In-vivo Surgical Model of Campylobacter-bacteriophage Dynamics in Chicken Caeca

Campylobacter infection continues to be a leading cause of bacterial gastroenteritis in people, reported Peter M. O'Kane of the UK's School of Biosciences at the University of Nottingham, adding that current intervention measures available at farm and abattoir level in the European Union are ineffective in preventing or reducing contamination.

Campylobacter-specific bacteriophages are known to reduce colonisation levels in broiler chickens. Precise timing of treatment may be important to achieve optimal reduction of Campylobacter colonisation pre slaughter. Previous studies have demonstrated a reduction in Campylobacter numbers within 24 hours of oral bacteriophage administration. The Nottingham experiment examines this effect in greater resolution.

Seven experimentally reared commercial broilers were pre-colonised with Campylobacter jejuni and terminally anaesthetised. In four birds, each caecum was ligated once, in three birds each caecum was ligated into four segments. Pre-treatment content samples were obtained, one caecum was phage-treated by injection of phage suspension, and the other caecum injected with water as control. Content samples were collected at two, four and six hours post-injection. All samples were enumerated for Campylobacter and bacteriophage.

An increase in Campylobacter counts was observed over time in the repeatedly sampled, singly ligated caeca. This was not observed in the segmentally ligated caeca. Limited exposure of the caecal contents to oxygen could be responsible for the increase in campylobacters. Phage replication was only evident six hours post-injection. Longer incubation times using segmentally ligated caeca will be required to measure Campylobacter reductions.

O'Kane summarised the experiment, saying that a viable model of real time in-vivo Campylobacter-bacteriophage dynamics has been developed to allow further studies over longer time periods.

Why is it Important to Control Campylobacter Infections?

Campylobacter is a common bacterial cause of intestinal infections in the United States and around the world. According to Robert Tauxe from the US National Center for Emerging and Zoonotic Infectious Diseases at CDC, the dominant pathogen is Campylobacter jejuni, which has a natural reservoir in birds.

In humans, it usually causes an acute gastroenteritis, with fever, bloody diarrhoea and painful abdominal cramps, that usually resolves in five to seven days but may become invasive in patients with impaired immune systems. About 1 in 1,000 patients develop a post–infectious paralysis called Guillain-Barre Syndrome, which can result in weeks of intensive care with ventilator support, before it slowly resolves.

Antibiotic treatment, usually with fluoroquinolones or azithromycin can shorten the duration of illness, and can be life-saving for the invasive infections. In human infections, after rising steadily, the resistance to fluoroquinolones has stabilised at approximately 23 per cent since 2007, and is more common in travel-associated infection; azithromycin resistance remains below two per cent.

In the United States, Campylobacter causes an estimated 1.3 million illnesses, 13,000 hospital admissions and 120 deaths each year. About one in 30 of these illnesses lead to a culture-confirmed case that is reported to public health surveillance. About 20 per cent of illnesses are thought to be related to travel to other countries where Campylobacter is also very common.

Most (80 per cent) Campylobacter infections are acquired by the foodborne route, and the remainder through contact with animals or contaminated water. Outbreaks of Campylobacter infection are uncommon, and almost all cases occur as individual sporadic cases, without evident links to other illnesses.

Investigations into the sources of Campylobacter infections in the United States and in other counties find an association with consumption of poultry as the leading risky exposure. Indirect contamination of other foods by fluids from raw poultry is also a likely source of many infections, though harder to quantify.

Cattle can also carry Campylobacter, and raw unpasteurised milk is a source of infection, and may cause outbreaks, for example when a group of children drink raw milk during a farm visit. In very young children, infection has been associated with riding in a shopping cart with packages of meat and poultry. The infectious dose for Campylobacter is relatively low, and likely to be below 500 organisms. In the United States, the frequency of Campylobacter infections as tracked in active surveillance through FoodNet, declined by 30 per cent in the late 1990s at a time when a number of measures were introduced to improve slaughter sanitation.

