Tomasz Seliwiorstow

Passive immunization to reduce Campylobacter jejuni colonization and transmission in broiler chickens

Campylobacter jejuni is the most common cause of bacterium-mediated diarrheal disease in humans worldwide. Poultry products are considered the most important source of C. jejuni infections in humans but to date no effective strategy exists to eradicate this zoonotic pathogen from poultry production. Here, the potential use of passive immunization to reduce Campylobacter colonization in broiler chicks was examined. For this purpose, laying hens were immunized with either a whole-cell lysate or the hydrophobic protein fraction of C. jejuni and their eggs were collected. In vitro tests validated the induction of specific ImmunoglobulinY (IgY) against C. jejuni in the immunized hens’ egg yolks, in particular. In seeder experiments, preventive administration of hyperimmune egg yolk significantly (P < 0.01) reduced bacterial counts of seeder animals three days after oral inoculation with approximately 104 cfu C. jejuni, compared with control birds. Moreover, transmission to non-seeder birds was dramatically reduced (hydrophobic protein fraction) or even completely prevented (whole-cell lysate). Purified IgY promoted bacterial binding to chicken intestinal mucus, suggesting enhanced mucosal clearance in vivo. Western blot analysis in combination with mass spectrometry after two-dimensional gel-electrophoresis revealed immunodominant antigens of C. jejuni that are involved in a variety of cell functions, including chemotaxis and adhesion. Some of these (AtpA, EF-Tu, GroEL and CtpA) are highly conserved proteins and could be promising targets for the development of subunit vaccines.

Campylobacter carcass contamination throughout the slaughter process of Campylobacter-positive broiler batches.

Campylobacter contamination on broiler carcasses of Campylobacter colonized flocks was quantified at seven sampling sites throughout the slaughter process. For this purpose, in four slaughterhouses samples were collected from twelve Campylobacter positive batches. Broilers from all visits carried high numbers of campylobacters in their caeca (≥7.9log10cfu/g). Campylobacter counts on feathers (up to 6.8log10cfu/g), positively associated with the breast skin contamination of incoming birds and carcasses after plucking, were identified as an additional source of carcass contamination. A high variability in Campylobacter carcass contamination on breast skin samples within batches and between batches in the same slaughterhouse and between slaughterhouses was observed. In slaughterhouses A, B, C and D Campylobacter counts exceeded a limit of 1000cfu/g on 50%, 56%, 78% and 11% of carcasses after chilling, respectively. This finding indicates that certain slaughterhouses are able to better control Campylobacter contamination than others. Overall, the present study focuses on the descriptive analysis of Campylobacter counts in different slaughterhouses, different batches within a slaughterhouse and within a batch at several sampling locations.

Discriminative power of Campylobacter phenotypic and genotypic typing methods.

The aim of this study was to compare different typing methods, individually and combined, for use in the monitoring of Campylobacter in food. Campylobacter jejuni (n=94) and Campylobacter coli (n=52) isolated from different broiler meat carcasses were characterized using multilocus sequence typing (MLST), flagellin gene A restriction fragment length polymorphism typing (flaA-RFLP), antimicrobial resistance profiling (AMRp), the presence/absence of 5 putative virulence genes; and, exclusively for C. jejuni, the determination of lipooligosaccharide (LOS) class. Discriminatory power was calculated by the Simpsons index of diversity (SID) and the congruence was measured by the adjusted Rand index and adjusted Wallace coefficient. MLST was individually the most discriminative typing method for both C. jejuni (SID=0.981) and C. coli (SID=0.957). The most discriminative combination with a SID of 0.992 for both C. jejuni and C. coli was obtained by combining MLST with flaA-RFLP. The combination of MLST with flaA-RFLP is an easy and feasible typing method for short-term monitoring of Campylobacter in broiler meat carcass.

Identification of risk factors for Campylobacter contamination levels on broiler carcasses during the slaughter process

Campylobacter carcass contamination was quantified across the slaughter line during processing of Campylobacter positive batches. These quantitative data were combined together with information describing slaughterhouse and batch related characteristics in order to identify risk factors for Campylobacter contamination levels on broiler carcasses. The results revealed that Campylobacter counts are influenced by the contamination of incoming birds (both the initial external carcass contamination and the colonization level of caeca) and the duration of transport and holding time that can be linked with feed withdrawal period. In addition, technical aspects of the slaughter process such as a dump based unloading system, electrical stunning, lower scalding temperature, incorrect setting of plucking, vent cutter and evisceration machines were identified as risk factors associated with increased Campylobacter counts on processed carcasses. As such the study indicates possible improvements of the slaughter process that can result in better control of Campylobacter numbers under routine processing of Campylobacter positive batches without use of chemical or physical decontamination. Moreover, all investigated factors were existing variations of the routine processing practises and therefore proposed interventions are practically and economically achievable.

Transfer of Campylobacter from a Positive Batch to Broiler Carcasses of a Subsequently Slaughtered Negative Batch: A Quantitative Approach

The present study was conducted to quantify Campylobacter cross-contamination from a positive batch of broiler chicken carcasses to a negative batch at selected processing steps and to evaluate the duration of this cross-contamination. During each of nine visits conducted in three broiler slaughterhouses, Campylobacter levels were determined on broiler carcasses originating from Campylobacter-negative batches processed immediately after Campylobacter-positive batches. Data were collected after four steps during the slaughter process (scalding, plucking, evisceration, and washing) at 1, 10, and 20 min after the start of the slaughter of the batches. Campylobacter levels in ceca of birds from Campylobacter-positive batches ranged from 5.62 to 9.82 log CFU/g. When the preceding positive batch was colonized at a low level, no (enumerable) carcass contamination was found in a subsequent negative batch. However, when Campylobacter levels were high in the positive batch, Campylobacter was found on carcasses of the subsequent negative batch but at levels significantly lower than those found on carcasses from the preceding positive batch. The scalding and the evisceration process contributed the least (< 1.5 log CFU/g) and the most (up to 4 log CFU/ g), respectively, to the Campylobacter transmission from a positive batch to a negative batch. Additionally, the number of Campylobacter cells transferred from positive to negative batches decreased over the first 20 min of sampling time. However, the reduction was slower than previously estimated in risk assessment studies, suggesting that pathogen transfer during crosscontamination is a complex process.

Relation between broiler and human C. jejuni strains isolated in Belgium from 2011 to 2013

This study inquires the relationship between Campylobacter jejuni isolated from broiler meat carcasses (n = 97) and human clinical samples (n = 72) in Belgium, from 2011 to 2013.