Optimizing pregnancy rates using frozen-thawed equine semen.
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Animals↗
Biomedical subjects
E S Metcalf
Publications and source records attributed to E S Metcalf.
The role of international transport of equine semen on disease transmission.
Despite the numerous benefits of having the capability to transport semen internationally, there are serious potential ramifications if that semen is contaminated with a communicable disease. Bacteria: Many commensal bacteria colonize the exterior of the stallion penis and are not regarded as pathogenic. They may be cultured from an ejaculate. Alterations of the normal bacterial flora on the exterior genitalia may cause the growth of opportunistic bacteria such as Klebsiella pneumonia, Pseudomonas aeruginosa, Streptococcus zooepidemicus, which, if inseminated, may cause infertility in susceptible mares. Contagious equine metritis (CEM), a highly transmissible, true venereal disease of horses, is caused by the gram-negative coccobacillis, Taylorella equigenitalis. Even with the use of rigorous testing protocols, the current techniques used may not ensure accuracy of results. Viruses: Equine coital exanthema (equine herpes virus type 3; EHV-3) is a highly contagious virus that causes painful lesions on the stallion's penis and mare's vulva. Although it is primarily transmitted through coitus, infected fomites have also been implicated in its spread. Therefore, it is possible that the virus can potentially be transmitted to the ejaculate through penile contact with an artificial vagina or sleeve. Equine arteritis virus appears to be becoming more prevalent in recent years. The most common method of transmission is through respiratory disease, but the organism can also be shed in the semen of asymptomatic stallions. Equine infectious anemia virus has also been found to be present in the semen of an infected stallion, although no evidence exists at this time that there is venereal transmission of this disease. Protozoa: Dourine, caused by Trympanosoma equiperidum, is a venereal disease found only in Africa, South and Central America and the Middle East. Serological testing using complement fixation is recommended for diagnosis. Piroplasmosis, a disease caused by Babesia equi or by a less severe strain, Babesia caballi, has received a great deal of attention in recent years due to the increased transfer of horses between countries. It is considered to be enzootic in many areas of the southern US, and is found throughout the world. The protozoal agent is most often spread by ticks, but mechanical transmission has also been documented; therefore, there is concern for venereal transmission if blood from an infected horse contaminates the semen.
Animals↗
Flagellar phase variation of Salmonella enterica serovar Typhimurium contributes to virulence in the murine typhoid infection model but does not influence Salmonella-induced enteropathogenesis.
Although Salmonella enterica serovar Typhimurium can undergo phase variation to alternately express two different types of flagellin subunit proteins, FljB or FliC, no biological function for this phenomenon has been described. In this investigation, we constructed phase-locked derivatives of S. enterica serovar Typhimurium that expressed only FljB (termed locked-ON) or FliC (termed locked-OFF). The role of phase variation in models of enteric and systemic pathogenesis was then evaluated. There were no differences between the wild-type parent strain and the two phase-locked derivatives in adherence and invasion of mouse epithelial cells in vitro, survival in mouse peritoneal macrophages, or in a bovine model of gastroenteritis. By contrast, the locked-OFF mutant was virulent in mice following oral or intravenous (i.v.) inoculation but the locked-ON mutant was attenuated. When these phase-locked mutants were compared in studies of i.v. kinetics in mice, similar numbers of the two strains were isolated from the blood and spleens of infected animals at 6 and 24 h. However, the locked-OFF mutant was recovered from the blood and spleens in significantly greater numbers than the locked-ON strain by day 2 of infection. By 5 days postinfection, a majority of the mice infected with the locked-OFF mutant had died compared with none of the mice infected with the locked-ON mutant. These results suggest that phase variation is not involved in the intestinal stage of infection but that once S. enterica serovar Typhimurium reaches the spleens of susceptible mice those organisms in the FliC phase can grow and/or survive better than those in the FljB phase. Additional experiments with wild-type S. enterica serovar Typhimurium, fully capable of switching flagellin type, supported this hypothesis. We conclude that organisms that have switched to the FliC(+) phase have a selective advantage in the mouse model of typhoid fever but have no such advantage in invasion of epithelial cells or the induction of enteropathogenesis.
