Salmonella typhimurium deletion mutants lacking adenylate cyclase and cyclic AMP receptor protein are avirulent and immunogenic.

Salmonella typhimurium SR-11 mutants with cya::Tn10 or crp::Tn10 mutations were found to be avirulent and immunogenic for BALB/c mice. Fusaric acid-resistant derivatives with deletions of the Tn10 and adjacent DNA sequences were constructed in S. typhimurium SR-11 strains with or without the virulence plasmid pStSR100. These delta cya delta crp strains grew more slowly than wild-type strains. They possessed wild-type ability to attach to, invade, and persist in gut-associated lymphoid tissue for up to a week but exhibited a diminished ability to reach mesenteric lymph nodes and the spleen. Mice 4 to 8 weeks old were resistant to oral infection with 10(9) cells of several different delta cya and delta cya delta crp strains (the equivalent to 10(4) 50% lethal doses of wild-type S. typhimurium SR-11) and 30 days after immunization became resistant to oral challenge with 10(3) to 10(4) 50% lethal doses of wild-type S. typhimurium SR-11.     

Recombinant avirulent Salmonella vaccine strains with stable maintenance and high level expression of cloned genes in vivo

Salmonella typhimurium strains with deletion (delta) of the adenylate cyclase (cya) and cyclic AMP receptor protein (crp) genes are avirulent for mice and induce a high level of protective immunity to oral challenge with up to 10,000 times what would be a lethal dose of wild-type virulent S. typhimurium cells. This immunity begins as early as seven days after immunization and lasts for at least four months. S. typhimurium delta cya delta crp mutants stably maintain plasmids and give high-level expression of cloned gene products; in this they appear superior to other avirulent S. typhimurium strains. S. typhimurium delta cya delta crp strains with a delta asd mutation (abolishing production of aspartate beta-semialdehyde dehydrogenase), have an obligate requirement for diaminopimelic acid (DAP). This strain can be used in conjunction with plasmid vectors lacking antibiotic resistance markers but having the wild-type asd+ gene from Streptococcus mutans to complement the delta asd chromosomal mutation. The Asd+ plasmid vector can be used to express a diversity of colonization and virulence antigens from other pathogens. In the delta cya delta crp delta asd S. typhimurium vaccine strain, the plasmid is completely stable in the absence of any exogenous selective pressure either in vitro or in vivo.     

Protection and immune responses induced by attenuated Salmonella typhimurium UK-1 strains

We previously reported that Salmonella typhimurium SR-11 mutants with deletion mutations in the genes encoding adenylate cyclase (cya) and the cAMP receptor protein (crp) are avirulent and protective in mice. Salmonella typhimurium UK-1 is highly virulent for chicks (oral LD50 of 3x10(3) CFU) and mice (oral LD50 of 8.5x10(3) CFU) and is capable of lethal infections in pigs, calves and horses. We postulated that attenuated derivatives of this lethal strain would probably induce a higher level of protective immunity than achieved with attenuated derivatives of less virulent S. typhimurium strains such as SR11. To test this hypothesis, we have constructed S. typhimurium UK-1 Deltacya-12Deltacrp-11 mutant strain chi3985 and its virulence plasmid cured derivative chi4095 to investigate their avirulence and immunogenicity in mice. We found that the mutants are avirulent and able to induce protective immune responses in BALB/c mice. These mutant strains retained wild-type ability to colonize the gut associated lymphoid tissue but reach and persist in spleen and liver at a significantly lower level than the wild-type parent strain. Mice survived oral infection with >1x10(9) CFU of chi3985 (the equivalent to 10(5) 50% lethal doses of wild-type S. typhimurium UK-1) and were fully protected against challenge with 10(5)times the LD50 of the wild-type parent. Immunized mice developed a high level of serum IgG titre to Salmonella LPS and delayed-type hypersensitivity (DTH) response to S. typhimurium outer membrane proteins. Compared to the virulence plasmid-containing strain chi3985, the virulence plasmid cured DeltacyaDeltacrp mutant strain chi4095 was more attenuated and less protective, as some mice immunized with chi4095 died when challenged with the wild-type UK-1 strain. This work demonstrates that S. typhimurium UK-1 Deltacrp Deltacya mutant strain may be a potential live vaccine to induce protective immunity against Salmonella infection or to deliver foreign antigens to the immune system.     

