Quantifying Transmission of Campylobacter jejuni in Commercial Broiler Flocks

Since meat from poultry colonized with Campylobacter spp. is a major cause of bacterial gastroenteritis, human exposure should be reduced by, among other things, prevention of colonization of broiler flocks. To obtain more insight into possible sources of introduction of Campylobacter into broiler flocks, it is essential to estimate the moment that the first bird in a flock is colonized. If the rate of transmission within a flock were known, such an estimate could be determined from the change in the prevalence of colonized birds in a flock over time. The aim of this study was to determine the rate of transmission of Campylobacter using field data gathered for 5 years for Australian broiler flocks. We used unique sampling data for 42 Campylobacter jejuni-colonized flocks and estimated the transmission rate, which is defined as the number of secondary infections caused by one colonized bird per day. The estimate was 2.37 ± 0.295 infections per infectious bird per day, which implies that in our study population colonized flocks consisting of 20,000 broilers would have an increase in within-flock prevalence to 95% within 4.4 to 7.2 days after colonization of the first broiler. Using Bayesian analysis, the moment of colonization of the first bird in a flock was estimated to be from 21 days of age onward in all flocks in the study. This study provides an important quantitative estimate of the rate of transmission of Campylobacter in broiler flocks, which could be helpful in future studies on the epidemiology of Campylobacter in the field.     

Effect of Bacteriophage Application on Campylobacter jejuni Loads in Commercial Broiler Flocks

Campylobacteriosis is the most frequent food-borne human enteritis. The major source for infection with Campylobacter spp. is broiler meat. Risk assessments consider the reduction of Campylobacter in primary production to be most beneficial for human health. The aim of this study was to test the efficacy of a bacteriophage application under commercial conditions which had proved to be effective in previous noncommercial studies under controlled experimental conditions. A phage cocktail for Campylobacter reduction was tested on three commercial broiler farms each with a control and an experimental group. Colonization of Campylobacter was confirmed prior to phage application in fecal samples. Subsequently, a phage cocktail was applied via drinking water in the experimental group (log₁₀ 5.8 to 7.5 PFU/bird). One day after phage application, Campylobacter counts of one experimental group were reduced under the detection limit (<50 CFU/g, P = 0.0140) in fecal samples. At slaughter, a significant reduction of >log₁₀ 3.2 CFU/g cecal content compared to the control was still detected (P = 0.0011). No significant reduction was observed in the experimental groups of the other trials. However, a significant drop in cecal Campylobacter counts occurred in a phage-contaminated control. These results suggest that maximum reduction of Campylobacter at the slaughterhouse might be achieved by phage application 1 to 4 days prior to slaughter.     

Genotypic Diversity among Campylobacter jejuni Isolates in a Commercial Broiler Flock

Analysis of nucleic acid polymorphism in the flagellin genes of Campylobacter jejuni was used to investigate genetic diversity among Campylobacter spp. in a commercial broiler flock. Three hundred single colonies of C. jejuni were isolated from fecal samples collected weekly for 3 weeks immediately before slaughter. Both the flaA and flaB genes were amplified by PCR, and the PCR product was digested with the restriction enzyme AluI. The fragments generated were then analyzed by agarose gel electrophoresis. Among the 300 recovered isolates, five different restriction fragment length polymorphism profiles were observed. Three of these profiles were dominant during the course of the study, and the other two profiles were detected at low frequency. Analysis of genetic variation in C. jejuni over the course of an experimental infection lasting 7 weeks indicated that there was no obvious drift in the flagellin gene type. These findings demonstrate that a range of bacterial genotypes can constitute the bacterial population within a commercial poultry flock, with the most likely sources of these types being multiple environmental exposure and/or genetic drift within the population. This degree of diversity must be considered in epidemiological analyses which utilize genetic typing methods that investigate Campylobacter contamination of any food source, including poultry, to ensure that the total gene pool for C. jejuni is evaluated.     

