Diversity of serotypes of Campylobacter jejuni and Campylobacter coli isolated in laboratory animals

One hundred nineteen isolates of Campylobacter jejuni and Campylobacter coli from nine laboratory animal species were serotyped using antisera to 20 Penner serotypes commonly isolated from cases of human enteric infections. Although C. jejuni and C. coli were isolated from laboratory animals with diarrhea, the majority were cultured from asymptomatic animals (81%). Seven of twenty-two isolates from animals with diarrhea were serotype 4 (32%) and three were serotype 1 (14%). Sixty-one of the 119 isolates (51%) were typeable using the 20 Penner antisera indicating that many of the isolates obtained from 29 nonhuman primates (five species), 20 ferrets, 7 hamsters, 15 cats and 48 dogs are serotypes commonly associated with human enteritis. Among typeable strains, 13 different serotypes were identified. Two particular serotypes, 4 and 19 were isolated from several species of animals and comprised 24% of the isolates studied. Since asymptomatic laboratory animals of several different species harbor serotypes of C. jejuni and C. coli that are potentially pathogenic to man, appropriate precautions should be instituted to minimize exposure of personnel to the organisms in laboratory animal feces. If suspected cases of zoonotic-related enteric campylobacteriosis involving laboratory animals do occur, serotyping of isolates would be a useful epidemiologic marker in studying the outbreak.     

