Comparison of Salmonella typhi and Salmonella typhimurium invasion,intracellular growth and localization in cultured human epithelial cells

Invasion of the cultured epithelial cell lines HeLa, Henle-407, and Caco-2 (polarized and non-polarized) by Salmonella typhi and Salmonella typhimurium were compared using conventional gentamicin invasion assays. Additionally, the mechanisms of invasion and intracellular trafficking by S. typhi and S. typhimurium were compared in HeLa cells using indirect immunofluorescence microscopy. S. typhi strain Ty2 was invasive in all human cell lines tested, including apical uptake into polarized Caco-2 cell monolayers. This strain also replicated at levels similar to S. typhimurium strain SL1344 inside HeLa and Henle-407 cells. Indirect immunofluorescence microscopy confirmed that S. typhi, like S. typhimurium, induced membrane ruffles and cytoskeletal rearrangements upon contact with HeLa cell surfaces. Ruffling induced by S. typhi and S. typhimurium was accompanied by macropinocytosis of the fluid phase endocytic marker fluorescein-dextran-sulphate and by aggregation of cell surface class I MHC heavy chain. Intracellular lysosomal trafficking of S. typhi and S. typhimurium in HeLa cells was also studied. The lysosomal membrane glycoprotein marker h-lamp-2 colocalized with S. typhi-containing vacuoles, as previously shown for S. typhimurium. The soluble lysosomal enzyme marker cathepsin D also was found within S. typhi-containing vacuoles to the same extent as previously published for S. typhimurium . The results from this study suggest that S. typhi and S. typhimurium use similar mechanisms for invasion and intracellular trafficking in cultured human epithelial cells.     

Virulence and vaccine potential of phoP mutants of Salmonella typhimurium

We have constructed Salmonella typhimurium phoP mutants and found them to be avirulent and able to induce a protective immune response. BALB/c mice survived challenge with phoP derivatives of the highly virulent S. typhimurium strains SR-11 and SL1344 when inoculated intraperitoneally and per oral with doses equivalent to 10⁴ 50% lethal doses (LD₅₀) of the parent virulent strains. The avirulent mutants were able to establish an infection of the Peyer's patches of orally infected animals for up to 10 days after inoculation but were very inefficient at reaching the spleens. Despite the low level of infectivity of these mutants, immunized animals developed a delayed-type hypersensitivity (DTH) response to Salmonella antigens and resisted challenge with up to 10⁴ LD₅₀ of the virulent parent strain 30 days after immunization.     

Differential Early Interactions between Salmonella enterica Serovar Typhi and Two Other Pathogenic Salmonella Serovars with Intestinal Epithelial Cells

Salmonella enterica serovar Typhi (hereafter referred to as S. typhi) is a host-restricted pathogen that adheres to and invades the distal ileum and subsequently disseminates to cause typhoid fever in humans. However, S. typhi appears to be avirulent in small animals. In contrast, other pathogenic salmonellae, such as S. enterica serovars Typhimurium and Dublin (S. typhimurium and S. dublin, respectively), typically cause localized gastroenteritis in humans but have been used as models for typhoid fever because these organisms cause a disease in susceptible rodents that resembles human typhoid. In vivo, S. typhi has been demonstrated to attach to and invade murine M cells but is rapidly cleared from the Peyer’s patches without destruction of the M cells. In contrast, invasion of M cells by S. typhimurium is accompanied by destruction of these M cells and subsequently sloughing of the epithelium. These data have furthered our view that the early steps in the pathogenesis of typhoidal and nontyphoidal Salmonella serovars are distinct. To extend this concept, we have utilized an in vitro model to evaluate three parameters of initial host-pathogen interactions: adherence of three Salmonella serovars to human and murine small intestinal epithelial cell (IEC) lines, the capacity of these salmonellae to invade IECs, and the ability of the bacteria to induce interleukin-6 (IL-6) in these cell lines as a measure of host cell activation and the host acute-phase response. The results demonstrate that S. typhi adheres to and invades human small IECs better than either S. typhimurium or S. dublin. Interestingly, invA and invE null mutants of S. typhi are able neither to adhere to nor to invade IECs, unlike S. typhimurium invA and invE mutants, which adhere to but cannot invade IECs. S. typhi also induces significantly greater quantities of IL-6 in human small IEC lines than either of the other two Salmonella serovars. These findings suggest that differential host cytokine responses to bacterial pathogens may play an important role in the pathological sequelae that follow infection. Importantly, S. typhimurium did not induce IL-6 in murine IECs. Since S. typhimurium infection in mice is often used as a model of typhoid fever, these findings suggest that, at least in this case, the mouse model does not reflect the human disease. Taken together, our studies indicate that (i) marked differences occur in the initial steps of S. typhi, S. typhimurium, and S. dublin pathogenesis, and (ii) conclusions about S. typhi pathogenesis that have been drawn from the mouse model of typhoid fever should be interpreted conservatively.     

