INFECTION AND IMMUNITY, JUlY 1990, p. 2283-2288 0019-9567/90/072283-06$02.00/0 Copyright C) 1990, Americn Society for Microbiology Vol. 58, No. 7 Immunity to Experimentl Fowl Typhoid in Chickens Induced by Virulence Plsmid-Cured Derivtive of Slmonell gllinrum P. A. BARROW AFRC Institute for Animl Helth, Houghton Lbortory, Houghton, Huntingdon, Cmbridgeshire PEJ7 2DA, United Kingdom Received 11 November 1989/Accepted 18 April 1990 Chickens were immunized by two intrmusculr inocultions t 1 nd 14 dys of ge with virulence plsmid-cured derivtives of Slmonell gulinrum nd were chllenged 14 dys lter by orl inocultion of c. 50 50% lethl doses (LD50) of fully virulent S. gllinrum 9. Mortlity in the nonimmunized nd immunized groups were 36 nd 3%, respectively. This difference ws highly significnt (P < 0.01). A significnt reduction in mortlity ws lso produced following orl chllenge with 5,000 LD50 doses. The LDso vlues by intrmusculr inocultion of the chllenge orgnism into nonimmunized nd immunized chickens were log1o (0.13 ± 1.57) nd (9.74 + 2.72), respectively. Immuniztion ws effective whether chickens were immunized t 1 nd 14 dys of ge or t 21 nd 35 dys of ge. Serum gglutinins were present in immunized chickens. Immuniztion with plsmid-cured SlmoneU pullorum gve less protection, nd immuniztion with Escherichi coli K-12 possessing the virulence plsmid of S. guinrum gve none. The plsmid-cured S. gllinrum ws mde both rough by virulent bcteriophge ctivity nd nlidixic cid resistnt (Nl') to produce strin designted 9Vp-4r Nlr. It ws compred with Nl' mutnt of the rough 9R vccine strin designted 9 Nlr for virulence nd immunogenicity. gvp-4r Nlr ws slightly less protective nd less virulent thn ws the 9R vccine strin. Fowl typhoid is still disese of worldwide significnce. It hs lrgely been erdicted from those countries which hve hd n intensive poultry industry for mny yers nd is now of prticulr economic importnce in those countries which re beginning to intensify their industry, e.g., countries in Ltin Americ, South Americ, the Middle Est, the Indin subcontinent, nd prts of Afric (18, 20). Recent work hs shown tht the very high virulence for chickens of Slmonell gllinrum, the custive gent of fowl typhoid, is relted to the possession of high-moleculr-weight (85-kilobse) plsmid (5). Brrow et l. (3, 5) found tht elimintion (curing) of this plsmid from strin gretly reduced its virulence. In 3-week-old chickens, the plsmid-cured derivtive ws virtully virulent, while in newly htched chickens, there ws some residul virulence. Reintroduction of the plsmid fully restored virulence. From pthogenesis studies, the plsmid ws shown to be involved both in invsiveness in vivo nd in the bility of the strin to survive nd multiply in the cells of the liver nd spleen. A similr plsmid ws lter shown to contribute to the virulence of Slmonell pullorum (2). Becuse of the economic importnce of fowl typhoid mny studies hve been crried out on the fesibility of vccintion to control it. Erly studies showed tht killed vccines were of little prcticl use (12, 15, 22, 31, 32). Smith (22) developed two ttenuted vccine strins, one of which ( rough strin, 9R) ws protective nd did not induce the production of significnt mounts of serum gglutinins. This ltter chrcteristic is of prticulr importnce where the disese is to be erdicted by using the whole-blood gglutintion test (8, 19). Lter work hs shown the vlue of the 9R strin (9, 14, 21, 27). A number of uthors hve indicted tht the strin still possesses some virulence for some breeds of chicken (9, 10, 21), lthough there is no evidence for reversion to full virulence in the field. The worldwide prevlence of fowl typhoid, combined with incresing numbers of reports of furzolidone resistnce in 2283 isoltes of S. gllinrum resulting from extensive prophylxis with this ntibiotic (11, 26, 29, 30), indictes the need for continuing work on