1. Summary, 121S 2. Introduction, 121S 3. Sources of Campylobacter infection, 121S 4. Materials and methods, 121S 4.1 Bacterial culture and speciation, 122S 4.2 Fla-typing analysis, 122S 5. Results, 122S 6. Spread of Campylobacter infection, 122S 7. Conclusions, 124S 8. References and Appendix, 125S Campylobacter organisms are present in the environment of the farm and it is accepted that the chance of infection transferring to chickens is very high. Sources of infection may include any of the standard requirements for poultry such as feed, water and litter. Any form of human intervention as a result of routine animal husbandry requirements may also introduce infection. It has been shown that on some farms it is possible to delay infection by various improvements to bio-security arrangements. The use of dedicated wellington boots for each poultry house and the regular use of foot dips were found to be important factors. The daily use of water sanitiser was also important in delaying the onset of infection. The efficiency of cleaning and disinfection and the construction of the buildings were less significant factors. If flocks were thinned, which involves entry by catching crews and equipment, the risk of infection was dramatically increased. After 42 d of age, the likelihood of infection was also much greater. The effectiveness of these intervention procedures applied in one integrated poultry company are described. Generally, it was felt that even the most stringent bio-security measures applied conscientiously would not be able to prevent infection occurring. Once infection has entered the house, all birds become Campylobacter carriers very quickly. A pen trial was set up to investigate this and the results are described. Most broiler (meat) chickens and breeders produced in the U.K. are reared in closed, environmentally controlled houses. House design will vary from farm to farm but all tend to have similar lighting, heating, feeding and watering systems. Feed is supplied by an automatic auger system with feed pans and water is provided through nipple and cup drinker systems, which have replaced the older, rather unhygienic, bell drinkers. Chicks are delivered as ‘day olds’ to the farm and placed on a litter of wood shavings or chopped straw. The number of chicks in a typical house would be about 30 000, but it may vary from 5000 to 50 000. Stocking density is calculated to ensure that a maximum of 36 kg m−2 is not exceeded at time of slaughter. Birds remain in the same house and will be killed usually between 40 and 53 d. Frequently, a house is ‘thinned’ once or twice, where a proportion of the birds are taken for slaughter before the remainder which are kept for heavier weights. Modern houses are ‘clear span’, without posts and roof supports, and this makes them more easy to clean. Birds are free to roam throughout the house. As specialist markets develop, a small number of chickens are kept as ‘free range’ or ‘organic’ where they must spend part of their time outside the houses on pasture. Usually one stock person looks after three or four houses or 100 000 birds. This paper describes observations made in the field as part of our ongoing quest to understand Campylobacter incidence, speed of dissemination and possible interventions that can be used to prevent or delay infection. The main source of infection is not known but possibilities include: feed, water, staff and visitors, equipment, litter, wild birds, rodents, insects and air. All observations have been made during the course of normal commercial growing practice. Campylobacter were isolated from cloacal swabs transported in Amie’s transport medium with charcoal (Bibby Sterilin Ltd. UK) and were cultured within 48 h of collection. The intervention techniques employed are largely self-explanatory and detailed in Appendix 1. Swabs were incubated in 10 ml Exeter medium for 48 h at 37°C under microaerobic conditions (6% O2, 10% CO2, 84% N2). A sample of 50 μl was then removed for plating on blood agar containing selective antibiotics with actidione (100 μg ml−1) and cefoperazone (30 mg ml−1). The plates were incubated microaerobically as before at 37°C for 2 d. All Campylobacter isolates were speciated by standard microbiological procedures. Identification was based on growth at 42°C, hippurate and indoxylacetate hydrolysis, catalase and oxidase activity, and resistance to nalidixic acid and cephalohtin. A single colony from each bird was stored in glycerol broth (10% v/v glycerol in 1% w/v proteose peptone) at − 70°C for subsequent molecular typing. PCR-RFLP of the flaA and flaB genes was performed according to the technique of 1 excepting that two separate digestion reactions were carried out using the restriction enzymes Ddel and Hinfl. In a survey involving 100 flocks in five integrated companies, it was found that 45% of farms were positive for Campylobacter at 3 weeks of age and 90% were positive by 7 weeks (2; 3). My own company was one of the five and there was no noticeable difference in the results between companies. There is no consistent pattern of infection on farms as shown in another integration where five farms were monitored over a 2-year period (Gooderham, personal communication) of 13 crops (Table 1). Each farm had 10 houses. It can be seen that some farms remained negative for Campylobacter throughout the crop, but no farms were either consistently positive or negative. A house was defined as positive if one or more birds tested positive. This was appropriate because infection spread so quickly once established. As a company, we took part in a national study involving two other companies to examine the effects of various interventions on Campylobacter incidence (4). The special measures focused on an improved cleaning and disinfection routine and a set procedure for all personnel entering a poultry house. The improved cleaning and disinfection procedure is detailed in the appendix. The results of this study are shown in Table 2. It can be seen that one flock in the intervention group stayed negative. In the other intervention