Use of Pulsed-Field Gel Electrophoresis and Flagellin Gene Typing in Identifying Clonal Groups of Campylobacter jejuni and Campylobacter coli in Farm and Clinical Environments

Although campylobacters have been isolated from a wide range of animal hosts, the association between campylobacters isolated from humans and animals in the farm environment is unclear. We used flagellin gene typing and pulsed-field gel electrophoresis (PFGE) to investigate the genetic diversity among isolates from animals (cattle, sheep, and turkey) in farm environments and sporadic cases of campylobacteriosis in the same geographical area. Forty-eight combined fla types were seen among the 315 Campylobacter isolates studied. Six were found in isolates from all four hosts and represented 50% of the total number of isolates. Seventy-one different SmaI PFGE macrorestriction profiles (mrps) were observed, with 86% of isolates assigned to one of 29 different mrps. Fifty-seven isolates from diverse hosts, times, and sources had an identical SmaI mrp and combined fla type. Conversely, a number of genotypes were unique to a particular host. We provide molecular evidence which suggests a link between campylobacters in the farm environment with those causing disease in the community.     

Prevalence of Campylobacter spp. in cattle in Finland and antimicrobial susceptibilities of bovine Campylobacter jejuni strains

The study investigated the prevalence of Campylobacter spp. in Finnish cattle at slaughter and carcass contamination after slaughter. During the period January to December 2003, bovine rectal fecal samples (n=952) and carcass surface samples (n=948) from 12 out of 15 Finnish slaughterhouses were examined. In total, campylobacters were detected in 31.1% of fecal samples and in 3.5% of carcass surface samples. Campylobacter jejuni was isolated from 19.5%, Campylobacter coli from 2.2%, and presumptive Campylobacter hyointestinalis from 10.8% of fecal samples. Campylobacters were detected in 4.4% and 37.4% of the fecal samples examined both by direct culture and by enrichment (n=730), respectively, suggesting a low level of campylobacters in the intestinal content. A slightly increasing trend was observed in the overall prevalence of campylobacters towards the end of summer and autumn. Seventeen different serotypes were detected among the fecal C. jejuni isolates using a set of 25 commercial antisera for serotyping heat-stable antigens (Penner) of C. jejuni by passive hemagglutination. The predominant serotypes, Pen2 and Pen4-complex, were isolated from 52% of the fecal samples. Subtyping by pulsed-field gel electrophoresis (SmaI) yielded 56 and 20 subtypes out of 330 fecal and 70 carcass C. jejuni isolates, respectively. MICs of ampicillin, enrofloxacin, erythromycin, gentamicin, nalidixic acid, and oxytetracycline for 187 C. jejuni isolates were determined using a commercial broth microdilution method. Sixteen (9%) of the isolates were resistant to at least one of the antimicrobials tested. Resistance to nalidixic acid was most commonly detected (6%). No multiresistance was observed.     

Evidence that certain clones of Campylobacter jejuni persist during successive broiler flock rotations

Through the national surveillance program for Campylobacter spp., nine broiler chicken farms that were infected with Campylobacter jejuni in at least five rotations in 1998 were identified. One additional farm, located at the island of Bornholm where divided slaughter is used extensively, was also selected. Twelve broiler houses located on 10 farms were included in the study. The C. jejuni isolates collected from the selected houses during the surveillance were typed using fla typing and macrorestriction profiling (MRP), and a subset of the isolates, representing each of the identified clones, was serotyped according to the Penner scheme. Pulsed-field gel electrophoresis typing using SmaI and KpnI revealed that the majority of houses (11 of 12) carried identical isolates in two or more broiler flocks. Such persistent clones were found in 63% of all flocks (47 of 75). The majority of persistent clones (7 of 13) had fla type 1/1, but MRPs distinguished between isolates from different houses, and fla type 1/1 clones belonged to different serotypes. Seven houses carried persistent clones that covered an interval of at least four broiler flock rotations, or at least one half year. The dominant fla type (1/1) was represented by 44% of isolates, or by at least one isolate from 31 of 62 broiler flocks. This significantly exceeded the prevalence of fla type 1/1 C. jejuni isolates that we have estimated from other studies and suggests that isolates carrying this fla type are overrepresented in flocks with recurrent Campylobacter problems. The MRPs of clones belonging to fla type 1/1 serotype O:2 isolated from persistently infected flocks shared a high percentage of bands compared to the remaining isolates, indicating that some clones that have the ability to cause persistent infections in broiler farms are highly related to each other.     

