948 resultados para CFU


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The aims of this investigation were to enumerate coliforms in fresh mangoes, puree, cheeks, and cheeks-in-puree in order to determine the source of these organisms in the processed products, to determine methods for their control, and to identify coliforms isolated from cheeks-in-puree to determine whether they have any public health significance. Product from four processors was tested on two occasions. The retail packs of cheeks-in-puree having the highest coliform counts were those in which raw puree was added to the cheeks. Coliform counts in these samples ranged between 1.4 × 103 and 5.4 × 104 cfu/g. Pasteurisation reduced the coliform count of raw puree to < 5 cfu/g. Forty-seven percent of the 73 colonies, isolated as coliforms on the basis of their colony morphology on violet red bile agar, were identified as Klebsiella pneumoniae using the ATB 32E Identification System. Klebsiella strains were tested for growth at 10 °C, faecal coliform response, and fermentation of -melizitose, to differentiate the three phenotypically similar strains, K. pneumoniae, K. terrigena and K planticola. Results indicated that 41% of K. pneumoniae isolates gave reactions typical of K. pneumoniae. A further 44% of strains gave an atypical reaction pattern for these tests and were designated ‘psychrotrophic’ K. pneumoniae. Klebsiella pneumoniae counts of between 2.1 × 103 and 4.9 × 104 cfu/g were predicted to occur in the retail packs of mango cheeks-in-puree produced by the processors who constituted this product with raw puree. In view of the opportunistic pathogenic nature of K. pneumoniae, its presence in these products is considered undesirable and steps, such as pasteurisation of puree, should be taken in order to inactivate it

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Aims: To assist in the development of safe piggery effluent re-use guidelines by determining the level of selected pathogens and indicator organisms in the effluent ponds of 13 south-east Queensland piggeries. Methods and Results: The numbers of thermotolerant coliforms, Campylobacter jejuni/coli, Erysipelothrix rhusiopathiae, Escherichia coli, Salmonella and rotavirus were determined in 29 samples derived from the 13 piggeries. The study demonstrated that the 13 final effluent ponds contained an average of 1Æ2 · 105 colony-forming units (CFU) 100 ml)1 of thermotolerant coliforms and 1Æ03 · 105 CFU 100 ml)1 of E. coli. The Campylobacter level varied from none detectable (two of 13 piggeries) to a maximum of 930 most probable number (MPN) 100 ml)1 (two of 13 piggeries). Salmonella was detected in the final ponds of only four of the 13 piggeries and then only at a low level (highest level being 51 MPN 100 ml)1). No rotavirus and no Erysip. rhusiopathiae were detected. The average log10 reductions across the ponding systems to the final irrigation pond were 1Æ77 for thermotolerant coliforms, 1Æ71 for E. coli and 1Æ04 for Campylobacter. Conclusions: This study has provided a baseline knowledge on the levels of indicator organisms and selected pathogens in piggery effluent. Significance and Impact of the Study: The knowledge gained in this study will assist in the development of guidelines to ensure the safe and sustainable re-use of piggery effluent.

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Aims: To investigate methods for the recovery of airborne bacteria within pig sheds and to then use the appropriate methods to determine the levels of heterotrophs and Escherichia coli in the air within sheds. Methods and Results: AGI-30 impingers and a six-stage Andersen multi-stage sampler (AMS) were used for the collection of aerosols. Betaine and catalase were added to impinger collection fluid and the agar plates used in the AMS. Suitable media for enumerating E. coli with the Andersen sampler were also evaluated. The addition of betaine and catalase gave no marked increase in the recovery of heterotrophs or E. coli. No marked differences were found in the media used for enumeration of E. coli. The levels of heterotrophs and E. coli in three piggeries, during normal pig activities, were 2Æ2 · 105 and 21 CFU m)3 respectively. Conclusions: The failure of the additives to improve the recovery of either heterotrophs or E. coli suggests that these organisms are not stressed in the piggery environment. The levels of heterotrophs in the air inside the three Queensland piggeries investigated are consistent with those previously reported in other studies. Flushing with ponded effluent had no marked or consistent effect on the heterotroph or E. coli levels. Significance and Impact of the Study: Our work suggests that levels of airborne heterotrophs and E. coli inside pig sheds have no strong link with effluent flushing. It would seem unlikely that any single management activity within a pig shed has a dominant influence on levels of airborne heterotrophs and E. coli

