Campylobacter in Broiler Chicken and Broiler Meat in Sri Lanka: Influence of Semi-Automated vs. Wet Market Processing on Campylobacter Contamination of Broiler Neck Skin Samples.

Broiler meat can become contaminated with Campylobacter of intestinal origin during processing. The present study aimed to identify the prevalence of Campylobacter in broiler flocks and meat contamination at retail shops, and determine the influence of semi-automated and wet market processing on Campylobacter contamination of neck skin samples. Samples were collected from semi-automated plants (n = 102) and wet markets (n = 25). From each batch of broilers, pooled caecal samples and neck skin samples were tested for Campylobacter. Broiler meat purchased from retail outlets (n = 37) was also tested. The prevalence of Campylobacter colonized broiler flocks was 67%. The contamination of meat at retail was 59%. Both semi-automated and wet market processing resulted to contaminate the broiler neck skins to the levels of 27.4% and 48%, respectively. When Campylobacter-free broiler flocks were processed in semi-automated facilities 15% (5/33) of neck skin samples became contaminated by the end of processing whereas 25% (2/8) became contaminated after wet market processing. Characterization of isolates revealed a higher proportion of C. coli compared to C. jejuni. Higher proportions of isolates were resistant to important antimicrobials. This study shows the importance of Campylobacter in poultry industry in Sri Lanka and the need for controlling antimicrobial resistance.

Campylobacter fetus infections in humans: exposure and disease.

Campylobacter fetus can cause intestinal illness and, occasionally, severe systemic infections. Infections mainly affect persons at higher risk, including elderly and immunocompromised individuals and those with occupational exposure to infected animals. Outbreaks are infrequent but have provided insight into sources. Source attribution of sporadic cases through case-control interviews has not been reported. The reservoirs for C. fetus are mainly cattle and sheep. Products from these animals are suspected as sources for human infections. Campylobacter fetus is rarely isolated from food, albeit selective isolation methods used in food microbiology are not suited for its detection. We hypothesize that the general population is regularly exposed to C. fetus through foods of animal origin, cross-contaminated foodstuffs, and perhaps other, as yet unidentified, routes. Campylobacter fetus infection should be suspected particularly in patients with nonspecific febrile illness who are immunocompromised or who may have been occupationally exposed to ruminants.

Evaluation of molecular assays for identification Campylobacter fetus species and subspecies and development of a C. fetus specific real-time PCR assay.

Phenotypic differentiation between Campylobacter fetus (C. fetus) subspecies fetus and C. fetus subspecies venerealis is hampered by poor reliability and reproducibility of biochemical assays. AFLP (amplified fragment length polymorphism) and MLST (multilocus sequence typing) are the molecular standards for C. fetus subspecies identification, but these methods are laborious and expensive. Several PCR assays for C. fetus subspecies identification have been described, but a reliable comparison of these assays is lacking. The aim of this study was to evaluate the most practical and routinely implementable published PCR assays designed for C. fetus species and subspecies identification. The sensitivity and specificity of the assays were calculated by using an extensively characterized and diverse collection of C. fetus strains. AFLP and MLST identification were used as reference. Two PCR assays were able to identify C. fetus strains correctly at species level. The C. fetus species identification target, gene nahE, of one PCR assay was used to develop a real-time PCR assay with 100% sensitivity and 100% specificity, but the development of a subspecies venerealis specific real-time PCR (ISCfe1) failed due to sequence variation of the target insertion sequence and prevalence in other Campylobacter species. None of the published PCR assays was able to identify C. fetus strains correctly at subspecies level. Molecular analysis by AFLP or MLST is still recommended to identify C. fetus isolates at subspecies level.

Genetic relationships among reptilian and mammalian Campylobacter fetus strains determined by multilocus sequence typing.

Reptile Campylobacter fetus isolates and closely related strains causing human disease were characterized by multilocus sequence typing. They shared approximately 90% nucleotide sequence identity with classical mammalian C. fetus, and there was evidence of recombination among members of these two groups. The reptile group represents a possible separate genomospecies capable of infecting humans.

