Colonization of mice by Campylobacter jejuni.

Both streptomycin-treated and untreated Swiss white mice were irregularly colonized when challenged orogastrically with between 1 and 10(11) viable organisms of either of two strains of Campylobacter jejuni. The organisms were occasionally recovered from portions of the intestinal tracts of these animals in numbers ranging from 10(1) to 10(3)/g when the challenge doses were 10(10) or more. When germfree mice were challenged with 10(8) organisms of either strain, the entire intestinal tracts of all the animals were colonized with C. jejuni in numbers ranging from 10(4) to 10(9)/g. The ceca were most heavily colonized. Both strains of C. jejuni multiplied anaerobically in brucella broth, except when the broth contained 60.80 mu eq of total volatile fatty acids (VFA) per ml at pH 6.75, simulating conditions in the ceca of untreated mice, or when it contained 21.63 mu eq/ml at pH 7.04, simulating conditions in the ceca of streptomycin-treated mice. Active multiplication occurred, however, in brucella broth without VFA at pH 7.02 that was incubated microaerobically, simulating conditions in the ceca of germfree mice. The results suggest that VFA operating under anaerobic conditions present in the intestinal tract of both streptomycin-treated and untreated conventional mice interfere with the multiplication of C. jejuni. The organisms actively multiply, on the other hand, in the absence of VFA at the higher oxidation-reduction potential of the intestinal tract of germfree mice.     

Detection of Campylobacter jejuni in healthy monkeys and monkeys with enteric infections by PCR

Campylobacter were detected by PCR in feces of monkeys of different species (clinically healthy, with diarrhea, and dead from acute enteric infections). High prevalence of these bacteria in monkeys was revealed. The incidence of C. jejuni DNA in monkeys with acute enteric infections was higher than in healthy animals (69.6 and 51.3%, respectively). The highest percentage (92.3) of positive results was observed in Macaca mulatta with enteric diseases and in macaque dead of these diseases. The presence of C. jejuni in monkeys with diarrhea and the absence of pathogenic enterobacteria (Shigella, Salmonella, Yersinia) in feces probably attest to etiological relationship of acute enteric infections with Campylobacter.     

motA基因在空肠弯曲菌致病性相关趋化和定植中的作用

目的 确定鞭毛马达蛋白(flagellar motor protein)MotA编码基因在空肠弯曲菌(Campylobacter jejuni)致病性相关趋化和定植中的作用.方法 采用PCR扩增motA基因以及用于motA基因敲除的Kan~r基因和plus-motA基因片段,目的扩增产物克隆后测序.根据同源重组原理,构建空肠弯曲菌NCTC11168株motA基因自杀质粒(pBlueskrit-Ⅱ-SK~(motA-kan))和motA基因敲除突变株(motA~-).采用半固体平板迁移试验、基于硬琼脂平板(hard agar plus,HAP)的脱氧胆酸钠(SDC)体外趋化试验、小鼠空肠定植试验,了解空肠弯曲菌motA~-突变株和野生株鞭毛动力、SDC趋化和BALB/c-ByJ小鼠空肠内定植能力差异性.结果 所克隆的空肠弯曲菌motA基因核苷酸和氨基酸序列与已报道的相应序列相似性为100%.PCR和测序及含抗生素培养基连续传代培养结果证实,自杀质粒和motA~-突变株构建成功.motA~-突变株在半固体琼脂平板上的菌斑直径、在HAP上对0.2moL/L SDC趋化聚集环直径、黏附于小鼠空肠黏膜表面以及空肠内容物中motA~-突变株数量均明显少于野生株(P<0.05).结论 本研究成功构建了空肠弯曲菌motA基因敲除突变株.motA基因是空肠弯曲菌鞭毛动力以及致病性相关趋化和定植的必需基因.     

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.     

Distribution and serotypes of Campylobacter jejuni and Campylobacter coli in enteric Campylobacter strains isolated from children in the Central African Republic.

One hundred eighty-five enteric Campylobacter strains isolated from diarrheic or healthy children in Bangui (Central African Republic) were studied to determine their species and serotypes. C. coli was identified in 38.9% of all strains and in 43.9% of strains from diarrheic children. By the hemagglutination technique for heat-stable antigens, 73.5% of the strains could be serotyped. Of the typeable strains, 75% were distributed among 13 more frequent serotypes. C. coli serotype Pen 37,56 was the most common serotype from diarrheic children.     

