Experimental infection of specific pathogen-free pigs with Campylobacter: excretion in faeces and transmission to non-inoculated pigs

Campylobacter species are leading agents of human bacterial gastroenteritis and consumption of food of animal origin is a major source of infection. Although pigs are known to frequently exhibit high counts of Campylobacter in their faeces, more information is needed about the dynamics of this excretion. An experimental trial was conducted to evaluate the faecal excretion of Campylobacter by 7-week-old specific pathogen-free piglets inoculated per os with three Campylobacter strains (one C. coli isolated from a pig, one C. coli and one C. jejuni from chickens) alone or simultaneously (5x10(7)CFU/strain). Non-inoculated pigs were housed in adjacent pens. Pigs were monitored for 80 days for clinical signs and by bacteriological analysis of faeces. Pigs inoculated with porcine C. coli or with a mix of the three strains excreted from 10(3) to 10(6)CFU/g of faeces with a slight decrease at the end of the trial. Animals inoculated with poultry C. coli or C. jejuni strain excreted a lower quantity and some of them stopped excreting. At the end of the trial, only C. coli was detected in the faeces of pigs inoculated simultaneously with the three bacteria. Moreover, the transmission of Campylobacter was noticed between pens for the two C. coli strains and all the neighbouring animals became shedders with a level of excretion similar to the inoculated pigs. Intermittence in the Campylobacter excretion was also observed. Finally, our study highlighted a host preference of Campylobacter, namely C. coli seems to have a higher colonization potential for pigs than C. jejuni.     

Isolation of Campylobacter from feral swine (Sus scrofa) on the ranch associated with the 2006 Escherichia coli O157:H7 spinach outbreak investigation in California

We report the isolation of Campylobacter species from the same population of feral swine that was investigated in San Benito County, California, during the 2006 spinach-related Escherichia coli O157:H7 outbreak. This is the first survey of Campylobacter in a free-ranging feral swine population in the United States. Campylobacter species were cultured from buccal and rectal-anal swabs, colonic faeces and tonsils using a combination of selective enrichment and antibiotic-free membrane filtration methods. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS, Bruker Daltonics, Inc., Billerica, MA, USA) was used to identify species followed by confirmatory multiplex PCR or 16S rRNA sequencing. Genetic relatedness of Campylobacter jejuni and Campylobacter coli strains was determined by multilocus sequence typing (MLST) and porA allele sequencing. Altogether, 12 (40%) of 30 feral swine gastrointestinal and oral cavity specimens were positive, and six species were isolated: Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalsis, Campylobacter jejuni, Campylobacter lanienae and Campylobacter sputorum. Campylobacter jejuni subtypes were closely related to MLST sequence type 21 (ST-21) and had identical porA sequences. Campylobacter coli subtypes were unrelated to isolates in the pubMLST/porA database. This feral swine population lived in close association with a 'grassfed' beef cattle herd adjacent to spinach and other leafy green row crop fields. The findings underscore the importance of protecting raw vegetable crops from faecal contamination by wild or feral animals. The study also illustrates a potential risk of Campylobacter exposure for hunters during handling and processing of wild swine meat.     

Use of embryonating eggs for isolation of Campylobacter species from intestines of swine with proliferative enteritis

Intestinal tissues from swine affected with proliferative enteritis were ground, filtered through a 0.65-micron pore membrane filter, diluted, and injected into 7-day-old embryonated hens' eggs via the yolk sac. At 2, 4, and 7 days later, yolk sac swab specimens taken from live embryos were cultured for Campylobacter species. Campylobacter hyointestinalis was recovered from eggs injected with tissues of swine with acute hemorrhagic proliferative enteritis at dilutions up to 10(-4). Campylobacter mucosalis was recovered from eggs injected with tissues of swine with chronic proliferative enteritis at dilutions up to 10(-6). Campylobacter coli was recovered from several specimens without lesions of proliferative enteritis and also from some specimens with lesions of proliferative enteritis. Two previously undescribed hemolytic Campylobacter species designed as hemolytic number 1 and hemolytic number 2 were recovered from normal and experimentally inoculated swine tissues. Few contaminating organisms grow in eggs and these were usually recovered at dilutions of 10(-2) or less. Recovery of Campylobacter species by use of these techniques was seldom successful in tissues stored at -70 C for more than 6 months.     