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A 5′ Taq nuclease assay utilising minor groove binder technology and targeting the 16S rRNA gene was designed to detect Pasteurella multocida (the causative agent of fowl cholera) in swabs collected from poultry. The assay was first evaluated using pure cultures. The assay correctly identified four P. multocida taxonomic type strains, 18 P. multocida serovar reference strains and 40 Australian field isolates (17 from poultry, 11 from pigs and 12 from cattle). Representatives of nine other Pasteurella species, 26 other bacterial species (18 being members of the family Pasteurellaceae) and four poultry virus isolates did not react in the assay. The assay detected a minimum of approximately 10 cfu of P. multocida per reaction. Of 79 poultry swabs submitted to the laboratory for routine bacteriological culture, 17 were positive in the 5′ Taq nuclease assay, but only 10 were positive by culture. The other 62 swabs were negative for P. multocida by both 5′ Taq nuclease assay and culture. The assay is suitable for use in diagnosing fowl cholera, is more rapid than bacteriological culture, and may also have application in diagnosing P. multocida infections in cattle and pigs.

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The current study was undertaken to enumerate Gram-positive bacteria in fresh sub-tropical marine fish and determine the effect of ambient storage (25°C) on the Gram-positive bacterial count. Total and Gram-positive bacteria were enumerated in the muscles, gills and gut of fresh and stored Pseudocaranx dentex, Pagrus auratus and Mugil cephalus on tryptone soya agar (TSA) and TSA with 0.25% phenylethyl alcohol (PEA), respectively. Initial studies indicated that PEA significantly reduced total aerobic bacterial count (TABC) whereas control Gram-positive bacteria were not affected by 0.25% PEA. TABC significantly increased in all fish body parts, whereas Gram-positive aerobic bacterial count (GABC) significantly increased only in the muscles and gills during ambient storage for 15 h. The TABC of the fish species increased from 4.00, 6.13 and 4.58 log cfu g-1, respectively in the muscles, gills, and gut to 6.31, 7.31 and 7.23 log cfu g-1 by the end of storage. GABC increased from 2.00, 3.52 and 2.20 log cfu g-1 to 4.70, 5.85 and 3.36 log cfu g-1. Within each species, TABC were significantly higher in the gills compared to that of muscles and gut; however, no significant differences were found in GABC between muscles and gills. This study demonstrated the potential importance of Gram-positive bacteria in sub-tropical marine fish and their spoilage.

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Sago starch is an important source of dietary carbohydrates in lowland Papua New Guinea. Over the past 30 years there have been sporadic reports of severe illness following consumption of sago starch. A common assumption is that fungal metabolites might be associated with the illness, leading to the need for a more thorough investigation of the mycoflora of sago starch. Sago starch was collected from areas of high sago consumption in Papua New Guinea for fungal analysis (69 samples). Storage methods and duration were recorded at the time of collection and pH on arrival at the laboratory. Yeasts were isolated from all samples except two, ranging from 1.2 × 103 to 8.3 × 107 cfu/g. Moulds were isolated from 65 of the 69 samples, ranging from 1.0 × 102 to 3.0 × 106 cfu/g. Of 44 samples tested for ergosterol content, 42 samples showed the presence of fungal biomass. Statistical analyses indicated that sago starch stored for greater than five weeks yielded significantly higher ergosterol content and higher numbers of moulds than sago stored for less than five weeks. The method of storage was also shown to influence mould numbers with storage in natural woven fibre containers returning significantly greater numbers than present in other storage methods tested. Potentially mycotoxigenic genera of moulds including Aspergillus and Penicillium were commonly isolated from sago starch, and as such storage factors that influence the growth of these and other filamentous fungi might contribute to the safety of traditional sago starch in PNG.