Adaptation of Campylobacter jejuni NCTC11168 to high-level colonization of the avian gastrointestinal tract

The genome sequence of the human pathogen Campylobacter jejuni NCTC11168 has been determined recently, but studies on colonization and persistence in chickens have been limited due to reports that this strain is a poor colonizer. Experimental colonization and persistence studies were carried out with C. jejuni NCTC11168 by using 2-week-old Light Sussex chickens possessing an acquired natural gut flora. After inoculation, NCTC11168 initially colonized the intestine poorly. However, after 5 weeks we observed adaptation to high-level colonization, which was maintained after in vitro passage. The adapted strain exhibited greatly increased motility. A second strain, C. jejuni 11168H, which had been selected under in vitro conditions for increased motility (A. V. Karlyshev, D. Linton, N. A. Gregson, and B. W. Wren, Microbiology 148:473-480, 2002), also showed high-level intestinal colonization. The levels of colonization were equivalent to those of six other strains, assessed under the same conditions. There were four mutations in C. jejuni 11168H that reduced colonization; maf5, flaA (motility and flagellation), and kpsM (capsule deficiency) eliminated colonization, whereas pglH (general glycosylation system deficient) reduced but did not eliminate colonization. This study showed that there was colonization of the avian intestinal tract by a Campylobacter strain having a known genome sequence, and it provides a model for colonization and persistence studies with specific mutations.     

The mechanism of protection of infant mice from intestinal colonisation with Campylobacter jejuni

BALB/c mice, vaccinated intraperitoneally with a heat-killed (62 degrees C) suspension of Campylobacter jejuni before mating, completely protect c. 90% of their own infants from intestinal colonisation. This protection has now been investigated further in fostering experiments. Fostering by vaccinated dams within the first 24 h of life prevented intestinal colonisation in 50% of infants from non-vaccinated dams, and reduced colonisation in a further 25%. Infants from vaccinated dams, even if allowed to receive their own mothers' colostrum and milk, became susceptible to challenge when subsequently fostered by non-vaccinated dams. Immunity in experimentally infected infant mice depended upon the consumption of immune milk at and after the time of challenge. High concentrations of IgG antibodies specific for C. jejuni were found in the serum and mammary secretion of vaccinated dams, but there was very little specific IgA antibody.     

Multilocus sequence typing of Campylobacter jejuni and Campylobacter coli to identify potential sources of colonization in commercial turkey farms

Poultry are the main reservoir for thermophilic Campylobacter spp., which is the most common causative agent of human bacterial gastroenteritis. The epidemiology of Campylobacter in poultry, particularly in turkeys, is not completely understood. This study aimed at identifying potential sources and transmission routes of thermophilic Campylobacter spp. in commercial turkey farms. C. jejuni and C. coli isolates from breeders (n?=?29, 20 C. jejuni and 9 C. coli) and their progeny (n?=?51, 18 C. jejuni and 33 C. coli) reared in two different farms for three sequential production cycles were analysed by multilocus sequence typing (MLST). Strains (n?=?88, 42 C. jejuni and 46 C. coli) isolated from environmental (i.e. anteroom and in-house overshoes), water (i.e. drinkers and water line), and pest (i.e. flies, Alphitobius diaperinus, and mice) sources were also examined. MLST of C. jejuni and C. coli isolates resulted in 13 and 12 different sequence types (STs) belonging to six and one previously-described clonal complexes (CCs), respectively. Three novel STs were identified. Genetic similarities were detected between isolates from fattening turkeys and the considered environmental, water, and pest sources, and with the breeders to a lesser extent. Source attribution analysis estimated that environmental and water sources accounted for most (～75%) of fattening turkey isolates and were therefore identified as the most likely sources of flock colonization, followed by pests (～20%) and breeders (～5%). These sources may thus be targeted by control measures to mitigate the risk of Campylobacter colonization in commercial turkeys.

RESEARCH HIGHLIGHTS

• High occurrence of C. jejuni and C. coli in commercial turkey flocks.

• High genetic diversity of C. jejuni and C. coli in commercial turkey flocks.