Campylobacter jejuni and Campylobacter coli inoculation of neonatal calves

Three groups of neonatal calves were inoculated orally with pathogenic strains of Campylobacter jejuni or C coli. The calves developed a mild, self-limiting enteritis characterized by thick mucoid feces. Bacteremia and fecal shedding of Campylobacter were sporadic in all inoculated calves. Two groups of calves were killed 1 to 3 weeks after inoculation to study the pathogenesis of infection. Postmortem culture of tissues revealed C jejuni or C coli most frequently in the ileum, cecum, colon, and blood. Clinical or pathologic differences between C jejuni-inoculated and C coli-inoculated calves were minimal.     

Species-specific DNA probes for Campylobacter species isolated from pigs with proliferative enteritis

Cloned, chromosomal DNA probes from porcine isolates of Campylobacter hyointestinalis and C. mucosalis were developed for the detection and identification of these putative swine enteric pathogens. High molecular weight chromosomal DNA from each species was used to construct genomic libraries in plasmids. Recombinants were selected which hybridized strongly to the homologous organism, but not to any other species of Campylobacter. Species-specific recombinants were labeled with phosphorus-32 and tested for sensitivity by dot blot hybridization to various dilutions of DNA and bacteria from each swine species, including C. hyointestinalis, C. mucosalis, C. coli and C. jejuni. Specificity was tested by hybridizing these probes against various strains of C. hyointestinalis or C. mucosalis, and against reference strains of all other described Campylobacter species. A C. hyointestinalis-specific probe and a C. mucosalis-specific probe were identified which were capable of detecting 1 ng of DNA or 10(4) cfu by bacterial spot blotting on nylon membranes. These probes hybridized to intestinal mucosal scrapings containing C. hyointestinalis and C. mucosalis obtained from pigs with proliferative enteritis, but not to material from normal pigs. Thus, cloned, chromosomal DNA probes may be useful in the detection and identification of bacteria involved in swine proliferative enteritis.     

Prevalence of Campylobacter spp isolated from the intestinal tract of pigs raised in an integrated swine production system

OBJECTIVE: To enumerate the prevalence of Campylobacter isolates in the intestinal tract of market-weight swine raised in an integrated swine operation in Texas. SAMPLE POPULATION: Samples of cecal contents were collected from 595 pigs (mean body weight, 110 kg [242 lb]) at time of slaughter. Pigs were off-spring of Yorkshire-Landrace sows and Duroc or Hampshire boars. Pigs originated from 4 farrow-to-finish farms. PROCEDURE: During a 9-month period, visits were made to a slaughter plant to remove cecal contents from market-weight hogs. Samples were obtained from 50 pigs/visit from designated farms so that samples were obtained 3 times from pigs of each of 4 farms. Isolation of Campylobacter spp was accomplished by use of enrichment broth and restrictive media, using microaerophilic conditions. RESULTS: Campylobacter spp were isolated from 70 to 100% of the pigs, depending on the farm and the date the samples were collected. Campylobacter coli was isolated from 20 to 100% (mean, 60%) of samples, and C jejuni was isolated from 0 to 76% (mean, 31%) of samples. Campylobacter lari was isolated from 2 pigs. Concentrations of C coli or C jejuni ranged from 10(3) to 10(7) colony-forming units/g of cecal content. CONCLUSIONS AND CLINICAL RELEVANCE: Campylobacter coli generally is accepted as a common inhabitant of the intestinal tract of swine. However, analysis of results of this study suggests that a relatively high prevalence of C jejuni may be found in pigs raised on specific farms.     

Production and some properties of cytotoxins produced by Campylobacter species isolated from proliferative enteropathy in swine.

Cytotoxin production by Campylobacter species isolated from proliferative enteropathy in swine was examined. Twenty-one of 29 strains of C. hyointestinalis, 10 of 27 strains of C. mucosalis and 10 of 10 strains of C. coli were cytotoxin positive. By the gel filtration chromatography of C. hyointestinalis culture filtrate, cytotoxin activities were observed in two peaks (fraction I and fraction II). Most of the cytotoxic activities lay in fraction I, which is heat-labile, trypsin-sensitive and the molecular weight was estimated at 40,000. On the other hand, fraction II cytotoxin was heat-stable, trypsin-insusceptible and molecular weight was approximately 1,000.     