The Evolution of Campylobacter jejuni and Campylobacter coli

The global significance of Campylobacter jejuni and Campylobacter coli as gastrointestinal human pathogens has motivated numerous studies to characterize their population biology and evolution. These bacteria are a common component of the intestinal microbiota of numerous bird and mammal species and cause disease in humans, typically via consumption of contaminated meat products, especially poultry meat. Sequence-based molecular typing methods, such as multilocus sequence typing (MLST) and whole genome sequencing (WGS), have been instructive for understanding the epidemiology and evolution of these bacteria and how phenotypic variation relates to the high degree of genetic structuring in C. coli and C. jejuni populations. Here, we describe aspects of the relatively short history of coevolution between humans and pathogenic Campylobacter, by reviewing research investigating how mutation and lateral or horizontal gene transfer (LGT or HGT, respectively) interact to create the observed population structure. These genetic changes occur in a complex fitness landscape with divergent ecologies, including multiple host species, which can lead to rapid adaptation, for example, through frame-shift mutations that alter gene expression or the acquisition of novel genetic elements by HGT. Recombination is a particularly strong evolutionary force in Campylobacter, leading to the emergence of new lineages and even large-scale genome-wide interspecies introgression between C. jejuni and C. coli. The increasing availability of large genome datasets is enhancing understanding of Campylobacter evolution through the application of methods, such as genome-wide association studies, but MLST-derived clonal complex designations remain a useful method for describing population structure.Campylobacter jejuni and Campylobacter coli remain among the most common causes of human bacterial gastroenteritis worldwide (Friedman et al. 2000). In high-income countries, Campylobacteriosis is much more common than gastroenteritis caused by Escherichia coli, Listeria, and Salmonella, and accounts for an estimated 2.5 million annual cases of gastrointestinal disease in the United States alone (Kessel et al. 2001). Infection with these bacteria is also a major cause of morbidity and mortality in low- and middle-income countries, although it is almost certainly underreported in these settings, especially as culture confirmation remains challenging. Poor understanding of the transmission of these food-borne pathogens to humans in all income settings has contributed to the failure of public health systems to adequately address this problem. As a consequence, over the past 20 years, much investment has been directed at understanding how these bacteria are transmitted from reservoir hosts to humans through the food chain.Although the disease was first recognized by Theodor Escherich in 1886, who described the symptoms of intestinal Campylobacter infections in children as “cholera infantum” (Samie et al. 2007) or “summer complaint” (Condran and Murphy 2008), difficulties in the culture and characterization of these organisms precluded their recognition as major causes of disease until the 1970s. Campylobacteriosis is usually nonfatal and self-limiting; however, the symptoms of diarrhea, fever, abdominal pain, and nausea can be severe (Allos 2001), and sequelae, including Guillain–Barre syndrome and reactive arthritis, can have serious long-term consequences. Subsequently, recognition of the very high disease burden of human Campylobacter infection stimulated research on these bacteria and their relatives. Since the 1970s, C. coli and C. jejuni have been isolated from a wide range of wild and domesticated bird and mammal species, in which, typically, they are thought to cause few if any disease symptoms. Humans are usually infected by the consumption of contaminated food (especially poultry meat), water, milk, or contact with animals or animal feces (Niemann et al. 2003).Most of what is known about these species comes from isolates obtained from humans with disease, the food chain, and the agricultural environment. It is, however, important to note that such isolates are by no means representative of natural Campylobacter populations, and it is becoming increasingly apparent that much of the diversity present among the Campylobacters is in strains that colonize wild animals. Increasing numbers of novel genotypes are being found as Campylobacter populations are analyzed in different animal species, especially wild birds (Carter et al. 2009; French et al. 2009); these populations undoubtedly contain many as-yet-undescribed lineages. Most human disease isolates from cases of gastroenteritis in countries, such as the United Kingdom and the United States, are C. jejuni, which typically accounts for 90% of cases in these settings, with the remaining ∼10% of cases mostly caused by C. coli. The majority of the genotypes isolated from human disease have also been isolated as commensal gastrointestinal inhabitants of domesticated and, especially, food animals. Furthermore, clinical isolates are a nonrandom subset of these strains. Asymptomatic carriage of C. jejuni and C. coli is thought to be rare in humans, especially among people in industrialized countries, suggesting that humans are not a primary host for these organisms in these settings and that people are sporadically, and frequently pathologically, infected via the food chain from animal reservoir hosts.An understanding of the relatively short history of coevolution between humans and pathogenic Campylobacters can be obtained by examining their population structure and ecology. This approach has formed the basis of many recent investigations of the cryptic epidemiology of these organisms (Lang et al. 2010; Müllner et al. 2010; Thakur et al. 2010; Hastings et al. 2011; Jorgensen et al. 2011; Kittl et al. 2011; Magnússon et al. 2011; Sheppard et al. 2011a,b; Sproston et al. 2011; Read et al. 2013) and will be the focus of this review. Such studies have included molecular epidemiological and evolutionary analyses and, in the past 15 years or so, the application of high-throughput DNA sequencing technologies of increasing capacity has enhanced the integration of these two areas of investigation to their mutual benefit.     

Genome maps of Campylobacter jejuni and Campylobacter coli.

Little information concerning the genome of either Campylobacter jejuni or Campylobacter coli is available. Therefore, we constructed genomic maps of C. jejuni UA580 and C. coli UA417 by using pulsed-field gel electrophoresis. The genome sizes of C. jejuni and C. coli strains are approximately 1.7 Mb, as determined by SalI and SmaI digestion (N. Chang and D. E. Taylor, J. Bacteriol. 172:5211-5217, 1990). The genomes of both species are represented by single circular DNA molecules, and maps were constructed by partial restriction digestion and hybridization of DNA fragments extracted from low-melting-point agarose gels. Homologous DNA probes, encoding the flaAB and 16S rRNA genes, as well as heterologous DNA probes from Escherichia coli, Bacillus subtilis, and Haemophilus influenzae, were used to identify the locations of particular genes. C. jejuni and C. coli contain three copies of the 16S and 23S rRNA genes. However, they are not located together within an operon but show a distinct split in at least two of their three copies. The positions of various housekeeping genes in both C. jejuni UA580 and C. coli UA417 have been determined, and there appears to be some conservation of gene arrangement between the two species.     