the development of n improved vccine. Becuse the plsmid-cured derivtive of S. gllinrum persisted in the reticuloendothelil system of the chicken for some time, it ws considered tht the strin might be used for vccintion purposes. This rticle reports on the bility of the 85-kilobse (kb) plsmid-cured derivtive of strin of S. gllinrum to protect chickens ginst fowl typhoid. In ddition, mutnt of this strin ws produced nd tested which ws rough (to prevent the production of serum gglutinins) nd resistnt to nlidixic cid (to fcilitte the mintennce of purity). The protective bility nd virulence of this mutnt were compred with these chrcteristics in Nlr mutnt of the 9R strin. MATERIALS AND METHODS Chickens. Slmonell-free Rhode Islnd Red chickens ged 1 dy or 3 weeks were used. They were kept under good hygienic conditions on wire-mesh floors in identiclly constructed pens in n niml house mintined t 21 C. The younger chickens were provided with extr heting from n infrred lmp suspended over ech pen. They were fed d libitum on diet of the following composition: whet mel, 40%; mize mel, 40%; British whitefish mel, 20%; minerl nd vitmin supplement, 0.25%. Bcteril strins. The strin used in most of the experiments ws n 85-kb plsmid-cured derivtive of S. gllinrum 9. The prent strin, which ws lso the chllenge strin, hs been mintined t the AFRC Institute for Animl Helth for mny yers nd is highly virulent for chickens. The method of "tgging" nd curing the plsmid hs been described previously (5). Additionl strins used included the 85-kb plsmid-cured derivtive of S. pullorum 3 (2) nd prototrophic Escherichi coli K-12 strin (proto) into which the 85-kb plsmid
2284 BARROW INFECT. IMMUN. TABLE 1. Immuniztion Mortlity following orl inocultion with S. gllinrum 9 fter immuniztion with virulence plsmid-cured derivtives of S. gllinrum nd S. pullorum No. of No. of chickens ded t dy (postchllenge): %No. chickens/ 9 grllinrum tlity x gent chickens 9egdorscl over 3 X2~ Lesions in Infected lenge dose 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 weeks liver nd livers spleen S. gllinrum plsmid 23 1o8b 0 0 1 1 3 3 4 5 10 10 10 10 10 11 11 11 11 48 10/12 3/12 cured 4.53 0.03 Nonimmunized 23 1o8b 0 2 10 14 16 18 18 18 18 18 18 18 18 18 18 18 18 78 0/5 0/5 S. gllinrum plsmid 30 106c 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 3 8/29 2/29 cured 9.92 <0.01 Nonimmunized 28 106 0 0 3 4 5 9 9 10 10 11 11 11 11 11 12 12 12 36 2/16 1/16 S. pullorum plsmid 30 106c 0 0 0 1 1 1 3 4 4 5 5 5 6 6 6 6 6 20 3/24 2/24 cured 2.84 0.1 Nonimmunized 30 106c 0 0 3 4 5 9 9 10 10 11 11 11 11 11 12 12 12 40 1/18 0/18 All chickens immunized intrmusculrly with 106 orgnisms t dy 1 (1 dy of ge) nd 101 orgnisms t dy 14, followed by chllenge t dy 28. b 108 = 5,000 x LD50 by the orl route. C 106 = 50 x LD50 by the orl route. ws mobilized from S. gllinrum 9 by the F plsmid. Briefly, this ws crried out s follows. A three-fctor mting ws used in which the donor ws S. gllinrum 9 contining the 85-kb plsmid which hd been tgged with trnsposon Tn3 (5). The mobilizing strin ws E. coli K-12 mutnt 711 (lc his pro trp) contining the F plsmid, nd the finl recipient ws Nlr mutnt of E. coli K-12 proto. The mting mixture ws plted on McConkey gr contining nlidixic cid (25,ig ml-') nd mpicillin (100,ug ml-'). Lctose-fermenting colonies were checked for the presence of the 85-kb plsmid nd for the presence of the F plsmid by plsmid nlysis (14). In one experiment, the 9R vccine strin developed by Smith from strin 9 (22) ws compred with the plsmidcured S. gllinrum strin. When nlyzed for the possession of plsmids (14), the 9R strin ws found to possess n 85-kb plsmid (unpublished dt). All cultures used were stored either on Dorset's egg slopes or in lyophilized condition. All broth cultures were mde in 10-ml volumes of nutrient broth (Oxoid CM 67) nd were incubted t 37 C for 24 h in shking wter bth (100 