Campylobacter fetus sap inversion occurs in the absence of RecA function

Phase variation of Campylobacter fetus surface layer proteins (SLPs) occurs by inversion of a 6.2-kb DNA segment containing the unique sap promoter, permitting expression of a single SLP-encoding gene. Previous work has shown that the C. fetus sap inversion system is RecA dependent. When we challenged a pregnant ewe with a recA mutant of wild-type C. fetus (strain 97-211) that expressed the 97-kDa SLP, 15 of the 16 ovine-passaged isolates expressed the 97-kDa protein. However, one strain (97-209) expressed a 127-kDa SLP, suggesting that chromosomal rearrangement may have occurred to enable SLP switching. Lack of RecA function in strains 97-211 and 97-209 was confirmed by their sensitivity to the DNA-damaging agent methyl methanesulfonate. Southern hybridization and PCR of these strains indicated that the aphA insertion into recA was stably present. However, Southern hybridizations demonstrated that in strain 97-209 inversion had occurred in the sap locus. PCR data confirmed inversion of the 6.2-kb DNA element and indicated that in these recA mutants the sap inversion frequency is reduced by 2 to 3 log(10) units compared to that in the wild type. Thus, although the major sap inversion pathway in C. fetus is RecA dependent, alternative lower-frequency, RecA-independent inversion mechanisms exist.     

Role of S-layer protein antigenic diversity in the immune responses of sheep experimentally challenged with Campylobacter fetus subsp. fetus

Surface layer proteins (SLPs) are essential for induction of abortion by Campylobacter fetus subsp. fetus in experimentally challenged ewes. These proteins are encoded by multiple sap genes and vary in size and antigenicity. The role of SLP antigenic variation during experimental ovine infection was investigated. Following subcutaneous challenge, the SLPs were highly antigenic, and antibodies were detected in serum, milk, bile, and urine. Fecal anti-SLP antibodies were detected only in animals challenged orally. Ewes challenged with wild-type strain 23D with variable SLPs developed detectable circulating anti-SLP immunoglobulin G (IgG) antibodies by 2 weeks postchallenge. In contrast, ewes challenged with mutants of 23D that had fixed expression of a single SLP developed antibodies within 1 week postchallenge, suggesting that antigenic variation in SLPs may delay the host antibody response. Although not statistically significant, the data from challenge experiments in which vaccinated ewes were used suggested that SLP-expressing vaccines could protect animals from abortion and that this effect was independent of the SLP expressed, indicating involvement of conserved epitopes in the SLP. The conserved 184-amino-acid N-terminal region of the SLP, identified from previously published sequences, was epitope mapped with rabbit anti-SLP antisera by using overlapping synthetic 20-mer peptides. Two putative epitopes were identified at amino acids 81 to 110 and 141 to 160. Amino acids 81 to 100 also bound serum IgG antibodies from experimentally challenged sheep. Conserved antigenic regions of the SLP that induce protective immune responses may enable development of synthetic vaccine candidates for C. fetus subsp. fetus-associated ovine abortion.     

Evidence that the Campylobacter fetus sap locus is an ancient genomic constituent with origins before mammals and reptiles diverged

Campylobacter fetus bacteria, isolated from both mammals and reptiles, may be either subsp. fetus or subsp. venerealis and either serotype A or serotype B. Surface layer proteins, expressed and secreted by genes in the sap locus, play an important role in C. fetus virulence. To assess whether the sap locus represents a pathogenicity island and to gain further insights into C. fetus evolution, we examined several C. fetus genes in 18 isolates. All of the isolates had 5 to 9 sapA or sapB homologs. One strain (85-387) possessed both sapA and sapB homologs, suggesting a recombinational event in the sap locus between sapA and sapB strains. When we amplified and analyzed nucleotide sequences from portions of housekeeping gene recA (501 bp) and sapD (450 bp), a part of the 6-kb sap invertible element, the phylogenies of the genes were highly parallel. Among the 15 isolates from mammals, serotype A and serotype B strains generally had consistent positions. The fact that the serotype A C. fetus subsp. fetus and subsp. venerealis strains were on the same branch suggests that their differentiation occurred after the type A-type B split. Isolates from mammals and reptiles formed two distinct tight phylogenetic clusters that were well separated. Sequence analysis of 16S rRNA showed that the reptile strains form a distinct phylotype between mammalian C. fetus and Campylobacter hyointestinalis. The phylogenies and sequence results showing that sapD and recA have similar G + C contents and substitution rates suggest that the sap locus is not a pathogenicity island but rather is an ancient constituent of the C. fetus genome, integral to its biology.     