Mice Are Protected from Helicobacter pylori Infection by Nasal Immunization with Attenuated Salmonella typhimurium phoPc Expressing Urease A and B Subunits

Live Salmonella typhimurium phoP^c bacteria were tested as mucosal vaccine vectors to deliver Helicobacter pylori antigens. The genes encoding the A and B subunits of H. pylori urease were introduced into S. typhimurium phoP^c and expressed under the control of a constitutive tac promoter (tac-ureAB) or a two-phase T7 expression system (cT7-ureAB). Both recombinant Salmonella strains expressed the two urease subunits in vitro and were used to nasally immunize BALB/c mice. The plasmid carrying cT7-ureAB was stably inherited by bacteria growing or persisting in the spleen, lungs, mesenteric or cervical lymph nodes, and Peyer’s patches of immunized mice, while the plasmid carrying tac-ureAB was rapidly lost. Spleen and Peyer’s patch CD4⁺ lymphocytes from mice immunized with S. typhimurium phoP^c cT7-ureAB proliferated in vitro in response to urease, whereas cells from mice given S. typhimurium phoP^c alone did not. Splenic CD4⁺ cells from mice immunized with phoP^c cT7-ureAB secreted gamma interferon and interleukin 10, while Peyer’s patch CD4⁺ cells did not secrete either cytokine. Specific H. pylori anti-urease immunoglobulin G1 (IgG1) and IgG2A antibodies were detected following immunization, confirming that both Th1- and Th2-type immune responses were generated by the live vaccine. Sixty percent of the mice (9 of 15) immunized with S. typhimurium phoP^c cT7-ureAB were found to be resistant to infection by H. pylori, while all mice immunized with phoP^c tac-ureAB (15 of 15) or phoP^c (15 of 15) were infected. Our data demonstrate that H. pylori urease delivered nasally by using a vaccine strain of S. typhimurium can trigger Th1- and Th2-type responses and induce protective immunity against Helicobacter infection.     

Evaluation of Two Salmonella typhimurium Hybrids as Challenge Organisms in a System for the Assay of Typhoid Vaccines

A mouse-virulent Salmonella typhimurium hybrid (H42), which expresses the Salmonella typhi Vi antigen in addition to S. typhi O antigens 9 and 12, and a mouse-virulent S. typhimurium hybrid (H1), which expresses only the 9 and 12 antigens of S. typhi, were compared in their behavior as challenge organisms in a system developed to assay the protective capabilities of typhoid vaccines. Swiss-Webster white mice, vaccinated intraperitoneally with live Escherichia coli hybrids expressing the S. typhi O antigens 9 and 12, were significantly protected against death from intraperitoneal challenge with each of the S. typhimurium hybrid strains. Vaccination with an E. coli hybrid expressing the S. typhi Vi antigen in addition to O antigens 9 and 12 was seen to confer no advantage in protection against either S. typhimurium hybrid challenge organism over that obtained by vaccination with an E. coli hybrid expressing only the O antigens of S. typhi. However, a notable difference in the behavior of the two S. typhimurium hybrids was seen in mice vaccinated with the parent of the E. coli hybrid vaccinating strains, E. coli F464, which expresses no surface antigens common to either of these S. typhimurium hybrid challenge organisms. A nonspecific (with respect to the vaccinating strain) protective effect, believed to be associated with Vi antigen expression by the challenge organism, was seen against the challenge with S. typhimurium hybrid H42 after F464 vaccination, whereas no protection was conferred by F464 vaccination against the challenge with Vi-nonexpressing S. typhimurium hybrid H1. Inasmuch as neither S. typhimurium hybrid discriminates between the expression or nonexpression of the Vi antigen in a vaccinating strain, it is concluded that the Vi-nonexpressing S. typhimurium hybrid H1, which more clearly indicates the vaccine-specific protective role of the S. typhi O antigens and does not exhibit the nonspecific protection response of hybrid H42, is the better choice as challenge organism for this vaccine assay system.     