Resistance to quinolones in Campylobacter jejuni and Campylobacter coli from Danish broilers at farm level

AIMS: To investigate the prevalence of quinolone resistance among Campylobacter jejuni and Camp. coli isolates from Danish poultry at the farm level, as well as for the whole country. METHODS AND RESULTS: Data and isolates were collected from a national surveillance of Campylobacter in poultry. Quinolone resistance was investigated by determination of minimum inhibitory concentration (MIC) to nalidixic acid and enrofloxacin. Among Camp. jejuni and Camp. coli combined, 7.5% were resistant to nalidixic acid. Quinolone resistance varied considerably from farm to farm, with 0% on some farms and almost 100% on others, but the resistance was evenly distributed geographically. With respect to isolates from farms where resistance was detected, quinolone resistance was higher among Camp. coli (28.7%) than among Camp. jejuni (11.3%). PFGE typing of quinolone-resistant and quinolone-susceptible isolates from four farms indicated that certain resistant isolates belonged to specific clones that were able to persist on the farms during several rotations, even in the absence of selective pressure. Some clones were present and repeatedly isolated in both a quinolone-susceptible and quinolone-resistant variant. CONCLUSIONS: Overall, quinolone resistance among Campylobacter isolates from Danish broilers was 7.5% in 1998 and 1999; it was higher among Camp. coli than Camp. jejuni. Genetic diversity among resistant isolates was lower than among susceptible isolates, and certain clones existed in both a resistant and a susceptible variant. Some resistant clones appeared to persist on the farms and were repeatedly isolated from poultry flocks. SIGNIFICANCE AND IMPACT OF THE STUDY: The study is important for the understanding of persistence and dynamics of Campylobacter in broiler houses. It also highlights the extent, farm-to-farm variation and persistence of quinolone-resistant Campylobacter in broiler houses.     

Pasteurella multocida from outbreaks of avian cholera in wild and captive birds in Denmark

An outbreak of avian cholera was observed among wild birds in a few localities in Denmark in 2001. The highest mortalities were among breeding eiders (Somateria mollissima) and gulls (Larus spp.). Pulsed-field gel electrophoresis (PFGE) was conducted using ApaI and SmaI as restriction enzymes and restriction enzyme analysis (REA) using HpaII. The Pasteurella multocida subsp. multocida strain isolated from birds in this outbreak was indistinguishable from a strain that caused outbreaks in 1996 and 2003. Most isolates from domestic poultry had other PFGE patterns but some were indistinguishable from the outbreak strain. Among 68 isolates from wild birds, only one PFGE and one REA pattern were demonstrated, whereas among 23 isolates from domestic poultry, 14 different SmaI, 12 different ApaI, and 10 different HpaII patterns were found. The results suggest that a P. multocida strain has survived during several years among wild birds in Denmark.     

Longitudinal study of Campylobacter jejuni bacteriophages and their hosts from broiler chickens

A longitudinal study of bacteriophages and their hosts was carried out at a broiler house that had been identified as having a population of Campylobacter-specific bacteriophages. Cloacal and excreta samples were collected from three successive broiler flocks reared in the same barn. Campylobacter jejuni was isolated from each flock, whereas bacteriophages could be isolated from flocks 1 and 2 but were not isolated from flock 3. The bacteriophages isolated from flocks 1 and 2 were closely related to each other in terms of host range, morphology, genome size, and genetic content. All Campylobacter isolates from flock 1 were genotypically indistinguishable by pulsed-field gel electrophoresis (PFGE). PFGE and multilocus sequence typing indicated that this C. jejuni type was maintained from flock 1 to flock 2 but was largely superseded by three genetically distinct C. jejuni types insensitive to the resident bacteriophages. All isolates from the third batch of birds were insensitive to bacteriophages and genotypically distinct. These results are significant because this is the first study of an environmental population of C. jejuni bacteriophages and their influence on the Campylobacter populations of broiler house chickens. The role of developing bacteriophage resistance was investigated as this is a possible obstacle to the use of bacteriophage therapy to reduce the numbers of campylobacters in chickens. In this broiler house succession was largely due to incursion of new genotypes rather than to de novo development of resistance.     