strokes min-'). These contined between 5 x 108 nd 1 x 109 CFU ml-1'. Production of rough mutnt. Spontneous rough mutnts were selected from broth cultures by their resistnce to bcteriophge 4BL isolted from humn sewge (4). Production of nlidixic cid-resistnt (Nlr) mutnts. Production of Nlr mutnts ws done by the method of Smith nd Gyles (24). Smith nd Tucker (25) showed tht Nlr mutnts of Slmonell strins were no less virulent for chickens thn were the ntibiotic-sensitive prent strins. However, Silv et l. (21) found tht Nlr mutnt of the 9R vccine strin ws less virulent thn ws the ntibioticsensitive form. Experimentl pln. In ll experiments, the chllenge orgnism ws S. gllinrum 9. In the first two experiments, the protective effect of vccinting chickens intrmusculrly with either the plsmid-cured derivtives of S. gllinrum 9 or S. pullorum 3 or n E. coli K-12 strin possessing the 85-kb S. gllinrum virulence plsmid, psg090, ws ssessed under different conditions. The rough, Nlr derivtive of the plsmid-cured S. gllinrum strin, designted 9VP-4) Nlr, nd the Nlr mutnt of the 9R vccine were compred for their protective bilities when inoculted vi different routes. The virulence of these two mutnts ws compred by inoculting chickens vi vriety of routes, followed by estimting vible counts of the strins in the spleens nd recording lesions in this orgn nd in the liver. For ll experiments, pproprite control groups were included. Fifty percent lethl dose (LD50) vlues were clculted by using the MLP sttisticl pckge (Rothmsted Experimentl Sttion, Hrpenden, United Kingdom), which follows conventionl methods of probit nlysis (7). Vible bcteril counts were estimted by the method of Miles, Misr, nd Irwin (16), plting the bcteri on McConkey gr (Oxoid CM7) contining 25,ug of nlidixic cid per ml. Detection of serum gglutinins. Chickens were bled from the wing vein. Serum ws obtined nd tested for the presence of gglutinins by slide gglutintion of bcteril colonies from nutrient gr (Oxoid CM 67) plte inoculted with S. gllinrum 9 nd incubted overnight t 37 C. RESULTS Immuniztion with plsmid-cured strin (orl chllenge). The effect of intrmusculr immuniztion with either the plsmid-cured S. gllinrum or S. pullorum strins on mortlity following orl chllenge with S. gllinrum 9 is shown in Tble 1. Considerble protection ws fforded by the plsmid-cured S. gllinrum. Following orl chllenge with 108 orgnisms, the mortlity recorded ws reduced from 78% (control chickens) to 48% (immunized chickens). The rte t which the immunized chickens died ws significntly lower (P < 0.01) thn tht of the control birds when exmined by the Wilcoxon-Mnn-Whitney two-smple rnk test (28). The difference in mortlity rtes ws of mrginl sttisticl significnce. When the chickens were chllenged with 106 orgnisms, the mortlity rte ws lower in both groups, 36 nd 3% in the control nd immunized chickens, respectively. This difference ws sttisticlly significnt. Although immuniztion with the plsmid-cured S. pullorum strin reduced the mortlity level produced by chllenge from 40 to 20%, this reduction ws not sttisticlly significnt. In ll of the groups, smll proportion of the surviving chickens hd smll res of necrosis in the livers nd spleens nd, in some cses, the chllenge strin ws reisolted from their livers. Immuniztion with plsmid-cured strins nd plsmid-contining E. coli K-12 (intrmusculr chllenge). The LD50 vlues of control nd immunized chickens chllenged intr-