Characterization and immunogenicity of Salmonella typhimurium SL1344 and UK-1 delta crp and delta cdt deletion mutants.

S. typhimurium SL1344 and UK-1 mutants with deletions of the crp (cyclic AMP receptor protein) and cdt (colonization of deep tissues) genes have been constructed and characterized, and their levels of virulence and immunogenicity have been determined for BALB/c mice. All Crp- Cdt- and Crp+ Cdt- mutants were avirulent, as mice survived oral doses of 10(9) cells without illness. All the mutants colonized the gut-associated lymphoid tissue efficiently, but capacities to colonize deeper tissues, such as those of the spleen and liver, and blood were significantly reduced for the Crp- Cdt- and Crp+ Cdt- mutants compared with the Crp- Cdt+ mutant and the wild-type parent strain. The Crp- Cdt- and Crp+ Cdt- SL1344 strains induced complete protection, as all mice immunized with the mutants survived challenge with approximately 10(4) times the 50% lethal dose of the wild-type SL1344 strain. The Crp- UK-1 strain was least attenuated yet induced the highest level of protective immunity against challenge with the wild-type UK-1 strain. The Crp+ Cdt- and Crp- Cdt- strains, although totally attenuated, differed in immunogenicity to challenge with the highly virulent UK-1 parent, with the apparently hyperattenuated Crp- Cdt- strain inducing a lower level of protective immunity than the Crp+ Cdt- strain. Nevertheless, these UK-1 Crp- Cdt- and Crp+ Cdt- strains induced complete protective immunity to challenge with the less-virulent SL1344 wild-type strain. Taken collectively, the results indicate that the attenuation of a highly virulent S. typhimurium strain can yield a vaccine that induces excellent protective immunity to challenge with less-virulent S. typhimurium strains.     

Hybrid hepatitis B virus core-pre-S proteins synthesized in avirulent Salmonella typhimurium and Salmonella typhi for oral vaccination.

Avirulent salmonellae expressing foreign genes are attractive for use as oral vaccine carriers. To facilitate the stable expression of heterologous genes without conferring antibiotic resistance, a deletion of the asdA1 gene was introduced into Salmonella typhimurium and S. typhi delta cya delta crp mutant vaccine strains. An asd-complementing plasmid expressing hybrid hepatitis B virus nucleocapsid-pre-S (HBcAg-pre-S) particles was constructed. These hybrid HBcAg-pre-S particle genes were stably expressed in S. typhimurium and S. typhi delta cya delta crp mutant vaccine strains in this balanced, lethal host-vector combination. A single oral immunization of BALB/c mice with a recombinant S. typhimurium delta cya delta crp mutant synthesizing hybrid HBcAg-pre-S elicited potentially virus-neutralizing anti-pre-S serum immunoglobulin G antibodies. In addition, serum immunoglobulin G recognizing S. typhimurium lipopolysaccharide was induced. Distribution in tissue after oral immunization was analyzed in one plasmid-strain combination. The recombinant S. typhimurium colonized the gut-associated lymphoid tissue and the spleen and persisted for over 4 weeks, retaining the HBcAg-pre-S expression plasmid. An isogenic virulence plasmid-cured S. typhimurium delta cya delta crp strain expressing the same HBcAg-pre-S gene had reduced immunogenicity for the carried antigen after oral immunization.     