In the last five years, the trend has reversed and as of 2012, the frequency of Campylobacter infection reported to FoodNet has increased by 14 per cent since 2006-2008.

This increase indicates that further prevention efforts are needed to reduce the frequency of this infection, concluded Dr Tauxe. Key prevention strategies, he added, include those that will reduce the level of contamination in poultry at retail, pasteurisation of milk and efforts to reduce Campylobacter infections worldwide.

Tracking Campylobacter Prevalence and Load in the Broiler Production System

Poultry is one of the most important sources of human foodborne Campylobacter infections in the United States, yet according to Randall S. Singer from the University of Minnesota, few studies have attempted to quantify the relationship between Campylobacter loads of broiler chicken flocks on the farm and at the time of processing.

The objective of this study was to determine if an on-farm sample set cultured for Campylobacter could predict Campylobacter loads in the same flock at the time of processing, including on the post-chill carcass.

Environmental samples were collected on farms during the two weeks immediately prior to harvest and cultured for Campylobacter using a quantitative serial-dilution method.

At processing, birds from each flock were collected from transport crates and from three post-harvest plant locations for whole-bird carcass rinses.

During processing, Campylobacter loads in carcass rinses decreased consistently as birds progressed through the plant.

In linear regression modeling, the coefficient of determination for the relationship between pre- and post-harvest sampling indicated that boot sock loads explained approximately 80 per cent of the variation in Campylobacter loads on birds arriving at the plant.

The Campylobacter boot sock loads also predicted the loads on the post-chill rinses from the same flock.

The distribution of Campylobacter species in 1,923 isolates was: 84 per cent C. jejuni, 10 per cent C. coli, and six per cent C. jejuni and C. coli.

Dr Singer concluded that there is a positive association between Campylobacter isolation on farms and at the processing plant, suggesting that the downstream effectiveness of on-farm interventions can be predicted with on-farm sampling. He added that in-plant HACCP procedures are effective in reducing Campylobacter contamination.

WHO Collaborating Center for Campylobacter and OIE Reference Laboratory for Campylobacteriosis

Dr Jaap A. Wagenaar from Utrecht University in the Netherlands reported on a WHO Expert meeting and Campylobacter Control in European Union.

Campylobacteriosis is one of the most important bacterial food-borne illnesses in humans worldwide (see ‘The Global view on Campylobacter ’ report of a WHO-expert meeting on Campylobacter')

The acute phase is characterised by gastrointestinal symptoms, and the major sequelae associated with Campylobacter infections are the Guillain-Barré Syndrome, reactive arthritis, post-Campylobacter irritable bowel syndrome and post-Campylobacter inflammatory bowel disease. The sequelae contribute considerably to the disease burden. Attribution studies have identified poultry as the major reservoir responsible for up to 80 per cent of the human infections. However, only an estimated 30 per cent of the human infections is associated with consumption and handling of poultry meat.

Other, yet unknown, routes of transmission of Campylobacter from the poultry reservoir towards humans contribute considerably.

Control of Campylobacter in the poultry meat production chain will reduce the human burden of illness; the control of the Campylobacter colonisation in broilers (primary production) would be the best approach. Broilers can easily become colonised with Campylobacter and, although risk factors for flocks to become colonised with Campylobacter have been identified, interventions in the poultry meat production chain have not been effectively introduced except for targeted interventions in Iceland and New Zealand.

Some intervention measures, e.g. bosecurity, have limited effect or are hampered by economic aspects or consumer acceptance.

Risk assessment studies were the basis of a paradigm shift in the last decade. Where the aim in the past was to produce Campylobacter-free products, efforts in the future will allow low levels of contamination. Elimination of highly contaminated products will have a considerable impact on the disease burden of Campylobacter.

Improved slaughter hygiene in combination with treatment of highly contaminated products will be part of a multi-level approach that will ultimately also include on-farm interventions.

According to Dr Wagenaar, some European countries - such as the UK and the Netherlands - are following this approach in their Campylobacter control programmes. Data from these countries can be used for risk management at a broader European level.