Experimental infection of the mouse mammary gland with Campylobacter coli.

Campylobacter coli strains of bovine and avian origin were inoculated into the mammary gland of mice. A bovine strain isolated from a case of mastitis produced gross and histological changes in most of the glands; one bovine and one avian faecal isolate did not. Histologically, lesions were characterised by neutrophil infiltration in the alveolar spaces and necrosis and oedema in the interalveolar tissue. On bacteriological examination, the bovine mastitis strain could be isolated from most of the glands, but neither of the faecal strains. The mouse, therefore, appears to provide a convenient model for studying campylobacter mastitis.     

Antimicrobial resistance of thermophilic Campylobacter spp. isolated from cattle in Poland

Campylobacter species are among the most frequently identified bacterial causes of human gastroenteritis. Because Campylobacter spp. harbored by cattle can be transmitted to humans, in this study we investigated antimicrobial resistance of thermophilic Campylobacter isolated from cows. Our study included 150 strains of Campylobacter (143 strains of C. jejuni and 7 strains of C. coli) isolated from cows in South-Western Poland. The minimal inhibitory concentration (MIC) to ciprofloxacin, erythromycin, gentamicin and tetracycline were determined using the agar dilution methodology. All strains of C. coli were susceptible to all four drugs studied. The most frequently detected resistance of C. jejuni was to ciprofloxacin (26 strains 18.2%). Resistance to tetracycline was observed in 5 strains (3.5%). All strains of C. jejuni were susceptible to erythromycin and gentamicin.     

Comparison of Agar Dilution and E-test for antimicrobial susceptibility testing of Campylobacter coli isolates recovered from 80 Ontario swine farms

The primary aim of this study was to evaluate the level of agreement of the E-test for in vitro antimicrobial susceptibility testing of Campylobacter coli using the agar dilution technique, which is the approved method. A convenience sample of 80 Ontario swine farms was chosen for this study; each farm was visited from January to June 2004. A total of 233 isolates of C. coli were tested for susceptibility to 10 antimicrobials by agar dilution and the E-test. Performance of the tests was evaluated using 7 quality control strains: Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, Campylobacter jejuni ATCC 33560, and Campylobacter coli ATCC 33559 for the E-test and E. coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and C. jejuni ATCC 33560 for the agar dilution test. Weighted Cohen's kappa and prevalence-adjusted bias-adjusted kappa (PABAK) tests were used for statistical analysis. The E-test and agar dilution test results had a strong agreement when resistance to streptomycin and tetracycline were evaluated (weighted kappa: 0.68 and 0.66, respectively). However, marked disagreement was detected when testing susceptibility to nalidixic acid and ampicillin (0.15 and 0.22, respectively). Almost perfect agreement was detected by PABAK when testing susceptibility to gentamicin (0.99). Agreement was found to be moderate for ciprofloxacin, azithromycin, clindamycin, erythromycin, and chloramphenicol. Although the level of agreement between the E-test and agar dilution depended on the antimicrobial being tested, the E-test always detected a lower proportion of resistant isolates compared to agar dilution.     

Prevalences and transmission routes of Campylobacter spp. strains within multiple pig farms

In this work, faecal samples were collected from 15 pig farms to determine the Campylobacter prevalences at different times during the rearing period and to visualize the exchange of strains among the pig population by genotyping specific isolates. All isolated strains were identified as C. coli. Whereas no Campylobacter were detectable in the faeces of piglets at the day of birth, the Campylobacter incidence rose within days to 32.8%. After transfer to the nursery unit the prevalence increased to 56.6%. Approximately two-thirds of the pigs remained C. coli shedders in the fattening unit. In contrast to most farms, one farm expressed a very low Campylobacter incidence during the whole rearing period. Amplified fragment length polymorphism (AFLP) analysis was performed on all C. coli isolates of one farm. Clonal strains were identified from the brood sows and their offsprings or neighbouring piglets. After moving to the nursery unit, new genotypes appeared in that pig group but the original C. coli strains largely remained within that group. C. coli genotypes, identified during the fattening period, replaced the previously isolated genotypes. Transportation to the abattoir had no significant influence on the shedding rate of C. coli. The detection rate before transportation was 79.1% and decreased slightly to 78.2% (n=330). Additionally, eleven of 1474 environmental samples from different sources of the pig farms were positive for C. coli. This study demonstrates the importance of pigs as a reservoir for C. coli. Maternal C. coli strains are the primary source of infection but non-related genotypes from different sources appear during the rearing period and these latter strains constitute largely the final C. coli flora.     