Virulence of and establishment of latency by genetically engineered deletion mutants of herpes simplex virus 1

We report the results of studies on the biologic properties of seven deletion mutants of herpes simplex virus 1 (HSV-1). The genes deleted from six of these mutants map in the S component of HSV-1 DNA and include those specifying the alpha protein 47, the glycoproteins G and E, the viral protein kinase, and two proteins whose functions are not yet known (open reading frames US2 and US11). The seventh virus [HSV-1(F) delta 305] contained a 700-bp deletion in the thymidine kinase gene. The results of intracerebral inoculation of Balb/c mice indicated that all but one of the deletion mutants in the S component were significantly attenuated. The PFU/LD50 ratios for these mutants ranged from 10(4)- to 10(5)-fold higher than that of the wild-type, HSV-1(F). The PFU/LD50 for mutant R7032, from which the glycoprotein E gene had been deleted, was less than 100-fold higher than that of the parent virus. All of the mutants, with one exception, were able to establish latency in mice; the exception, HSV-1(F) delta 305, was able to establish latency in rabbits.     

Control of colonization by virulent Salmonella typhimurium by oral immunization of chickens with avirulent delta cya delta crp S. typhimurium

Oral immunization with a delta cya delta crp Salmonella typhimurium strain has been shown to preclude colonization by wild-type, virulent S. typhimurium and induces humoral and cellular immune response in chickens. Intestinal tract colonization by the virulent challenge strain was used to determine the level of protection conferred by immunization with the delta cya delta crp mutant. The associated humoral and cellular immune responses were measured by ELISA and delayed-type hypersensitivity (DTH) tests, respectively. The levels of colonization by both Salmonella strains were determined by enumeration of viable cells in the intestinal tract. A reduction in faecal excretion of the wild-type strain was observed with a single oral immunization with the delta cya delta crp mutant, but caecal colonization was not affected. However, double oral immunization with the delta cya delta crp mutant precludes caecal colonization by the virulent strain. IgM, IgA and IgG were detected against sonicated Salmonella whole-cell antigens. Outer membrane and flagella proteins induced DTH responses, whereas lipopolysaccharide failed to do so. The effectiveness of the delta cya delta crp strain in reducing caecal colonization by the highly virulent challenge strain in chickens demonstrates that oral vaccination with the delta cya delta crp S. typhimurium should aid in eliminating Salmonella carriers in chickens. The elimination of these carriers on the poultry farm should help to control Salmonella contamination of poultry products, therapy improving public health.     

Essential role for cyclic AMP and its receptor protein in Yersinia enterocolitica virulence

Insertion mutations were isolated in cya and crp of Yersinia enterocolitica, which encode adenylate cyclase and the cyclic AMP (cAMP) receptor protein (CRP). The cya and crp mutants were affected for the production of proteins exported by the Ysc, Ysa, and flagellar type III secretion systems (TTSS). Protein production by each TTSS was restored when the respective mutation was complemented by a plasmid-encoded copy of the wild-type gene. Both cya and crp mutants exhibited reduced virulence for orally infected BALB/c mice in a 50% lethal dose analysis. Examination of bacterial survival in host tissues showed that cya and crp mutants colonized Peyer's patches and, to a lesser extent, mesenteric lymph nodes. However, the mutants did not appear to disseminate to the liver and spleen of infected mice. An initial examination of the effectiveness of Y. enterocolitica cya and crp mutants to stimulate protective immunity against subsequent challenge with virulent bacteria in mice was promising. The results indicate that the cAMP-CRP regulatory system is required for Y. enterocolitica virulence.     

Safety and immunogenicity in humans of an attenuated Salmonella typhi vaccine vector strain expressing plasmid-encoded hepatitis B antigens stabilized by the Asd-balanced lethal vector system.