What’s Old is Again New: Difficulty with Culturing for Campylobacter

The methods used by many diagnostic laboratories to isolate Campylobacter from samples are not standardised and, therefore, efficacy varies considerably, according to Dr Margie D. Lee from the University of Georgia.

Many of the culture approaches for detecting Campylobacter in faeces, processed poultry or meat, focus on an initial enrichment step, using a broth medium, and then the use of a selective isolation medium.

The broth enrichments are usually composed of a rich assortment of nutrients but enrichment of Campylobacter in broths can be enhanced by the addition of antimicrobials that suppress the growth of contaminating organisms but allow replication of Campylobacter.

The successful cultivation of Campylobacter requires the careful selection of an enrichment broth that will culture the Campylobacter species of interest. Not all enrichment broths will grow all species or strains of Campylobacter.

Furthermore, enrichment broths that are effective for the cultivation of C. jejuni from fecal samples may not be suitable for isolation of campylobacters from environmental samples because bacterial cells that are stressed or damaged may require resuscitation in order to grow on selective enrichment broths or agars.

Dr Lee stressed that it is important to select a culture strategy that has been tested for the samples of interest.

What is Known About Campylobacter Control in the Processing Plant Environment

Introduction

Over many decades, poultry processors have attempted to control Salmonella on their finished products, repotred Professor Scott M. Russell of the Univeristy of Georgia. A variety of intervention strategies during breeding, hatching, grow-out and processing have proven effective for controlling this important pathogen. However, these strategies are often ineffective against Campylobacter. The question is why not? The answer lies in the fact that they are very different bacteria, adapted to very different environments.

Comparison with Salmonella

Salmonella is a Gram-negative bacterium that attaches to and colonises the intestines of chickens. It is a hearty organism that may remain active on dry fluff for weeks.

Campylobacter is also a Gram-negative bacterium but it is considered a thermophilic, obligate and microaerophilic bacterium that is ubiquitous in temperate environments. Because avian species have a higher body temperature than mammals, Campylobacter is particularly suited to colonise them. However, Campylobacter resides in a commensalistic relationship with chickens.

Campylobacter Colonisation

In chickens, Campylobacter colonises the mucus overlying the epithelial cells primarily in the caeca and the small intestine but may also be recovered from elsewhere in the gut and from the spleen and liver. Experimentally, the dose of viable Campylobacter required to colonise chicks can be as low as 40 colony-forming units (CFU).

Once colonisation is established, Campylobacter can rapidly reach extremely high numbers in the caecal contents (as high as 10⁹ CFU in both experimentally challenged and naturally contaminated birds). One billion CFU is an extremely high level of colonisation and it is a very difficult task to reduce the numbers of Campylobacter on these chickens to zero.

Campylobacter is readily detectable in the faeces of colonised birds. Chickens may become colonised by consumption of contaminated faeces from other birds. Once flock colonisation is detected, bird-to-bird transmission within flocks is extremely rapid, and the majority - up to 100 per cent - of birds in a positive flock are colonised within only a few days.

These colonisation kinetics indicate that measures to reduce the 'within-flock prevalence' at slaughter are likely to be unsuccessful, which means the Salmonella approach used in Europe (i.e. elimination of negative birds) will not work.

Numbers Game

The key to reducing and eliminating Campylobacter is multiple intervention strategies. Each time a carcass is treated in a way that either physically removes Campylobacter or chemically kills Campylobacter, the numbers are reduced. By exposing the bacteria to numerous interventions, it is possible to lower Campylobacter from 10⁶ and even 10⁹ CFU per ml to zero on many carcasses (Figure 1). Figure 1 shows how Campylobacter prevalence tracks from the grow out operation (76 per cent contaminated on average in the US and EU) through the scalder (lowers by 50 per cent on average), increases through picking, then is greatly reduced by the effect of chemical intervention in the online reprocessing system (OLR), the chiller and the post-chill dip (finishing chiller) to 10.66 per cent (national average).

