirulent when inoculatedorally into chickens of both ages and intramuscularly into3-week-old chickens. In newly hatched chickens inoculatedintramuscularly, E. coli K-12(pSP3) and E. coli K-12(pSG9)were slightly more virulent than was E. coli K-12 proto; thisincrease was not statistically significant.

Survival of S. gallinarum, S. pulorum, E. coli K-12 proto,and their derivatives in chicken tissues. To assess how S.gallinarum 9, S. pullorum 3, E. coli K-12 proto, and theirderivatives behaved in the tissues of chickens, i0' (Salmo-nella strains) or 108 (E. coli K-12 strains) organisms wereinoculated intravenously into 3-week-old chickens and thenumbers of bacteria in the tissues were counted. Severalpatterns of behavior were observed (Table 3).

TABLE 3. Survival of S. gallinarum 9, S. pullorum 3,E. coli K-12 proto, and their derivatives in the

tissues of 21-day-old chickens

Log1o viable countsb of strains/g of tissue atStrain and tissue following time postinoculation

samplealh 3 5 7 10 17 24

days days days days days daysS. gallinarum 9cBlood Nd 2.0 3.8 de d d dLiver 2.9 5.6 7.3 d d d dSpleen 4.9 6.0 7.1 d d d dMyocardium N 4.3 6.2 d d d dBreast muscle N 2.6 4.2 d d d d

S. gallinarum 9 VP-Liver 3.1 3.4 2.9 N N N fSpleen 5.9 5.4 5.0 4.5 4.6 2.5Myocardium N 3.0 2.5 2.3 N N

S. pullorum 3gLiver 3.2 4.2 3.7 3.3 3.2 N NSpleen 4.6 4.3 3.7 4.2 4.7 2.3 3.0Myocardium N N 2.0 2.3 4.3 3.2 4.4

S. pullorum 3 VP-Liver 3.3 2.6 N N N - -Spleen 4.6 4.2 4.6 4.1 2.5 2.7 N

E. coli K-12 protohLiver 4.0 N N - - - -Spleen 5.5 2.0 2.0 N -

a The full set of samples was tested for S. gallinarum 9. Those tissues notshown had viable counts of <2.0 at all times.

b Median count from three chickens.c Similar results seen with S. gallinarum 9(pSP3).d N, <2.0 viable counts.I d, Dead.f-, Not done.9 Similar results seen with S. pullorum 3(pSG9).h Similar results seen with E. coli K-12(pSP3) and E. coli K-12(pSG9).

At 1 h after inoculation, the bacteria had been clearedfrom the blood and were isolated from the liver and spleenonly. S. gallinarum 9 and S. gallinarum 9(pSP3) multipliedrapidly in the liver and spleen and spread to the other tissuessampled, and all the chickens died between 5 and 7 dayspostinoculation. By contrast, S. gallinarum 9 VP- graduallydecreased in numbers in the tissues, and the chickensremained healthy. S. pullorum 3 and S. pullorum 3(pSG9)persisted for a considerable period in the liver and spleen.They disappeared from the liver by day 17 postinoculation,even though lesions were present in this organ until theexperiment ended. The numbers of organisms of these twostrains in the myocardium increased throughout the experi-ment, and large, necrotic lesions appeared. The chickens,however, appeared healthy. S. pullorum 3 VP- was clearedfrom the liver and spleen more rapidly than was S. pullorum3 or S. pullorum 3(pSG9), and it was not detected in themyocardium. The chickens remained healthy and no lesionswere seen. Chickens inoculated with E. coli K-12 proto or itsderivatives remained healthy, and no lesions were seen. Theorganisms persisted for a few days in the liver and thespleen, even though they are highly serum sensitive. Organ-isms isolated from the livers of the chickens inoculated withE. coli K-12(pSP3), E. coli K-12(pSG9), or S. pullorum3(pSG9) were ampicillin resistant and hence had not losttheir Tn3-tagged plasmids.Because E. coli K-12(pSP3) and E. coli K-12(pSG9) were

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TABLE 4. Virulencea of S. gallinarum 9, S. pullorum 3, andS. typhimurium F98 and its derivatives for mice