Animals↗
Absence of all components of the flagellar export and synthesis machinery differentially alters virulence of Salmonella enterica serovar Typhimurium in models of typhoid fever, survival in macrophages, tissue culture invasiveness, and calf enterocolitis.
In this study, we constructed an flhD (the master flagellar regulator gene) mutant of Salmonella enterica serovar Typhimurium and compared the virulence of the strain to that of the wild-type strain in a series of assays that included the mouse model of typhoid fever, the mouse macrophage survival assay, an intestinal epithelial cell adherence and invasion assay, and the calf model of enterocolitis. We found that the flhD mutant was more virulent than its parent in the mouse and displayed slightly faster net growth between 4 and 24 h of infection in mouse macrophages. Conversely, the flhD mutant exhibited diminished invasiveness for human and mouse intestinal epithelial cells, as well as a reduced capacity to induce fluid secretion and evoke a polymorphonuclear leukocyte response in the calf ligated-loop assay. These findings, taken with the results from virulence assessment assays done on an fljB fliC mutant of serovar Typhimurium that does not produce flagellin but does synthesize the flagellar secretory apparatus, indicate that neither the presence of flagella (as previously reported) nor the synthesis of the flagellar export machinery are necessary for pathogenicity of the organism in the mouse. Conversely, the presence of flagella is required for the full invasive potential of the bacterium in tissue culture and for the influx of polymorphonuclear leukocytes in the calf intestine, while the flagellar secretory components are also necessary for the induction of maximum fluid secretion in that enterocolitis model. A corollary to this conclusion is that, as has previously been surmised but not demonstrated in a comparative investigation of the same mutant strains, the mouse systemic infection and macrophage assays measure aspects of virulence different from those of the tissue culture invasion assay, and the latter is more predictive of findings in the calf enterocolitis model.
Animals↗
Pro- and anti-inflammatory gene expression in the murine small intestine and liver after chronic exposure to alcohol.
BACKGROUND: Endotoxin has been proposed to play a primary role in ALD, by initiating an inflammatory cascade within the liver. Although the source of these cytokines has been presumed to be circulating monocytes or tissue macrophages, ethanol-induced, nonhepatic sources of soluble mediators recently have been identified. One potential, but not clearly defined, extrahepatic source of cytokines in ALD is the intestine. In the current study, we hypothesized that alcohol would alter cytokine expression within the small intestine of mice exposed to ethanol and that LPS would alter levels of cytokine expression even more dramatically. METHODS: Mice were fed a modified Lieber-DeCarli liquid ethanol or control diet for up to 14 days prior to injecting either saline or LPS. Plasma alanine aminotransferase (ALT) and cytokine levels, histology, and RT-PCR of pro- and anti-inflammatory cytokine gene expression were determined from distal ileum and liver samples. Translocation of intestinal bacterial flora also was assessed. RESULTS: Ethanol exposure upregulated basal gene expression of IL-1 beta, TNF-alpha, IL-6, and iNOS in the distal ileum, but similar effects of ethanol on the liver were not observed. In contrast, LPS challenge of ethanol-exposed mice increased intestinal gene expression of some cytokines, but decreased expression of others. These effects were not associated with bacterial translocation. Also, ethanol alone induced a modest increase in both ICAM-1 and TLR4 mRNA expression in the intestine, but expression of both molecules was inhibited in mice that received both ethanol and LPS. Finally, whereas basal levels of hepatic IL-11 mRNA were not elevated by exposure to ethanol, intestinal IL-11 mRNA levels were increased more than 100-fold. CONCLUSIONS: These studies are the first to show that ethanol affects cytokine gene expression in the ileum and identifies the ileum as a potential target for ethanol effects. In addition, our results suggest that IL-11 expression may be enhanced in the intestine to help repair or protect this organ from alcohol-induced damage. Collectively, these studies suggest that both pro- and anti-inflammatory soluble mediators in the intestine maintain and exacerbate the local hepatic response to ethanol.
Alanine Transaminase↗
Experimental Salmonella typhi infection in the domestic pig, Sus scrofa domestica.