Attenuated Salmonella typhi organisms which express genes encoding protective antigens of other pathogens have been developed for use as experimental oral vaccines. A delta asd S. typhi strain attenuated by deletions in cya, crp, and cdt which contains hepatitis B core (HBc) and pre-S genes encoded on an Asd+ pBR-based plasmid vector was constructed. Healthy adult volunteers ingested a single dose of 5 x 10(5) to 5 x 10(8) CFU of strain chi4073 (delta cya delta crp delta cdt S. typhi Ty2), 6 x 10(7) or 1 x 10(9) CFU of strain chi4632(pYA3149), a further derivative of chi4073 deleted in asd and containing the Asd+ vector without the HBc-pre-S fusion, or 3 x 10(7) or 7 x 10(8) CFU of strain X4632(pYA3167), a derivative containing the vector with the HBc-pre-S fusion. Chi4073 was generally well tolerated by 22 volunteers. No volunteer had fever or positive blood cultures; 4 of 22 volunteers shed vaccine organisms in the stool in the first 48 h only. Two of 18 volunteers who received one of the plasmid-containing derivatives of chi4073 developed low-grade fevers on day 10 or 12 after ingestion. One of these volunteers had positive blood cultures on days 7 and 8. Seven of these 18 volunteers had vaccine organisms detected in their stools in the first 48 h only. Most volunteers developed S. typhi-specific serum responses and developed S. typhi-specific antibody-secreting cells. However, no volunteer developed serum antibody to hepatitis pre-S or pre-S-specific antibody-secreting cells. Although the parent strain chi4073 was well tolerated, induced immunoglobulin G seroconversion to S. typhi lipopolysaccharide in 80 to 100% of vaccinees and stimulated specific IgA-secreting lymphocytes in 80 to 100% of vaccinees given a single oral dose of 2 x 10(7) and 5 x 10(8) CFU, chi4073 derivatives containing the Asd+ vector with and without sequences encoding the HBc-pre-S fusion caused occasional febrile reactions at high doses and did not stimulate detectable immune responses to hepatitis B antigens.     

Are appR and katF the same Escherichia coli gene encoding a new sigma transcription initiation factor?

The phenotype of Escherichia coli appR pleiotropic mutants has been compared with that of mutants in the katF gene, which lies in the same region and controls expression of catalase HPII (katE) and exonuclease III (xth). All the described characters of appR mutants--reduced pH 2.5 acid phosphatase level, overexpression of alkaline phosphatase and ability of crp or cya mutants to utilize some CAP + cAMP-dependent carbon sources--were reproduced by a katF:: Tn10 insertion. In all cases, the wild-type phenotype was restored by the presence of a plasmid-borne katF+ gene. Conversely, spontaneous appR mutants were hypersensitive to H2O2 to the same degree as katF mutants. We conclude that the appR gene is identical to katF, which encodes a putative new sigma factor (Mulvey and Loewen, 1989).     

Test of the virulence and live-vaccine efficacy of auxotrophic and galE derivatives of Salmonella choleraesuis.

Aromatic compound-dependent (aro) derivatives of three mouse-virulent strains of Salmonella choleraesuis (Salmonella cholerae-suis) were constructed and shown to be nonvirulent for mice (intraperitoneal [i.p.] 50% lethal dose [LD50], greater than 5 X 10(6) CFU). A pur derivative, and a thy derivative, each of a different virulent parent, remained moderately virulent (i.p. LD50S for BALB/c mice, ca. 10(5) and 5 X 10(4) CFU, respectively). Tested as live vaccines i.p., the aro strains were ineffective in salmonella-susceptible BALB/c and C57BL/6 mice but were somewhat effective in salmonella-resistant CBA/J mice and in outbred CD-1 mice. The pur and thy strains were effective as live vaccines in BALB/c mice when given in sublethal doses. Two previously isolated nonvirulent galE derivatives of S. choleraesuis (i.p. LD50 in BALB/c mice, greater than 10(6) CFU) were also ineffective as live vaccines in BALB/c and C57BL/6 mice. The main antigenic difference between S. choleraesuis (O-6,7) and S. typhimurium (O-4,12) is in O-antigen character, thought to largely determine the specificity of protection in salmonellosis. Paired, nearly isogenic O-6,7 and O-4,12 derivatives were constructed from an aro S. typhimurium strain of proven efficacy as a live vaccine. Used as live vaccines, the O-4,12 member protected BALB/c mice against challenge with virulent S. typhimurium, whereas the O-6,7 member did not protect against virulent S. choleraesuis. However, BALB/c mice vaccinated with the O-6,7 member and mice vaccinated with an aro S. choleraesuis strain were protected against challenge with a moderately virulent (LD50, 5 X 10(4) CFU) O-6,7 derivative of an S. typhimurium strain.     