• Horizontal transmission responsible for Campylobacter colonization of commercial turkey flocks.

• Environmental and water sources involved in Campylobacter colonization of commercial turkey flocks.

• Strategies for prevention and control of Campylobacter colonization of commercial turkey flocks are needed.

     

Susceptibility to enteric botulinum colonization of antibiotic-treated adult mice.

The relationship between the indigenous intestinal microflora of adults and their resistance to the enteric botulinum infection of infant botulism was studied. Orogastric challenges of 10(5) type A Clostridium botulinum spores were given to adult mice whose gut flora had been altered by feedings of a mixture of erythromycin and kanamycin sulfate. From 80 to 100% of mice became infected when challenged 15 to 60 h after antibiotic administration. The mean infective dose of 2 X 10(4) spores per mouse for challenges given 23 h after antibiotic administration contrasted with the failure of 10(6) spores to infect control mice. Botulinum-colonized mice remained asymptomatic, although colonization lasted up to 5 days, and total botulinum toxin in the gut on days 3 and 4 postchallenge averaged 3,400 and 2,200 mouse intraperitoneal mean lethal doses. The mean infective dose for inocula placed in the colon of antibiotic-treated mice was 10(3) spores per mouse, and C. botulinum multiplied in the cecum as well as in the colon.     

C57BL/6 and congenic interleukin-10-deficient mice can serve as models of Campylobacter jejuni colonization and enteritis

Campylobacter jejuni is a globally distributed cause of human food-borne enteritis and has been linked to chronic joint and neurological diseases. We hypothesized that C. jejuni 11168 colonizes the gastrointestinal tract of both C57BL/6 mice and congenic C57BL/6 interleukin-10-deficient (IL-10(-/-)) mice and that C57BL/6 IL-10(-/-) mice experience C. jejuni 11168-mediated clinical signs and pathology. Individually housed mice were challenged orally with C. jejuni 11168, and the course of infection was monitored by clinical examination, bacterial culture, C. jejuni-specific PCR, gross pathology, histopathology, immunohistochemistry, and anti-C. jejuni-specific serology. Ceca of C. jejuni 11168-infected mice were colonized at high rates: ceca of 50/50 wild-type mice and 168/170 IL-10(-/-) mice were colonized. In a range from 2 to 35 days after infection with C. jejuni 11168, C57BL/6 IL-10(-/-) mice developed severe typhlocolitis best evaluated at the ileocecocolic junction. Rates of colonization and enteritis did not differ between male and female mice. A dose-response experiment showed that as little as 10(6) CFU produced significant disease and pathological lesions similar to responses seen in humans. Immunohistochemical staining demonstrated C. jejuni antigens within gastrointestinal tissues of infected mice. Significant anti-C. jejuni plasma immunoglobulin levels developed by day 28 after infection in both wild-type and IL-10-deficient animals; antibodies were predominantly T-helper-cell 1 (Th1)-associated subtypes. These results indicate that the colonization of the mouse gastrointestinal tract by C. jejuni 11168 is necessary but not sufficient for the development of enteritis and that C57BL/6 IL-10(-/-) mice can serve as models for the study of C. jejuni enteritis in humans.     

Effect of endotoxin on colonisation of Campylobacter jejuni in infant mice

An infant mouse model has been used to investigate the colonisation of the intestine by Campylobacter jejuni and the effect of endotoxin (Escherichia coli O26: B6) on the initial stage of this process. Endotoxin injected 1 or 16 h before the bacterial challenge had no effect on the growth of campylobacters but endotoxin injected 4 to 10 h before the bacterial challenge caused a bacteriostatic effect on the growth of campylobacters which lasted for one day. The bacteriostatic effect was evident both in the small intestine and in the distal part of the intestine containing caecum and colon. The mechanism of the bacteriostatic effect of endotoxin could not be explained in the study, but is thought to be non-immunological because it developed so rapidly. Oral and parenteral iron administered as ammonium ferric citrate or iron dextran, respectively, were used in an attempt to reverse the bacteriostatic effect. High oral doses of iron (0.5 mg per animal) were effective but small doses (0.5 mg per animal) were ineffective. Parenteral iron administration had a delayed effect on the reversal of the bacteriostatic effect of endotoxin. Transferrin administered orally caused a clear bacteriostatic effect in both endotoxin pretreated and untreated mice. Campylobacter counts were always lower in the small intestine than in the large intestine both in control and in endotoxin pretreated mice. This indicates that the large intestine is the primary ecological niche where campylobacters colonise mice.     