Sources of Campylobacter spp. Colonizing Housed Broiler Flocks during Rearing

The study aimed to identify sources of campylobacter in 10 housed broiler flocks from three United Kingdom poultry companies. Samples from (i) the breeder flocks, which supplied the broilers, (ii) cleaned and disinfected houses prior to chick placement, (iii) the chickens, and (iv) the environments inside and outside the broiler houses during rearing were examined. Samples were collected at frequent intervals and examined for Campylobacter spp. Characterization of the isolates using multilocus sequence typing (MLST), serotyping, phage typing, and flaA restriction fragment length polymorphism typing was performed. Seven flocks became colonized during the growing period. Campylobacter spp. were detected in the environment surrounding the broiler house, prior to as well as during flock colonization, for six of these flocks. On two occasions, isolates detected in a puddle just prior to the birds being placed were indistinguishable from those colonizing the birds. Once flocks were colonized, indistinguishable strains of campylobacter were found in the feed and water and in the air of the broiler house. Campylobacter spp. were also detected in the air up to 30 m downstream of the broiler house, which raises the issue of the role of airborne transmission in the spread of campylobacter. At any time during rearing, broiler flocks were colonized by only one or two types determined by MLST but these changed, with some strains superseding others. In conclusion, the study provided strong evidence for the environment as a source of campylobacters colonizing housed broiler flocks. It also demonstrated colonization by successive campylobacter types determined by MLST during the life of a flock.     

Campylobacter jejuni Strains Compete for Colonization in Broiler Chicks

Campylobacter jejuni isolates possess multiple adhesive proteins termed adhesins, which promote the organism's attachment to epithelial cells. Based on the proposal that one or more adhesins are shared among C. jejuni isolates, we hypothesized that C. jejuni strains would compete for intestinal and cecal colonization in broiler chicks. To test this hypothesis, we selected two C. jejuni strains with unique SmaI pulsed-field gel electrophoresis macrorestriction profiles and generated one nalidixic acid-resistant strain (the F38011 Nal^r strain) and one streptomycin-resistant strain (the 02-833L Str^r strain). In vitro binding assays revealed that the C. jejuni F38011 Nal^r and 02-833L Str^r strains adhered to LMH chicken hepatocellular carcinoma epithelial cells and that neither strain influenced the binding potential of the other strain at low inoculation doses. However, an increase in the dose of the C. jejuni 02-833L Str^r strain relative to that of the C. jejuni F38011 Nal^r strain competitively inhibited the binding of the C. jejuni F38011 Nal^r strain to LMH cells in a dose-dependent fashion. Similarly, the C. jejuni 02-833L Str^r strain was found to significantly reduce the efficiency of intestinal and cecal colonization by the C. jejuni F38011 Nal^r strain in broiler chickens. Based on the number of bacteria recovered from the ceca, the maximum number of bacteria that can colonize the digestive tracts of chickens may be limited by host constraints. Collectively, these data support the hypothesis that C. jejuni strains compete for colonization in chicks and suggest that it may be possible to design novel intervention strategies for reducing the level at which C. jejuni colonizes the cecum.     

Comparison of Genotypes and Serotypes of Campylobacter jejuni Isolated from Danish Wild Mammals and Birds and from Broiler Flocks and Humans

The incidence of human infection with Campylobacter jejuni is increasing in most developed countries and the reason for this is largely unknown. Although poultry meat is considered to be a major source, it is evident that other reservoirs exist, possibly common to humans and poultry. Environmental sources are believed to be important reservoirs of Campylobacter infection in broiler chicken flocks. We investigated the potential importance of wildlife as a source of infection in commercial poultry flocks and in humans by comparing the serotype distributions, fla types, and macrorestriction profiles (MRPs) of C. jejuni isolates from different sources. The serotype distribution in wildlife was significantly different from the known distributions in broilers and humans. Considerable sero- and genotype diversity was found within the wildlife collection, although two major groups of isolates within serotype O:12 and the O:4 complex were found. Common clonal lines among wildlife, chicken, and/or human isolates were identified within serotype O:2 and the O:4 complex. However, MRPs of O:12 and O:38 strains isolated from wildlife and other sources indicated that some clonal lines propagated in a wide selection of animal species but were not detected in humans or broilers in this study. The applied typing methods successfully identified different clonal groups within a strain collection showing large genomic diversity. However, the relatively low number of wildlife strains with an inferred clonal relationship to human and chicken strains suggests that the importance of wildlife as a reservoir of infection is limited.     