Conservation and diversity of sap homologues and their organization among Campylobacter fetus isolates

Campylobacter fetus surface layer proteins (SLPs), encoded by sapA homologues, are important in virulence. In wild-type C. fetus strain 23D, all eight sapA homologues are located in the 54-kb sap island, and SLP expression reflects the position of a unique sapA promoter in relation to the sapA homologues. The extensive homologies in the sap island include both direct and inverted repeats, which allow DNA rearrangements, deletion, or duplication; these elements confer substantial potential for genomic plasticity. To better understand C. fetus sap island diversity and variation mechanisms, we investigated the organization and distribution of sapA homologues among 18 C. fetus strains of different subspecies, serotypes, and origins. For all type A strains, the boundaries of the sap island were relatively consistent. A 187-bp noncoding DNA insertion near the upstream boundary of the sap island was found in two of three reptile strains studied. The sapA homologue profiles were strain specific, and six new sapA homologues were recognized. Several homologues from reptile strains are remarkably conserved in relation to their corresponding mammalian homologues. In total, the observed differences suggest that the sap island has evolved differing genotypes that are plastic, perhaps enabling colonization of varied niches, in addition to antigenic variation.     

Surface array proteins of Campylobacter fetus block lectin-mediated binding to type A lipopolysaccharide.

Campylobacter fetus strains with type A lipopolysaccharide (LPS) and a surface array protein layer (S+) have been found to be pathogenic in humans and animals. Spontaneous laboratory mutants that lack surface array proteins (S-) are sensitive to the bactericidal activity of normal human serum. The ability of lectins to determine the presence of the S-layer and differentiate LPS type was assessed. We screened 14 lectins and found 3 (wheat germ agglutinin, Bandeiraea simplicifolia II, and Helix pomatia agglutinin) that agglutinated S- C. fetus strains with type A LPS but not S- strains with type B or type C LPS or S+ strains. However, the S+ type A strains were agglutinated after sequential water extraction, heat, or pronase treatment, all of which remove the S-layer, whereas there was no effect on the control strains. Specific carbohydrates for each lectin and purified LPS from a type A C. fetus strain specifically inhibited agglutination of an S- type A strain. In a direct enzyme-linked lectin assay, binding to the S- type A LPS strain was significantly greater than binding to the S+ strain (P = 0.01) or to a Campylobacter jejuni strain (P = 0.008). Consequently, these results indicate that the three lectins bind to the O side chains of C. fetus type A LPS but that the presence of the S-layer on intact cells blocks binding.     

Correlation between molecular size of the surface array protein and morphology and antigenicity of the Campylobacter fetus S layer.

The correlation between the molecular size of the surface layer protein (S protein) and both structure and antigenicity of the Campylobacter fetus surface layer (S layer) was investigated in several clinical strains and their spontaneous variants which produce S proteins of molecular weights (MW) different from those of the parents. Only three molecular sizes of the S proteins were observed (98, 127, and 149 kDa) in the parental and variant strains. Immunologically, the 98-kDa protein and the 149-kDa protein but not the 127-kDa protein were cross-reactive. Freeze-etching analysis showed that the 98-kDa S protein formed a hexagonal arrangement with a 24-nm center-to-center space and that the S proteins with larger MW (127 or 149 kDa) formed tetragonal ones with an 8-nm center-to-center space. Thus, the MW changes of the S proteins seen in the variant strains were associated with both morphological and antigenic changes in S layer. These observations support the hypothesis that the pattern and antigenicity of the C. fetus S layer is determined by the particular type of S protein. Furthermore, the presence of the two different S layer patterns on a single bacterial cell indicates that multiple S proteins can be produced and expressed in a single cell.     