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Sago starch is an important dietary carbohydrate in lowland Papua New Guinea (PNG). An investigation was conducted to determine whether microbes play a role in its preservation using traditional methods. In 12 stored sago samples collected from PNG villages, lactic acid bacteria (LAB) were present (>= 3.6 x 10(4) cfu/g) and pH ranged from 6.8 to 4.2. Acetic and propionic acids were detected in all samples, while butyric, lactic and valeric acids were present in six or more. In freshly prepared sago, held in sealed containers in the laboratory at 30 degrees C, spontaneous fermentation by endogenous microflora of sago starch was observed. This was evident by increasing concentrations of acetic, butyric and lactic acids over 4 weeks, and pH reducing from 4.9 to 3.1: both LAB and yeasts were involved. Survival of potential bacterial pathogens was monitored by seeding sago starch with similar to 10(4)/g of selected organisms. Numbers of Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus fell to <30/g within 7 days. Salmonella sp. was present only in low numbers after 7 days (<36/g), but Escherichia coli was still detectable after three weeks (>10(2)/g). Fermentation appeared to increase the storability and safety of the product.

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This study assessed the levels of two key pathogens, Salmonella and Campylobacter, along with the indicator organism Escherichia coli in aerosols within and outside poultry sheds. The study ranged over a 3-year period on four poultry farms and consisted of six trials across the boiler production cycle of around 55 days. Weekly testing of litter and aerosols was carried out through the cycle. A key point that emerged is that the levels of airborne bacteria are linked to the levels of these bacteria in litter. This hypothesis was demonstrated by E. coli. The typical levels of E. coli in litter were similar to 10(8) CFU g(-1) and, as a consequence, were in the range of 10(2) to 10(4) CFU m(-3) in aerosols, both inside and outside the shed. The external levels were always lower than the internal levels. Salmonella was only present intermittently in litter and at lower levels (10(3) to 10(5) most probable number [MPN] g(-1)) and consequently present only intermittently and at low levels in air inside (range of 0.65 to 4.4 MPN m(-3)) and once outside (2.3 MPN m(-3)). The Salmonella serovars isolated in litter were generally also isolated from aerosols and dust, with the Salmonella serovars Chester and Sofia being the dominant serovars across these interfaces. Campylobacter was detected late in the production cycle, in litter at levels of around 107 MPN g(-1). Campylobacter was detected only once inside the shed and then at low levels of 2.2 MPN m(-3). Thus, the public health risk from these organisms in poultry environments via the aerosol pathway is minimal.

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1. Litter samples were collected at the end of the production cycle from spread litter in a single shed from each of 28 farms distributed across the three Eastern seaboard States of Australia. 2. The geometric mean for Salmonella was 44 Most Probable Number (MPN)/g for the 20 positive samples. Five samples were between 100 and 1000 MPN/g and one at 105 MPN/g, indicating a range of factors are contributing to these varying loads of this organism in litter. 3. The geometric mean for Campylobacter was 30 MPN/g for the 10 positive samples, with 7 of these samples being 100 MPN/g. The low prevalence and incidence of Campylobacter were possibly due to the rapid die-off of this organism. 4. E. coli values were markedly higher than the two key pathogens (geometric mean 20 x 105 colony forming units (cfu)/g) with overall values being more or less within the same range across all samples in the trial, suggesting a uniform contribution pattern of these organisms in litter. 5. Listeria monocytogenes was absent in all samples and this organism appears not to be an issue in litter. 6. The dominant (70% of the isolates) Salmonella serovar was S. Sofia (a common serovar isolated from chickens in Australia) and was isolated across all regions. Other major serovars were S. Virchow and S. Chester (at 10%) and S. Bovismorbificans and S. Infantis (at 8%) with these serovars demonstrating a spatial distribution across the major regions tested. 7. There is potential to re-use litter in the environment depending on end use and the support of relevant application practices and guidelines.

