Inactivation of Cryptosporidium parvum oocysts in fresh apple cider by UV irradiation.

This study evaluated the efficacy of UV irradiation on the inactivation of Cryptosporidium parvum oocysts in fresh apple cider. Cider was inoculated with oocysts and exposed to 14.32 mJ of UV irradiation/cm(2). Oocyst viability was assessed with the gamma interferon gene knockout (GKO) mouse and infant BALB/cByJ mouse models. All GKO mice challenged with UV-treated cider demonstrated no morbidity or mortality, and infant BALB/c mice challenged with treated cider were negative for the presence of C. parvum. In contrast, the GKO mice challenged with non-UV-treated inoculated cider died and the parasite was detected in the ileums of all challenged infant mice. This study shows that UV irradiation can be used to inactivate C. parvum in fresh apple cider.

New Zealand white rabbit as a nonsurgical experimental model for Salmonella enterica gastroenteritis.

Rabbits orally challenged with Salmonella enterica developed a dose-dependent diarrheal disease comparable to human salmonellosis. Viable Salmonella organisms recovered from the intestine and deep tissues indicate local and systemic infections. Therefore, results show that the rabbit can be used as a model for diarrheal disease and sequelae associated with salmonellosis.

A phase-variable capsule is involved in virulence of Campylobacter jejuni 81-176.

Campylobacter jejuni strain 81-176 (HS36, 23) synthesizes two distinct glycan structures, as visualized by immunoblotting of proteinase K-digested whole-cell preparations. A site-specific insertional mutant in the kpsM gene results in loss of expression of a high-molecular-weight (HMW) glycan (apparent Mr 26 kDa to > 85 kDa) and increased resolution of a second ladder-like glycan (apparent Mr 26-50 kDa). The kpsM mutant of 81-176 is no longer typeable in either HS23 or HS36 antisera, indicating that the HMW glycan structure is the serodeterminant of HS23 and HS36. Both the kpsM-dependent HMW glycan and the kpsM-independent ladder-like structure appear to be capsular in nature, as both are attached to phospholipid rather than lipid A. Additionally, the 81-176 kpsM gene can complement a deletion in Escherichia coli kpsM, allowing the expression of an alpha2,8 polysialic acid capsule in E. coli. Loss of the HMW glycan in 81-176 kpsM also increases the surface hydrophobicity and serum sensitivity of the bacterium. The kpsM mutant is also significantly reduced in invasion of INT407 cells and reduced in virulence in a ferret diarrhoeal disease model. The expression of the kpsM-dependent capsule undergoes phase variation at a high frequency.

Involvement of a plasmid in virulence of Campylobacter jejuni 81-176.

Campylobacter jejuni strain 81-176 contains two, previously undescribed plasmids, each of which is approximately 35 kb in size. Although one of the plasmids, termed pTet, carries a tetO gene, conjugative transfer of tetracycline resistance to another strain of C. jejuni could not be demonstrated. Partial sequence analysis of the second plasmid, pVir, revealed the presence of four open reading frames which encode proteins with significant sequence similarity to Helicobacter pylori proteins, including one encoded by the cag pathogenicity island. All four of these plasmid-encoded proteins show some level of homology to components of type IV secretion systems. Mutation of one of these plasmid genes, comB3, reduced both adherence to and invasion of INT407 cells to approximately one-third that seen with wild-type strain 81-176. Mutation of comB3 also reduced the natural transformation frequency. A mutation in a second plasmid gene, a virB11 homolog, resulted in a 6-fold reduction in adherence and an 11-fold reduction in invasion compared to the wild type. The isogenic virB11 mutant of strain 81-176 also demonstrated significantly reduced virulence in the ferret diarrheal disease model. The virB11 homolog was detected on plasmids in 6 out of 58 fresh clinical isolates of C. jejuni, suggesting that plasmids are involved in the virulence of a subset of C. jejuni pathogens.

Evaluation of a truncated recombinant flagellin subunit vaccine against Campylobacter jejuni.