Serotyping of Campylobacter jejuni, Campylobacter coli, and Campylobacter laridis from domestic and wild animals

By using 50 unabsorbed antisera, we were able to serotype 272 (65.7%) of 414 thermotolerant campylobacters from wild and domestic animals, on the basis of heat-stable antigens identified by means of passive hemagglutination. Forty-two serotypes were recognized. The pattern of serotypes detected in the various animal species was compared to human clinical isolates by using the Czekanowski index (proportional similarity index). The highest degree of similarity to the clinical isolates was observed for the poultry isolates, followed by strains from wild birds, flies, and pigs (in order of decreasing similarity). The serotypes recovered most frequently from poultry (LAU 1 and LAU 2) were also most prevalent in Norwegian patients. In contrast, serotype LAU 35/44, the predominant porcine serotype, was never recovered from human clinical specimens. Flies captured in chicken farms and in piggeries harbored serotypes which were also commonly seen in chickens and pigs, respectively. Nine of the strains included in this study could not be ascribed to any defined species. All of these were resistant to nalidixic acid and did not produce H2S.     

Antibiotic resistance and resistance mechanisms in Campylobacter jejuni and Campylobacter coli

Campylobacter jejuni and Campylobacter coli are recognized as the most common causative agents of bacterial gastroenteritis in the world and infections with these organisms occur more frequently than do infections due to Salmonella species, Shigella species, or Escherichia coli 0157:H7. The incidence of human Campylobacter infections has increased markedly in both developed and developing countries worldwide and, more significantly, so has the rapid emergence of antibiotic-resistant Campylobacter strains, with evidence suggesting that the use of antibiotics, in particular the fluoroquinolones, as growth promoters in food animals and the veterinary industry is accelerating this trend. In this minireview, the patterns of emerging resistance to the antimicrobial agents useful in treatment of the disease are presented and the mechanisms of resistance to these drugs in Campylobacter spp are discussed.     

Serotyping of Campylobacter jejuni, Campylobacter coli, and Campylobacter laridis from domestic and wild animals.

By using 50 unabsorbed antisera, we were able to serotype 272 (65.7%) of 414 thermotolerant campylobacters from wild and domestic animals, on the basis of heat-stable antigens identified by means of passive hemagglutination. Forty-two serotypes were recognized. The pattern of serotypes detected in the various animal species was compared to human clinical isolates by using the Czekanowski index (proportional similarity index). The highest degree of similarity to the clinical isolates was observed for the poultry isolates, followed by strains from wild birds, flies, and pigs (in order of decreasing similarity). The serotypes recovered most frequently from poultry (LAU 1 and LAU 2) were also most prevalent in Norwegian patients. In contrast, serotype LAU 35/44, the predominant porcine serotype, was never recovered from human clinical specimens. Flies captured in chicken farms and in piggeries harbored serotypes which were also commonly seen in chickens and pigs, respectively. Nine of the strains included in this study could not be ascribed to any defined species. All of these were resistant to nalidixic acid and did not produce H2S.     

PCR-ELISAs for the detection of Campylobacter jejuni and Campylobacter coli in poultry samples

Campylobacter species, primarily Campylobacter jejuni and Campylobacter coli, are regarded as a major cause of human gastrointestinal disease, commonly acquired by eating undercooked chicken. We describe a PCR-ELISA for the detection of Campylobacter species and the discrimination of C. jejuni and C. coli in poultry samples. The PCR assay targets the 16S/23S ribosomal RNA intergenic spacer region of Campylobacter species with DNA oligonucleotide probes designed for the specific detection of C. jejuni, C. coli, and Campylobacter species immobilized on Nucleo-Link wells and hybridized to PCR products modified with a 5' biotin moiety. The limit of detection of the PCR-ELISA was 100-300 fg (40-120 bacterial cells) for C. jejuni and C. coli with their respective species-specific oligonucleotide probes and 10 fg (4 bacterial cells) with the Campylobacter genus-specific probe. Testing of poultry samples, which were presumptive positive for Campylobacter following culture on the Malthus V analyzer, with the PCR-ELISA determined Campylobacter to be present in 100% of samples (n = 40) with mixed cultures of C. jejuni/C. coli in 55%. The PCR-ELISA when combined with culture pre-enrichment is able to detect the presence of Campylobacter and definitively identify C. jejuni and C. coli in culture-enriched poultry meat samples.     