VOL. 58, 1990 PLASMID-CURED S. GALLINARUM AND FOWL TYPHOID IMMUNITY 2285 TABLE 2. Intrmusculr chllenge with S. gllinrum 9 following immuniztion with virulence plsmid-cured derivtives of S. gllinrum nd S. pullorum nd with E. coli K-12 contining the S. gllinrum virulence plsmid LD50 vlue (log1o) + SE in: Immunizing strin Immuniztion schedule Immunized Nonimmunized X2 P chickens chickens S. gllinrum plsmid cured 106 CFU 1 dy of ge, 108 CFU 14 dys 9.74 + 2.72 0.13 + 1.57 24.12 <0.01 of ge, chllenge 28 dys of ge (7.52 + 1.55)b (0.80 + 1.91)b 12.29 <0.01 S. gllinrum plsmid curedc 107 CFU 21 dys of ge, 107 CFU 35 9.89 + 2.58 0.80 + 1.01 20.34 <0.01 dys of ge, chllenge 49 dys of ge S. pullorum plsmid cured 105 CFU 1 dy of ge, 108 CFU 14 dys 3.77 ± 0.73 0.07 ± 1.10 7.34 <0.01 of ge, chllenge 28 dys of ge E. coli contining S. gllinrum 108 CFU 1 dy of ge, 108 CFU 14 dys 1.23 ± 0.64 0.71 ± 0.66 0.35 0.6 plsmid of ge, chllenge 28 dys of ge All immuniztions were by the intrmusculr route; five chickens were inoculted per dilution of culture. b First vlue represents replicte one; vlue in prentheses represents replicte two. c Serum gglutinins present in 8 of 10 chickens t time of chllenge. musculrly re shown in Tble 2. Lrge increses in the LD50 vlues were observed in the chickens which hd been immunized with the plsmid-cured S. gllinrum strin when compred with the vlues observed with the nonimmunized controls. In the chickens which were immunized t 1 dy old, the log1o of the increse ws greter thn 9 on one occsion nd greter thn 6 on nother, nd in chickens immunized first t 3 weeks of ge, it ws lso greter thn 9. These differences were sttisticlly significnt. Immuniztion of newly htched chicks with the plsmid-cured S. pullorum strin produced log1o increse in the LD50 of the chllenge strin of greter thn 3; this difference ws lso highly significnt, even though the degree of protection ws considerbly less thn tht fforded by the plsmid-cured S. gllinrum strin. Immuniztion with the E. coli K-12 strin possessing the S. gllinrum virulence plsmid hd no protective effect, the log1o increse in LD50 in the immunized chickens compred with tht in the control birds being less thn 1. Of 10 chickens immunized with S. gllinrum t 21 dys old, 8 possessed gglutinins of sufficient titer to be detected by slide gglutintion. Immuniztion with vccine strins 9R Nlr or 9VP4r Nir (orl chllenge). The effects of single orl immuniztion with either the derived strins 9VP-4) Nlr or 9R NlF nd of prenterl immuniztion by different routes with 9Vp-4r Nlr on mortlity following orl chllenge re shown in Tble 3. When compred with the mortlity in the nonimmunized control group (65%), orl immuniztion with 9R Nlr produced considerbly better protection (8% mortlity) thn did TABLE 3. immuniztion with 9VP-4ir Nlr (42%). The degree of protection produced by the former strin only ws sttisticlly significnt, nd the differences in degrees of protection produced by the two strins were lso sttisticlly significnt. Immuniztion with 9VP-+r Nlr by either intrmusculr or subcutneous routes produced better protection (24 nd 27% mortlity, respectively) thn did immuniztion with this strin by orl dministrtion. Although dministrtion of 9R Nlr by the orl route induced better protection, the difference between this nd the protection induced by intrmusculr immuniztion of 9VP-4) Nlr ws not sttisticlly significnt. Immuniztion with vccine strins 9R Nl' or gvp-+r Nlr (intrmusculr chllenge). The protective effects of single intrmusculr immuniztion with 9R Nlr or 9Vp-(r Nlr were compred (Tble 4). In both cses, considerble protection ws produced when compred with the protection produced with n nonimmunized group of chickens. The log1o increse in the LD50 vlues produced by 9R Nlr nd strin gvp-4r Nlr were greter thn 11 nd greter thn 9, respectively. Both increses were sttisticlly highly significnt. The difference between the two LD50 vlues in the immunized chickens ws of mrginl sttisticl significnce. None of the 20 serum smples exmined showed ny trce of gglutintion with S. gllinrum cells by slide gglutintion. When compred with those of the control group, the stndrd errors of the LD50 vlues for immunized chickens were high. This ws cused by the unusul distribution of Mortlity following orl inocultion with S. gllinrum 9 fter immuniztion with vccine strins 9VP-4/ Nlr or 9R