Intestinal colonization of neonatal animals by Campylobacter fetus subsp. jejuni.

Neonatal mice (2.3 to 2.8 g) were inoculated intragastrically with different human isolates of Campylobacter fetus subsp. jejuni. At weekly intervals thereafter, mice were sacrificed and dilution plate counts were performed on segments of the gastrointestinal tract. Mice were uniformly colonized by some strains for 2 weeks, whereas other strains were being cleared at that time. One strain (BO216) persisted in some mice for 3 weeks. The greatest number of organisms (10(7)) was recovered from the cecum and large intestine. The small intestine had from 10(2) to 10(5) colony-forming units. Colonization of the stomach was not found consistently. One strain killed 13% of the infected mice. Deaths occurred between 1 and 5 days postinfection. Two other strains killed a smaller percentage of challenged animals, and two additional strains killed none. Retarded weight gain was noticed in some, but not all, of the infected mice. The intestines of neonatal rats and rabbits were colonized much the same as those of mice, whereas hamsters were resistant to colonization. Preweanling mice, up to about 6.5 to 7.0 g, could be colonized with C. fetus subsp. jejuni after intragastric challenge, but weanling mice of larger weight (9.8 g) and young adult mice (18.3 g) could not. Scanning electron photomicrographs of the lower ileum showed campylobacters in and below the dried mucous gel that lines the intestines. The use of this model for additional studies is discussed.     

Veterinary and medical aspects of abortion in Danish sheep

The Danish sheep population totals around 144,000 animals, but little is known of the causes and prevalance of diseases. This study focuses on the causes of abortion in Danish sheep. During one breeding season, aborted foetuses and stillbirths with signs of intrauterine death or malformation were submitted for laboratory examination from a population of 3,758 breeding ewes. Samples from 24 incidents of abortion and 21 ewes delivering malformed lambs or lambs with ante partum decomposition were submitted. A specific aetiology was established in 66.7% and 14.3% of the cases, respectively. Bacterial pathogens were the most prevalent cause of abortion. Several of the abortifacients were zoonotic microorganisms, for example Listeria monocytogenes, Campylobacter fetus subsp. fetus, Yersinia pseudotuberculosis and Toxoplasma gondii. The identified microorganisms probably represent the most common causes of abortion in Danish sheep but occurrence in Denmark of other pathogens such as Coxiella burnetii and Chlamydophila abortus cannot be excluded. Due to the high prevalence of zoonotic microorganisms, precautions must be taken in handling abortions or assisting lambing, especially for pregnant women.     

Virus-induced abortion. Studies of equine herpesvirus 1 (abortion virus) in hamsters.

A hamster-adapted strain of equine herpesvirus-1 (equine abortion virus) caused severe hepatic degeneration in both pregnant and nonpregnant hamsters and, in addition, regularly induced abortion in pregnant hamsters inoculated at midgestation. In nonpregnant hamsters, the only consistently affected organ was the liver despite a prolonged viremia. Newborn animals usually died 1 to 2 days after inoculation; adults died 5 to 9 days after inoculation. In pregnant hamsters, the virus had a tropism for the placenta as well as the liver. The placental infection was confined almost exclusively to one cell type in the fetal portion of the placenta: the trophoblast cells of the syncytiotrophoblast zone. Necrosis of this zone led to fetal death and abortion. Infection of the fetus did not occur.     