Factors affecting the lethality of Campylobacter fetus subspecies jejuni in mice

Intraperitoneal injection of Campylobacter fetus ss. jejuni into HAM/1CR mice was lethal, but viable counts of bacteria from whole body homogenates, organs and blood indicated that death was not due to sustained bacterial multiplication. Heat-killed organisms (5 X 10(9) cfu) injected into 7-day-old mice caused death within 24 h and this was shown to be due to endotoxin. Both ferric iron and heterologous lipopolysaccharide enhanced virulence; the LD50 was lowered from 1.8 X 10(9) cfu to 2.7 X 10(7) cfu when both were used. Three-day-old or adult animals survived challenge with Campylobacter fetus without clinical symptoms when challenged orally or by intravenous or intraperitoneal routes.     

Mononuclear cell response in the liver of mice infected with hepatotoxigenic Campylobacter jejuni.

Intragastric inoculation with hepatotoxigenic strains of Campylobacter jejuni led to the death of mice during the late phase of infection. Histological study disclosed a massive infiltration of mononuclear cells in the liver, mimicking intrahepatic hypersensitivity. Neither enterotoxigenic nor enteroinvasive Escherichia coli induced such a lesion. However, the same histopathological change was induced by injecting the hepatotoxic factor of hepatotoxigenic C. jejuni intravenously on two occasions separated by 14 days. Neither a single injection of an increased dose of the hepatotoxic factor nor two injections, the second of which was heat-inactivated, induced this change. Pre-treatment with rabbit antibody to the hepatotoxic factor inhibited the development of the hepatic lesion. These results suggest that C. jejuni-induced hepatic lesions in mice may be caused, at least in part, by the active moiety of the hepatotoxic factor. The possible mechanisms by which the toxic factor induces hepatitis as a consequence of hypersensitivity are discussed in relation to Guillain-Barré syndrome and Reiter's syndrome associated with C. jejuni enteritis.     

A tolerogenic mucosal immune response leads to persistent Campylobacter jejuni colonization in the chicken gut

Campylobacter enteritis is the most reported zoonotic disease in many developed countries where it imposes a serious health burden. Campylobacter transmission to humans occurs primarily through the chicken vector. Chicks are regarded as a natural host for Campylobacter species and are colonized with C. jejuni in particular. But despite carrying a very high bacterial load in their gastrointestinal tract, these birds, in contrast to humans, do not develop pathological signs. It seems that in chickens C. jejuni principally harbors in the cecal mucosal crypts, where an inefficient inflammatory response fails to clear the bacterium from the gut. Recent intensive research resulted in an increased insight into the cross talk between C. jejuni and its avian host. This review discusses the chicken intestinal mucosal immune response upon C. jejuni entrance, leading to tolerance and persistent cecal colonization. It might in addition provide a solid base for further research regarding this topic aiming to fully understand the host-bacterium dynamics of C. jejuni in chicks and to develop effective control measures to clear this zoonotic pathogen from poultry lines.     

Isolation of nonchemotactic mutants of Campylobacter jejuni and their colonization of the mouse intestinal tract.

Three nonchemotactic mutants (D54, Y14, and N74) of Campylobacter jejuni were isolated from wild-type strain FUM158432 by either the negative swarming or liquid gradient method with brucella broth as the attractive substance. Strains D54 and Y14 were isolated after mutagenesis with methyl methanesulfonate, and N74 was isolated from a nonmutagenized culture. These mutants all failed to swarm on a semisolid medium and did not show any chemotactic behavior in the hard-agar plus assay method for any of the chemicals which act as attractants for the wild-type strain. They had intact flagella and were actively motile. Swimming behavior examined by a video tracking technique showed that the mutants swim only straight, without any tumbling. When suckling mice were challenged orally with approximately 10(5) CFU of these mutant strains, all of the mutants were cleared from the intestinal tract by 48 h. In contrast, the wild-type strain colonized the intestinal tracts of all mice challenged with 10(2) CFU. We concluded that chemotactic movement is important for colonization of the intestinal tract of suckling mice by C. jejuni.     