Nlr given by different routes Chickens No. of chickens ded t dy (post-chllenge): % Mortlity Groups P vlue from no. chickens immunized imnzto over 3 compred 2 with: 7 8 9 10 11 12 13 14 15 16 17 18 weeks (x2 vlue) x comprison Group No. of Route of 1 26 gvp-or Nlr Orl 0 0 4 6 6 10 11 11 11 11 11 11 42 1 nd 5 (2.8) 0.1 2 nd 1 (8.3) <0.01 2 26 9R Nlr Orl 0 0 0 0 1 2 2 2 2 2 2 2 8 2 nd 3 (2.7) 0.1 2 nd 5 (18.6) <0.01 3 25 9Vp-(r Nlr Intrmusculr 0 0 1 4 4 4 5 5 6 6 6 6 24 3 nd 5 (8.8) <0.01 4 26 9Vp-4r Nlr Subcutneous 0 1 2 2 3 5 5 7 7 7 7 7 27 4 nd 5 (7.7) <0.01 5 26 Nothing b 0 3 6 9 11 13 15 15 15 15 17 17 65 All chickens immunized orlly with 3 x 108 orgnisms in 0.3 ml or intrmusculrly with 1 x 108 orgnisms in 0.1 ml t 3 weeks of ge followed by orl chllenge with 3 x 108 orgnisms 3 weeks lter. b-, Not relevnt.
2286 BARROW INFECT. IMMUN. TABLE 4. Intrmusculr chllenge with S. gllinrum 9 fter intrmusculr immuniztion with nlidixic-resistnt mutnts of either the 9R vccine strin or rough mutnt of the virulence plsmid-cured derivtive of S. gllinrum No. of birds with serum Immunizing Immuniztion schedule gglutinins present t LD50 vlue X2 p strin time of SE (log10)x chllenge/totl 9R Nlr 107 orgnisms dy 21, chllenge dy 42 0/10 11.85 ± 4.70 19.56 <0.01 SG9VP- (r Nlr 107 orgnisms dy 21, chllenge dy 42 0/10 9.8 + 4.06 8.48 <0.01 Nonimmunized chllenge dy 42 NDb 0.80 1.24 x2 vlues clculted by comprison with nonimmunized control. x2 vlue for comprison between the two immunized groups ws 5.77; P = 0.02. b ND, Not done. deths in the groups of immunized chickens. For the LD50 estimtions, chickens were divided into groups of five nd inoculted with seril dilutions of the chllenge strin. The numbers of chickens which died in the groups inoculted with (log10) 8, 7, 6, 5, 4, 3, 2, 1, nd 0 orgnisms were 0, 0, 2, 0, 0, 0, 0, 0, nd 0, respectively, in the cse of 9R Nlr nd 2, 1, 0, 2, 2, 1, 0, nd 1, respectively, in the cse of 9VP-4r Nlr. These results reflected the outbred nture of the experimentl chickens. Isoltion of chllenge strin from spleens fter immuniztion with vccine strins. The vible numbers of S. gllinrum 9 Nlr in the spleens of chickens which hd been vccinted twice intrmusculrly with either 9Vp-(r Nlr or 9R Nlr nd subsequently chllenged with the prent strin re shown in Tble 5. Selected bcteril colonies were ll smooth when tested for gglutintion with 0.2% criflvine nd were therefore the prent strin. Within few dys of chllenge, lrge numbers of the chllenge orgnisms were isolted from the spleens of the nonimmunized control group. Deths were observed in this group, depleting the number of birds vilble for smpling. The numbers of orgnisms of the chllenge strin in the spleens of chickens immunized with 9VP-(r Nlr were initilly low but rose to levels comprble with those in the control group, despite the protection produced by gvp-4r Nlr. No detectble chllenge orgnisms (vible counts of log1o <2) were found in the spleens of chickens immunized with 9R Nlr. Virulence of vccine strins. The virulence of 9R Nlr nd 9Vp-4r Nlr for newly htched chickens ws clculted by LD50 estimtions obtined by intrmusculr inocultion. The log10 LD50 vlue + stndrd error for 9R Nlr ws 6.85 ± 0.71 nd for 9Vp-4r Nlr ws 8.19 + 0.86. The persistence of 9R Nlr nd 9Vp-4r Nlr in the spleens of chickens inoculted with either of these strins by TABLE 5. Isoltion of S. gllinrum 9 from the spleens of chickens chllenged with this orgnism fter immuniztion with 9Vp-(r Nlr or 9R Nlr Dys Log1o medin vible Slmonell count (rnge) with:b postchllenge 9VP4,Or Nlr 9R Nlr Nonimmunized 4 <2 (<2-4.0) <2 (<2) 4.0 (<2-4.6) 7 4.9 (<2-6.1) <2 (<2) 5.5 (3.4-5.8) 14 4.4 (<2-5.0) <2 (<2) 4.4c 21 1.7 (<2-3.1) <2 (<2) 2.0c Chickens immunized by the intrmusculr route 5 nd 3 weeks before chllenge with 107 orgnisms in 0.1 ml. Chickens chllenged orlly with 3 x 108 orgnisms in 0.3 ml. b Counts nd rnges re from spleens of five chickens t vrious times fter inocultion with S. gllinrum 9 in chickens immunized with the given strins. c Log1o men count of two chickens only. number of routes is shown in Tble 6. Following intrmusculr inocultion, the vible counts of the two strins were initilly comprble. However, wheres 9VP-4)r Nlr ws not detectble t 2 weeks postinocultion 9R Nlr persisted for week longer. Following orl inocultion, no orgnisms of 9VP-4r Nlr were detected t ll in the spleen wheres 9R Nlr persisted for t lest 2 weeks. 