The domestic pig, Sus scrofa domestica, was examined as a model for typhoid fever, a severe and systemic disease of humans caused by Salmonella typhi. Six pigs were inoculated 1 week post-weaning with approximately 10(10)colony forming units (cfu) of wild type Salmonella typhi strain ISP1820 intranasally and observed for 3 weeks. S. typhi was cultured from the tonsils of 50% of the pigs at necropsy. Cultures from all other organs analysed (ileum, colon, spleen and liver) were negative. No clinical or histopathological signs of disease were observed. Pigs inoculated in parallel with swine-virulent S. choleraesuis all exhibited signs of systemic salmonellosis indicating that the parameters of the experimental infection with S. typhi (e.g. route) were appropriate. Whereas the pig has a gastrointestinal tract that is very similar to humans, our results indicated that the unique features of host and microbe interaction needed to produce typhoid fever were not mimicked in swine. Nevertheless, our observation of tonsillar involvement was consistent with former observations of S. choleraesuis and S. typhimurium infections in swine and supports a role for the tonsil in all porcine salmonella infections.
Animals↗
Gram-negative pathogens and molecular mimicry: is there a case for mistaken identity? Response.
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Animals↗
Molecular mimicry mediated by MHC class Ib molecules after infection with gram-negative pathogens.
The development of many autoimmune diseases has been etiologically linked to exposure to infectious agents. For example, a subset of patients with a history of Salmonella infection develop reactive arthritis. The persistence of bacterial antigen in arthritic tissue and the isolation of Salmonella or Yersinia reactive CD8+ T cells from the joints of patients with reactive arthritis support the etiological link between Gram-negative bacterial infection and autoimmune disease. Models proposed to account for the link between infection and autoimmunity include inflammation-induced presentation of cryptic self-epitopes, antigen persistence and molecular mimicry. Several studies support molecular mimicry as a mechanism for the involvement of class II epitopes in infectious disease-induced self-reactivity. Here, we have identified an immunodominant epitope derived from the S. typhimurium GroEL molecule. This epitope is presented by the mouse H2-T23-encoded class Ib molecule Qa-1 and was recognized by CD8+ cytotoxic T lymphocytes induced after natural infection. S. typhimurium-stimulated cytotoxic T lymphocytes recognizing the GroEL epitope cross-reacted with a peptide derived from mouse heat shock protein 60 and recognized stressed macrophages. Our results indicate involvement of MHC class Ib molecules in infection-induced autoimmune recognition and indicate a mechanism for the etiological link between Gram-negative bacterial infection and autoimmunity.
Amino Acid Sequence↗
T cell responses to Gram-negative intracellular bacterial pathogens: a role for CD8+ T cells in immunity to Salmonella infection and the involvement of MHC class Ib molecules.
Despite being a major group of intracellular pathogens, the role of class I-restricted T cells in the clearance of Gram-negative bacteria is not resolved. Using a murine typhoid model, a role for class I-restricted T cells in the immune response to the Gram-negative pathogen Salmonella typhimurium is revealed. Class I-deficient beta2-microglobulin-/- mice show increased susceptibility to infection with S. typhimurium. Following infection, CD8+ CTLs specific for Salmonella-infected targets can be readily detected. The Salmonella-specific CTLs recognize infected H-2-mismatched targets, suggesting the involvement of shared class Ib molecules. Studies using transfectants expressing defined class Ia and class Ib molecules indicate the involvement of the class Ib molecule, Qa-1. Ab-blocking studies and the measurement of bacteria-specific CTL frequencies identified Qa-1 as a dominant restricting element. The Qa-1-restricted CTL recognition depends on TAP and proteasome functions. Surprisingly, Qa-1-restricted CTLs recognized cells infected with other closely related Gram-negative bacteria. Taken together, these observations indicate that Salmonella-specific CTLs recognize a cross-reactive epitope presented by Qa-1 molecules and, as such, may be novel targets for vaccine development.
Animals↗
Environmental regulation of Salmonella typhi invasion-defective mutants.