Virulence attenuation and live vaccine potential of aroA, crp cdt cya, and plasmid-cured mutants of Salmonella enterica serovar Abortusovis in mice and sheep

Three live vaccine candidates of Salmonella enterica subspecies I serotype Abortusovis (aroA, cya crp cdt, and plasmid-cured strains) have been developed, and their efficacies in inducing humoral antibodies and protecting against abortion after challenge with wild-type strain SS44 were evaluated in sheep. Following estrus synchronization, animals were immunized 3 weeks after fertilization and boosted once 3 weeks later. Following challenge with wild-type SS44, pregnancy failure of vaccinated ewes was reduced compared to that of nonimmunized controls. Attenuation of each vaccine was also assessed in challenge experiments with nonimmunized pregnant ewes and in BALB/c mice. All three vaccine candidates appear to be safe for use in sheep and provide a model for the development of live vaccine candidates against naturally occurring ovine salmonellosis.     

Comparison of the safety and immunogenicity of delta aroC delta aroD and delta cya delta crp Salmonella typhi strains in adult volunteers.

Three attenuated Salmonella typhi strains have been constructed by introducing deletions in aroC and aroD or deletions in cya and crp into one of two wild-type parent strains, Ty2 or ISP1820. These mutant strains were designated CVD 906 (ISP1820 delta aroC delta aroD), CVD 908 (Ty2 delta aroC delta aroD), and chi 3927 (Ty2 delta cya delta crp). Two studies were conducted with 36 healthy adult inpatient volunteers to determine in a double-blind fashion the safety and immunogenicity of approximately 5 x 10(4) and 5 x 10(5) CFU of each of these three vaccine candidates given as a single dose. No statistically significant difference in the incidence of reactions among vaccinees was observed. Fever (oral temperature greater than or equal to 38.2 degrees C) occurred in 2 of 12 volunteers who received CVD 906, in 0 of 12 who received CVD 908, and in 1 of 12 who received chi 3927. Vaccine bacteremia without symptoms occurred in 1 of 12 vaccinees who received CVD 906, in 0 of 12 who received CVD 908, and in 2 of 12 who received chi 3927. Overall, 19 (53%) of 36 vaccinees developed immunoglobulin G antibody to S. typhi lipopolysaccharide after vaccination, with no statistically significant differences in the rate of seroconversion among volunteers in the three groups. We conclude that defined mutations in the aromatic biosynthetic pathway and in the cyclic AMP global regulatory system attenuate S. typhi. Mutant strains CVD 906, CVD 908, and chi 3927 are highly (and approximately equally) immunogenic but possibly differ in their propensity to induce fever. Further studies are needed to document the apparent relative safety of CVD 908 as a typhoid vaccine and as a vaccine carrier of foreign antigens.     