Examination of the early conceptus

The pathologist encounters the early, first-trimester conceptus as a product of spontaneous or surgical abortion or as a specimen from ectopic, usually tubal, gestation. In the vast majority of cases of spontaneous abortion, the embryo/fetus has been dead for 1 week to several weeks and is most often lost in the process of uterine emptying; the placenta, accordingly, stands as the main "witness" of the abortive process. The fact of embryonic/fetal death is established and dated through an interpretation of gross and microscopic changes in the villous stroma, including its vessels and the embryonic erythrocytes therein. Elective abortions performed for social reasons occasionally yield abnormal findings that suggest gestations otherwise destined to eventual spontaneous abortion. Other therapeutic abortions are indicated by abnormal sonographic or cytogenetic findings that may be correlated with the morphologic features of the evacuated conceptus. Cytogenetic abnormalities correlate to a high degree with embryonic growth disorganization and with early death, usually before the fetal stage (30 mm) is reached. Adequate sampling of the placenta, its membranes, and of the embryo/fetus is recommended. For cytogenetic studies, clean, viable specimens must be obtained; these are most often required in cases of habitual abortion.     

A mutation in the sap operon attenuates survival of nontypeable Haemophilus influenzae in a chinchilla model of otitis media

Bacteria have evolved strategies to resist killing by antimicrobial peptides (APs), important effectors of innate immunity. The sap (sensitivity to antimicrobial peptides) operon confers resistance to AP-mediated killing of Salmonella. We have recently shown that sapA gene expression is upregulated in the middle ear in a chinchilla model of nontypeable Haemophilus influenzae (NTHI)-induced otitis media. Based on these findings, we constructed an NTHI strain containing a Lux reporter plasmid driven by the sapA promoter and demonstrated early yet transient expression of the sap operon within sites of the chinchilla upper airway upon infection. We hypothesized that the sap operon products mediate NTHI resistance to APs. In order to test this hypothesis, we constructed a nonpolar mutation in the sapA gene of NTHI strain 86-028NP, a low-passage-number clinical isolate. The sapA mutant was approximately eightfold more sensitive than the parent strain to killing by recombinant chinchilla beta-defensin 1. We then assessed the ability of this mutant to both colonize and cause otitis media in chinchillas. The sapA mutant was significantly attenuated compared to the parent strain in its ability to survive in both the nasopharynx and the middle ear of the chinchilla. In addition, the mutant was impaired in its ability to compete with the parent strain in a dual-strain challenge model of infection. Our results indicate that the products of the sap operon are important for resisting the activity of APs and may regulate, in part, the balance between normal carriage and disease caused by NTHI.     

Lack of the extracellular 19-kilodalton fibrinogen-binding protein from Staphylococcus aureus decreases virulence in experimental wound infection.

A mutant deficient for the 19-kDa extracellular fibrinogen-binding protein (Fib) from Staphylococcus aureus has been constructed. The gene was inactivated by allele replacement. A 2.0-kb fragment from transposon Tn4001 carrying the gene for gentamicin resistance was inserted into the gene encoding Fib (fib). The genotype was verified by PCR analysis, and the loss of Fib was demonstrated by Western blotting (immunoblotting). The mutation has not altered the ability of the strain to bind to fibrinogen or fibronectin compared with that of the isogenic parental strain, FDA486. The mutant, designated K4.3, was compared with strain FDA486 in a wound infection model in rats. Sixty-eight percent of the rats challenged with parental strain FDA486 developed severe clinical signs of wound infection, whereas only 29% of the animals challenged with isogenic mutant K4.3 showed severe symptoms (P < 0.01). The weight loss of animals infected with the wild type was also significantly different from that of animals infected with the mutant strain. The result demonstrates that the extracellular 19-kDa fibrinogen-binding protein from S. aureus contributes to the virulence in wound infection and delays the healing process.     

MtuA,a lipoprotein receptor antigen from Streptococcus uberis,is responsible for acquisition of manganese during growth in milk and is essential for infection of the lactating bovine mammary gland