Salmonella typhi is the etiologic agent of human typhoid. During infection, S. typhi adheres to and invades epithelial and M cells that line the distal ileum. To survive in the human host, S. typhi must overcome numerous complex extracellular and intracellular environments. Since relatively little is known about S. typhi pathogenesis, studies were initiated to identify S. typhi genes involved in the early steps of interaction with the host and to evaluate the environmental regulation of these genes. In the present study, TnphoA mutagenesis was used to study these early steps. We isolated 16 Salmonella typhi TnphoA mutants that were defective for both adherence and invasion of the human small intestinal epithelial cell line Int407. Twelve of sixteen mutations were identified in genes homologous to the S. typhimurium invG and prgH genes, which are known to be involved in the type III secretion pathway of virulence proteins. Two additional insertions were identified in genes sharing homology with the cpxA and damX genes from Escherichia coli K-12, and two uncharacterized invasion-deficient mutants were nonmotile. Gene expression of TnphoA fusions was examined in response to environmental stimuli. We found that the cpxA, invG, and prgH genes were induced when grown under conditions of high osmolarity (0.3 M NaCl). Expression of invG and prgH genes was optimal at pH 6.5 and strongly reduced at low pH (5.0). Transcription of both invG and prgH TnphoA gene fusions was initiated during the late logarithmic growth phase and was induced under anaerobic conditions. Finally, we show that both invG and prgH genes appear to be regulated by DNA supercoiling, a mechanism influenced by environmental factors. These results are the first to demonstrate that in S. typhi, (i) the prgH and cpxA genes are osmoregulated, (ii) the invG gene is induced under low oxygen conditions, (iii) the invG gene is pH regulated and growth phase dependent, and (iv) the prgH gene appears to be regulated by DNA supercoiling. Since our experimental conditions were designed to mimic the in vivo environmental milieu, our results suggest that specific environmental conditions act as signals to induce the expression of S. typhi invasion genes.
DNA Transposable Elements↗
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.
Animals↗
Salmonella typhi stimulation of human intestinal epithelial cells induces secretion of epithelial cell-derived interleukin-6.
Interleukin 6 (IL-6) is a multifunctional cytokine that has been shown to be associated with both systemic and tissue-specific responses within the host. Moreover, IL-6 is produced by both lymphoid and nonlymphoid cells and has been identified as a growth-inducing, growth-inhibiting, and differentiation-inducing factor for these cells. Recent studies of uropathogenic and upper respiratory pathogens have suggested that epithelial cell-derived IL-6 plays a role in mucosal host-parasite interactions. Since many mucosal enteric pathogens enter the host through the epithelial cells of the distal small intestine, a role for intestinal epithelial cell-derived IL-6 in the initial interaction between bacteria and host might also be predicted. However, no studies to date have determined whether the interaction of any bacteria with the epithelial cells that line the small intestine of the host can induce IL-6. To address this issue, we have established an in vitro model to evaluate the capacity of the gram-negative bacterium Salmonella typhi to induce IL-6 in the small intestine epithelial cell line Int407 and in other intestinal epithelial cell lines. The results demonstrate that both wild-type and live, attenuated S. typhi vaccine strains induce small and large intestine epithelial cells to secrete IL-6, and kinetic analysis suggests that IL-6 may be one of the earliest responses following adherence and invasion of enteric organisms. Thus, these studies suggest a physiologic role for epithelial cell-derived IL-6 in the initial interactions between host and bacterium in the small intestine.
Bacterial Vaccines↗
Osmolarity and growth phase overlap in regulation of Salmonella typhi adherence to and invasion of human intestinal cells.
The study of the effects of osmolarity and growth phase on Salmonella typhi adherence to and invasion of Henle 407 epithelial cells provides the first evidence of a clear overlap between these two environmental stimuli. High-osmolarity conditions are required in the late-log phase for optimum induction of the adherent and invasive phenotypes.
Bacterial Adhesion↗
Modulation of mouse complement receptors 1 and 2 suppresses antibody responses in vivo.
A mAb, 7G6, that binds to mouse CR1 and CR2 and down-modulates their expression on splenic B cells in vivo, was used to determine whether a decrease in CR1 and CR2 expression affects antibody responses to different T-dependent and T-independent Ag. Injection of mice with the mAb 7G6 prior to immunization with FITC haptenated Salmonella typhimurium (SH5771), Salmonella montevideo (SH5770), SRBC, or Ficoll dramatically decreased subsequent antibody responses to FITC. Although both IgM and IgG primary antibody responses were affected similarly, the antibody levels were most inhibited during early phases of the response. In contrast, down-modulation of the CR did not affect memory antibody responses, because injection of mice with 7G6 before a second immunization with FITC-SH5771 had no effect on subsequent anti-FITC antibody production. Moreover, polyclonal in vivo activation of the mouse immune system by anti-mouse IgD antibodies was not affected by previous administration of 7G6, because anti-IgD-induced increases in Ia expression and serum IgG1 levels were not affected. Taken together, these observations suggest that CR1 and CR2 may play an important role in enhancing primary antibody responses to many T-dependent and T-independent Ag and may contribute to a host's response to naturally occurring antigens such as bacteria.
