Pathogenesis of Campylobacter fetus infections. Role of surface array proteins in virulence in a mouse model.
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We developed a mouse model to compare the virulence of Campylobacter fetus strains with (S-plus) and without (S-minus) surface array protein (S-protein) capsules. In adult HA/ICR mice pretreated with ferric chloride, the LD50 for S-plus strain 84-32 was 43.3 times lower than its spontaneous S-minus mutant 84-54. Seven strains of inbred mice were no more susceptible than the outbred strain. In contrast to the findings with Salmonella typhimurium by others, 3 X 10(7) CFU of strain 84-32 caused 90% mortality in C3H/HeN (LPSn) mice and 40% mortality in C3H/HeJ (LPSd) mice. High-grade bacteremia in HA/ICR mice occurred after oral challenge with S-plus C. fetus strains and continued for at least 2 d, but was not present in any mice challenged with S-minus strains. Bacteremia at 30 min after challenge was 51.6-fold lower in mice pretreated with 10 microliters of rabbit antiserum to purified S-protein than after pretreatment with normal rabbit serum. Challenge of mice with a mixture of S-minus strain 84-54 and free S-proteins at a concentration 31.1-fold higher than found in wild-type strain 84-32 caused 30% mortality, compared with 0% with strain 84-54 or S-protein alone. These findings in a mouse model point toward the central role of the S-protein in the pathogenesis of C. fetus infection. The S-protein is not toxic per se, but enhances virulence when present on the bacterial cell surface as a capsule. (+info)
Application of a multiplex PCR assay for Campylobacter fetus detection and subspecies differentiation in uncultured samples of aborted bovine fetuses.
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Campylobacter (C.) fetus (epsilonproteobacteria) is an important veterinary pathogen. This species is currently divided into C. fetus subspecies (subsp.) fetus (Cff) and C. fetus subsp. venerealis (Cfv). Cfv is the causative agent of bovine genital Campylobacteriosis, an infectious disease that leads to severe reproductive problems in cattle worldwide. Cff is a more general pathogen that causes reproductive problems mainly in sheep although cattle can also be affected. Here we describe a multiplex PCR method to detect C. fetus and differentiate between subspecies in a single step. The assay was standardized using cultured strains and successfully used to analyze the abomasal liquid of aborted bovine fetuses without any pre-enrichment step. Results of our assay were completely consistent with those of traditional bacteriological diagnostic methods. Furthermore, the multiplex PCR technique we developed may be easily adopted by any molecular diagnostic laboratory as a complementary tool for detecting C. fetus subspecies and obtaining epidemiological information about abortion events in cattle. (+info)
Evaluation of a Campylobacter fetus subspecies venerealis real-time quantitative polymerase chain reaction for direct analysis of bovine preputial samples.
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The detection and subspeciation of Campylobacter fetus subsp. venerealis (CFV) from veterinary samples is important for both clinical and economic reasons. Campylobacter fetus subsp. venerealis is the causative agent of bovine genital campylobacteriosis, a venereal disease that can lead to serious reproductive problems in cattle, and strict international regulations require animals and animal products to be CFV-free for trade. This study evaluated methods reported in the literature for CFV detection and reports the translation of an extensively tested CFV-specific polymerase chain reaction (PCR) primer set; including the VenSF/VenSR primers and a real-time, quantitative PCR (qPCR) platform using SYBR Green chemistry. Three methods of preputial sample preparation for direct qPCR were evaluated and a heat lysis DNA extraction method was shown to allow for CFV detection at the level of approximately one cell equivalent per reaction (or 1.0 x 10(3) CFU/mL) from prepuce. The optimized sample preparation and qPCR protocols were then used to evaluate 3 western Canadian bull cohorts, which included 377 bulls, for CFV. The qPCR assay detected 11 positive bulls for the CFV-specific parA gene target. DNA sequence data confirmed the identity of the amplified product and revealed that positive samples were comprised of 2 sequence types; one identical to previously reported CFV parA gene sequences and one with a 9% sequence divergence. These results add valuable information towards our understanding of an important CFV subspeciation target and offer a significantly improved format for an internationally recognized PCR test. (+info)
A domestic ferret model of immunity to Campylobacter jejuni-induced enteric disease.