Attenuation and Immunogenicity of Δcya Δcrp Derivatives of Salmonella choleraesuis in Pigs

Six different isogenic Deltacya Deltacrp derivatives of a strain of Salmonella choleraesuis var. kunzendorf-chi3246 virulent for pigs were constructed by transposon-mediated deletion mutagenesis. These strains were evaluated for virulence and ability to elicit a protective immune response in young weaned pigs after oral administration and were compared to a commercially available vaccine which lacks the 50-kb virulence plasmid (vpl(-)). These derivatives were Deltacya Deltacrp vpl(+), Deltacya Deltacrp vpl(-), Deltacya Delta(crp-cdt) vpl(+), Deltacya Delta(crp-cdt) vpl(-), Deltacya Deltacrp pmi-3834 vpl(+), and Deltacya Delta(crp-cdt) pmi-3834. In experiments to evaluate safety, no significant adverse effects of any of the vaccine constructs were observed, except that two of the strains which carried the virulence plasmid (vpl(+)) caused a small, short-term elevation in maximum temperature compared to pretreatment temperature values. Orally immunized animals, except for those vaccinated with the Deltacya Deltacrp pmi-3834 vpl(+) strain or SC-54, developed significant serum antibody responses 21 days postvaccination as measured by enzyme-linked immunosorbent assay. No cell-mediated immune responses to heat-killed S. choleraesuis were noted at the same time point as measured with heat-killed bacteria as antigen in a lymphocyte proliferation assay. In an oral challenge exposure model with a highly virulent heterologous strain of S. choleraesuis, the Deltacya Deltacrp strains with deletions in pmi were not protective. As measured by morbidity scores, the responses to challenge of the pigs vaccinated with the other four Deltacya Deltacrp derivatives were significantly better than those of the nonvaccinated, challenged group. With the exception of temperature elevation and slight differences in diarrhea scores postchallenge, none of these strains differed significantly from each other in the other clinical parameters analyzed. While the commercial vaccine was protective by most of the parameters measured, it was not fully protective against challenge with virulent S. choleraesuis as judged by diarrhea scores and temperature elevation. Collectively, these data demonstrate that Deltacya Deltacrp derivatives, with or without the virulence plasmid but not with deletions in the pmi gene, are candidates for vaccines for protection against salmonellosis in pigs.     

Salmonella typhimurium mutants lacking flagella or motility remain virulent in BALB/c mice

Nonmotile flagellated (mot) and nonflagellated (fla) mutants of Salmonella typhimurium LT-2 were isolated from a collection of mutants with random Tn10-insertion mutations. Both classes of mutants were resistant to infection by the flagellotropic bacteriophage chi. The nonflagellated (fla::Tn10) mutants did not react with H antigen-specific antisera and did not possess flagella when examined by electron microscopy, and sheared-cell extracts were devoid of flagellin. The nonmotile (mot::Tn10) mutants reacted with H-specific antisera and expressed paralyzed flagella that were indistinguishable from wild-type flagella. The Tn10 insertions in strain LT-2 were mapped to loci in regions II (flh and mot) and III (fli) of the flagellar genes, and the mutations were transduced into the mouse-virulent S. typhimurium strains SR-11 and SL1344. Lack of motility reduced the ability of S. typhimurium to invade Henle cells in vitro, yet the virulence in mice of the nonmotile mutants of SR-11 and SL1344 was unaffected by the inactivity or loss of flagella. Wild-type SR-11 had a 50% lethal dose (LD50) in BALB/c mice following oral (p.o.) challenge of 2.4 x 10(4) CFU. The p.o. LD50 of the SR-11 fli-8007::Tn10 mutant was 4.5 x 10(4) CFU. The mot-8008::Tn10 mutation in SR-11 conferred paralyzed flagella and increased the p.o. LD50 in mice to 2.2 x 10(5) CFU, but this was not statistically significant. A similar increase in the p.o. LD50 was observed when the SL1344 mot-8008::Tn10 mutant was tested in mice. Wild-type SR-11 and the isogenic nonflagellated and nonmotile mutants were equally virulent in mice challenged via intraperitoneal injection.     

Intact motility as a Salmonella typhi invasion-related factor.