Campylobacter hyointestinalis subsp. hyointestinalis, a common Campylobacter species in reindeer

AIMS: To study the prevalence of Campylobacter spp. in the faecal material of reindeer, and to identify the isolates by means of a polyphasic approach. In addition, to study the genetic diversity of Camp. hyointestinalis subsp. hyointestinalis reindeer isolates by pulsed-field gel electrophoresis (PFGE). METHODS AND RESULTS: The material, collected during the slaughter period in autumn 1998, comprised 399 faecal contents from the reindeer (Rangifer tarandus), a semi-domesticated, meat-producing ruminant of northern Finland. These samples came from 16 herds in the areas of eight reindeer slaughterhouses. Samples were cultured by methods suitable for isolation of fastidious Campylobacter species. Of all samples, 6% (24/399) were Campylobacter-positive. Phenotypic characteristics, SDS-PAGE protein patterns, dot blot DNA-DNA hybridization, 23S rDNA restriction fragment polymorphism analysis and PFGE identified the isolates as Camp. hyointestinalis subsp. hyointestinalis. CONCLUSIONS: Campylobacter hyointestinalis subsp. hyointestinalis was the only Campylobacter species isolated from reindeer in this study. The isolates showed high genomic diversity in PFGE with the restriction enzymes SmaI and KpnI. SIGNIFICANCE AND IMPACT OF THE STUDY: PFGE analysis is a useful subtyping method for epidemiological studies. Contaminated reindeer meat can be a source for human infections.     

Occurrence and strain diversity of thermophilic campylobacters in cattle of different age groups in dairy herds

AIMS: To investigate the occurrence and numbers of thermophilic campylobacters excreted by cattle in dairy herds, and to assess the strain diversity within herds. METHODS AND RESULTS: Faecal samples from 15 animals at each of 24 cattle farms were cultured quantitatively for thermophilic campylobacters and 23% of animals and 83% of farms were positive for Campylobacter jejuni. Young animals had a higher prevalence and higher faecal concentration than older animals. Serotyping and pulsed field gel electrophoresis (PFGE) of isolates showed that the most common serotypes were 2, 4-complex and 11. Serotype 2 was especially prevalent among calves (68% of the positive calves). In eight of the 20 positive herds, all isolates had the same sero- and PFGE type while, in the other herds, two to five different types were isolated. CONCLUSIONS: Significant differences were found between age groups in relation to the prevalence and numbers of excreted campylobacters, serotype distribution and strain diversity. The relatively few different strains in each herd indicate that transmission between animals is common. SIGNIFICANCE AND IMPACT OF THE STUDY: The high prevalence on cattle farms of the human pathogen C. jejuni and the wide distribution of serotype 2, the most common serotype among Danish patients, indicate that cattle might be an important reservoir for human infections. The ability of this serotype to colonize calves in high numbers further indicates that serotype 2 strains may have an advantage over other serotypes.     

Characterization of Listeria monocytogenes recovered from 41 cases of sporadic listeriosis in Austria by serotyping and pulsed-field gel electrophoresis