Epidemiology of ovine Campylobacter infection determined by numerical analysis of electrophoretic protein profiles

The relationship of 50 Campylobacter strains isolated from aborted ovine foetuses, and the faeces of sheep, cattle and chickens were determined by numerical analysis of electrophoretic (SDS-PAGE) protein profiles. Comparison of protein patterns by numerical methods revealed differences between C. fetus ssp. fetus, C. jejuni, and C. coli strains as well as heterogeneity among isolates from different outbreaks. Isolates from each farm produced a distinct cluster and flocks from different locations were found to be infected with relatively different strains. In most cases, protein patterns of ovine foetal isolates were very similar to those of ovine faecal isolates. Ovine isolates of C. fetus ssp. fetus, C. jejuni and C. coli gave similar protein patterns to the corresponding Campylobacter species isolated from cattle or chicken, on the same farm. Thus, it was concluded that certain protein types of ovine Campylobacter strains were more likely associated with local areas, and Campylobacter strains causing ovine abortions are distributed in the environment more widely than assumed to date.     

Antimicrobial susceptibility in thermophilic Campylobacter species isolated from pigs and chickens in South Africa

Campylobacter jejuni is one of the leading causes of sporadic food-borne bacterial disease in humans. In intensive poultry and pig rearing systems the use of oral antibiotics is essential to maintain health. Consequently, there is a high risk for the thermophilic Campylobacter jejuni and C. coli resident in the intestinal tract of food animals to develop resistance to commonly used antibiotics. Contamination of meat or eggs with pathogenic strains of resistant Campylobacter could, therefore, result in a form of campylobacteriosis in humans that is difficult to treat. The aim of this investigation was to determine the antimicrobial susceptibility of thermophilic Campylobacter spp. isolated from pigs and poultry by the broth microdilution minimum inhibitory concentration (MIC) test. A total of 482 samples from the Western Cape and Gauteng provinces was collected and analysed. Thirty-eight Campylobacter isolates were obtained. Analysis of data revealed that C. jejuni strains mainly of poultry origin were more resistant to the fluoroquinolones, macrolides and tetracyclines and the C. coli strains were more resistant to the macrolides and lincosamides. Multi-resistance was also detected in 4 Campylobacter strains from the Western Cape. With the exception of tetracyclines, strains from high health Gauteng broiler farms were susceptible to antibiotics used to treat Campylobacter infections.     

Disinfectant susceptibility testing of avian and swine Campylobacter isolates by a filtration method

The susceptibility testing of disinfectants against Campylobacter jejuni and Campylobacter coli strains from broilers and pigs was investigated. The filtration method European standard EN 1040 was adapted to Campylobacter cultures and validated with reference strains. Two disinfectants were tested: 1% benzalkonium chloride active matter, as quaternary ammonium compound, and 0.63% sodium hypochlorite as chlorine-releasing agent. Both disinfectants were effective against the 34 Campylobacter strains tested after 5 min exposure under in vitro conditions. No link between resistance to disinfectants and antibiotics could be observed.     

Detection of thermophilic Campylobacter from sparrows by multiplex PCR: the role of sparrows as a source of contamination of broilers with Campylobacter

The best combination of primers and the annealing temperature of multiplex PCR for Campylobacter jejuni, Campylobacter coli, and Campylobacter lari were examined. The multiplex PCR was able to detect type strains of the three species. All results of identification of wild strains (30 strains of C. jejuni, 20 strains of C. coli, and 4 strains of C. lari) by the multiplex PCR coincided with those of the conventional biochemical identification tests, suggesting that the multiplex PCR can simultaneously differentiate C. jejuni, C. coli, and C. lari from wild strains of campylobacters easily and rapidly. Campylobacters were detected from sparrow feces by the multiplex PCR and antimicrobial sensitivities of the strains were determined to discuss the role of sparrows in contamination of broilers with C. jejuni. Three out of 13 strains of C. jejuni isolated from sparrow feces showed quinolone resistance. From the frequent use of quinolones for treatment of industrial animals like chickens, pigs, and cows, the three strains of quinolone-resistant C. jejuni in sparrows must have been originated from those industrial animals. Sparrows that have quinolone-resistant C. jejuni were considered to have contacted with industrial animals or thier feed. It may be presumed, on the contrary, that C. jejuni in sparrows could be a potential source of contamination of broilers.     