A possible mechanism for host-specific pathogenesis ofSalmonellaserovars

We have identified the complement receptors on human and murine macrophages involved in the recognition ofSalmonellaserovars, and investigated their relevance to the intracellular survival.S. typhiwas capable of surviving within human monocyte-derived macrophages, whereasS. typhimuriumwas not. Conversely,S. typhimurium, but notS. typhi, resisted intracellular killing by murine macrophages, demonstrating that the intracellular survival ofSalmonellaserovars is host-dependent. In the presence of serum opsonin, human monocyte-derived macrophages recognizedS. typhiandS. typhimuriumvia complement receptor type 1 (CR1) and type 3 (CR3), respectively. In contrast, murine macrophages recognizedS. typhiandS. typhimuriumvia CR3 and CR1, respectively. These findings demonstrate that the intracellular fate ofSalmonellaserovars following phagocytosis may depend on the type of complement receptors involved in their recognition, in that CR1-mediated recognition is closely correlated to subsequent intracellular survival. The Tn5 insertion mutant ofS. typhimuriumwhich lacks the ability to interact with CR1 was sensitive to intracellular killing by murine macrophagesin vitro, and was much less virulent to micein vivo, confirming the relevance of CR1-mediated bacterial recognition to the pathogenicity ofS. typhimuriumfor mice. These results suggest that selective recognition ofSalmonellaserovars through CR1 may lead to their subsequent intracellular survival, and is responsible for the host-specific pathogenesis ofSalmonellaserovars.     

Physical Limitations on Salmonella typhi Entry into Cultured Human Intestinal Epithelial Cells

Kinetic studies of Salmonella typhi invasion of INT407 cells at different multiplicities of infection (MOIs) have revealed a strict physical limitation on S. typhi entry at MOIs of ≥40. Staining of infected monolayers to distinguish intracellular from extracellular bacteria revealed that all monolayer cells are susceptible to infection and that internalized bacteria are typically contained in one to three separate clusters per cell during the first 60 min. Scanning and transmission electron microscopic analyses of time course-infected monolayers showed that at early times postinfection, bacteria bind to shortened, coalesced microvilli in one to three focal aggregate structures per host cell surface. As reported previously for S. typhimurium, focal aggregates progress to conical membrane ruffles that appear to engulf one or a few centrally contained S. typhi cells by a macropinocytic process, which enhanced the entry of simultaneously added Escherichia coli HB101 about 30-fold. Additionally, kinetic studies showed that at an MOI of 400, maximal S. typhi entry is virtually completed within 30 to 35 min. Monolayers pretreated with S. typhi for 30 min to saturate the entry process were severely reduced in the ability to internalize subsequently added kanamycin-resistant strains of S. typhi or S. typhimurium, but E. coli HB101(pRI203) expressing the cloned Yersinia inv gene was not reduced in entry. In invasion inhibition assays, anti-β1 integrin antibodies markedly reduced E. coli HB101(pRI203) invasion efficiency but did not reduce S. typhi entry. Collectively, these data provide direct physical and visual evidence which indicates that S. typhi organisms are internalized at a limited number (i.e., two to four) of sites on host cells. S. typhi and S. typhimurium likely share INT407 cell entry receptors which do not appear to be members of the β1 integrin superfamily.     

A Salmonella enterica serovar Typhi plasmid induces rapid and massive apoptosis in infected macrophages

pR_ST98 is a chimeric plasmid isolated from Salmonella enterica serovar Typhi (S. typhi) that mediates the functions of drug resistance and virulence. Previously, we reported that Salmonella plasmid virulence (spv) genes were present in S. typhi. In our current study, we investigated whether plasmid pR_ST98 exhibits significant cytotoxicity in macrophages. pR_ST98 was transferred into the avirulent Salmonella enterica serovar Typhimurium (S. typhimurium) strain RIA to create the transconjugant pR_ST98/RIA. The standard S. typhimurium virulent strain SR-11, which carries a 100-kb virulence plasmid, was used as a positive control. The bacterial strains were incubated with a murine macrophage-like cell line (J774A.1) in vitro. Apoptosis of J774A.1 cells was examined by electron microscopy and flow cytometry after annexin-V/propidium iodide labeling, and the survival of Salmonella strains in J774A.1 cells was determined. Results showed that macrophages infected with strain pR_ST98/RIA displayed greater levels of apoptosis than those infected with RIA and that pR_ST98 may increase bacterial survival in macrophages. Further studies showed that the pR_ST98-induced death of macrophages was associated with the loss of mitochondrial membrane potential and that pR_ST98 may activate caspase-9 and then caspase-3. The research data indicate that the virulence of bacteria that contain the pR_ST98 plasmid is enhanced; the presence of this plasmid increases the survival of the bacterial pathogen and acts through the mitochondrial pathway to mediate macrophage apoptosis.     