Isolation, characterization, and serotyping of Campylobacter jejuni and Campylobacter coli from slaughter cattle

A total of 525 specimens from 100 slaughter beef cattle were examined for the presence of Campylobacter jejuni and Campylobacter coli by direct plating and enrichment techniques. Isolates were identified by cultural, biochemical, antibiotic sensitivity, and immunofluorescence tests and further characterized with the aid of recently developed biotyping and serotyping methods. Fifty animals were positive for C. jejuni; only one was positive for C. coli. The distribution pattern of C. jejuni-positive animals, in decreasing order, was steers (55%), bulls (40%), heifers (40%), and cows (22%). Significantly higher isolation rates were obtained from the gall bladders (33%), large intestines (35%), and small intestines (31%) than from the livers (12%) or the lymph nodes (1.4%). C. jejuni isolation by the enrichment technique was 40.2% more frequent than by direct plating; 24-h enrichment resulted in 24% more isolations than 48-h enrichment. Eighty-four of 105 C. jejuni cultures were typable serologically and represented 13 serogroups. Biotype I accounted for 71% of biotyped cultures. Serogroup 7 biotype I was the most commonly encountered (24%) isolate. About one in three positive animals had C. jejuni strains representing more than one serogroup. C. jejuni serogroups encountered in slaughter cattle were similar to those commonly isolated from human sources.     

Improved method for the isolation of Campylobacter jejuni and Campylobacter coli bacteriophages

A centrifugation and filtration method of isolating Campylobacter phages has been developed. Forty-nine Campylobacter phages were isolated from 272 effluent samples of which 42 produced lysis with Campylobacter jejuni strains and seven with C. coli strains. Phages were recovered from pig manure, abattoir effluents, human faeces, sewage and poultry manure. Phages were not isolated from water samples, cattle and sheep faeces or farm pasture soil.     

The bactericidal activity of tea against Campylobacter jejuni and Campylobacter coli

Extracts of black and green tea inhibited the growth of clinical isolates of Campylobacter jejuni and C. coli. Tea extracts killed C. jejuni and C. coli within 4 h. Heat treatment of extracts did not affect inhibitory or bactericidal activity.     

Prevalence of Campylobacter jejuni,Campylobacter lari,and Campylobacter coli in different ecological guilds and taxa of migrating birds

A total of 1,794 migrating birds trapped at a coastal site in southern Sweden were sampled for detection of Campylobacter spp. All isolates phenotypically identified as Campylobacter jejuni and a subset of those identified as non-C. jejuni were identified to the species level by PCR-based techniques. C. jejuni was found in 5.0% of the birds, Campylobacter lari was found in 5.6%, and Campylobacter coli was found in 0.9%. An additional 10.7% of the tested birds were infected with hippurate hydrolysis-negative Campylobacter spp. that were not identified to the species level. The prevalence of Campylobacter spp. differed significantly between ecological guilds of birds. Shoreline-foraging birds feeding on invertebrates and opportunistic feeders were most commonly infected (76.8 and 50.0%, respectively). High prevalence was also shown in other ground-foraging guilds, i.e., ground-foraging invertebrate feeders (11.0%), ground-foraging insectivores (20.3%), and plant-eating species (18.8%). Almost no Campylobacter spp. were found in ground-foraging granivores (2.3%), arboreal insectivores (0.6%), aerial insectivores (0%), or reed- and herbaceous plant-foraging insectivores (3.5%). During the autumn migration, a high proportion of samples from juveniles were positive (7.1% in passerines, 55.0% in shorebirds), indicating transmission on the breeding grounds or during the early part of migration. Prevalence of Campylobacter spp. was associated with increasing body mass among passerine bird species. Furthermore, prevalence was higher in short-distance migrants wintering in Europe than in long-distance migrants wintering in Africa, the Middle East, or Asia. Among ground-foraging birds of the Muscicapidae, those of the subfamily Turdinae (i.e., Turdus spp.) showed a high prevalence of Campylobacter spp., while the organism was not isolated in any member of the subfamily Muscicapinae (i.e., Erithacus and Luscinia). The prevalence of Campylobacter infection in wild birds thus seems to be linked to various ecological and phylogenetic factors, with great variations in carriership between different taxa and guilds.     