41 clinical Listeria monocytogenes strains recovered from seven feto-maternal and 34 non-pregnancy associated cases of human listeriosis documented between 1997 and 2000 underwent serotyping and typing by pulsed-field gel electrophoresis (PFGE) applying the enzymes AscI, ApaI and SmaI. The pulsotypes of the clinical strains were compared to the pulsotypes of three L. monocytogenes strains isolated from healthy fecal carriers and nine reference strains isolated from seven outbreaks in Europe and the USA. The 41 clinical strains of Austrian provenance showed 37 pulsotypes. Five sets of two Austrian strains each were indistinguishable by PFGE typing. Epidemiological links were absent between these indistinguishable isolates. One unique pulsotype (AB) was found in three fecal isolates. Five pulsotypes (A, Q, R, AC and AD) were distinguished among the strains associated with outbreaks. Clusters consisting of two, five and six Austrian strains each were indistinguishable from the outbreak-associated pulsotypes A, Q and R, respectively, after PFGE analysis with AscI. Three strains of AscI pulsotype Q and five strains of AscI pulsotype R could be further differentiated by restriction with ApaI and SmaI. One strain each from sporadic cases shared a combined pulsotype with the outbreak strains of pulsotypes A and R, respectively. These PFGE data suggest that a similar genetic background can be found in strains which have been contributing to outbreaks world-wide and in isolates associated with sporadic listeriosis in Austria.     

Prevalence of Campylobacter spp. in Cattle in Finland and Antimicrobial Susceptibilities of Bovine Campylobacter jejuni Strains

The study investigated the prevalence of Campylobacter spp. in Finnish cattle at slaughter and carcass contamination after slaughter. During the period January to December 2003, bovine rectal fecal samples (n = 952) and carcass surface samples (n = 948) from 12 out of 15 Finnish slaughterhouses were examined. In total, campylobacters were detected in 31.1% of fecal samples and in 3.5% of carcass surface samples. Campylobacter jejuni was isolated from 19.5%, Campylobacter coli from 2.2%, and presumptive Campylobacter hyointestinalis from 10.8% of fecal samples. Campylobacters were detected in 4.4% and 37.4% of the fecal samples examined both by direct culture and by enrichment (n = 730), respectively, suggesting a low level of campylobacters in the intestinal content. A slightly increasing trend was observed in the overall prevalence of campylobacters towards the end of summer and autumn. Seventeen different serotypes were detected among the fecal C. jejuni isolates using a set of 25 commercial antisera for serotyping heat-stable antigens (Penner) of C. jejuni by passive hemagglutination. The predominant serotypes, Pen2 and Pen4-complex, were isolated from 52% of the fecal samples. Subtyping by pulsed-field gel electrophoresis (SmaI) yielded 56 and 20 subtypes out of 330 fecal and 70 carcass C. jejuni isolates, respectively. MICs of ampicillin, enrofloxacin, erythromycin, gentamicin, nalidixic acid, and oxytetracycline for 187 C. jejuni isolates were determined using a commercial broth microdilution method. Sixteen (9%) of the isolates were resistant to at least one of the antimicrobials tested. Resistance to nalidixic acid was most commonly detected (6%). No multiresistance was observed.     

Effects of subtherapeutic administration of antimicrobial agents to beef cattle on the prevalence of antimicrobial resistance in Campylobacter jejuni and Campylobacter hyointestinalis

The influence of antimicrobial agents on the development of antimicrobial resistance (AMR) in Campylobacter isolates recovered from 300 beef cattle maintained in an experimental feedlot was monitored over a 315-day period (11 sample times). Groups of calves were assigned to one of the following antimicrobial treatments: chlortetracycline and sulfamethazine (CS), chlortetracycline alone (Ct), virginiamycin, monensin, tylosin phosphate, and no antimicrobial agent (i.e., control treatment). In total, 3,283 fecal samples were processed for campylobacters over the course of the experiment. Of the 2,052 bacterial isolates recovered, 92% were Campylobacter (1,518 were Campylobacter hyointestinalis and 380 were C. jejuni). None of the antimicrobial treatments decreased the isolation frequency of C. jejuni relative to the control treatment. In contrast, C. hyointestinalis was isolated less frequently from animals treated with CS and to a lesser extent from animals treated with Ct. The majority (> or =94%) of C. jejuni isolates were sensitive to ampicillin, erythromycin, and ciprofloxacin, but more isolates with resistance to tetracycline were recovered from animals fed Ct. All of the 1,500 isolates of C. hyointestinalis examined were sensitive to ciprofloxacin. In contrast, 11%, 10%, and 1% of these isolates were resistant to tetracycline, erythromycin, and ampicillin, respectively. The number of animals from which C. hyointestinalis isolates with resistance to erythromycin and tetracycline were recovered differed among the antimicrobial treatments. Only Ct administration increased the carriage rates of erythromycin-resistant isolates of C. hyointestinalis, and the inclusion of CS in the diet increased the number of animals from which tetracycline-resistant isolates were recovered. The majority of C. hyointestinalis isolates with resistance to tetracycline were obtained from cohorts within a single pen, and most of these isolates were recovered from cattle during feeding of a forage-based diet as opposed to a grain-based diet. The findings of this study show that the subtherapeutic administration of tetracycline, alone and in combination with sulfamethazine, to feedlot cattle can select for the carriage of resistant strains of Campylobacter species. Considering the widespread use of in-feed antimicrobial agents and the high frequency of beef cattle that shed campylobacters, the development of AMR should be monitored as part of an on-going surveillance program.     