A mutant strain of Streptococcus uberis (AJS001) that was unable to grow in bovine milk was isolated following random insertional mutagenesis. The level of growth in milk was restored to that of the parental strain (strain 0140J) following addition of MnSO(4) but not following addition of other metal ions. The mutant contained a single insertion within mtuA, a homologue of mtsA and psaA, which encode metal-binding proteins in Streptococcus pyogenes and Streptococcus pneumoniae, respectively. Strain AJS001 was unable to infect any of eight quarters on four dairy cows following intramammary challenge with 10(5) CFU. Bacteria were never recovered directly from milk of these animals but were detected following enrichment in Todd-Hewitt broth in three of eight milk samples obtained within 24 h of challenge. The animals showed no inflammatory response and no signs of mastitis. Three mammary quarters on two different animals simultaneously challenged with 600 CFU of the parental strain, strain 0140J, became colonized, shed high numbers of S. uberis organisms in milk, displayed a marked inflammatory response to infection, and showed overt signs of mastitis. These data indicate that mtuA was required for efficient uptake of Mn(2+) during growth in bovine milk and infection of the lactating bovine mammary gland.     

Surface Structure, Hydrophobicity, Phagocytosis, and Adherence to Matrix Proteins of Bacillus cereus Cells with and without the Crystalline Surface Protein Layer

Nonopsonic phagocytosis of Bacillus cereus by human polymorphonuclear leukocytes (PMNs) with particular attention to bacterial surface properties and structure was studied. Two reference strains (ATCC 14579^T and ATCC 4342) and two clinical isolates (OH599 and OH600) from periodontal and endodontic infections were assessed for adherence to matrix proteins, such as type I collagen, fibronectin, laminin, and fibrinogen. One-day-old cultures of strains OH599 and OH600 were readily ingested by PMNs in the absence of opsonins, while cells from 6-day-old cultures were resistant. Both young and old cultures of the reference strains of B. cereus were resistant to PMN ingestion. Preincubation of PMNs with the phagocytosis-resistant strains of B. cereus did not affect the phagocytosis of the sensitive strain. Negatively stained cells of OH599 and OH600 studied by electron microscopy had a crystalline protein layer on the cell surface. In thin-sectioned cells of older cultures (3 to 6 days old), the S-layer was observed to peel off from the cells. No S-layer was detected on the reference strains. Extraction of cells with detergent followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major 97-kDa protein from the strains OH599 and OH600 but only a weak 97-kDa band from the reference strain ATCC 4342. One-day-old cultures of the clinical strains (hydrophobicity, 5.9 to 6.0%) showed strong binding to type I collagen, laminin, and fibronectin. In contrast, reference strains (hydrophobicity, −1.0 to 4.2%) as well as 6-day-old cultures of clinical strains (hydrophobicity, 19.0 to 53.0%) bound in only low numbers to the proteins. Gold-labelled biotinylated fibronectin was localized on the S-layer on the cell surface as well as on fragments of S-layer peeling off the cells of a 6-day-old culture of B. cereus OH599. Lactose, fibronectin, laminin, and antibodies against the S-protein reduced binding to laminin but not to fibronectin. Heating the cells at 84°C totally abolished binding to both proteins. Benzamidine, a noncompetitive serine protease inhibitor, strongly inhibited binding to fibronectin whereas binding to laminin was increased. Overall, the results indicate that changes in the surface structure, evidently involving the S-layer, during growth of the clinical strains of B. cereus cause a shift from susceptibility to PMN ingestion and strong binding to matrix and basement membrane proteins. Furthermore, it seems that binding to laminin is mediated by the S-protein while binding to fibronectin is dependent on active protease evidently attached to the S-layer.     

Humoral and cell-mediated immunity in mice to a 17-kilodalton lipoprotein of Francisella tularensis expressed by Salmonella typhimurium.

A 17-kDa lipoprotein, TUL4, of the facultative intracellular bacterium Francisella tularensis is one of several membrane proteins that induce an in vitro response in T cells from F. tularensis-primed humans. A DNA fragment of the live vaccine strain F. tularensis LVS encoding TUL4 was cloned into Salmonella typhimurium chi 4072, an attenuated delta cya delta crp mutant. Expression of the protein by the recombinant S. typhimurium chi 4072 (pTUL4-15) was maintained after passage in BALB/cJ mice. When mice were immunized with S. typhimurium chi 4072(pTUL4-15), some animals showed an antibody response and a T-cell response to TUL4. When the immunized mice were challenged with the live vaccine strain F. tularensis LVS, bacterial counts in the liver and spleen were lower than in animals immunized with S. typhimurium chi 4072. Immunization with F. tularensis LVS caused a much stronger protection against the challenge than did immunization with S. typhimurium chi 4072(pTUL4-15). The present study demonstrated that the 17-kDa lipoprotein TUL4 of F. tularensis is involved in a protective immunity to tularemia. Possibly, several T-cell-reactive proteins of the organism have to contribute for optimal protection to be achieved.     