Evaluation of methods for the enumeration of Campylobacter jejuni and Campylobacter coli bacteriophages

The Spiral Plater System used for enumeration of bacteria was evaluated for the titration of Carnpylobacter phages. Twelve phages of C. jejuni were titrated using the conventional surface droplet technique, soft agar overlay-pour plate technique, and the Spiral Plater System. Phage counts obtained by the three methods were similar but the Spiral Plater System showed greater precision than the other methods.     

Isolation, characterization, and serotyping of Campylobacter jejuni and Campylobacter coli from slaughter cattle.

A total of 525 specimens from 100 slaughter beef cattle were examined for the presence of Campylobacter jejuni and Campylobacter coli by direct plating and enrichment techniques. Isolates were identified by cultural, biochemical, antibiotic sensitivity, and immunofluorescence tests and further characterized with the aid of recently developed biotyping and serotyping methods. Fifty animals were positive for C. jejuni; only one was positive for C. coli. The distribution pattern of C. jejuni-positive animals, in decreasing order, was steers (55%), bulls (40%), heifers (40%), and cows (22%). Significantly higher isolation rates were obtained from the gall bladders (33%), large intestines (35%), and small intestines (31%) than from the livers (12%) or the lymph nodes (1.4%). C. jejuni isolation by the enrichment technique was 40.2% more frequent than by direct plating; 24-h enrichment resulted in 24% more isolations than 48-h enrichment. Eighty-four of 105 C. jejuni cultures were typable serologically and represented 13 serogroups. Biotype I accounted for 71% of biotyped cultures. Serogroup 7 biotype I was the most commonly encountered (24%) isolate. About one in three positive animals had C. jejuni strains representing more than one serogroup. C. jejuni serogroups encountered in slaughter cattle were similar to those commonly isolated from human sources.     

Genotyping of clinical and chicken isolates of Campylobacter jejuni and Campylobacter coli

Genomic DNA from 58 strains of Campylobacter made up of 48 Campylobacter jejuni and ten Campylobacter coli were digested with Sma I and analysed by pulsed-field gel electrophoresis (PFGE). The cleavage of DNA by Sma I gave 22 distinct hybridization patterns. The two Campylobacter species were subtyped by PFGE. The average genomic size for C. jejuni by Sma I digestion was 1.73 Mb, while that of C. coli gave 1.7 Mb. Results from this study indicate that PFGE analysis by Sma I digested genomic DNA provides a reliable means of differentiating between and within species of Campylobacter and provides a practical approach to epidemiological studies of Campylobacter.     

Clinical aspects of Campylobacter jejuni infections in adults.

Campylobacter jejuni is an almost ubiquitous, microaerophilic, gram-negative rod. Outbreaks have been associated with drinking raw milk or contaminated water and eating poultry. Campylobacter jejuni accounts for 3.2% to 6.1% of cases of diarrheal illness in the general population of the United States, and infected patients frequently present with abdominal pain and fever. Less frequently, C jejuni is responsible for bacteremia, septic arthritis, septic abortion, and other extraintestinal infections. Reactive arthritis, Reiter''s syndrome, the Guillain-Barré syndrome, and pancreatitis may accompany or follow C jejuni enterocolitis. Campylobacter jejuni is an important cause of diarrheal illness and is a more commonly identified stool organism than Salmonella or Shigella species. Recurrent and chronic infection is generally reported in immunocompromised hosts.     