Bacteriophage Therapy To Reduce Campylobacter jejuni Colonization of Broiler Chickens

Colonization of broiler chickens by the enteric pathogen Campylobacter jejuni is widespread and difficult to prevent. Bacteriophage therapy is one possible means by which this colonization could be controlled, thus limiting the entry of campylobacters into the human food chain. Prior to evaluating the efficacy of phage therapy, experimental models of Campylobacter colonization of broiler chickens were established by using low-passage C. jejuni isolates HPC5 and GIIC8 from United Kingdom broiler flocks. The screening of 53 lytic bacteriophage isolates against a panel of 50 Campylobacter isolates from broiler chickens and 80 strains isolated after human infection identified two phage candidates with broad host lysis. These phages, CP8 and CP34, were orally administered in antacid suspension, at different dosages, to 25-day-old broiler chickens experimentally colonized with the C. jejuni broiler isolates. Phage treatment of C. jejuni-colonized birds resulted in Campylobacter counts falling between 0.5 and 5 log₁₀ CFU/g of cecal contents compared to untreated controls over a 5-day period postadministration. These reductions were dependent on the phage-Campylobacter combination, the dose of phage applied, and the time elapsed after administration. Campylobacters resistant to bacteriophage infection were recovered from phage-treated chickens at a frequency of <4%. These resistant types were compromised in their ability to colonize experimental chickens and rapidly reverted to a phage-sensitive phenotype in vivo. The selection of appropriate phage and their dose optimization are key elements for the success of phage therapy to reduce campylobacters in broiler chickens.     

Molecular Epidemiology of Campylobacter jejuni in Broiler Flocks Using Randomly Amplified Polymorphic DNA-PCR and 23S rRNA-PCR and Role of Litter in Its Transmission

Poultry has long been cited as a reservoir for Campylobacter spp., and litter has been implicated as a vehicle in their transmission. Chicks were raised on litter removed from a broiler house positive for Campylobacter jejuni. Litter was removed from the house on days 0, 3, and 9 after birds were removed for slaughter. Chicks were raised on these three litters under controlled conditions in flocks of 25. None of these birds yielded C. jejuni in their cecal droppings through 7 weeks. Two successive flocks from the same Campylobacter-positive broiler house were monitored for Campylobacter colonization. Campylobacter jejuni prevalence rates were determined for each flock. Randomly amplified polymorphic DNA (RAPD)-PCR and 23S rRNA-PCR typing methods were used to group isolates. A high prevalence (60%) of C. jejuni in flock 1 coincided with the presence of an RAPD profile not appearing in flock 2, which had a lower rate of prevalence (28%). A 23S rRNA-PCR typing method was used to determine if strains with different RAPD profiles and different prevalence rates contained different 23S sequences. RAPD profiles detected with higher prevalence rates contained a spacer in the 23S rRNA region 100% of the time, while RAPD profiles found with lower prevalence rates contained an intervening sequence less than 2% of the time. Data suggest varying colonizing potentials of different RAPD profiles and a source other than previously used litter as a means of transmission of C. jejuni. These molecular typing methods demonstrate their usefulness, when used together, in this epidemiologic investigation.     