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Flax and hemp have traditionally been used mainly for textiles, but recently interest has also been focused on non-textile applications. Microbial quality throughout the whole processing chain of bast fibres has not previously been studied. This study concentrates on the microbial quality and possible microbial risks in the production chain of hemp and flax fibres and fibrous thermal insulations. In order to be able to utilize hemp and flax fibres, the bast fibres must be separated from the rest of the plant. Non-cellulosic components can be removed with various pretreatment processes, which are associated with a certain risk of microbial contamination. In this study enzymatic retting and steam explosion (STEX) were examined as pretreatment processes. On the basis of the results obtained in this study, the microbial contents on stalks of both plants studied increased at the end of the growing season and during the winter. However, by processing and mechanical separation it is possible to produce fibres containing less moulds and bacteria than the whole stem. Enzymatic treatment encouraged the growth of moulds in fibres. Steam explosion reduced the amount of moulds in fibres. Dry thermal treatment used in this study did not markedly reduce the amount of microbes. In this project an emission measurement chamber was developed which was suitable for measurements of emissions from both mat type and loose fill type insulations, and capable of interdisciplinary sampling. In this study, the highest amounts of fungal emissions were in the range of 10^3 10^5 cfu/m^3 from the flax and hemp insulations at 90% RH of air. The fungal emissions from stone wool, glass wool and recycled paper insulations were below 10^2 cfu/m^3 even at 90% RH. Equally low values were obtained from bast fibrous materials in lower humidities (at 30% and 80% RH of air). After drying of moulded insulations at 30% RH, the amounts of emitted moulds were in all cases higher compared to the emissions at 90% RH before drying. The most common fungi in bast fibres were Penicillium and Rhizopus. The widest variety of different fungi was in the untreated hemp and linseed fibres and in the commercial loose-fill flax insulation. Penicillium, Rhizopus and Paecilomyces were the most tolerant to steam explosion. According to the literature, the most common fungi in building materials and indoor air are Penicillium, Aspergillus and Cladosporium, which were all found in some of the bast fibre materials in this study. As organic materials, hemp and flax fibres contain high levels of nutrients for microbial growth. The amount of microbes can be controlled and somewhat decreased by the processing methods presented.

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The objective of the present study was to establish a valid transformation method of Haemophilus parasuis, the causative agent of Glasser's disease in pigs, using a novel H. parasuis-Escherichia coli shuttle vector. A 4.2 kb endogenous plasmid pYC93 was extracted from an H. parasuis field isolate and completely sequenced. Analysis of pYC93 revealed a region approximately 800 bp showing high homology with the defined replication origin oriV of pLS88, a native plasmid identified in Haemophilus ducreyi. Based on the origin region of pYC93, E. coli cloning vector pBluescript SK(+) and the Tn903 derived kanamycin cassette, a shuttle vector pSHK4 was constructed by overlapping PCR strategy. When electroporation of the 15 H. parasuis serovar reference strains and one clinical isolate SH0165 with pSHK4 was performed, only one of these strains yielded transformants with an efficiency of 8.5 x 10(2) CFUhlg of DNA. Transformation efficiency was notably increased (1.3 x 10(5) CFU/mu g of DNA) with vector DNA reisolated from the homologous transformants. This demonstrated that restriction-modification systems were involved in the barrier to transformation of H. parasuis. By utilizing an in vitro DNA modification method with cell-free extracts of the host H. parasuis strains, 15 out of 16 strains were transformable. The novel shuttle vector pSHK4 and the established electrotransformation method constitute useful tools for the genetic manipulation of H. parasuis to gain a better understanding of the pathogen. (C) 2011 Elsevier B.V. All rights reserved.