A recombinant protein comprising the maltose-binding protein (MBP) of Escherichia coli fused to amino acids 5 to 337 of the FlaA flagellin of Campylobacter coli VC167 was evaluated for immunogenicity and protective efficacy against challenge by a heterologous strain of campylobacter, Campylobacter jejuni 81-176, in two murine models. The sequence of the flaA gene of strain 81-176 revealed a predicted protein which was 98.1% similar to that of VC167 FlaA over the region expressed in the fusion protein. Mice were immunized intranasally with two doses of 3 to 50 microgram of MBP-FlaA, given 8 days apart, with or without 5 microgram of the mutant E. coli heat-labile enterotoxin (LT(R192G)) as a mucosal adjuvant. The full range of MBP-FlaA doses were effective in eliciting antigen-specific serum immunoglobulin G (IgG) responses, and these responses were enhanced by adjuvant use, except in the highest dosing group. Stimulation of FlaA-specific intestinal secretory IgA (sIgA) responses required immunization with higher doses of MBP-FlaA (>/=25 microgram) or coadministration of lower doses with the adjuvant. When vaccinated mice were challenged intranasally 26 days after immunization, the best protection was seen in animals given 50 microgram of MBP-FlaA plus LT(R192G). The protective efficacies of this dose against disease symptoms and intestinal colonization were 81.1 and 84%, respectively. When mice which had been immunized with 50 microgram of MBP-FlaA plus LT(R192G) intranasally were challenged orally with 8 x 10(10), 8 x 10(9), or 8 x 10(8) cells of strain 81-176, the protective efficacies against intestinal colonization at 7 days postinfection were 71.4, 71.4, and 100%, respectively.

CheY-mediated modulation of Campylobacter jejuni virulence.

Four motile, non-adherent and non-invasive mutants of Campylobacter jejuni 81-176 generated by a site-specific insertional mutagenesis scheme were characterized at the molecular level and all contained a duplication of the same region of the chromosome. When this region was cloned from wild-type 81-176 and transferred into 81-176 on a shuttle plasmid, the same non-invasive phenotype as the original mutants was observed, suggesting that the region contained a repressor of adherence and invasion. The smallest piece of DNA identified which was capable of repressing adherence and invasion was a 0.8 kb fragment encoding the cheY gene of C.jejuni. To confirm further that CheY was responsible for the observed non-adherent and non-invasive phenotypes, the cheY gene was inserted into the arylsulfatase gene of 81-176 to generate a strain with two chromosomal copies of cheY. This diploid strain displayed the same non-adherent and non-invasive phenotype as the original mutants. Insertional inactivation of the cheY gene in 81-176 resulted in an approx. threefold increase in adherence and invasion in vitro, but this strain was unable to colonize or cause disease in animals. The diploid cheY strain, although able to colonize mice, was attenuated in a ferret disease model.

An environmentally regulated pilus-like appendage involved in Campylobacter pathogenesis.

Examination of strains of Campylobacter jejuni, Campylobacter coli, and Campylobacter fetus by electron microscopy revealed that they produced peritrichous pilus-like appendages when the bacteria were grown in the presence of bile salts. Various bile-salt supplements were used and it was found that deoxycholate and chenodeoxycholic acid caused a significant enhancement of pilus production and resulted in a highly aggregative phenotype. Morphologically, the pili were between 4 and 7 nm in width and were greater than 1 micron in length. A gene, termed pspA, which encodes a predicted protein resembling protease IV of Escherichia coli, was identified in C. jejuni strain 81-176. A site-specific insertional mutation within this gene resulted in the loss of pilus synthesis as determined by electron microscopy. Insertions upstream and downstream of the gene had no effect on pilus production. The non-piliated mutant of strain 81-176 showed no reduction in adherence to or invasion of INT 407 cells in vitro. However, this mutant, while still possessing the ability to colonize ferrets, caused significantly reduced disease symptoms in this animal model.

Identification and characterization of genes required for post-translational modification of Campylobacter coli VC167 flagellin.

Two genes have been identified in Campylobacter coli VC167 which are required for the biosynthesis of post-translational modifications on flagellin proteins. The ptmA gene encodes a protein of predicted M(r) 28,486 which shows significant homology to a family of alcohol dehydrogenases from a variety of bacteria. The ptmB gene encodes a protein of predicted M(r) 26,598 with significant homology to CMP-N-acetylneuraminic acid synthetase enzymes involved in sialic acid capsular biosynthesis in Neisseria meninigitidis and Escherichia coli K1. Site-specific mutation of either ptmA or ptmB caused loss of reactivity with antisera specific to the post-translational modifications and a change in the isoelectric focusing fingerprints relative to the parent strains. Mutation of ptmB, but not of ptmA, caused a change in apparent M(r) of the flagellin subunit in SDS-PAGE gels. The ptmA and ptmB genes are present in other strains of Campylobacter. In a rabbit model the ptmA mutant showed a reduced ability to elicit protection against subsequent challenge with heterologous strains of the same Lior serotype compared to the parental wild-type strain. This suggests that the surface-exposed post-translational modifications may play a significant role in the protective immune response.

Flagellar serotypes of Salmonella typhi in Indonesia: relationships among motility, invasiveness, and clinical illness.