Impact of a Single Phage and a Phage Cocktail Application in Broilers on Reduction of Campylobacter jejuni and Development of Resistance

Campylobacteriosis is currently the most frequent foodborne zoonosis in many countries. One main source is poultry. The aim of this study was to enhance the knowledge about the potential of bacteriophages in reducing colonization of broilers with Campylobacter , as there are only a few in vivo studies published. Commercial broilers were inoculated with 10⁴ CFU/bird of a Campylobacter jejuni field strain. Groups of 88 birds each were subsequently treated with a single phage or a four-phage cocktail (10⁷ PFU/bird in CaCO₃ buffered SM-Buffer). Control birds received the solvent only. Afterwards, subgroups of eleven birds each were examined for their loads with phages and Campylobacter on day 1, 3, 7, 14, 21, 28, 35 and 42 after phage application. The susceptibility of the Campylobacter population to phage infection was determined using ten isolates per bird. In total 4180 re-isolates were examined. The study demonstrated that the deployed phages persisted over the whole investigation period. The Campylobacter load was permanently reduced by the phage-cocktail as well as by the single phage. The reduction was significant between one and four weeks after treatment and reached a maximum of log₁₀ 2.8 CFU/g cecal contents. Phage resistance rates of initially up to 43% in the single phage treated group and 24% in the cocktail treated group later stabilized at low levels. The occurrence of phage resistance influenced but did not override the Campylobacter reducing effect. Regarding the reduction potential, the cocktail treatment had only a small advantage over the singe phage treatment directly after phage administration. However, the cocktail moderated and delayed the emergence of phage resistance.     

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.     

Analysis of Recombination in Campylobacter jejuni from MLST Population Data

We analyze recombination in C. jejuni using MLST data from isolates taken from wild birds, cattle, wild rabbits, and water in a 100-km² study region in Cheshire, UK. We use a recent approximate likelihood method for inference, based on combining likelihood information from all pairs of segregating (polymorphic) sites in the data. We find substantial evidence for recombination, but only for recombination with short tract lengths, of around 225–750 bp. We estimate that the rate of recombination is of a similar magnitude to the rate of mutation.[Reviewing Editor: Dr. Magnus Nordborg]     

Genotype and Antibiotic Resistance Analyses of Campylobacter Isolates from Ceca and Carcasses of Slaughtered Broiler Flocks