Campylobacter jejuni isolated from retail poultry meat, bovine feces and bile, and human diarrheal samples in Japan: comparison of serotypes and genotypes

To determine the significance of poultry and bovine as infectious sources of Campylobacter jejuni in Japan, the serotype distribution and pulsed-field gel electrophoresis (PFGE) patterns of poultry and bovine isolates were compared with those of isolates from patients with diarrhea in Akita (Japan). Serotypes O:2 and O:4-complex were common in human, poultry, and bovine isolates, and serotype O:23,36,53 was common in human and bovine isolates. SmaI PFGE patterns of isolates belonging to these serotypes were generated. Eight PFGE patterns were shared by poultry and human isolates and three patterns were shared by human and bovine isolates. Further analysis of the isolates having the same SmaI PFGE pattern by KpnI PFGE confirmed that four patterns and two patterns were still shared by poultry and human isolates, and bovine and human isolates, respectively. Thus, serotypic and genotypic data indicated a possible link between sporadic human campylobacteriosis and C. jejuni from retail poultry and bovine bile and feces, suggesting that bovine serves as an infectious source of C. jejuni in Japan, as is observed in other countries.     

Effect of incubation temperature on isolation of Campylobacter jejuni genotypes from foodstuffs enriched in Preston broth

Preston broth and agar incubated at either 37 or 42 degrees C have been widely used to isolate campylobacters from foodstuffs. The consequences of using either incubation temperature were investigated. Retail packs of raw chicken (n = 24) and raw lamb liver (n = 30) were purchased. Samples were incubated in Preston broth at 37 and 42 degrees C and then streaked onto Preston agar and incubated as before. Two Campylobacter isolates per treatment were characterized. Poultry isolates were genotyped by random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE), and flagellin PCR-restriction fragment length polymorphism, and lamb isolates were genotyped by RAPD only. In total, 96% of the poultry and 73% of the lamb samples yielded campylobacters. The lamb isolates were all Campylobacter jejuni, as were 96% of the poultry isolates, with the remainder being Campylobacter lari. The incubation temperature had no significant effect on the number of positive samples or on the species isolated. However, genotyping of the C. jejuni isolates revealed profound differences in the types obtained. Overall (from poultry and lamb), the use of a single incubation temperature, 37 degrees C, gave 56% of the total number of RAPD C. jejuni genotypes, and hence, 44% remained undetected. The effect was especially marked in the poultry samples, where incubation at 37 degrees C gave 47% of the PFGE genotypes but 53% were exclusively recovered after incubation at 42 degrees C. Thus, the incubation temperature of Preston media selects for certain genotypes of C. jejuni, and to detect the widest range, samples should be incubated at both 37 and 42 degrees C. Conversely, genotyping results arising from the use of a single incubation temperature should be interpreted with caution.     