Significance of flagella in colonization resistance of rabbits immunized with Campylobacter spp.

Cross-protection among different Lior and Penner serogroups of Campylobacter spp. was studied. Rabbits were orally immunized by gastric feeding with Campylobacter spp., and 27 to 30 days later, they were challenged with matched or unmatched serogroups by the removable intestinal tie adult rabbit diarrhea (RITARD) procedure. When immunized animals were challenged with different Lior serotypes, no protection against colonization was seen; however, when challenged with homologous Lior serogroups, protection was demonstrated. Immune animals were colonized for an average of 1 day or less versus at least 6 days for nonimmune animals. Rabbits challenged with matched Penner-unmatched Lior strains showed only marginal protection. Our study also demonstrated that flagella are important in initiating colonization and eliciting protective immunity. Campylobacter coli VC167B3, an isogenic, nonflagellated mutant, did not colonize rabbits regardless of the route of administration. Single feeding of the mutant strain did not protect the host, whereas three feedings, 48 h apart, resulted in complete protection against the flagellated parent strain. When mutant strain immunized rabbits were challenged with other strains of the same Lior serotype, marginal protection was obtained. Immunogold labeling indicated that there is one or more antigens on the cell surface of the nonflagellated mutant which reacts with a polyclonal antiserum from organisms of the same Lior serogroup. These data implicated the flagellum as the cross-strain protective component of the Lior antigen complex.     

Coxsackievirus B infection in pregnant mice and transplacental infection of the fetus.

Direct instillation of coxsackievirus B1 into the gastrointestinal tracts of albino mice caused viremia in more than 85% of the animals within 1 day. In pregnant mice infected early in gestation (7 days), the geometric mean titer of virus in the blood was lower (P = 0.02) and the duration of viremia was shorter (P = 0.07) than in nonpregnant female mice, but infection of the heart, liver, and uterus did not differ on each of 5 days after infection. Although transplacental infection of the placenta or fetus or both occurred, the high spontaneous abortion rate (48%) obviated comparison of transplacental infection in these mice with mice infected later in gestation. Pregnant mice infected in the third trimester had significantly greater geometric mean titers of virus in the blood, heart, liver, and uterus, and infection persisted longer than in nonpregnant mice (P = 0.04). A very high geometric mean titer of virus was recovered from the uteri of these mice for 3 days after infection, whereas simultaneous geometric mean titers of virus in the placentas and fetuses were lower. In the majority of third trimester pregnant mice, virus was found in low titers in the fetuses at 2 and 3 days after maternal infection, and virus was not detected after day 3. We conclude that coxsackievirus B1 infection in late gestational pregnant mice is more severe than in mice at earlier gestational stages and in nonpregnant mice and that transplacental infection of the fetus occurs transiently during maternal infection. This model will prove useful in the study of perinatal enterovirus infection and in examination of the numerous factors that may influence outcome of infection of perinatally infected newborn infants.     

Pregnancy failure following vaginal infection of sheep with Chlamydia psittaci prior to breeding.