4型菌毛、毒素及蛋白酶在动物源气单胞菌的检测

将来源于鱼、猪、犬等９种动物的１８株气单胞菌分离株通过ＡＤ（氨苄青霉素－糊精）培养基纯化。另以大肠杆菌束状菌毛相关（ｂｆｐ－ｒｅｌａｔｅｄ）基因的ＰＣＲ引物对ＥＢ１（ＧＡＴＴＧＡＡＴＣＴＧＣＡＡＴＧＧＴＧＣ）—ＥＢ２（ＧＧＡＴＴＡＣＴＧＴＣＣＴＣＡＣＡＴＡＴ）扩增,用ｄｉｇｏｘｉｎｇｅｎｉｎ－１１－ｄＵＴＰ标记,构建核酸探针,作斑点杂交试验,用以检测上述气单胞菌菌株的４型菌毛,同时以溶血试验检测其毒素,用脱脂奶溶蛋白圈试验检测其蛋白酶。结果显示,在１８个菌株中,４型菌毛、毒素和蛋白酶均为阳性者为１０株（５５．６％）;３项指标均无肯定的阳性结果者４株,两者合计共１４株,占检测总数约８０％（１４／１８）,提示这３种毒力因子存在较高的相关性。所有鱼源株（５株）三项指标皆为阳性;４株猪分离株三项指标均为阳性者占一半;其它动物分离株检测结果无明显规律性     

Comparison of Four Enrichment Media in the Recovery of Campyloracter Jejuni

The effectiveness of 4 enrichment media for the recovery of low levels of inoculated cells of Campylobacter jejuni was evaluated. The media contained antibiotics or antibiotics and bile acids as selective compounds. Three of the media recovered most of the inoculated low numbers of 6 C. jejuni strains. In the 3 media the growth rate of 3 strains, indicated by the increase in the log number of cells during 24 h or 48 h incubation at 42 ° C, was about the same as in the control medium without selective compounds. The same 3 media also recovered a low number of Campylobacter cells from artificially contaminated raw milk or ground meat samples. The enrichment medium B containing 40 I.U. Colistin, 5 μg novobiocin, 2 mg Na-cholic acid and 50 mg cycloheximide per ml was inhibitory for most Campylobacter strains studied.     

Profiles of toxin production by thermophilic Campylobacter of animal origin.

Seventy-five strains of Campylobacter jejuni and C. coli, which were isolated from a variety of animal species, primarily poultry, were examined for production of toxin. Polymyxin extracts were tested in in vitro assays using CHO-KI, FCL (foetal calf lung), Vero, HeLa and CEF (chicken embryo fibroblast) cells. The toxic effects observed were cell rounding and death. Extracts from almost all C. jejuni and C. coli strains were toxic to both CHO-KI and FCL cells and 69.0% of C. jejuni isolates and 75% of C. coli isolates were also toxic to CEF cells. 50.7% of C. jejuni extracts were toxic to Vero cells and 46.5% toxic to HeLa cells. None of the C. coli isolates were toxic to either of these cell lines. None of the strains tested produced cytotonic enterotoxin. No differences in toxigenicity patterns were evident between Campylobacter isolated from different sources.     

Isolation of Campylobacter jejuni and Campylobacter coli from the gall bladder samples of sheep and identification by polymerase chain reaction

In this study, 100 gall bladder samples of sheep slaughtered at an abattoir in Elazi? province were examined for Campylobacter jejuni and Campylobacter coli by culture and polymerase chain reaction (PCR). Preston Campylobacter Agar supplemented with 7% horse blood and Preston Selective Supplement (Oxoid, Hampshire, UK) were used for isolation of the agents. Campylobacter spp. were isolated in 66 samples, and they were identified as 34% C. jejuni and 32% C. coli. A multiplex PCR based upon the use of ceuE gene-specific primers was applied on DNA samples extracted from C. jejuni and C. coli isolates. All C. jejuni and C. coli strains that were positive by culture were also detected to be positive by PCR. This study shows that PCR can be used an alternative, rapid and sensitive test for the identification of C. jejuni and C. coli which threaten human and animal health.