Comparison of sludge and clinical isolates of Listeria monocytogenes

AIMS: Listeria monocytogenes strains isolated in the same geographical area from sewage sludge and from patients presenting with listeriosis were compared. METHODS AND RESULTS: All isolates were typed by serotyping, phage typing and SmaI/ApaI pulsed-field gel electrophoresis (PFGE). Among the sludge isolates (n=32), 22 subtypes could be distinguished by the combination of all typing methods. The human isolates (n=11) were distributed into 10 subtypes which clearly differed from those observed among sludge isolates, except for one cluster formed by two related human isolates which showed high similarity in PFGE patterns (SmaI: 92%; ApaI: 89.5%) with one sludge isolate. CONCLUSION: These results suggest the existence of an epidemiological link between sludge and human isolates, but they may also be reflecting the distribution of L. monocytogenes types within the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Sludge and human L. monocytogenes may be related but further epidemiological studies are necessary to elucidate this point.     

The heterogeneity of Campylobacter jejuni and Campylobacter coli strains isolated from healthy cattle

AIMS: To identify campylobacters isolated from clinically healthy cattle at species level by a multiplex polymerase chain reaction (m-PCR). The heterogeneity among Campylobacter jejuni and Campylobacter coli isolates was also investigated by using a restriction fragment length polymorphism (RFLP) analysis of flagellin (flaA) gene. METHODS AND RESULTS: Samples of intestinal contents, gall bladders, liver and faeces were collected from a total number of 1154 healthy cattle. The samples were inoculated onto Preston enrichment broth and agar. Of 1154 samples, 301 (26.1%) were positive for Campylobacter spp. Using an m-PCR assay for species identification, 179 (59.5%) were positive with C. jejuni specific primers while 30 (10%) were positive with C. coli specific primers. None of the liver samples examined was positive for C. jejuni or C. coli by mPCR. All the isolates identified as C. jejuni and C. coli were successfully subtyped by flaA typing. Of the 209 isolates tested, 28 different flaA types were found. Twenty-three flaA types were identified among 179 C. jejuni isolates and the remaining five from C. coli isolates. CONCLUSIONS: Although the overall results suggest that the degree of heterogeneity among the flaA genes of thermophilic Campylobacter strains isolated from healthy cattle is relatively high, they should be treated cautiously as the number of band types for C. coli was low and band type 8 in C. jejuni was represented by a high percentage (%58). SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of the present study suggest that healthy cattle can play role in the contamination of environment and human food chain by Campylobacter spp.     

Longitudinal Study of Campylobacter jejuni Bacteriophages and Their Hosts from Broiler Chickens

A longitudinal study of bacteriophages and their hosts was carried out at a broiler house that had been identified as having a population of Campylobacter-specific bacteriophages. Cloacal and excreta samples were collected from three successive broiler flocks reared in the same barn. Campylobacter jejuni was isolated from each flock, whereas bacteriophages could be isolated from flocks 1 and 2 but were not isolated from flock 3. The bacteriophages isolated from flocks 1 and 2 were closely related to each other in terms of host range, morphology, genome size, and genetic content. All Campylobacter isolates from flock 1 were genotypically indistinguishable by pulsed-field gel electrophoresis (PFGE). PFGE and multilocus sequence typing indicated that this C. jejuni type was maintained from flock 1 to flock 2 but was largely superseded by three genetically distinct C. jejuni types insensitive to the resident bacteriophages. All isolates from the third batch of birds were insensitive to bacteriophages and genotypically distinct. These results are significant because this is the first study of an environmental population of C. jejuni bacteriophages and their influence on the Campylobacter populations of broiler house chickens. The role of developing bacteriophage resistance was investigated as this is a possible obstacle to the use of bacteriophage therapy to reduce the numbers of campylobacters in chickens. In this broiler house succession was largely due to incursion of new genotypes rather than to de novo development of resistance.     