Animals↗
Altered expression of the Salmonella typhimurium-specific B-cell repertoire in mice chronically treated with antibodies to immunoglobulin D.
Using a modification of the splenic focus assay, we analyzed the Salmonella typhimurium-specific B-cell repertoire in salmonella-susceptible BALB/c mice. Although these mice normally succumbed to salmonella infection before antibody was produced, they appeared to have splenic S. typhimurium-specific B-cell precursors that could be activated to differentiate and secrete antibody in a manner which was quantitatively and qualitatively identical to that of salmonella-resistant mouse strains. We also analyzed the primary S. typhimurium-specific B-cell repertoire in BALB/c mice that had been chronically treated with antibodies to immunoglobulin D (IgD) and therefore had no surface IgD-positive B cells. Although the frequency of S. typhimurium-specific precursors in these mice was similar to that of control mice, there was an apparent alteration in the isotype distribution pattern in anti-IgD-treated mice. Control mice generated a significantly greater proportion of IgG-secreting clones than did anti-IgD-treated mice. In addition, a greater proportion of S. typhimurium-specific clones from control mice secreted IgG2 than secreted IgG1, and those clones that secreted IgG2 but not IgM, IgG3, or IgG1 were greater than 20-fold more common in control than in anti-IgD-treated mice. Finally, we analyzed the immune response of control and anti-IgD-treated mice to a live avirulent vaccine, S. typhimurium SL3235. Although both groups were protected after challenge with a live virulent S. typhimurium strain, only the control mice made serum antibodies to this vaccine. Taken together, these results show that (i) salmonella-susceptible BALB/c mice have S. typhimurium-specific B cells, (ii) the S. typhimurium-specific B cells in anti-IgD-treated mice may have a restricted capacity to switch heavy-chain classes, (iii) the similarity observed in the frequency of the S. typhimurium-specific precursors for these two groups of BALB/c mice is not reflected in the serum, and (iv) the failure of anti-IgD-treated mice to generate a serum antibody response to SL3235 in the face of complete protection suggests that this model may be used to study cell-mediated immune mechanisms in the apparent absence of humoral immunity.
Animals↗
The diversity of the secondary Salmonella typhimurium-specific B cell repertoire.
This report describes the first analysis of the expressed B cell repertoire specific for a bacterium. In this study, responses to an acetone-killed and dried preparation of Salmonella typhimurium strain TML (AKD-TML) are described. The results show that AKD-TML can stimulate splenic B cells from primed CBA/Ca mice over a wide dose range. The average frequency of secondary TML-specific B cells is 16.4 per 10(5) splenic B cells. This frequency is similar to that observed for another complex, natural antigen, the hemagglutinin of influenza virus. The majority of all secondary TML-specific B cells (greater than 70%) secrete immunoglobulin M, but most of these clones also secrete other isotypes of which immunoglobulins G2 and A are the most prevalent. Analysis of the specificity of secondary TML-specific B cells showed that the vast majority of these B cells were specific for the lipopolysaccharide (LPS) molecule. Moreover, fine specificity analysis demonstrated that approximately two-thirds of these anti-LPS-specific B cell clones are directed against the core polysaccharides or lipid A regions of the LPS molecule, while only about one-third are directed toward the O antigen region. Since anti-S. typhimurium serum antibodies are directed primarily against the O antigens, these studies suggest that the serum levels of antibodies to a given epitope on a bacterial antigen may not be a true reflection of the expressed B cell repertoire when analyzed at the single B cell level. These studies also suggest that the role of antibodies to lipid A molecules in the development of protective immunity to S. typhimurium be reevaluated.
Animals↗
Clonal analysis of primary B cells responsive to the pathogenic bacterium Salmonella typhimurium.