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Oral or intravenous inoculation of previously unexposed juvenile and adult ferrets with Campylobacter jejuni uniformly resulted in intestinal colonization lasting 2 to 12 days. Disease varied from mild to moderate diarrhea, which resolved in 2 to 3 days. Orally infected animals developed agglutinin titers of 8 to 256 within 3 weeks, while those infected intravenously developed titers of 256 to 2,048. Ferrets which had recovered from campylobacteriosis all developed high titers of agglutinating and bacterial antibodies but were readily colonized by subsequent oral inoculation with the same strain of C. jejuni. Orally infected ferret kits 3 to 6 weeks of age exhibited the same general pattern of infection and disease as adults, but diarrhea was somewhat more severe. Kits resolved their diarrhea in 1 to 6 days and developed agglutinin titers in serum of 16 to 32 within 3 weeks. A series of five oral or rectal inoculations of kits during the 5- to 9-week age interval resulted in progressively shorter clearance times and eventual strain-specific resistance against infection, as well as disease. Gnotobiotic adults showed the same pattern of strain-specific accelerated clearance and resistance to disease. Kits born to immune dams with high levels of whey antibodies had passively acquired serum agglutinin titers of 256 to 2,048. These kits showed no resistance to colonization with the homologous strain of C. jejuni but were completely refractory to diarrhea. These observations suggest that (i) some form(s) of specific immunity, rather than factors relating solely to age or normal flora, is responsible for resistance to C. jejuni colonization and disease production and (ii) humoral immunity at a level that does not prevent colonization can protect against enteric disease caused by this organism. (+info)
Bismuth subsalicylate in the prevention of colonization of infant mice with Campylobacter jejuni.
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Infant mice were used for the evaluation of the efficacy of bismuth subsalicylate (BSS) in the prevention of the growth of Campylobacter jejuni in the intestine. The MIC90 of ten C. jejuni strains was 900 micrograms/ml. Of three dosage regimens tested, continuous treatment before and after the bacterial challenge, mimicking the way BSS is used in the prevention of traveller's diarrhoea, was the most effective. Growth inhibition was dose dependent; the high dose of 2000 micrograms per day was more effective than 300 micrograms per day. After cessation of treatment, campylobacter counts increased to the same level as in the control animals. (+info)
Evidence of reinfection with multiple strains of Campylobacter jejuni and Campylobacter coli in Macaca nemestrina housed under hyperendemic conditions.
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A prospective bacteriologic study of 18 infant pig-tailed macaques (Macaca nemestrina) housed in a nursery facility in which Campylobacter spp. are endemic was undertaken to determine the epidemiology of infection and reinfection. The isolates of Campylobacter jejuni and C. coli cultured from 8 of the 18 infants were characterized by serotyping, DNA hybridization, and polyacrylamide gel electrophoresis protein profiles. The chronology of infection was indicative of multiple reinfections with different strains of C. jejuni and C. coli during the 12-month study of each infant. The duration of infection with a particular strain was 3 to 4 weeks. Infants were also infected with nalidixic acid-resistant campylobacters. These observations indicated that long-term infections under endemic conditions are caused by continual reinfection. C. jejuni or C. coli infection correlated with diarrhea in 5 of the 18 infants at 1 to 4 months of age. (+info)
Infection of adult Syrian hamsters with flagellar variants of Campylobacter jejuni.
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Two variants of Campylobacter jejuni IN1 differing in the expression of flagella, IN1 (Fla+ Mot+, wild type) and IN1-NM (Fla- Mot-), were tested for their ability to establish infection in adult hamsters. Animals were challenged intracecally with 2 X 10(9) to 5 X 10(10) CFU and monitored for evidence of infection. None of the challenged animals developed illness. There was a significant difference, however, in the ability of IN1 to infect hamsters (35 of 43) compared with that of IN1-NM (1 of 42) (P less than 0.01). Additionally, eight animals challenged with IN1-NM excreted only the campylobacters of Fla+ phenotype, which were shown to be identical with the parental Fla+ Mot+ type. Both groups of animals developed serum immunoglobulin G antibodies to outer membrane proteins of IN1 as measured by an enzyme-linked immunosorbent assay; however, only animals that excreted Fla+ organisms developed antiflagellin antibodies. In vitro reversibility from Fla+ to Fla- occurred with a frequency of 9.2 X 10(-6) per cell per generation; however, reversion from Fla- to Fla+ could not be detected in vitro. Further characterization of IN1-NM showed that it produced a cytoplasmic 62K flagellin subunit protein, but this protein lacked six epitopes detected in IN1 and also differed in its two-dimensional gel electrophoresis pattern. The results of these studies support the concept that an intact flagellum is necessary for intestinal infection with C. jejuni and that this model may be useful for studying the immune response to C. jejuni. (+info)
Partial purification and characterization of the enterotoxin produced by Campylobacter jejuni.
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Campylobacter jejuni enterotoxin was partially purified from culture supernatant. The purified fraction after gel filtration indicated three bands at 68, 54, and 43 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This fraction enhanced the adenylate cyclase activity of HeLa cell membranes by 1.5-fold over that of the control. The study with anti-cholera toxin immunoglobulin G (IgG) and ganglioside affinity column chromatographies revealed that the eluent from the anti-cholera toxin IgG column chromatography exhibited a single band (68 kDa) on SDS-PAGE and native PAGE, whereas the eluent from ganglioside column chromatography exhibited two bands (68 and 54 kDa) on SDS-PAGE. These suggest that the 68-kDa polypeptide should have an immunological relationship with cholera toxin, and the 68- and 54-kDa polypeptides might be responsible for the recognition of ganglioside. (+info)