To obtain genetic information about Campylobacter jejuni and Campylobacter coli from broilers and carcasses at slaughterhouses, we analyzed and compared 340 isolates that were collected in 2008 from the cecum right after slaughter or from the neck skin after processing. We performed rpoB sequence-based identification, multilocus sequence typing (MLST), and flaB sequence-based typing; we additionally analyzed mutations within the 23S rRNA and gyrA genes that confer resistance to macrolide and quinolone antibiotics, respectively. The rpoB-based identification resulted in a distribution of 72.0% C. jejuni and 28.0% C. coli. The MLST analysis revealed that there were 59 known sequence types (STs) and 6 newly defined STs. Most of the STs were grouped into 4 clonal complexes (CC) that are typical for poultry (CC21, CC45, CC257, and CC828), and these represented 61.8% of all of the investigated isolates. The analysis of 95 isolates from the cecum and from the corresponding carcass neck skin covered 44 different STs, and 54.7% of the pairs had matching genotypes. The data indicate that cross-contamination from various sources during slaughter may occur, although the majority of Campylobacter contamination on carcasses appeared to originate from the slaughtered flock itself. Mutations in the 23S rRNA gene were found in 3.1% of C. coli isolates, although no mutations were found in C. jejuni isolates. Mutations in the gyrA gene were observed in 18.9% of C. jejuni and 26.8% of C. coli isolates, which included two C. coli strains that carried mutations conferring resistance to both classes of antibiotics. A relationship between specific genotypes and antibiotic resistance/susceptibility was observed.Campylobacteriosis is the leading food-borne bacterial gastroenteritis worldwide (12, 15). In Switzerland, the number of registered campylobacteriosis cases has rapidly increased to more than 100 per 100,000 inhabitants in the past few years (14), and this trend has also been observed in the European Union (EU) (12). However, the real number of cases is likely higher, because not all cases are reported due to the self-limiting nature of the disease and its potentially mild symptoms.Campylobacter jejuni and Campylobacter coli are commonly associated with human infection, and they can be detected in up to 85% and 15% of cases, respectively (33). Despite the important role that C. jejuni and C. coli play as zoonotic pathogens worldwide, there is little information regarding the route(s) of transmission (17). Numerous case-control and modeling studies on the infection sources of C. jejuni and C. coli have suggested that handling and consumption of contaminated poultry meat are associated with a risk of human campylobacteriosis (17, 45, 47, 49, 51). Initial meat contamination with C. jejuni or C. coli from the chicken intestine may occur during commonly used automated slaughter processing through several routes, such as the air, water, previously slaughtered flocks, or machinery (19, 36, 37).Precise genotyping and continuous comparison of the strains obtained from, e.g., the production site, flocks, slaughterhouse, retail meat, and infected humans would help to trace the source of infection and might indicate possible intervention strategies for the contaminated site.DNA sequence-based typing methods, such as multilocus sequence typing (MLST), are well suited for this purpose (28), and MLST has become the method of choice for genotyping of Campylobacter (6, 8). Moreover, extension of the classical MLST technique for C. jejuni and C. coli with sequencing of the short variable region (SVR) within the flagellin-encoding gene flaB allows a more precise differentiation among strains that have the same MLST sequence type (ST) (9, 29). An extended MLST work flow was recently developed that reduces the associated time and cost (24). In addition, the new approach allows genetic determination of antibiotic resistance to quinolones and macrolides. Resistance to these antibiotics is a worldwide issue of concern, as an increasing number of Campylobacter isolates are resistant to them. Strikingly, a number of strains are resistant to ciprofloxacin (a quinolone) and, to a lesser extent, erythromycin (a macrolide), which is problematic, because these drugs are typically used to treat campylobacteriosis. Resistance to quinolones is mainly associated with a point mutation in the DNA gyrase gene (gyrA) at position C257T, and a transition in the 23S rRNA gene at position A2075G is commonly responsible for macrolide resistance (1). Simple sequence-based analysis of these common mutational positions can therefore provide information about the antibiotic susceptibility or resistance of a strain. Besides the prudent use of antibiotics, knowledge about the genetic composition of the infectious agent can be helpful to both treat the disease and prevent the spread of resistant strains.In the current study, MLST, flaB typing, and sequence-based determination of quinolone and macrolide resistances were used to investigate the genetic background of C. jejuni and C. coli isolates collected from Swiss broilers in a spatiotemporal study in 2008. We addressed the following three aspects: (i) the diversity of Campylobacter isolates that were recovered from pooled cecum samples and the carcass neck skin, (ii) the possible impact of cross- and self-contamination during slaughter, and (iii) the antibiotic resistance of Campylobacter strains from the broiler flocks and chicken carcasses. All of the data, including the strain information and trace files, were entered into a commercial Web-based Campylobacter MLST database (SmartGene, Zug, Switzerland). This database allows users to retrieve and compare information for any analyzed strain for monitoring purposes (24).     

Effects of Climate on Incidence of Campylobacter spp. in Humans and Prevalence in Broiler Flocks in Denmark

Campylobacter infections are increasing and pose a serious public health problem in Denmark. Infections in humans and broiler flocks show similar seasonality, suggesting that climate may play a role in infection. We examined the effects of temperature, precipitation, relative humidity, and hours of sunlight on Campylobacter incidence in humans and broiler flocks by using lag dependence functions, locally fitted linear models, and cross validation methods. For humans, the best model included average temperature and sunlight 4 weeks prior to infection; the maximum temperature lagged at 4 weeks was the best single predictor. For broilers, the average and maximum temperatures 3 weeks prior to slaughter gave the best estimate; the average temperature lagged at 3 weeks was the best single predictor. The combined effects of temperature and sunlight or the combined effects of temperature and relative humidity predicted the incidence in humans equally well. For broiler flock incidence these factors explained considerably less. Future research should focus on elements within the broiler environment that may be affected by climate, as well as the interaction of microclimatic factors on and around broiler farms. There is a need to quantify the contribution of broilers as a source of campylobacteriosis in humans and to further examine the effect of temperature on human incidence after this contribution is accounted for. Investigations should be conducted into food consumption and preparation practices and poultry sales that may vary by season.     