9VP-4r Nlr ws not detected in the spleens of chickens inoculted subcutneously; no comprison ws mde with 9R Nlr by this route. DISCUSSION These experiments show tht chickens cn be protected ginst orl or intrmusculr chllenge with fowl typhoid by intrmusculr or subcutneous immuniztion with the 85-kb virulence plsmid-cured derivtive of S. gllinrum. Not surprisingly, protection ws less effective ginst lrge chllenge doses. Protection ws not complete, s ws pprent from the deth of some immunized chickens, from the presence of lesions in the liver nd spleen nd the isoltion of the chllenge orgnism from the liver. Incomplete protection ws lso induced by the 9R vccine strin. This my hve been reflection of the fct tht young chickens were used in the experiments, since Gordon, Grside, nd Tucker (9) found tht protection ws better in older birds. Protection ginst chllenge with S. gllinrum ws not s good when chickens were immunized with plsmid-cured derivtive of S. pullorum. This strin persists in the tissues for s long s does the plsmid-cured S. gllinrum strin (3). Thus, the degree of exposure of the host to the ntigens is the sme. It is possible tht different ntigens re possessed by these two biotypes. Smith (22) showed tht fully virulent S. pullorum would protect chickens ginst chllenge with S. gllinrum, but it is likely tht the strin used by Smith possessed its virulence plsmid nd this my be necessry to induce full protection. Mice cn be protected ginst Slmonell enteritidis nd Slmonell dublin infection by being immunized with strins of these serotypes cured of their virulence-ssocited plsmids (6, 17). It is therefore not surprising tht good protection in chickens ws produced by strin of S. gllinrum lcking virulence plsmid. Plsmid-encoded ntigens re clerly not essentil for the production of good protective immunity. The 9R vccine strin possesses virulence plsmid, though it is not cler whether it is functionl. The fct tht the plsmid-cured strin ws less immunogenic thn ws the 9R strin suggests tht the virulence plsmid does contribute to complete immunogenicity. This ws true for both prenterl nd orl vccintion. The plsmid-cured strin ws not expected to perform well by orl dministrtion since, unlike S. dublin nd S. typhimurium, its virulence plsmid contributes to in vitro invsiveness. However, this
VOL. 58, 1990 PLASMID-CURED S. GALLINARUM AND FOWL TYPHOID IMMUNITY 2287 TABLE 6. Persistence of 9VP-(j Nlr nd 9R Nlr vccine strins when inoculted into chickens by vrious routes Log1o medin vible Slmonell count (rnge) fter inocultionb postinocultion Intrmusculrly with: 9VP -r Nlr Orlly with: 9Vp-4r Nlr 9R Nlr (subcutneous) gvp-4,r Nlr 9R Nlr 4 2.7 (<2-3.8) 3.2 (2.3-3.5)c <2 (<2) <2 (<2) 2.5 (<2-3.7)c 7 2.3 (<2-3.4) 2.5 (<2-4.0)c <2 (<2) <2 (<2) 3.0 (<2-3.7)c 14 <2 (<2) 2.5 (<2-3.9)c <2 (<2) <2 (<2) 2.5 (2.3-3.3) 21 <2 (<2) <2 (<2) <2 (<2) <2 (<2) <2 (<2) Chickens were inoculted t 21 dys of ge with 108 orgnisms in 0.1 ml (intrmusculr nd subcutneous) or 3 x 108 orgnisms in 0.3 ml (orl). b Counts nd rnges re from spleens of five chickens t vrious times fter inocultion vi the given routes with the given strins. Liver lesions present. is of little prcticl significnce t the moment since, in the field, most chickens re vccinted prenterlly. It is pprent tht possession of the plsmid in the bsence of s yet unchrcterized chromosoml genes does not give