Contributions to the study of Salmonella: II. Protein determinants shared by S. typhi, S. enteritidis and S. typhimurium

The antigenic relationship between S. typhi and S. enteritidis, that at present are known to share only the polysaccharide factors 9 and 12, has been investigated with a view to confirming the existence of common protein O specificity.

A serum to S. enteritidis absorbed with the polysaccharide of S. typhi O-901 and a serum to S. enteritidis in which the polysaccharide antibodies were present in extremely small amounts (undetectable in agar diffusion tests) precipitated with extracts containing proteins of S. typhi.

Additional evidence of the existence of common protein O factors was obtained by precipitin tests in agar in which sera to proteins from S. typhi reacted with proteins of S. enteritidis and S. typhimurium (whose H determinants differ from one another); the precipitations of heterologous proteins in agar-gel varied in intensity according to the antibody content of the antiprotein sera tested and to the proportion of O determinants of the respective protein. Heterologous precipitin reactions of the sera to S. enteritidis with antigens from S. typhi were stronger than those with the homologous antigen, whereas the antigens from S. enteritidis were but weakly precipitated by the sera to S. typhi.

This appears to indicate an intrinsically higher antigenic potency of S. typhi, compared with S. enteritidis.

     

Salmonella enterica serovar Typhi plasmid pRST98-mediated inhibition of autophagy promotes bacterial survival in infected fibroblasts

pR_ST98 is a chimeric plasmid isolated from Salmonella enterica serovar typhi (S. typhi) and mediates both drug-resistance and virulence of S. typhi. Autophagy has been recently reported as an important component of the innate immune response against intracellular pathogen. In this study, we investigated the effect of pR_ST98 on cellular autophagy, apoptosis and bacterial survival in infected fibroblasts. S. typhi strain ST₈ carrying pR_ST98, Salmonella typhimurium strain SR-11 carrying a 100 Kb virulent plasmid, and avirulent S. typhi strain ST₁₀ without plasmid were tested in this experiment. Results showed that embryonic fibroblasts infected with ST₈ containing pR_ST98 had decreased autophagy accompanied by increased bacterial survival and apoptosis. Further study showed that autophagy inducer rapamycin reversed pR_ST98-mediated inhibition of autophagy and reduced apoptosis in infected fibroblasts. Our data indicate that pR_ST98 can inhibit autophagy, thus facilitating S. typhi survival and promoting apoptosis of host cells. This study contributes to understanding the underlying mechanism of pR_ST98-mediated virulence in S. typhi.     

Trafficking of Porin-Deficient Salmonella typhimurium Mutants inside HeLa Cells: ompR and envZ Mutants Are Defective for the Formation of Salmonella-Induced Filaments

Outer membrane porin genes of Salmonella typhimurium, including ompC, ompF, and tppB, are regulated by the products of ompB, a two-component regulatory locus encoding OmpR and EnvZ. S. typhimurium ompR mutants are attenuated in mice, but to date no one has studied the intracellular trafficking of S. typhimurium porin-deficient mutants. In this study, isogenic transposon mutants of S. typhimurium with insertions in ompR, envZ, ompF, ompC, ompD, osmZ, and tppB were compared with wild-type SL1344 for trafficking in the human epithelial cell line HeLa. We found that ompR and envZ mutants were reduced or completely inhibited for the formation of Salmonella-induced filaments (Sifs). This result was confirmed with an ompB deletion mutant. Sifs are tubular structures containing lysosomal glycoprotein which are induced specifically by intracellular Salmonella. Genetic analysis showed that the ompR mutation could be complemented in trans by cloned ompR to restore its ability to induce Sifs. In contrast, mutations in the known ompR-regulated genes ompF, ompC, and tppB (as well as the ompR-independent porin gene, ompD) had no effect on Sif formation relative to that of wild-type SL1344, thus indicating that OmpR does not exert its role on these genes to induce Sif formation. The omp mutants studied were able to invade and replicate in HeLa cells at levels comparable to those in wild-type SL1344. We conclude that OmpR and EnvZ appear to regulate Sif formation triggered by intracellular S. typhimurium.     