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.     

Phenotypic and Genotypic Characterizations of Campylobacter jejuni Isolated from the Broiler Meat Production Process

A set of C. jejuni isolates of different origins and flaA-genotypes obtained throughout the broiler meat production chain was tested in this study for a possible correlation of their origin, phylogenetic relationship, and phenotypic properties. Interestingly, the results showed a correlation of the origin and the phylogenetic relationship between the C. jejuni isolates and their ability to form biofilm, but not in their ability to survive at -18, 5, 20, and 48?°C. Two strains, a broiler cloacae isolate and a broiler fillet isolate, were unable to develop biofilm, while most of the C. jejuni isolates originating from meat and surfaces of the slaughterhouse readily formed biofilms after both 24, 48, and 72?h. Interestingly, these biofilm-forming strains were closely related. Furthermore, two strains that were isolated after disinfection developed significantly more biofilms after 24?h of incubation than the remaining strains. A comparative genomic analysis using DNA microarrays showed that the gene contents of strains that efficiently formed biofilms were different from those that did not. The study suggests that biofilm formation might be a lineage specific property, allowing C. jejuni to both survive environmental stress at the slaughterhouse and to attach to the surface of meat.     

Primary Isolation Strain Determines Both Phage Type and Receptors Recognised by Campylobacter jejuni Bacteriophages

In this study we isolated novel bacteriophages, infecting the zoonotic bacterium Campylobacter jejuni. These phages may be used in phage therapy of C. jejuni colonized poultry to prevent spreading of the bacteria to meat products causing disease in humans. Many C. jejuni phages have been isolated using NCTC12662 as the indicator strain, which may have biased the selection of phages. A large group of C. jejuni phages rely on the highly diverse capsular polysaccharide (CPS) for infection and recent work identified the O-methyl phosphoramidate modification (MeOPN) of CPS as a phage receptor. We therefore chose seven C. jejuni strains each expressing different CPS structures as indicator strains in a large screening for phages in samples collected from free-range poultry farms. Forty-three phages were isolated using C. jejuni NCTC12658, NCTC12662 and RM1221 as host strains and 20 distinct phages were identified based on host range analysis and genome restriction profiles. Most phages were isolated using C. jejuni strains NCTC12662 and RM1221 and interestingly phage genome size (140 kb vs. 190 kb), host range and morphological appearance correlated with the isolation strain. Thus, according to C. jejuni phage grouping, NCTC12662 and NCTC12658 selected for CP81-type phages, while RM1221 selected for CP220-type phages. Furthermore, using acapsular ∆kpsM mutants we demonstrated that phages isolated on NCTC12658 and NCTC12662 were dependent on the capsule for infection. In contrast, CP220-type phages isolated on RM1221 were unable to infect non-motile ∆motA mutants, hence requiring motility for successful infection. Hence, the primary phage isolation strain determines both phage type (CP81 or CP220) as well as receptors (CPS or flagella) recognised by the isolated phages.     

Darkling Beetles (Alphitobius diaperinus) and Their Larvae as Potential Vectors for the Transfer of Campylobacter jejuni and Salmonella enterica Serovar Paratyphi B Variant Java between Successive Broiler Flocks