In the present study, a modification of the splenic focus system is used to analyze the S. typhimurium strain TML (TML)-specific B cell repertoire. The results show that the frequency of primary TML-specific splenic B cells in CBA/Ca mice is approximately 1 per 10(5) B cells and less than 30% of these B cells are specific for LPS. In contrast, the frequency of memory TML-specific cells is approximately 1 per 5-8 X 10(3) splenic B cells and greater than 95% of these B cells are specific for LPS. These results suggest that the frequency of primary TML-specific B cells is extremely low and that it expands 15-20-fold after antigen exposure. It is interesting that less than 30% of the primary B cells are specific for the LPS molecule since it is considered to be the major antigenic determinant on Salmonella organisms. Furthermore, the majority of the LPS-specific anti-TML antibody-producing clones are directed against the LPS O antigen region. Conversely, more than half to two-thirds of the memory LPS-specific anti-TML B cell clones are directed against the KDO or lipid A region of the LPS molecule. These results indicate that the preferential expansion of LPS-specific B cell clones observed after immunization resides primarily in the B cell subsets responsive to the KDO/lipid A moieties on the LPS molecule. Finally, unlike B cell responses to chemically defined antigens, TML stimulates very little IgG1 antibody. IgG2 and IgA isotypes appear to play a predominant role in anti-TML antibody responses, although all H chain classes are produced to some extent. Collectively, these findings are consistent with the responses reported for two other natural antigens, HA and PC. Hence, the pattern of stimulation by infectious agents, such as S. typhimurium, appears to be distinct from that of synthetic antigens. Thus, the studies presented herein have begun to provide insights into those subsets of B cells responsive to S. typhimurium and other infectious disease organisms.
Animals↗
Antibody-defective, genetically susceptible CBA/N mice have an altered Salmonella typhimurium-specific B cell repertoire.
CBA/N mice, which express the X-linked immunodeficiency gene xid, are susceptible to Salmonella typhimurium. The basis for this susceptibility is currently unknown. However, previous studies (10) from this laboratory have provided evidence that susceptibility may be due to a defective anti-S. typhimurium antibody response. In that report we hypothesized that the defective antibody response may be a reflection of an altered S. typhimurium-specific B cell repertoire. In the studies described here, we have investigated this hypothesis using a modification of the in vitro splenic focus system. The frequency and characteristics of salmonella-specific B cells in normal, innately resistant, CBA/Ca mice have been compared with those of salmonella-susceptible, anti-S. typhimurium antibody-defective CBA/N mice. The results show that CBA/N mice express no primary or secondary S. typhimurium-specific B cell precursors after stimulation with an acetone-killed and dried (AKD) preparation of S. typhimurium strain TML. However, after three immunizations, the CBA/N tertiary frequency of 15.4 per 10(6) splenic B cells was similar to the primary precursor frequency in immunologically normal CBA/Ca mice, but 23-fold lower than the tertiary precursor frequency in CBA/Ca control mice. Moreover, CBA/N mice had an altered isotype distribution pattern after stimulation with AKD-TML. Greater than 70% of the tertiary CBA/N TML-specific B cells secreted IgG2, in contrast to either nonimmune or primed control mice. In addition, 80% of the CBA/N TML-specific B cells secreted only a single isotype, whereas the majority of B cells from primed normal mice secreted multiple isotypes. Fine specificity analysis of the TML-specific B cells indicated that the array of antigenic determinants to which CBA/N B cells could respond was restricted. Although the majority of primed CBA/Ca and primed CBA/N B cells were specific for LPS, the fine specificity pattern exhibited by CBA/N B cells was similar to that observed in unprimed normal mice, i.e., the vast majority were specific for the O antigen region of the LPS molecule. In contrast, a major portion of the LPS-specific B cells in primed CBA/Ca mice were directed against the KDO/lipid A region of the LPS molecule. Therefore, it appears that CBA/N mice lack or are unable to stimulate the B cell subset that predominates in primed, normal mice. Taken together, these studies indicate that the basis for susceptibility of CBA/N mice to S. typhimurium is multifactorial and suggests that the inability of some animals to respond to some infectious agents may be related to holes in their B cell repertoire.
Animals↗