Strain Loglo LD50bS. gallinarum 9.5.38S. pullorum 3.6.11S. typhimurium F98.4.07S. typhimurium F98 VP.4.92S. typhimurium F98(pSG9).3.92S. typhimurium F98(pSP3).4.16

a Differences in virulence between strains were calculated by the method ofWalters (26). S. typhimurium F98 - S. gallinarum 9, P < 0.02; S. typhimu-rium F98 - S. pullorum 3, P < 0.02; S. typhimurium F98 - S. typhimuriumF98 VP-, P < 0.05; S. typhimurium F98 - S. typhimurium F98(pSG9), notsignificant; S. typhimurium F98 - S. typhimurium F98(pSP3), not significant;S. gallinarum 9 - S. typhimurium F98(pSG9), P < 0.02; S. pullorum 3 - S.typhimurium F98(pSP3), P = <0.02.

b Not possible to calculate standard errors. LD50, 50% lethal dose.

slightly more virulent than were E. coli K-12 proto and E.coli K-12 F+ when inoculated intramuscularly into newlyhatched chickens (Table 2), the survival of these strains inthe tissues of chickens of this age was monitored afterintramuscular inoculation of 108 organisms in 0.1 ml. Thedata (not shown) indicated no differences in behavior amongthe four strains.

Virulence of S. gallinarum-, S. pullorum-, and S. typhimu-rium-derived strains for mice. The effect of replacing pTM980in S. typhimurium F98 with plasmid pBL001::Tn3 orpSG090: :Tn3 from S. pullorum 3 and S. gallinarum 9,respectively, on the virulence for mice following intravenousinoculation is shown in Table 4. Elimination of pTM980 fromS. typhimurium F98 produced a relatively small increase inthe median lethal dose (of 0.85), as occurred with S. pul-lorum 3 in chickens. The increase was, however, reproduc-ible and statistically significant (P < 0.05). Introduction ofpBL001::Tn3 or pSG090::Tn3 into S. typhimurium F98 VP-by mobilization with F fully restored virulence. By contrast,S. pullorum 3 and S. gallinarum 9 were considerably lessvirulent than was S. typhimurium F98 (P < 0.02 for both) orS. typhimurium F98(pSP3) or F98(pSG9) (P < 0.02 for both).All strains except S. pullorum 3 produced splenomegaly andareas of necrosis in the liver. None of the strains, includingS. typhimurium F98, was virulent for mice by the oral route.Some mice died when they were given oral streptomycin,but there was no difference in the number of mice that diedafter inoculation with the different strains (data not shown).

Survival of S. pullorum- and S. typhimurium-derived strainsin mouse tissues. Counts were performed on S. typhimuriumF98, F98 VP-, and F98(pSP3) and S. pullorum 3 in mousetissues following intravenous inoculation with 105 organ-isms. The results are shown in Table 5. Initially the highestnumbers of bacteria were isolated from the liver and spleen.S. typhimurium F98 and F98(pSP3) numbers increased rap-idly in all the tissues, and the mice had all died by 6 dayspostinoculation. S. typhimurium F98 VP- multiplied in theliver, spleen, and myocardium, and then the numbers beganto fall by 17 days. The mice remained healthy. The numbersof S. pullorum 3 organisms fell rapidly, so that the mice werecleared of detectable bacteria by day 8 postinoculation. Themice remained healthy.

DISCUSSION

Previous work has indicated the importance of high-molecular-weight plasmids to the virulence of S. gallinarum(4), S. pullorum (2), and S. typhimurium (1, 12, 13, 16).

TABLE 5. Survival of S. typhimurium F98 and its derivativesand S. pullorum 3 in the tissue of mice

Log10 viable countsb of strains in tissues at

Strain and tissue following time postinoculationsamplea 2 4 6 8 17 24

1 h days days days days days days

S. typhimurium F98CBlood 2.5 3.6 6.4 dd d d dLiver 4.0 5.9 7.9 d d d dSpleen 4.5 6.6 8.5 d d d dMyocardium 2.3 4.1 6.8 d d d dGastrocnemius 2.0 3.1 5.9 d d d d

S. typhimurium F98 VP-Blood No 2.0 N N N N NLiver 3.9 5.2 5.2 5.0 5.0 4.0 2.0Spleen 4.4 5.1 5.6 5.5 5.5 3.8 3.0Myocardium 2.8 2.0 4.3 4.0 4.2 2.3 NGastrocnemius N N N N N N N

S. pullorum 3Blood 2.5 N N N N N NLiver 4.2 2.0 N N N N NSpleen 4.6 2.6 2.3 2.0 N N N

a The full set of samples was tested for S. typhimurium F98. Those tissuesnot shown had viable counts of <2.0 at all times.

b Median count from three chickens.Similar results seen with S. typhimurium F98(pSP3).

dd, Dead.I N, <2.0 viable counts.