Figure 1. Average prevalence of chickens/carcasses with Campylobacter at each stage of production/processing in the US

This means that processors are able to start with 76 per cent of their birds highly contaminated (9,000CFU per mL national average to as high as 10⁹ CFU per mL) and completely eliminate Campylobacter from 86 per cent of the chickens. This is a herculean feat.

However, in Europe, because chemical in terventions are not used, extremely high levels of Campylobacter-positive chickens reach consumers (76 per cent - see Figure 2).

Figure 2. Average prevalence of chickens/carcasses with Campylobacter at each stage of production/processing in the EU

It should be noted that, even though processors are able to eliminate Campylobacter to very low levels at certain times of the year, from June to September (the hottest months) are by far the worst for Campylobacter. This holds true in the US as well.

Processors will have the most difficult time with Campylobacter during the warmest months of the year, as Campylobacter is considered a thermophile. Thus, it is essential that multiple interventions be used during this time to prevent a Campylobacter Performance Standard violation (>10.4 per cent positive).

Physical Intervention

Bird brush systems are effective for lowering the amount of faeces on the outside of the carcass, which lowers the amount of Campylobacter entering the scalder.

Additionally, the dilution effect of the scalder is often able to reduce pathogen numbers and prevalence if run in a counter-current direction. However, because Campylobacter are often found in high numbers in the caeca, as the birds are picked, the caecal material is expressed, allowing high numbers of Campylobacter to be spread from carcass to carcass during picking.

This is why a post-pick spray with a disinfectant is recommended.

Additional Chemical Interventions

In many plants, the only chemical interventions used are the OLR, the chiller and, in some cases, the finishing chiller. Each of these systems may be capable of 1– to 2-log₁₀ CFU reductions in Campylobacter; however, if a 6-log₁₀ CFU reduction is required to bring the carcass to zero and lower prevalence, then these interventions are generally, not sufficient.

The OLR system will generally be able to reduce pathogens by 1-log₁₀ CFU because of the short contact time. The chiller, depending on the organic load, chemical level and pH is generally able to produce a 1- to 3-log₁₀ CFU reduction. The finishing chiller is also able to provide a 1- to 2-log₁₀ CFU reduction.

In the summer months, a scalder intervention and post-pick spray disinfection may produce an additional 3-log₁₀ CFU reduction, thereby enabling processors to meet the Performance Standard.

Figure 3 shows that when carcasses are treated in a strong acid mixture during scalding, then treated with a post-pick spray, the total number of bacteria on the carcass is reduced by 3-log₁₀ CFU at the post-chill location.

Figure 3. The effect of a strong acid mixture on total aerobic plate count (APC) on chicken carcasses

While APC reductions cannot be related directly to Campylobacter reductions, the effect of strong acids on Salmonella prevalence using this method is dramatic.

Studies on Campylobacter on Chicken Carcasses during Pocessing

Son et al. (2006) reported on the prevalence of Campylobacter at three sites along the processing line: pre-scald, pre-chill and post-chill. Campylobacter was isolated from 92 per cent of pre-scald carcasses, 100 per cent of pre-chill carcasses and 52 per cent of post-chill carcasses.

In total, Campylobacter was isolated from 78.5 per cent (255 of 325) of the carcasses from the three collection sites. The most common Campylobacter species identified was C. jejuni (87.6 per cent) followed by C. coli (12.4 per cent). The authors reported that Campylobacter reductions were more difficult for Campylobacter than for Arcobacter during processing (Son et al., 2006). It is important to realise that, in this plant, the chiller was able to make 48 per cent of the chickens go from positive to zero Campylobacter. While that is an extremely admirable feat, it would not enable the plant to meet the requirement for fewer that 10.4 per cent positive carcasses.

Berrang et al. (2007) conducted a study to examine the prevalence and number of Campylobacter on broiler chicken carcasses in commercial processing plants in the United States. Carcass samples were collected from each of 20 US plants four times during the year, roughly approximating the four seasons of 2005.

At each plant on each sample day, 10 carcasses were collected at re-hang (prior to evisceration), and 10 carcasses from the same flock were collected post-chill.