Molecular epidemiology of Campylobacter fetus subsp. fetus on bovine artificial insemination stations using pulsed field gel electrophoresis.

The presence of Campylobacter fetus subspecies fetus (Cff) on bovine artificial insemination (AI)-stations can have major economical consequences. More knowledge on the epidemiology of C. fetus is needed to control Cff infections at AI-stations. We assessed the epidemiology of Cff on AI-stations and the molecular relationship between Cff strains isolated from outbreaks on AI-stations. Thirteen Cff strains (two Cff strains per outbreak and one sporadic case) isolated from bulls housed on different AI-stations were selected and compared with ten unrelated bovine and ovine Cff isolates from different geographical regions. Molecular typing by pulsed field gel electrophoresis (PFGE) with the restriction enzymes SmaI, SalI and KpnI, yielded unique profiles for most unrelated strains but indistinguishable profiles for all isolates from the same outbreak. Computer aided analysis using a composite data set of SmaI, SalI and KpnI restriction profiles revealed separate clusters for outbreak strains. Thus, PFGE profiling of Cff strains is a valuable tool to discriminate between strains derived from separate outbreaks and to identify routes of infection.

Clonal nature of Campylobacter fetus as defined by multilocus sequence typing.

Campylobacter fetus can be divided into the subspecies C. fetus subsp. fetus and C. fetus subsp. venerealis. C. fetus subsp. fetus causes sporadic infections in humans and abortion in cattle and sheep and has been isolated from a variety of sites in different hosts. C. fetus subsp. venerealis is host restricted, being isolated mainly from the genital tracts of cattle, and is the causative agent of bovine genital campylobacteriosis. Despite differences in niche preference, microbiological subspecies differentiation has proven difficult. Different typing methods divided C. fetus isolates into different subgroups, depending on the methods used. The relative value of these methods can be assessed by the evolutionary relationship of isolates belonging to the genus; therefore, we developed a multilocus sequence typing (MLST) scheme for C. fetus. This scheme was applied to 140 C. fetus isolates previously typed by amplified fragment length polymorphism (AFLP) analysis. A total of 14 different sequence types (STs) were identified, and these exhibited low levels of inter-ST genetic diversity, with only 22 variable sites in 3,312 nucleotides. These MLST data indicate that C. fetus is genetically homogeneous compared to the homogeneity of other Campylobacter species. The two C. fetus subspecies were extremely closely related genetically, but ST-4 was associated only with C. fetus subsp. venerealis, which represents a "bovine" clone. The C. fetus subsp. fetus isolates studied were more diverse in terms of their STs, and the STs correlated with epidemiological relationships. Congruence was observed among C. fetus subspecies, sap type, and ST; therefore, MLST confirms that mammalian C. fetus is genetically stable, probably as result of the introduction of a single ancestral clone into a mammalian niche.

Phage therapy reduces Campylobacter jejuni colonization in broilers.

The effect of phage therapy in the control of Campylobacter jejuni colonization in young broilers, either as a preventive or a therapeutic measure, was tested. A prevention group was infected with C. jejuni at day 4 of a 10-day phage treatment. A therapeutic group was phage treated for 6 days, starting 5 days after C. jejuni colonization of the broilers had been established. Treatment was monitored by enumerating Campylobacter colony forming units (CFU) and phage plaque forming units (PFU) from caecal content. Counts were compared with control birds not receiving phage therapy. A clear 3 log decline in C. jejuni counts was initially observed in the therapeutic group, however, after 5 days bacterial counts stabilized at a level 1 log lower than that of the control group. Colonization of C. jejuni in the prevention group was delayed by the treatment and after an initial 2 log reduction, colonization stabilized within a week at levels comparable to the therapeutic group. The CFU and PFU counts displayed opposing highs and lows over time, indicative of alternating shifts in amplification of bacteria and phages. There were no adverse health effects from the phage treatment. Two different phages were combined as therapeutic treatment of Campylobacter positive chickens challenged at the age approaching broiler harvest. This again resulted in a significant decrease in Campylobacter colonization. We conclude that phage treatment is a promising alternative for reducing C. jejuni colonization in broilers.