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B. cereus is a gram-positive bacterium that possesses two different forms of life:the large, rod-shaped cells (ca. 0.002 mm by 0.004 mm) that are able to propagate and the small (0.001 mm), oval shaped spores. The spores can survive in almost any environment for up to centuries without nourishment or water. They are insensitive towards most agents that normally kill bacteria: heating up to several hours at 90 ºC, radiation, disinfectants and extreme alkaline (≥ pH 13) and acid (≤ pH 1) environment. The spores are highly hydrophobic and therefore make them tend to stick to all kinds of surfaces, steel, plastics and live cells. In favorable conditions the spores of B. cereus may germinate into vegetative cells capable of producing food poisoning toxins. The toxins can be heat-labile protein formed after ingestion of the contaminated food, inside the gastrointestinal tract (diarrhoeal toxins), or heat stable peptides formed in the food (emesis causing toxin, cereulide). Cereulide cannot be inactivated in foods by cooking or any other procedure applicable on food. Cereulide in consumed food causes serious illness in human, even fatalities. In this thesis, B. cereus strains originating from different kinds of foods and environments and 8 different countries were inspected for their capability of forming cereulide. Of the 1041 isolates from soil, animal feed, water, air, used bedding, grass, dung and equipment only 1.2 % were capable of producing cereulide, whereas of the 144 isolates originating from foods 24 % were cereulide producers. Cereulide was detected by two methods: by its toxicity towards mammalian cells (sperm assay) and by its peculiar chemical structure using liquid-chromatograph-mass spectrometry equipment. B. cereus is known as one of the most frequent bacteria occurring in food. Most foods contain more than one kind of B. cereus. When randomly selected 100 isolates of B. cereus from commercial infant foods (dry formulas) were tested, 11% of these produced cereulide. Considering a frequent content of 103 to 104 cfu (colony forming units) of B. cereus per gram of infant food formula (dry), it appears likely that most servings (200 ml, 30 g of the powder reconstituted with water) may contain cereulide producers. When a reconstituted infant formula was inoculated with >105 cfu of cereulide producing B. cereus per ml and left at room temperature, cereulide accumulated to food poisoning levels (> 0.1 mg of cereulide per serving) within 24 hours. Paradoxically, the amount of cereulide (per g of food) increased 10 to 50 fold when the food was diluted 4 - 15 fold with water. The amount of the produced cereulide strongly depended on the composition of the formula: most toxin was formed in formulas with cereals mixed with milk, and least toxin in formulas based on milk only. In spite of the aggressive cleaning practices executed by the modern dairy industry, certain genotypes of B. cereus appear to colonise the silos tanks. In this thesis four strategies to explain their survival of their spores in dairy silos were identified. First, high survival (log 15 min kill ≤ 1.5) in the hot alkaline (pH >13) wash liquid, used at the dairies for cleaning-in-place. Second, efficient adherence of the spores to stainless steel from cold water. Third, a cereulide producing group with spores characterized by slow germination in rich medium and well preserved viability when exposed to heating at 90 ºC. Fourth, spores capable of germinating at 8 ºC and possessing the psychrotolerance gene, cspA. There were indications that spores highly resistant to hot 1% sodium hydroxide may be effectively inactivated by hot 0.9% nitric acid. Eight out of the 14 dairy silo tank isolates possessing hot alkali resistant spores were capable of germinating and forming biofilm in whole milk, not previously reported for B. cereus. In this thesis it was shown that cereulide producing B. cereus was capable of inhibiting the growth of cereulide non-producing B. cereus occurring in the same food. This phenomenon, called antagonism, has long been known to exist between B. cereus and other microbial species, e.g. various species of Bacillus, gram-negative bacteria and plant pathogenic fungi. In this thesis intra-species antagonism of B. cereus was shown for the first time. This brother-killing did not depend on the cereulide molecule, also some of the cereulide non-producers were potent antagonists. Interestingly, the antagonistic clades were most frequently found in isolates from food implicated with human illness. The antagonistic property was therefore proposed in this thesis as a novel virulence factor that increases the human morbidity of the species B. cereus, in particular of the cereulide producers.