While the H1-d flagellar serotype of Salmonella typhi has been found worldwide, the H1-j serotype occurs only in Indonesia. A cross-sectional survey in Indonesia compared epidemiologic, clinical, and pathogenetic characteristics of these two serotypes. S. typhi isolates were collected from patients with acute typhoid fever in four Indonesian cities. Flagellar serotype was determined by polymerase chain reaction amplification of the fliC locus of the flg gene. Of 321 isolates, 51 (15.9%) were H1-j. Patients with H1-j infection were older than those with H1-d (P < .001). Among 30 patients with known clinical outcomes, H1-j infection was associated with milder clinical illness than H1-d (P = .06). In vitro, H1-j isolates were both less motile on semi-solid agar plates (P = .004) and less invasive of HEp-2 cells (P = .002) than H1-d isolates. The association of decreased severity of illness with decreased motility and invasiveness suggests that flagellar properties are a component of S. typhi's virulence.

Isolation of motile and non-motile insertional mutants of Campylobacter jejuni: the role of motility in adherence and invasion of eukaryotic cells.

A method of insertional mutagenesis for naturally transformable organisms has been adapted from Haemophilus influenzae and applied to the study of the pathogenesis of Campylobacter jejuni. A series of kanamycin-resistant insertional mutants of C. jejuni 81-176 has been generated and screened for loss of ability to invade INT407 cells. Eight noninvasive mutants were identified which showed 18-200-fold reductions in the level of invasion compared with the parent. Three of these eight show defects in motility, and five are fully motile. The three mutants with motility defects were further characterized to evaluate the method. One mutant, K2-32, which is non-adherent and non-invasive, has an insertion of the kanamycin-resistance cassette into the flaA flagellin gene and has greatly reduced motility and a truncated flagellar filament typical of flaA mutants. The adherent non-invasive mutants K2-37 and K2-55 are phenotypically paralysed, i.e. they have a full-length flagellar filament but are non-motile. All three mutants show an aberration in flagellar structure at the point at which the filament attaches to the cell. Mutants K2-37 and K2-55 represent overlapping deletions affecting the same gene, termed pflA (paralysed flagella). This gene encodes a predicted protein of 788 amino acid residues and a molecular weight of 90,977 with no significant homology to known proteins. Site-specific insertional mutants into this open reading frame result in the same paralysed flagellar phenotype and the same invasion defects as the original mutants. The differences in adherence between the two classes of flagellar mutant suggest that flagellin can serve as a secondary adhesion, although other adhesins mediate a motility-dependent internalization process. Characterization of the mutants at the molecular level and in animal models should further contribute to our understanding of the pathogenicity of these organisms.

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.

Etiology of acute diarrhea among United States military personnel deployed to South America and west Africa.

A study of acute diarrhea was conducted from 1985 to 1987 among U.S. military personnel participating in routine shipboard exercises in South America and West Africa and ground troops deployed to coastal Ecuador. An enteropathogen was identified in 146 (51%) of 289 acute cases of diarrhea. Enterotoxigenic Escherichia coli, found in 50 (17%) patients with diarrhea, was the most commonly identified enteropathogen. Viral enteropathogens were also found in a high percentage of acute cases of diarrhea: rotavirus was detected in 11% of the patients and Norwalk virus infection in 10%. Most enteric pathogens were acquired in equal frequencies in South America and West Africa, except for rotavirus infection which was identified more often in West Africa and enteroaggregative E. coli infection which was identified more often in South America. Bacterial enteropathogens were frequently resistant to trimethoprim/sulfamethoxazole, but no resistance to quinolone drugs was observed, indicating that quinolone drugs have become important agents for the treatment of diarrhea in South America and West Africa.

Specific detection of Campylobacter jejuni and Campylobacter coli by using polymerase chain reaction.

Development of a routine detection assay for Campylobacter jejuni and Campylobacter coli in clinical specimens was undertaken by using the polymerase chain reaction (PCR). An oligonucleotide primer pair from a conserved 5' region of the flaA gene of C. coli VC167 was used to amplify a 450-bp region by PCR. The primer pair specifically detected 4 strains of C. coli and 47 strains of C. jejuni; but it did not detect strains of Campylobacter fetus, Campylobacter lari, Campylobacter upsaliensis, Campylobacter cryaerophila, Campylobacter butzleri, Campylobacter hyointestinalis, Wolinella recta, Helicobacter pylori, Escherichia coli, Shigella spp., Salmonella spp., Vibrio cholerae, Citrobacter freundii, or Aeromonas spp. By using a nonradioactively labeled probe internal to the PCR product, the assay could detect as little as 0.0062 pg of purified C. coli DNA, or the equivalent of four bacteria. In stools seeded with C. coli cells, the probe could detect between 30 and 60 bacteria per PCR assay. The assay was also successfully used to detect C. coli in rectal swab specimens from experimentally infected rabbits and C. jejuni in human stool samples.