Invasiveness of Salmonella typhi was investigated. At first, we introduced Tn5 into the chromosome of a wild-type S. typhi strain, GIFU 10007, and screened the independent Tn5 insertion mutants for noninvasive (Inv-) strains. During the first half of this work, we obtained 4 Inv- strains from 1,338 independent Tn5 mutants. The four were either nonflagellate (Fla-), nonmotile (Mot-), or nonchemotactic (Che-). We then isolated more Fla-, Mot-, or Che- mutants and examined the invasiveness of these mutants. Sixty-three spontaneous or Tn5 insertion motility mutants, i.e., Fla-, Mot-, or Che-, were independently isolated from the wild-type strain GIFU 10007; all of them were noninvasive. Motile revertants isolated from some of these mutants showed the same invasiveness as the parent strain. P22-mediated transductional crosses were carried out between some of the motility mutants (as the recipients) and the Fla- reference strains of S. typhimurium with known deletion sites on the genome (as the donors). The mutational sites of the S. typhi mutants were assigned almost evenly to the three flagellar gene regions (regions I, II, and III) of S. typhimurium. The invasiveness of the motile recombinants obtained from the transduction assays was examined. The restoration of intact motility resulted in the restoration of invasiveness. Thus, we conclude that intact motility is an invasion-related factor of S. typhi. The relationship of Vi antigen to the invasiveness of S. typhi was also studied. Vi-negative mutants with intact motility remained invasive, whereas all 63 Inv- spontaneous or Tn5 mutants were Vi positive. Therefore, Vi antigen was not related to the invasiveness of S. typhi.     

Attenuation, persistence, and vaccine potential of an Edwardsiella ictaluri purA mutant.

In this study, an adenine-auxotrophic strain of Edwardsiella ictaluri was constructed and its virulence, tissue persistence, and vaccine efficacy were evaluated. A clone containing the purA gene was isolated from an E. ictaluri genomic library, sequenced, and shown to have an overall sequence identity of 79.3% at the nucleotide level and 85.7% at the amino acid level with the Escherichia coli purA gene. The cloned E. ictaluri purA gene was mutated by deleting a 598-bp segment of the gene and inserting the kanamycin resistance gene from Tn903 into the gap. The delta purA::Km(r) gene was subcloned into the suicide plasmid pGP704, and the resulting plasmid was used to deliver the modified gene into a virulent strain of E. ictaluri by conjugation. Homologous recombination replaced the chromosomal purA gene with the mutated gene to create an adenine-auxotrophic strain (LSU-E2). Compared to wild-type E. ictaluri, LSU-E2 was highly attenuated by the injection, immersion, and oral routes of exposure. By the injection route, LSU-E2 had a 50% lethal dose (LD50) that was greater than 5 logs10 higher than the LD50 for wild-type E. ictaluri. In a tissue persistence study, LSU-E2 was able to invade channel catfish by the immersion route and persist in internal organs for at least 48 h. Channel catfish that were vaccinated with a single immersion dose of LSU-E2 had mortality significantly lower (P < 0.01) following a wild-type E. ictaluri challenge than that of nonvaccinated fish.     

Oral immunization of mice and swine with an attenuated Salmonella choleraesuis [delta cya-12 delta(crp-cdt)19] mutant containing a recombinant plasmid.

Salmonella choleraesuis chi 3781, an attenuated [delta cya-12 delta(crp-cdt)19] mutant, was electroporated with the plasmid pBA31-R7, which codes for the expression of a 31-kDa protein from Brucella abortus (BCSP31). Recombinant S. choleraesuis chi 3781 stably maintained the pBA31-R7 plasmid and continued to express the cloned protein following recovery of the organism from orally inoculated animals. Unlike previous studies using S. typhimurium chi 4064(pBA31-R7), S. choleraesuis chi 3781(pBA31-R7) was able to colonize both the gut mucosa and deep tissues of both BALB/cByJ mice and crossbred swine. Orally inoculated mice developed serum antibodies to both the cloned 31-kDa protein (rBCSP31) and to S. choleraesuis chi 3781 endotoxin. These mice also developed a local intestinal antibody response to Salmonella endotoxin but not to rBCSP31. Similarly, mice inoculated with recombinant S. choleraesuis chi 3781 did not develop a delayed-type hypersensitivity (DTH) footpad response following injection with rBCSP31; however, these mice did respond to S. choleraesuis chi 3781 soluble antigen. Conversely, orally inoculated swine did not develop significant serum or intestinal antibody responses to cloned protein or Salmonella endotoxin, but DTH responses to both cloned protein and S. choleraesuis chi 3781 soluble antigen were strongly positive. The cell-mediated nature of these DTH responses was confirmed by histological examination. Results suggest that S. choleraesuis chi 3781 may be a suitable choice for further studies of vaccine efficacy in swine, especially for diseases which require cell-mediated immunity for resolution.     