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Megasphaera cerevisiae, Pectinatus cerevisiiphilus, Pectinatus frisingensis, Selenomonas lacticifex, Zymophilus paucivorans and Zymophilus raffinosivorans are strictly anaerobic Gram-stain-negative bacteria that are able to spoil beer by producing off-flavours and turbidity. They have only been isolated from the beer production chain. The species are phylogenetically affiliated to the Sporomusa sub-branch in the class "Clostridia". Routine cultivation methods for detection of strictly anaerobic bacteria in breweries are time-consuming and do not allow species identification. The main aim of this study was to utilise DNA-based techniques in order to improve detection and identification of the Sporomusa sub-branch beer-spoilage bacteria and to increase understanding of their biodiversity, evolution and natural sources. Practical PCR-based assays were developed for monitoring of M. cerevisiae, Pectinatus species and the group of Sporomusa sub-branch beer spoilers throughout the beer production process. The developed assays reliably differentiated the target bacteria from other brewery-related microbes. The contaminant detection in process samples (10 1,000 cfu/ml) could be accomplished in 2 8 h. Low levels of viable cells in finished beer (≤10 cfu/100 ml) were usually detected after 1 3 d culture enrichment. Time saving compared to cultivation methods was up to 6 d. Based on a polyphasic approach, this study revealed the existence of three new anaerobic spoilage species in the beer production chain, i.e. Megasphaera paucivorans, Megasphaera sueciensis and Pectinatus haikarae. The description of these species enabled establishment of phenotypic and DNA-based methods for their detection and identification. The 16S rRNA gene based phylogenetic analysis of the Sporomusa sub-branch showed that the genus Selenomonas originates from several ancestors and will require reclassification. Moreover, Z. paucivorans and Z. raffinosivorans were found to be in fact members of the genus Propionispira. This relationship implies that they were carried to breweries along with plant material. The brewery-related Megasphaera species formed a distinct sub-group that did not include any sequences from other sources, suggesting that M. cerevisiae, M. paucivorans and M. sueciensis may be uniquely adapted to the brewery ecosystem. M. cerevisiae was also shown to exhibit remarkable resistance against many brewery-related stress conditions. This may partly explain why it is a brewery contaminant. This study showed that DNA-based techniques provide useful tools for obtaining more rapid and specific information about the presence and identity of the strictly anaerobic spoilage bacteria in the beer production chain than is possible using cultivation methods. This should ensure financial benefits to the industry and better product quality to customers. In addition, DNA-based analyses provided new insight into the biodiversity as well as natural sources and relations of the Sporomusa sub-branch bacteria. The data can be exploited for taxonomic classification of these bacteria and for surveillance and control of contaminations.

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Bacterial proliferation in both vase solutions and in cut flower stems has been implicated in reducing the vase life of numerous genera. Boronia heterophylla F. Muell. (Red Boronia) vase life was assessed at two stages of floral maturity for nine vase solution treatments covering a pH range of 2.5-5.7. Vase life for advanced harvest maturity stems ranged from 4.2 d in 10 mM citric acid + 50 mg L-1 chlorine (pH 2.5) to 12.9 d after STS pulsing (pH 5.7). For normal harvest maturity stems, the corresponding range was 5.8-19.0 d, respectively. Vase solutions containing 50 mg L-1 chlorine biocide resulted in decreased longevity. In contrast, pulsing with the ethylene-binding inhibitor, STS, significantly increased vase life. The number of bacteria in the vase solutions after 11 d was determined in stems of advanced maturity. The solution with the greatest number of bacteria, 4.0 x 10(10) cfu mL(-1), was water used after STS pulsing and in which the flowers lasted longest. Vase solution bacteria were enumerated on days 0,3, 6, 9 and 12 of the vase period with stems of normal harvest maturity. There was no relationship between vase life and vase solution bacterial numbers ((R) over bar (2) = 0.000). Moreover, there was a negative relationship between numbers of bacteria in basal 0-5 cm stem segments and vase life. As no correlations were evident between longevity and either the pH or vase solution bacterial numbers, B. heterophylla vase life was evidently limited principally by ethylene action. (C) 2013 Elsevier B.V. All rights reserved.

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Campylobacter is an important food borne pathogen, mainly associated with poultry. A lack of through-chain quantitative Campylobacter data has been highlighted within quantitative risk assessments. The aim of this study was to quantitatively and qualitatively measure Campylobacter and Escherichia coli concentration on chicken carcasses through poultry slaughter. Chickens (n = 240) were sampled from each of four flocks along the processing chain, before scald, after scald, before chill, after chill, after packaging and from individual caeca. The overall prevalence of Campylobacter after packaging was 83% with a median concentration of 0.8 log10 CFU/mL. The processing points of scalding and chilling had significant mean reductions of both Campylobacter (1.8 and 2.9 log10 CFU/carcase) and E. coli (1.3 and 2.5 log10 CFU/carcase). The concentration of E. coli and Campylobacter was significantly correlated throughout processing indicating that E. coli may be a useful indicator organism for reductions in Campylobacter concentration. The carriage of species varied between flocks, with two flocks dominated by Campylobacter coli and two flocks dominated by Campylobacter jejuni. Current processing practices can lead to significant reductions in the concentration of Campylobacter on carcasses. Further understanding of the variable effect of processing on Campylobacter and the survival of specific genotypes may enable more targeted interventions to reduce the concentration of this poultry associated pathogen.