Plasmid curing was associated with loss or reduction ofvirulence, which could be completely restored by reintro-duction of the plasmid. The present results show that viru-lence can be restored by reintroduction of the 85-kbp viru-lence-associated plasmids from heterologous Salmonellaserotypes or biotypes in addition to the plasmid from thehomologous strain. The full restoration of virulence wasdemonstrated by oral or parenteral parameters of virulenceand by the ability of plasmid-containing strains to surviveand multiply in the tissues, particularly in the liver andspleen, following intravenous inoculation. This indicates aconsiderable degree of functional homology between theplasmids of S. gallinarum, S. pullorum, and S. typhimurium,in addition to similarities indicated by Southern hybridiza-tion (4, 28).The results also indicate the importance of chromosomal

determinants to the full expression of either oral or parent-eral virulence. This was shown by the avirulence of E. coliK-12 strains possessing the virulence-associated plasmidsfrom either S. gallinarum 9 or S. pullorum 3. In addition,when these plasmids were transferred to plasmid-curedderivatives of S. pullorum 3, 5. gallinarum 9, and S. typhi-murium F98, the transconjugants retained the virulencecharacteristics of the recipient parent strain. The low viru-lence of the E. coli K-12 strains possessing the virulenceplasmids from the S. pullorum and S. gallinarum strains wasunlikely to be the result of interference in expression of thevirulence plasmids by the F plasmid, since F did not affectvirulence in the parent strains. Neither was it likely to besolely a reflection of the serum sensitivity of the E. coli K-12strains, since these strains survived for several days in thespleen, whereas they survive for less than 1 h in chickenserum (unpublished data).The importance of chromosomal genes to the ability of S.

typhimurium organisms to survive and multiply in the cellsof the reticuloendothelial system of mice is now being

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SALMONELLA VIRULENCE PLASMID 3141

reanalyzed (7, 8, 12). It has now been shown that bothchromosomal and plasmid genes contribute to virulence in S.gallinarum and S. pullorum, although the relative contribu-tions of these have not yet been determined. The virulenceand other differences between these two biotypes cannot beaccounted for solely by virulence plasmid differences. Theplasmids do not appear to contribute to intracellular survivalwithin avian splenic macrophages in vitro (N. Bumstead andP. A. Barrow, unpublished data).The ability of the S. gallinarum and S. pullorum plasmids

to restore the virulence of a plasmid-cured S. typhimurium inmice is more difficult to explain, since differences in hostspecificity are also involved and degrees of expression of asingle characteristic are perhaps less likely to be the expla-nation. It is clear, however, that host specificity is chromo-somally mediated.There is also the possibility that all the virulence charac-

teristics are chromosomally mediated and that the plasmidacts mainly in a regulatory capacity. However, it wouldseem wasteful for the bacterium to maintain such a largeplasmid for such a simple function unless it has other, as yetunrecognized, functions.The virulence plasmids of the S. gallinarum and S. pul-

lorum strains are similar to F in that although they do notbelong to incompatibility group FIA, they suppress sex pilusformation by F and are thusfi+. This indicates that they havea gene homologous to the inactivated fin-O gene of the Fplasmid (27). The plasmid from the S. typhimurium strainwas notfi+, in contrast to similar plasmids from S. typhimu-rium (18, 22). Unlike F, the plasmids of S. gallinarum 9 andS. pullorum 3 are not self-transmissible either in broth or onagar and could not be transduced with naturally occurringlysogenic phages, although laboratory transducing phageswere not tested (10). Since easily characterized metaboliccharacteristics or nutrient requirements were not plasmidmediated, virulence must be expressed in some other way. Itwould seem that cloning the relevent plasmid genes offersthe best opportunity to study this.

ACKNOWLEDGMENTS

We thank B. Wells, H. Vickery, and V. Peters for assistance invarious ways and D. E. Walters for performing the Bayesian statis-tical comparisons.

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