Broiler flocks often become infected with Campylobacter and Salmonella, and the exact contamination routes are still not fully understood. Insects like darkling beetles and their larvae may play a role in transfer of the pathogens between consecutive cycles. In this study, several groups of beetles and their larvae were artificially contaminated with a mixture of Salmonella enterica serovar Paratyphi B Variant Java and three C. jejuni strains and kept for different time intervals before they were fed to individually housed chicks. Most inoculated insects were positive for Salmonella and Campylobacter just before they were fed to the chicks. However, Campylobacter could not be isolated from insects that were kept for 1 week before they were used to mimic an empty week between rearing cycles. All broilers fed insects that were inoculated with pathogens on the day of feeding showed colonization with Campylobacter and Salmonella at levels of 50 to 100%. Transfer of both pathogens by groups of insects that were kept for 1 week before feeding to the chicks was also observed, but at lower levels. Naturally contaminated insects that were collected at a commercial broiler farm colonized broilers at low levels as well. In conclusion, the fact that Salmonella and Campylobacter can be transmitted via beetles and their larvae to flocks in successive rearing cycles indicates that there should be intensive control programs for exclusion of these insects from broiler houses.     

Reduction of Campylobacter jejuni in Broiler Chicken by Successive Application of Group II and Group III Phages

Background

Bacteriophage treatment is a promising tool to reduce Campylobacter in chickens. Several studies have been published where group II or group III phages were successfully applied. However, these two groups of phages are different regarding their host ranges and host cell receptors. Therefore, a concerted activity of group II and group III phages might enhance the efficacy of a treatment and decrease the number of resistant bacteria.

Results

In this study we have compared the lytic properties of some group II and group III phages and analysed the suitability of various phages for a reduction of C. jejuni in broiler chickens. We show that group II and group III phages exhibit different kinetics of infection. Two group III and one group II phage were selected for animal experiments and administered in different combinations to three groups of chickens, each containing ten birds. While group III phage CP14 alone reduced Campylobacter counts by more than 1 log₁₀ unit, the concomitant administration of a second group III phage (CP81) did not yield any reduction, probably due to the development of resistance induced by this phage. One group of chickens received phage CP14 and, 24 hours later, group II phage CP68. In this group of animals, Campylobacter counts were reduced by more than 3 log₁₀ units.

Conclusion

The experiments illustrated that Campylobacter phage cocktails have to be carefully composed to achieve the best results.     

Genomic Diversity of Campylobacter coli and Campylobacter jejuni Isolates Recovered from Free-Range Broiler Farms and Comparison with Isolates of Various Origins

In many industrialized countries, the incidence of campylobacteriosis exceeds that of salmonellosis. Campylobacter bacteria are transmitted to humans mainly in food, especially poultry meat products. Total prevention of Campylobacter colonization in broiler flocks is the best way to reduce (or eliminate) the contamination of poultry products. The aim of this study was to establish the sources and routes of contamination of broilers at the farm level. Molecular typing methods (DNA macrorestriction pulsed-field gel electrophoresis and analysis of gene polymorphism by PCR-restriction fragment length polymorphism) were used to characterize isolates collected from seven broiler farms. The relative genomic diversity of Campylobacter coli and Campylobacter jejuni was determined. Analysis of the similarity among 116 defined genotypes was used to determine clusters within the two species. Furthermore, evidence of recombination suggested that there were genomic rearrangements within the Campylobacter populations. Recovery of related clusters from different broiler farms showed that some Campylobacter strains might be specifically adapted to poultry. Analysis of the Campylobacter cluster distribution on three broiler farms showed that soil in the area around the poultry house was a potential source of Campylobacter contamination. The broilers were infected by Campylobacter spp. between days 15 and 36 during rearing, and the type of contamination changed during the rearing period. A study of the effect of sanitary barriers showed that the chickens stayed Campylobacter spp. free until they had access to the open area. They were then rapidly colonized by the Campylobacter strains isolated from the soil.     

Colonization of Broilers with Campylobacter in Conventional Broiler-Chicken Flocks

Eight of 16 conventional broiler-chicken flocks examined contained Campylobacter. All isolates were identified as C. jejuni except from 1 flock were C. coli was isolated. One herd consisting of 6 different houses where Campylobacter regularly has been isolated was continuously examined. It was not possible to isolate Campylobacter from newly hatched chickens or from environmental samples and cloacal swabs during the 2 first weeks of growth.