The overall mean number of Campylobacter detected on carcasses at rehang was 2.66 log₁₀ CFU per ml of carcass rinse.

In each plant, the Campylobacter numbers were significantly reduced by broiler processing: the mean concentration after immersion chilling was 0.43 log₁₀ CFU per ml. Overall prevalence was also reduced by processing from a mean of 75 per cent positive carcasses at rehang to 35 per cent positive carcasses at the post-chill location.

The authors noted that the use of a chlorinated carcass wash before evisceration did not affect the post-chill Campylobacter numbers (Berrang et al., 2007). However, use of chlorine in the chill tank was related to lower numbers on post-chill carcasses.

The conclusion reached in the study was that, in the US, commercial poultry slaughter operations are successful in significantly lowering the prevalence and number of Campylobacter on broiler carcasses during processing.

Conclusions

These studies demonstrate the need to use the number of Campylobacter on carcasses as an indicator of the success of a plant’s interventions as opposed to prevalence, according to Dr Russell.

It is possible to achieve an 8-log₁₀ CFU reduction of Campylobacter from the pre-scald location to the post-finishing chiller location and still fail the USDA-FSIS Performance Standard. This equates to a 99.999999 per cent reduction, resulting in a fail. This standard should be reassessed, he added.

References

• Berrang, M.E., J.S. Bailey, S.F. Altekruse, B. Patel, W.K. Shaw Jr., R. Meinersman and P.J. Fedorka-Cray, 2007. Prevalence and numbers of Campylobacter on broiler carcasses collected at rehang and postchill in 20 US processing plants. Journal of Food Protection 70 (7):1556-1560.
• European Food Safety Authority, 2010. Analysis of the baseline survey on the prevalence of Campylobacter in broiler batches and of Campylobacter and Salmonella on broiler carcasses in the EU. Part A: Campylobacter and Salmonella prevalence estimates for 2008. Scientific Report of the European Food Safety Authority. EFSA Journal, 8(3):1503, Parma, Italy.
• Son, I., M.D. Englen, M. Berrang, P.J. Fedorka-Cray and M.A. Harrison, 2006. Prevalence of Arcobacter and Campylobacter on broiler carcasses during processing. International Journal of Food Microbiology, 113(1):16-22.
• United States Department of Agriculture, Food Safety Inspection Service, 2008. The Nationwide Microbiological Baseline Data Collection Program: Young Chicken Survey. Office of Public Health Science, Microbiology Division, July 2007 – July 2008.

How the Incidence of Human Cases of Campylobacteriosis in New Zealand was Reduced by 50 Per Cent - Roles Played by Government, Industry and Academia

Nigel P. French of Massey University reported that, in New Zealand, the number of campylobacteriosis notifications increased markedly between 2000 and 2007, before a major intervention targeted at the poultry supply was introduced. What followed was a dramatic reduction in case rates, from around 16,000 to around 8,000 per annum. This lower rate has been sustained to the present day, resulting in considerable savings to the country’s economy.

A sentinel surveillance site was established in 2005 and identified poultry as the predominant source of human infection using multilocus sequence typing and modelling. Differences were detected in the probability and level of contamination and the relative frequency of genotypes for individual poultry suppliers and humans.

The dominant human sequence type in New Zealand (ST-474), was found almost exclusively in isolates from one poultry supplier.

The reduction in human illness cases followed the introduction of performance targets based on enumerated levels of Campylobacter spp. on poultry carcasses sampled at the end of primary processing, and the imposition of escalating regulatory responses when targets were not met.

Reduced contamination of broiler meat and the subsequent marked decline in human cases were attributed to a variety of interventions, including improvements in hygienic practices throughout production and processing and alterations to the immersion-chiller conditions.

Although the decline in campylobacteriosis in New Zealand is impressive, it is difficult to extrapolate this success to other parts of the world, according to Dr French. Prior to 2007, New Zealand had an extremely high incidence, and even after the marked improvements, the incidence is still high compared to other parts of the world.

December 2013