Incidence and ecology of Campylobacter jejuni and coli in animals

Since its initial emergence in the 1970s, Campylobacter has become one of the most common causative agents of bacterial foodborne illness. Campylobacter species readily colonize the gastrointestinal tracts of domestic, feral and wild animals and while they rarely cause clinical disease in food animals, they can produce severe acute gastroenteritis in humans. Prevalence of Campylobacter in food animals can exceed 80% thus challenging processors to employ post-harvest pathogen reduction strategies. Reduction of pathogens before arrival to the abattoir is also of interest because the implementation of pre-harvest interventions may compliment existing post-harvest control techniques to further diminish possible retail sources of infection. Such multiple hurdle approaches that simultaneously utilize pre- and post-harvest control techniques are expected to be the most effective approach for decreasing human illness associated with foodborne pathogens.     

Effect of iron concentration on toxin production in Campylobacter jejuni and Campylobacter coli

The effect of iron concentrations in culture media on supernatant yields of campylobacter cytotonic toxin (CCT) was studied. Of the 118 Campylobacter spp. strains surveyed, 78.8% produced toxin in brucella broth or in casamino acids--yeast extract (CYE) broth. When the iron concentration of CYE was increased from 0.44 microgram/mL (7.9 microM) to 0.65 microgram/mL (11.6 microM) by the addition of ferric chloride, 94.9% of the strains were positive for toxin in a ganglioside GM1 based, enzyme-linked immunosorbent assay, using antibody to affinity-purified CCT. The addition of iron as ferrous sulfate was less effective. When four toxin-positive strains were grown in a deferrated medium of conalbumin-treated CYE with 0.04-0.08 microgram iron/mL (0.72-1.43 microM), two of the culture supernatants became negative (absorbance at 410 nm, less than 0.1 and less than 10 ng CCT/mL), and two produced about 90% less CCT but were still classified as positive (absorbance, greater than or equal to 0.1 and greater than or equal to 10 ng CCT/mL). It was therefore concluded that the production of CCT by Campylobacter spp. is influenced by iron concentration.     

Comparison of the survival of Campylobacter jejuni and Campylobacter coli in culturable form in surface water

Six Campylobacter jejuni and six Campylobacter coli strains were isolated from cows and pigs, and their survival in lake water was compared by viable counts. Campylobacter jejuni survived longer in culturable form than C. coli in untreated and membrane-filtered water both at 4 and 20 degrees C. This difference in survival time may be a reason why C. jejuni is generally isolated from surface waters more frequently than C. coli. Both species survived better in filtered than in untreated water. This suggests that predation and competition for nutrients affect the survival of both Campylobacter species in the aquatic environment.     

Immunogens of interest for the diagnosis of Campylobacter jejuni infections

In order to identify the C. jejuni immunogens of interest for the diagnosis of Campylobacter infections, we analyzed the humoral response of 153 patients by using complement fixation (CF) and western blot assays. A first group of 79 sera was from C. jejuni infected patients suffering from enteritis (n=16), Guillain-Barré syndrome (GBS) (n=40) and arthritis (n=23). A second group of 49 sera was from healthy blood donors and a third group consisted of 25 sera from children under 4 years old. Using the CF test, 88.6% of the C. jejuni infected patients were seropositive versus 28.5% of the healthy blood donors and none of the children. The Western blot assay allowed detection of antibodies directed against seven selected antigens ranging from 14 to 67 kDa. Three of these antigens with a molecular size of 29, 37 and 43 kDa were detected by 86.0%, 84.8% and 91.1% of the C. jejuni infected patients, respectively. These three antigens seem to be good candidates for the development of assays suitable for direct and indirect diagnosis of Campylobacter infections.