protection, since the E. coli K-12 strin possessing the 85-kb S. gllinrum plsmid ws not protective. The presence of the F plsmid is unlikely to hve ffected the expression of the virulence plsmid, since its presence in fully virulent S. gllinrum nd S. pullorum strins does not ffect virulence (3). However, E. coli K-12 persists for very short time in the liver nd spleen of chickens nd this my contribute to the poor immunogenicity. It is cler tht for optiml immunogenicity nd protection, expression of both plsmid nd chromosoml genes re required. Although the plsmid-cured strin induced protection in the chickens, it did not prevent multipliction of the chllenge strin in the spleen except in the first few dys fter chllenge. This suggests tht the min mechnism of protection my be different from tht induced by the 9R vccine. Studies to chrcterize this mechnism more fully would require the use of inbred chickens. The heterogeneous response to chllenge in some groups of the Rhode Islnd Red chickens ws n interesting nd importnt prcticl observtion. Since there is vrition between breeds in susceptibility to fowl typhoid (23; N. Bumsted nd P. A. Brrow, unpublished results) nd Rhode Islnd Reds re outbred, this response is not surprising. It suggests tht in some breeds of chicken bred primrily for performnce, heterogeneity of response to vccintion might be expected in the field (9, 21). It is importnt tht live vccines ginst fowl typhoid do not express somtic ntigens, so tht ntibodies ginst these do not interfere with the whole-blood gglutintion. Regrdless, the somtic ntigens re obviously not essentil for protection. It would lso be useful to hve vccine expressing genetic mrker which incresed the ese of purifiction nd isoltion. It ws thus of prcticl significnce tht the rough Nlr mutnt of the plsmid-cured derivtive, designted gvp-4r Nlr, ws lso protective nd ws of slightly reduced virulence for chickens when compred with 9R Nlr. How these two strins would compre under field conditions is uncler, but further work is necessry on the efficcy of the SG9VP-4r Nlr strin in breeding chickens housed under field conditions. The degree of protection induced by 9VP-4r Nir ws less thn tht induced by 9R under experimentl conditions, but the former strin hs the dvntge of being less virulent. 9VP-,V Nlr might therefore be used on its own where the incidence of fowl typhoid is low, or it might be used to vccinte more susceptible hevy breeds (23) prior to immuniztion with 9R, where the virulence of the ltter strin might be problem. ACKNOWLEDGMENTS The uthor would like to thnk M. A. Lovell for excellent technicl ssistnce nd B. Wells, V. Peters, nd H. Vickery for dditionl ssistnce. LITERATURE CITED 1. Bird, G. D., E. J. Mnning, nd P. W. Jones. 1985. Evidence for relted virulence sequences in plsmids of Slmonell dublin nd Slmonell typhimurium. J. Gen. Microbiol. 131:181% 1823. 2. Brrow, P. A., nd M. A. Lovell. 1988. The ssocition between lrge moleculr mss plsmid nd virulence in strin of Slmonell pullorum. J. Gen. Microbiol. 134:2307-2316. 3. Brrow, P. A., nd M. A. Lovell. 1989. Functionl homology of virulence plsmids in Slmonell gllinrum, S. pullorum, nd S. typhimurium. Infect. Immun. 57:3136-3141. 4. Brrow, P. A., J. M. Simpson, nd M. A. Lovell. 1988. Intestinl coloniztion in the chicken by food-poisoning Slmonell serotypes: microbil chrcteristics ssocited with fecl excretion. Avin Pthol. 17:571-588. 5. Brrow, P. A., J. M. Simpson, M. A. Lovell, nd M. M. Binns. 1987. Contribution of Slmonell gllinrum lrge plsmid towrd virulence in fowl typhoid. Infect. Immun. 55:388-392. 6. Fierer, J., G. Chikmi, L. Htlen, E. J. Heffernn, nd D. Guiney. 1988. Active immuniztion with LD842, plsmidcured strin of Slmonell dublin, protects mice ginst Group D nd Group B Slmonell infection. J. Infect. Dis. 158: 460-463. 7. Finney, D. J. 1964. Sttisticl method in biologicl ssy. Griffin, London. 