Genetic characterization and antibiotic resistance of Campylobacter spp. isolated from poultry and humans in Senegal

AIMS: The main objectives of this study were to investigate the diversity of Campylobacter genotypes circulating in Senegal and to determine the frequency of antibiotic resistance. METHODS AND RESULTS: Strains of Campylobacter jejuni isolated from poultry (n = 99) and from patients (n = 10) and Campylobacter coli isolated from poultry (n = 72) were subtyped by pulsed-field gel electrophoresis (PFGE). The pulsotypes obtained after digestion by SmaI and KpnI revealed a significant genetic diversity in both species, but without any predominant pulsotypes. However, farm-specific clones were identified in the majority of poultry houses (76.5%). Human and poultry isolates of C. jejuni had common PFGE patterns. High quinolone-resistance rates were observed for C. jejuni (43.4%) and C. coli (48.6%) isolates obtained from poultry. CONCLUSIONS: The results showed a genetic diversity of Campylobacter between farms indicating multiple sources of infection; but specific clones had the ability to colonize the broiler farms. The antimicrobial resistance patterns were not related to any specific PFGE pattern suggesting that resistance was due to the selective pressure of antibiotic usage. Campylobacter with similar genotypes were circulating in both human and poultry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is important for the understanding of the epidemiology of Campylobacter in broiler farms in Senegal. It also emphasizes the need for a more stringent policy in the use of antimicrobial agents in food animals.     

Genomic relatedness within five common Finnish Campylobacter jejuni pulsed-field gel electrophoresis genotypes studied by amplified fragment length polymorphism analysis, ribotyping, and serotyping

Thirty-five Finnish Campylobacter jejuni strains with five SmaI/SacII pulsed-field gel electrophoresis (PFGE) genotypes selected among human and chicken isolates from 1997 and 1998 were used for comparison of their PFGE patterns, amplified fragment length polymorphism (AFLP) patterns, HaeIII ribotypes, and heat-stable (HS) serotypes. The discriminatory power of PFGE, AFLP, and ribotyping with HaeIII were shown to be at the same level for this selected set of strains, and these methods assigned the strains into the same groups. The PFGE and AFLP patterns within a genotype were highly similar, indicating genetic relatedness. The same HS serotypes were distributed among different genotypes, and different serotypes were identified within one genotype. HS serotype 12 was only associated with the combined genotype G1 (PFGE-AFLP-ribotype). These studies using polyphasic genotyping methods suggested that common Finnish C. jejuni genotypes form genetic lineages which colonize both humans and chickens.     

Effects of Subtherapeutic Administration of Antimicrobial Agents to Beef Cattle on the Prevalence of Antimicrobial Resistance in Campylobacter jejuni and Campylobacter hyointestinalis

The influence of antimicrobial agents on the development of antimicrobial resistance (AMR) in Campylobacter isolates recovered from 300 beef cattle maintained in an experimental feedlot was monitored over a 315-day period (11 sample times). Groups of calves were assigned to one of the following antimicrobial treatments: chlortetracycline and sulfamethazine (CS), chlortetracycline alone (Ct), virginiamycin, monensin, tylosin phosphate, and no antimicrobial agent (i.e., control treatment). In total, 3,283 fecal samples were processed for campylobacters over the course of the experiment. Of the 2,052 bacterial isolates recovered, 92% were Campylobacter (1,518 were Campylobacter hyointestinalis and 380 were C. jejuni). None of the antimicrobial treatments decreased the isolation frequency of C. jejuni relative to the control treatment. In contrast, C. hyointestinalis was isolated less frequently from animals treated with CS and to a lesser extent from animals treated with Ct. The majority (≥94%) of C. jejuni isolates were sensitive to ampicillin, erythromycin, and ciprofloxacin, but more isolates with resistance to tetracycline were recovered from animals fed Ct. All of the 1,500 isolates of C. hyointestinalis examined were sensitive to ciprofloxacin. In contrast, 11%, 10%, and 1% of these isolates were resistant to tetracycline, erythromycin, and ampicillin, respectively. The number of animals from which C. hyointestinalis isolates with resistance to erythromycin and tetracycline were recovered differed among the antimicrobial treatments. Only Ct administration increased the carriage rates of erythromycin-resistant isolates of C. hyointestinalis, and the inclusion of CS in the diet increased the number of animals from which tetracycline-resistant isolates were recovered. The majority of C. hyointestinalis isolates with resistance to tetracycline were obtained from cohorts within a single pen, and most of these isolates were recovered from cattle during feeding of a forage-based diet as opposed to a grain-based diet. The findings of this study show that the subtherapeutic administration of tetracycline, alone and in combination with sulfamethazine, to feedlot cattle can select for the carriage of resistant strains of Campylobacter species. Considering the widespread use of in-feed antimicrobial agents and the high frequency of beef cattle that shed campylobacters, the development of AMR should be monitored as part of an on-going surveillance program.     