Loss of core fucosylation suppressed the humoral immune response in Salmonella typhimurium infected mice

BackgroundThe humoral immune response is pivotal to protect the host from Salmonella typhimurium (S. typhimurium) infection. Previously, we found that core fucosylation catalyzed by core fucosyltransferase (Fut8) could regulate the immune responses. However, the role of core fucosylation during S. typhimurium infection remains unclear.MethodsTo demonstrate the role of Fut8 in S. typhimurium infection, we infected Fut8^+/+ and Fut8^−/− mice using S. typhimurium. The production of antiserum against the S. typhimurium was detected. The expression of T and B cell activation-related genes during S. typhimurium infection was analyzed. The role of core fucosylation on CD4⁺ T-B cell interaction and B cell generation was investigated during S. typhimurium infection. The production of sIgA was compared between Fut8^+/+ and Fut8^−/− mice.ResultsCompared to Fut8^+/+ mice, the number of S. typhimurium colonized in the cecum was markedly increased in Fut8^−/− mice. The production of the IgG and sIgA specific for S. typhimurium was significantly decreased in Fut8^−/− mice. Moreover, loss of Fut8 decreased the induction of Th2-type cytokines from splenic cells of Fut8^−/− mice during S. typhimurium infection. In addition, we found that the core fucosylation regulated the interaction between B and T cells in the lipid raft formation.ConclusionCore fucosylation plays important roles in host defence against S. typhimurium infection.     

Cell-Contact-Stimulated Formation of Filamentous Appendages by Salmonella typhimurium Does Not Depend on the Type III Secretion System Encoded by Salmonella Pathogenicity Island 1

The formation of filamentous appendages on Salmonella typhimurium has been implicated in the triggering of bacterial entry into host cells (C. C. Ginocchio, S. B. Olmsted, C. L. Wells, and J. E. Galán, Cell 76:717–724, 1994). We have examined the roles of cell contact and Salmonella pathogenicity island 1 (SPI1) in appendage formation by comparing the surface morphologies of a panel of S. typhimurium strains adherent to tissue culture inserts, to cultured epithelial cell lines, and to murine intestine. Scanning electron microscopy revealed short filamentous appendages 30 to 50 nm in diameter and up to 300 nm in length on many wild-type S. typhimurium bacteria adhering to both cultured epithelial cells and to murine Peyer’s patch follicle-associated epithelia. Wild-type S. typhimurium adhering to cell-free culture inserts lacked these filamentous appendages but sometimes exhibited very short appendages which might represent a rudimentary form of the cell contact-stimulated filamentous appendages. Invasion-deficient S. typhimurium strains carrying mutations in components of SPI1 (invA, invG, sspC, and prgH) exhibited filamentous appendages similar to those on wild-type S. typhimurium when adhering to epithelial cells, demonstrating that formation of these appendages is not itself sufficient to trigger bacterial invasion. When adhering to cell-free culture inserts, an S. typhimurium invG mutant differed from its parent strain in that it lacked even the shorter surface appendages, suggesting that SPI1 may be involved in appendage formation in the absence of epithelia. Our data on S. typhimurium strains in the presence of cells provide compelling evidence that SPI1 is not an absolute requirement for the formation of the described filamentous appendages. However, appendage formation is controlled by PhoP/PhoQ since a PhoP-constitutive mutant very rarely possessed such appendages when adhering to any of the cell types examined.     

Superinfection exclusion by λ prophage in lysogens ofSalmonella typhimurium

Lysogens of coliphage λ inSalmonella typhimurium excludeSalmonella phages P22 and L. The λrex function is responsible for exclusion of superinfecting P22, since P22 is not excluded by lysogens of λrex^? mutants. However, λrex^? lysogens still exclude phage L, apparently because they retain another superinfection exclusion mechanism which is identical in specificity to the P22sie B exclusion mechanism. Mutants of λ defective in this new exclusion system (sie^? mutants) have been isolated. The phenotypes of λbio deletion/substitution phages indicate that thesie gene maps between thecIII andN genes, at a position on the λ genome analogous to that of thesie B gene on the P22 genome.     