8. Gordon, R. F., nd G. C. Brnder. 1942. The vlue of the rpid whole blood-stined ntigen gglutintion test in the erdiction of pullorum disese. Vet. Rec. 54:265-279. 9. Gordon, R. F., J. S. Grside, nd J. F. Tucker. 1959. The use of living ttenuted vccines in the control of fowl typhoid. Vet. Rec. 71:300-305. 10. Gordon, W. A. M., nd D. Luke. 1959. A note on the use of the 9R fowl typhoid vccine in poultry breeding flocks. Vet. Rec. 71:926-927. 11. Hll, C. F., nd H. T. Crtrite. 1961. Observtion on strins of Slmonell gllinrum pprently resistnt to furzolidone. Avin Dis. 5:382-392. 12. Hll, W. J., A. D. McDonld, nd D. H. Legenhusen. 1949. Studies on fowl typhoid. II. Control of the disese. Poult. Sci. 28:789-801. 13. Hrbourne, J. F., B. M. Willims, W. H. Prker, nd I. H. Finchm. 1963. The prevention of fowl typhoid in the field using freeze-dried 9R vccine. Vet. Rec. 75:858-861. 14. Kdo, C. I., nd S. T. Liu. 1981. Rpid procedure for detection nd isoltion of lrge nd smll plsmids. J. Bcteriol. 145: 1365-1373. 15. McNutt, S. H. 1926. Vccintion of poultry. J. Am.Vet. Med. Assoc. 69:472-477. 16. Miles, A. A., S. S. Misr, nd J. 0. Irwin. 1938. The estimtion of the bctericidl power of the blood. J. Hyg. 38:732-749. 17. Nkmur, M., S. Sto, T. Ohy, S. Suzuki, S. Iked, nd T.
2288 BARROW INFECT. IMMUN. Koed. 1985. Plsmid-cured Slmonell enteritidis AL1192 s cndidte for live vccine. Infect. Immun. 50:586-587. 18. Pomeroy, B. S. 1984. Fowl typhoid. In M. S. Hofstd, H. J. Brries, B. W. Clnek, W. M. Reid, nd H. W. Yoder, Jr. (ed.), Diseses of poultry, 8th ed. Iow Stte University Press, Ames, Iow. 19. Schffer, J. M., A. D. McDonld, W. J. Hll, nd H. Bunye. 1931. A stined ntigen for the rpid whole blood test for pullorum disese. J. Am. Vet. Med. Assoc. 79:236-240. 20. Silv, E. N. 1985. The Slmonell gllinrum problem in centrl nd South Americ, p. 150-156. In G. H. Snoeyenbos (ed.), Proceedings of the Interntionl Symposium on Slmonell, New Orlens, Louisin, 1984. Americn Assocition of Avin Pthologists, Inc., Phildelphi. 21. Silv, E. N., G. H. Snoeyenbos, 0. M. Weinck, nd C. F. Smyser. 1981. Studies on the use of 9R strin of Slmonell gllinrum s vccine in chickens. Avin Dis. 25:38-52. 22. Smith, H. W. 1956. The use of live vccines in experimentl Slmonell gllinrum infection in chickens with observtions on their interference effect. J. Hyg. 54:419-432. 23. Smith, H. W. 1956. The susceptibility of different breeds of chickens to experimentl Slmonell gllinrum infection. Poult. Sci. 35:701-705. 24. Smith, H. W., nd C. L. Gyles. 1970. The reltionship between different trnsmissible plsmids introduced by F into the sme strin of Escherichi coli K12. J. Gen. Microbiol. 62:277-285. 25. Smith, H. W., nd J. F. Tucker. 1980. The virulence of slmonell strins for chickens: their excretion by infected chickens. J. Hyg. 84:479-488. 26. Smith, H. W., J. F. Tucker, nd M. A. Lovell. 1981. Furzolidone resistnce in Slmonell gllinrum: the reltionship between in vitro nd in vivo determintion of resistnce. J. Hyg. 87:71-81. 27. Smith, I. M. 1969. Protection ginst experimentl fowl typhoid by vccintion with strin 9R reconstituted from the freezedried stte. J. Comp. Pthol. 79:197-205. 28. Snedecor, G. W., nd W. G. Cochrn. 1978. Sttisticl methods. 6th ed. Iow Stte University Press, Ames, Iow. 29. Sturt, E. E., R. D. Keenum, nd H. W. Bruins. 1962. Experimentl studied on n isolte of Slmonell gllinrum pprently resistnt to furzolidone. Avin Dis. 7:294-303. 30. Sturt, E. E., R. D. Keenum, nd H. W. Bruins. 1967. The emergence of furzolidone resistnt strin of Slmonell gllinrum. Avin Dis. 11:139-145. 31. Wilson, J. E. 1946. Fowl typhoid. Certin spects of the experimentlly produced disese. Vet. Rec. 58:269-271. 32. Wilson, J. E. 1956. Fowl typhoid-the effect of vccintion on the nturl nd experimentl disese. Vet. Rec. 68:664-668. Downloded from http://ii.sm.org/ on October 4, 2018 by guest