flocks there was generally a delay in the time of infection compared with the control farms. As a result of analysis of questionnaires, the authors found that the use of boot dips and changing the boot dip solution had the biggest effect on delaying infection. The daily use of water sanitiser and the efficiency of poultry house cleaning were also major factors. The risk of infection rose sharply after 42 d, which probably related to the entry of catching crews to ‘thin’ the flocks. We had the opportunity within a trials house to investigate the spread of Campylobacter between groups of birds penned separately. The results are reported by 7. The trial houses consisted of 72 pens of 100 birds. Five birds were sampled weekly in each of 12 pens as shown in Fig. 1. All birds remained free of Campylobacter to 32 d of age, when six of the 60 sampled were found to be colonized. There were four positive in pen 64 and two in pen 70, both situated towards the rear of the house. One week later 56 of the 60 birds sampled were positive, as shown in Table 3. Plan of broiler house and sampling strategy for flock 1. □ Pens sampled; pens containing the first positive birds detected with strain fla type 1·9; pens containing the first positive birds detected with strain fla type 3·7 The strains isolated at 32 & 39 d were all C. jejuni, with identical subtypes: serotype LEP 6, fla-type 1·9. On the next sampling at 46 d, some of the birds in five of the pens (40, 52, 58, 64 & 70) were found to be colonized with a different strain, C. jejuni, LEP 23, fla-type 3·7. All other birds sampled at this time were colonized with the first strain. In the second flock (Fig. 2) infection occurred at 35 d, when 42 out of 80 birds were positive with C. jejuni, fla-type 1·1. The pens with positive birds were again towards the rear of the house. By 42 d, all birds were positive with the same strain of organism which persisted to slaughter at 49 d. Plan of broiler house an sampling strategy for flock 2. □ Pens sampled; pens containing the first positive birds detected with strain fla type 1·1 It was interesting that infection seemed to start at the near of the house and may have related to occasional opening of door A, which was adjacent to the site incinerator. These and other studies indicate that it is difficult and probably impossible to guarantee keeping Campylobacter infection out of poultry houses, and also that, once infection is present in a small number of birds, it spreads very quickly even if birds are separated by wire pens. Bio-security measures on poultry farms have improved in many ways in recent years and have undoubtedly helped with control of infectious disease and Salmonella in particular. The use of foot dips and hygiene barriers at the entrance to houses is becoming standard practice. In addition, the daily use of a water sanitiser is more widely practised, yet Campylobacter infection still occurs. It may be unrealistic to keep out an organism which is so widespread and colonizes poultry so readily. 6, as in this report, have noted that the standard of cleanout is important. In addition, these authors observed that if the houses were left empty for more than 14 d, Campylobacter was less likely in the subsequent flock. This is probably due to the adverse effect of drying on the organisms. Allowing time to dry between washing and disinfection during cleanout is also a critical factor. 6 also reported that addition of bought-in wheat rather than home-produced wheat to feed also increases the risk of infection, as also do the presence of other livestock on the farm. The operation of an effective hygiene barrier is thus the single most significant intervention. Figure 3 shows that there should be a step-over bench in the demarcation zone between the outside and inside of the house. This acts as a physical barrier and stops items such as a wheelbarrow crossing the demarcation line. Separate wellington boots and overalls should be used outside and inside, dipping each time in disinfectant solution. The efficiency of boot dipping can be improved by cleaning and washing the boots each time using a hose and brush. In reality this is a rather time-consuming regime and requires great conscientiousness on the part of the manager. It is probably unrealistic to expect farmers to operate this system for all staff effectively day in and day out. As soon as the barrier has to be broken, for example for thinning, the risk of infection increases markedly. Hygiene barrier • Approved insecticide band sprayed at the time of depopulation. • Dust removal from all surfaces including the floor, by blowing. • All internal surfaces washed with a sanitiser sold for the purpose (defined dilution and application rate), thorough wetting achieved and allowed to soak for at least 1 h; special attention paid to soaking drinker cups. • Minimum overnight drying period between washing and disinfection. • Inspection of house before disinfection; any pools of water swept out. • All internal surfaces disinfected with a specified product at a defined dilution (MAFF ‘General Order’) and application rate (quaternary ammonium/glutaraldehyde/formaldehyde). • Brooding chick equipment washed and disinfected in main house at the same time, if not disposable. • Adjoining rooms to poultry house hand washed and disinfected if not included in main wash/disinfection programme. • Water system (header tank and lines) cleaned and then disinfected for a minimum of 24 h with an iodine-based disinfectant at MAFF General Orders dilution rate. • Approved insecticide band sprayed before litter placed. • Concrete areas on the site disinfected before litter placed. • Dip boots* on entry to anteroom. • Changed into dedicated boots and overalls (used only in study house). • Move into separate (chalk line or bench) clean area of anteroom. • Sanitize hands. • Dip dedicated boots* before entry to main house. * Specified disinfectant (blend of organic acids and surfactants) with dilution (MAFF ‘TB orders’ rate) and frequency of replacement (twice weekly) also specified, used in all boot dips.