Molecular and Functional Characterization of Salmonella enterica Serovar Typhimurium poxA Gene: Effect on Attenuation of Virulence and Protection

Salmonella enterica poxA mutants exhibit a pleiotropic phenotype, including reduced pyruvate oxidase activity; reduced growth rate; and hypersensitivity to the herbicide sulfometuron methyl, α-ketobutyrate, and amino acid analogs. These mutants also failed to grow in the presence of the host antimicrobial peptide, protamine. In this study, PoxA⁻ mutants of S. enterica serovar Typhimurium (S. typhimurium) were found to be 10,000-fold attenuated in orally inoculated BALB/c mice and 1,000-fold attenuated in intraperitoneally inoculated BALB/c mice, compared to wild-type S. typhimurium UK-1. In addition, poxA mutants were found to be capable of colonizing the spleen, mesenteric lymph nodes, and Peyer’s patches; to induce strong humoral immune responses; and to protect mice against a lethal wild-type Salmonella challenge. A 2-kb DNA fragment was isolated from wild-type S. typhimurium UK-1 based on its ability to complement an isogenic poxA mutant. The nucleotide sequence of this DNA fragment revealed an open reading frame of 325 amino acids capable of encoding a polypeptide of 36.8 kDa that was confirmed in the bacteriophage T7 expression system. Comparison of the translated sequence to the available databases indicated high homology to a family of lysyl-tRNA synthetases. Our results indicate that a mutation of poxA has an attenuating effect on Salmonella virulence. Further, poxA mutants are immunogenic and could be useful in designing live vaccines with a variety of bacterial species. To our knowledge, this is the first report on the effect of poxA mutation on bacterial virulence.     

Antimicrobial Efficacy and Phytochemical Analysis of Indigofera Trita Linn.

An in vitro antimicrobial activity and phytochemical analysis of various extracts of Indigofera trita L. viz. petroleum ether, chloroform, acetone, ethanol and aqueous extracts were carried out. A total of 21 microorganisms (19 bacteria and 2 fungal strains) were used for antimicrobial activity by disc diffusion method and a standard procedure was used to identify the phytochemical constituents. Petroleum ether extract showed moderate inhibitory activity against Staphylococcus aureus (14.40 mm), S. epidermidis (14.20 mm), Salmonella paratyphi A (12.80 mm), Streptococcus mutans (12.20 mm), Escherichia coli, Proteus vulgaris, S. typhi and Burkholderia cepacia (12.00 mm). The chloroform extract also showed antimicrobial activity against S. epidermidis (14.20 mm), S. typhimurium (12.60 mm), S. paratyphi A, S. brunei and Yersinia enterocolitica (12.00 mm). The acetone extract of I. trita showed considerable inhibitory activity against S. epidermidis (18.20 mm), S. typhimurium (14.60 mm), S. infantis (13.80 mm), S. aureus (13.40 mm), Y. enterocolitica (13.00 mm) and Enterobacter aerogenes (12.00 mm) were documented. Ethanol extract showed significant antimicrobial activity against S. epidermidis (18.60 mm), S. paratyphi A (14.60 mm), Y. enterocolitica (13.40 mm), S. typhi (12.40 mm), S. aureus, E. aerogenes, S. typhimurium and S. infantis (12.00 mm). Aqueous extract of I. trita considerably inhibited S. epidermidis (13.80 mm), S. paratyphi A and Y. enterocolitica (12.20 mm), E. aerogenes and Haemophilus parahaemolyticus (12.00 mm). All the five extracts showed a minimal antifungal activity when compared to antibacterial activity. The result revealed that the antimicrobial properties of I. trita might be associated with the presence of phenolic compounds, flavonoids, tannins, glycosides, saponins, phytosterols and alkaloids.     

Contributions on the study of Salmonella: Further studies on the protein determinants shared by Salmonellae of groups B and D; variations in the immune response of different hybrid rabbits

The immunization of hybrid rabbits under subtropical climatic conditions, confirmed previous findings indicating the existence of common `O' protein determinants of Salmonella typhi, Salmonella enteritidis and Salmonella typhimurium. The antisera from these rabbits however, cross-reacted differently from those previously, under European climatic conditions, with the same Salmonella species. The previously found pattern of stronger precipitation with heterologous than with homologous antigens of the sera to S. enteritidis was not obtained in the present experiments, and the sera to S. typhi—in contrast to the previous results—reacted in agar gel with the antigens of S. enteritidis and Salmonella dublin.