Chickens and Cattle as Sources of Sporadic Domestically Acquired Campylobacter jejuni Infections in Finland

A substantial sampling among domestic human campylobacter cases, chicken process lots, and cattle at slaughter was performed during the seasonal peak of human infections. Campylobacter jejuni isolates (n = 419) were subtyped using pulsed-field gel electrophoresis with SmaI, and isolates representing overlapping types (n = 212) were further subtyped using KpnI for restriction. The SmaI/KpnI profiles of 55.4% (97/175) of the human isolates were indistinguishable from those of the chicken or cattle isolates. The overlapping SmaI/KpnI subtypes accounted for 69.8% (30/43) and 15.9% (32/201) of the chicken and cattle isolates, respectively. The occurrence of identical SmaI/KpnI subtypes with human C. jejuni isolates was significantly associated with animal host species (P < 0.001). A temporal association of isolates from chickens and patients was possible in 31.4% (55/175) of the human infections. Besides chickens as sources of C. jejuni in the sporadic infections, the role of cattle appears notable. New approaches to restrict the occurrence of campylobacters in other farm animals may be needed in addition to hygienic measures in chicken production. However, only about half of the human infections were attributable to these sources.The incidence of human enteric infections caused by campylobacters is highest in the summer months, showing a consistent peak at the end of July in Finland (www.ktl.fi/attachments/suomi/julkaisut/julkaisusarja_b/2008/2008b09.pdf), as well as in other Nordic countries (16, 33). Almost 70% of campylobacter infections detected in July and August in Finland are domestically acquired, whereas the annual average proportion of domestic cases is about 30%, and most of them are caused by Campylobacter jejuni (30). The prevalence of campylobacters in Finnish broiler flocks peaks simultaneously with the human cases (7), and similar sero- and genotypes have been reported among human and poultry strains isolated in Finland and in other countries (5, 8, 21-23). Several epidemiological studies have identified the handling and consumption of raw or undercooked poultry meat as a major risk factor for campylobacter enteritis (for example, see references 18, 20, and 41), whereas opposite conclusions about the significance of the consumption of chicken meat were drawn from the Swedish case-control study among young children (2) and an extensive Danish register-based study (6).Data derived from the genotyping studies of C. jejuni isolates from human infections and animals support the current suggestion that poultry is the most important single source of sporadic campylobacteriosis (12, 22, 29). However, several reports on genotype comparisons suggest that poultry may be a less significant source of campylobacters than generally thought, and other animal reservoirs should also be considered notable sources of campylobacters pathogenic to humans (3, 8, 17, 27, 31). Studies of the temporal occurrence of campylobacters in human infections and poultry flocks have revealed that the peak in prevalence, as well as some of the overlapping sero- and genotypes, is detected in humans prior to being detected in poultry (21, 28).Although cattle are well-known carriers of campylobacters, the survival of these fragile organisms in beef is poor (39, 42). In recent years, some authors (1, 4, 10) have raised the question of an indirect association between cattle and human cases. In a Finnish study combining data from the multilocus sequence typing of campylobacters isolated from production animals and from epidemiological studies of human cases, significant associations emerged between certain sequence-type complexes from human infections and contact with cattle, the consumption of unpasteurized milk, or the tasting or consumption of raw minced meat (23).The aim of this study was to investigate the contributions of poultry and cattle as sources of human C. jejuni infections in Finland by comparing over a limited time frame the macrorestriction profiles obtained from pulsed-field gel electrophoresis (PFGE) analysis of a geographically representative collection of C. jejuni isolates from domestically acquired sporadic human infections, chicken process lots, and cattle.