Hepatotoxic activity of Campylobacter jejuni

Hepatotoxic factor(s) were isolated from whole-cell lysates of Campylobacter jejuni GIFU 8734 and purified by chromatography. A single intravenous injection of 10 micrograms of this factor reproducibly produced hepatitis in mice, as determined by histology and liver function tests. The hepatic lesions were very similar to those evoked by C. jejuni infection. Tissue-culture studies with mouse hepatocytes demonstrated that low concentrations of the factor caused release of hepatic enzymes into the medium without appreciable cytolysis. High concentrations of the factor induced cytolysis. These effects were neutralised by antiserum to the factor, but not by antisera to the lipopolysaccharide of C. jejuni or to the heat-labile enterotoxin of Escherichia coli. Among 20 clinical isolates of C. jejuni, only four evoked hepatitis in mice and produced the hepatotoxic factor.     

The influence of feeding crimped kernel maize silage on growth performance and intestinal colonization with Campylobacter jejuni of broilers

An infection trial and a production trial over 35 days were conducted in parallel to study the influence of feeding crimped kernel maize silage (CKMS) on the intestinal Campylobacter jejuni colonization and broiler performance, respectively. The CKMS was used at dietary inclusion levels of 15% and 30% in maize-based diets. Broilers were orally inoculated with 2?×?10⁵?log?cfu/ml C. jejuni on day 14. Four birds from each pen were randomly selected and killed by cervical dislocation on days 3, 6, 9, 14 and 21 post infection and intestinal contents from ileum, caeca and rectum as well as liver samples were taken. Body weight and feed consumption of broilers were registered on days 13, 22 and 35. On day 35, litter dry matter (DM) was measured and the condition of the foot pads was evaluated. There was no significant effect of CKMS on the colonization of C. jejuni. Body weight of the broilers supplemented with 15% CKMS was comparable with the control maize-based feed, whereas addition of 30% CKMS reduced broiler body weight (P?Campylobacter colonization, but improved the foot pad health of broilers.     

Immunogenicity and protective efficacy of recombinant Campylobacter jejuni flagellum-secreted proteins in mice

Immunogenicity and protective efficacy of three Campylobacter jejuni flagellum-secreted proteins, FlaC, FspA1, and FspA2, were compared by use of a mouse model. Mice were immunized intranasally with each protein with or without LTR192G as the adjuvant and challenged intranasally with C. jejuni 81-176 or CG8486. All three proteins were immunogenic, although FspA1 induced the highest levels of serum immunoglobulin G (IgG) and fecal IgA. Although immunogenic, FlaC provided only 18% protection against disease from C. jejuni 81-176. Immunization with FspA1 resulted in 57.8% protection without adjuvant or 63.8% protection with adjuvant against homologous challenge with 81-176. Alternatively, immunization with FspA2 provided 38.4% (without adjuvant) or 47.2% (with adjuvant) protection against disease from homologous challenge with CG8486. In contrast to FspA2, FspA1 provided some heterologous protection against C. jejuni CG8486 when delivered with (31.2%) or without (44.8%) LTR192G. These results suggest that FspA1 may be a good subunit vaccine candidate against C. jejuni disease.     

Interaction of Cryptosporidium parvum and Campylobacter jejuni in experimentally infected neonatal mice

By the method of scanning electron microscopy (SEM), the inner mucosal surface of the ileum, ceacum and colon was studied in inbred BALB/c mice. Two-day-old mice were infected with either 10(6) oocysts of Cryptosporidium parvum and 10(8) CFU of porcine and human strains of the bacterium Campylobacter jejuni or with a combination of both enteropathogens. Pathological changes in infection with C. parvum were related to enterocytes and villous atrophy appeared. In infection with C. jejuni, pathological changes were related to goblet cells. In combined infections, pathological changes were similar to those in monoinfections and occurred simultaneously within the intestine. Synergistic interaction of C. parvum and C. jejuni manifested itself morphologically in a more intense colonization of the inner surface of the small and large intestine by C. jejuni, in a more intense infection of the caecum and colon by C. parvum, and in prolongation of severe, massive infection of the small and large intestine, and also a prolongation of the patent period.