Susceptibility of Lipopolysaccharide Mutants to the Bactericidal Action of Human Neutrophil Lysosomal Fractions

Acetate extracts of purified human neutrophil granules (a mixed population containing specific and azurophil granules) were dialyzed against phosphate-buffered saline (pH 7.0) and tested for bactericidal activity against smooth parent and rough mutant, gram-negative bacteria. Rough (Re) mutants of Escherichia coli, Salmonella typhimurium, and Salmonella minnesota were exquisitely more sensitive to extracts of human polymorphonuclear leukocyte granules than were their smooth (S) parents. The mean lethal dose (LD(50)) for the parent strains was 25 to 50 mug of granule protein per ml. As much as 500 mug of extract per ml failed to kill 100% of the S parents. The LD(50) for the rough mutants was 1.5 to 2.0 mug of the same granule extract per ml; 100% killing occurred with 5 to 10 mug of lysosomal protein per ml. Conditions affecting the growth of the bacteria greatly affected their sensitivity to the granule extracts. Granule extract killed bacteria grown with aeration to log phase 10 to 15 times more efficiently than the same bacteria grown to stationary phase under static conditions. The bactericidal incubation mixture also influenced results, in that greater killing occurred with tryptone than with phosphate or N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid-buffered saline. Bactericidal activity depended on lysosomal protein concentration, time, and temperature. Boiled lysosomal fractions failed to kill the S parents but retained 20 to 50% of their ability to kill the Re mutants. Parents (smooth) were killed more efficiently at pH 5 to 6, whereas their Re mutants were killed more efficiently at pH 7 to 8.     

Poxvirus deletion mutants: virulence and immunogenicity

Post-vaccinial encephalitis and disseminated vaccinia are major concerns with the use of vaccinia virus recombinants as immunization vectors in man. To identify and characterize possible attenuated poxvirus vectors, rabbitpox virus (RPV) (closely related to vaccinia) and four deletion mutants of RPV were studied for organ tropism, neurovirulence, and protection from wild-type challenge in BALB/c mice. Intraperitoneal (IP) inoculation with 10(7) PFU wild-type (wt) RPV or with two mutants 8 sm and 28 (containing approximately 12 kilobase deletions) showed titers of greater than 10(3) PFU/g tissue in multiple organs. In contrast, IP inoculation of 10(7) PFU of mutants 31 or 23 (containing approximately 30 kilobase deletions) showed markedly reduced growth in all organs. Neurovirulence of wt and mutant RPV was determined by intracerebral (IC) inoculation of mice. Wt and mutants 8 sm, and 28 RPV had LD50 less than 10(2) PFU; in contrast, 31 and 23 had LD50 greater than 10(5) PFU. Finally, 10(6) PFU of mutants 31 or 23, were administered to mice by scarification, the normal route of vaccinia immunization. Both 31 and 23 grew locally in the skin and protected mice challenged IC at 21 days with 100 LD50 of wt RPV, while all unimmunized controls died. We conclude that deletion mutants 31 and 23 demonstrate markedly reduced invasiveness and neurovirulence while retaining immunogenicity. Similar deletion mutations in vaccinia may create avirulent, but effective vaccine vectors for man.