Norfloxacin compared to trimethoprim/sulfamethoxazole for the treatment of travelers' diarrhea among U.S. military personnel deployed to South America and West Africa.

A randomized treatment trial of travelers' diarrhea was carried out among U.S. military personnel participating in routine exercises in several port cities in South America and West Africa. A 5-day, twice daily course of either norfloxacin (400 mg) or trimethoprim/sulfamethoxazole (TMP/SMX, 160/800 mg) was given to 142 volunteers. At the end of 5 days of treatment, diarrhea had resolved in 100% of 73 patients receiving norfloxacin and 97.1% (67/69) receiving TMP/SMX. A probable bacterial pathogen was determined in 44% of 142 subjects: 49% of the norfloxacin group and 39% of the TMP/SMX group. The most common pathogens detected were enterotoxigenic Escherichia coli in 20% of cases and rotavirus in 15%. Resistance to TMP/SMX was present in 20 (27%) bacterial isolates, while no resistance to norfloxacin was found. Eight of 10 patients in the TMP/SMX treatment group who had TMP/SMX-resistant bacterial enteropathogens improved clinically. Both norfloxacin and TMP/SMX were clinically effective in the treatment of travelers' diarrhea in this military population.

In vivo antigenic variation of Campylobacter flagellin.

Campylobacter coli VC167 cells producing either antigenic phase 1 (P1) or phase 2 (P2) flagellins (as determined by characteristic protein and DNA patterns) were used to infect rabbits by the removable intestinal tie-adult rabbit diarrhea (RITARD) procedure. Rabbits infected with P2 cells shed predominantly P2 cells throughout the infection; in rabbits infected with P1 cells, a transition of fecal isolates from P1 to P2 was observed.

Mucosal and systemic immunity to Campylobacter jejuni in rabbits after gastric inoculation.

The mucosal and systemic immune responses to Campylobacter jejuni were studied in rabbits receiving gastric inoculation with live organisms. A lavage procedure was used to facilitate repeated monitoring of the intestinal immune response to C. jejuni. Immunity to C. jejuni was determined by secondary challenge by using the removable intestinal tie adult rabbit diarrhea (RITARD) model and monitoring for resistance to colonization and bacteremia. Oral-gastric inoculation of normal rabbits produced a transient intestinal colonization without diarrhea. C. jejuni serotypes differed in their ability to colonize the intestines of rabbits and to stimulate primary intestinal and serum antibody responses. Animals previously colonized were resistant to recolonization and the development of bacteremia after homologous challenge by the RITARD procedure but were not resistant to heterologous challenges. Anticampylobacter intestinal and serum IgA titers before this secondary infection were the most reliable predictors of resistance to colonization and bacteremia.

Intestinal mucus gel and secretory antibody are barriers to Campylobacter jejuni adherence to INT 407 cells.

An in vitro mucus assay was developed to study the role of mucus gel and secretory immunoglobulin A (sIgA) in preventing attachment of Campylobacter jejuni to INT 407 cells. An overlay of rabbit small intestinal mucus was found to impede the attachment of C. jejuni to a monolayer of INT 407 cells. Mucus from rabbits previously colonized with C. jejuni was found to completely inhibit bacterial adherence to the underlying cells. Anti-Campylobacter sIgA was readily detected in mucus samples from previously exposed rabbits and was responsible for eliminating bacterial adherence to the INT 407 cells. This was shown by loss of inhibition after mucus absorption with Campylobacter cells. sIgA-containing mucus caused aggregation of the C. jejuni cells within the mucus layer of the assay system. Nonimmune mucus and sIgA alone were unable to cause bacterial aggregation, suggesting a cooperative role for mucus and sIgA. Antibodies responsible for adhesion inhibition were cross-reactive among several Campylobacter strains and were not directed solely against flagellar antigens.

Gastric lavage: a simple method to obtain IgA-rich intestinal secretions from the rabbit.

A lavage procedure was developed to obtain intestinal secretions from rabbits. The procedure facilitated the repeated monitoring of the intestinal IgA immune response of these animals to enteric infection with Campylobacter jejuni. This non-invasive technique was easily performed, reproducible and yielded consistent levels of IgA from rabbit intestinal secretions. It is anticipated that this procedure will aid in the study of the intestinal immune response of rabbits to other enteric pathogens.