Enzootic abortion in sheep, caused by Chlamydia psittaci, has been associated with pregnancy failure in most sheep-producing countries. Late-term abortions or the birth of weak low-birth-weight lambs occurred following primary C. psittaci infection in pregnant ewes. However, the mode by which C. psittaci can be transmitted among sheep has not been established. The present study was designed to determine whether the vaginal tracts of nonpregnant ewes were susceptible to C. psittaci infection and whether such infections had an impact during the next pregnancy. At day 0 of the estrus cycle, the vaginal tracts of 10 nonpregnant ewes were inoculated with C. psittaci and 10 ewes were exposed by subcutaneous injection. The ewes were bred 6 weeks postinfection. Five ewes from the vaginally infected group and four from the subcutaneously infected group were reinfected by subcutaneous injection at day 60 of gestation. Pregnancy outcomes and antibody responses to infection were compared with that of ewes that were infected with C. psittaci, either subcutaneously or intravaginally, for the first time during pregnancy and with that of noninfected control ewes. Subcutaneous infection of nonpregnant ewes did not cause subsequent pregnancy failure; rather, this provided protection against abortion following reinfection during pregnancy. As expected, abortions or the birth of weak lambs was observed in those ewes that received primary C. psittaci infection by either route during pregnancy. Similarly, abortion or the birth of weak lambs was a consequence of vaginal inoculation prior to breeding, thereby confirming the susceptibility of the vaginal mucosa to infection and demonstrating the potential for venereal transmission.     

The role of the eaeA gene in diarrhea and neurological complications in a gnotobiotic piglet model of enterohemorrhagic Escherichia coli infection.

We reported previously that mutation of the chromosomal gene eaeA from enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 prevented bacterial attachment in vivo. Attachment was restored when the EHEC or enteropathogenic E. coli (EPEC) eaeA gene was introduced into the mutant on a plasmid. In this communication we have compared in gnotobiotic piglets the pathogenicities of wild-type O157:H7 strain 86-24 and its eaeA mutant UMD619 with those of the two plasmid-complemented strains expressing IntiminO157 (EHEC) and IntiminO127 (EPEC). 86-24 colonized the surface and glandular epithelium of the large intestine and induced diarrhea, while UMD619 did not colonize any intestinal site and induced little or no diarrhea. Surprisingly, strain UMD619 expressing IntiminO127 behaved in pigs more like EPEC than EHEC strains; it colonized the distal half of the small intestine and the surface of the large intestine, inducing serious diarrhea. In contrast, strain UMD619 expressing IntiminO157 colonized the colon extremely poorly, inducing little or no diarrhea. While only the two strains causing extensive attachment--86-24 and UMD619 expressing IntiminO127--induced diarrhea, neurological symptoms attributed to Shiga-like toxin II occurred equally in all four groups of animals. The intimate bacterial attachment and mucosal damage were not a prerequisite for Shiga-like toxin II translocation from the gut lumen into the circulation. IntiminO127 appears not only to facilitate intimate attachment to cells but also to influence the site of intestinal colonization and other characteristics of EPEC infection.     

Development and characterization of recA mutants of Campylobacter jejuni for inclusion in attenuated vaccines.

Isogenic recA mutants of Campylobacter jejuni have been constructed for evaluation of their usefulness in attenuated vaccines against this major worldwide cause of diarrhea. The recA+ gene of C. jejuni 81-176 was cloned by using degenerate primers to conserved regions of other RecA proteins in a PCR. The C. jejuni recA+ gene encodes a predicted protein with an M(r) of 37,012 with high sequence similarity to other RecA proteins. The termination codon of the recA+ gene overlaps with the initiation codon of another open reading frame which encodes a predicted protein which has > 50% identity with the N terminus of the Escherichia coli enolase protein. A kanamycin resistance gene was inserted into the cloned recA+ gene in E. coli and returned to C. jejuni VC83 by natural transformation, resulting in allelic replacement of the wild-type recA gene. The resulting VC83 recA mutant displayed increased sensitivity to UV light and a defect in generalized recombination as determined by natural transformation frequencies. The mutated recA gene was amplified from VC83 recA by PCR, and the product was used to transfer the mutation by natural transformation into C. jejuni 81-176 and 81-116, resulting in isogenic recA mutants with phenotypes similar to VC83 recA. After oral feeding, strain 81-176 recA colonized rabbits at levels comparable to wild-type 81-176 and was capable of eliciting the same degree of protection as wild-type 81-176 against subsequent homologous challenge in the RITARD (removable intestinal tie adult rabbit diarrhea) model.