THERMOPHILIC CAMPYLOBACTER SPP. IN POULTRY MEAT PRODUCTION

Th ermophilic Campylobacter spp. are the leading cause of zoonotic enteric disease in Europe and USA. In Serbia, it has an upward trend in human population. Th e disease is usually indirectly transmitted to humans through the consumption of food contaminated by the faeces of infected animals. Th e aim of this paper was to analyze data on the prevalence of Campylobacter spp. in poultry meat production chain and the risk for the development of the disease in humans. Th e Campylobacter jejuni/coli was identifi ed at farm level in 73.3% of poultry, 66.6% calves and 58.3% pig samples of already ill or suspected cases. Clinical manifestation of the disease in birds can be expected if an additional immunosuppressive factor is present. Artifi cial infection of healthy chickens with 6.77 log cfu C. jejuni per chicken on day 21st of life leads to 5.26 log cfu/g faeces aft er only fi ve days with a tendency to decrease during the next 18 days. Although chilling and freezing may signifi cantly reduce Campylobacter contamination of carcasses, it cannot completely eliminate the initial contamination. According to our experimental results the prevalence of Campylobacter contaminated chickens from positive fl ock appears to drop from 100% live birds (with 3.02 log cfu/g faeces) to 50% of chicken carcasses. Contamination of the carcasses depends on initial contamination of live birds, good hygiene practices and good manufacturing practices. Th erefore, high variability in contamination of carcasses can be considered; prevalence range from 11.43 to 90.00% of carcases was established in various slaughterhouses. At retail, Campylobac1∗ Corresponding author: jelena@niv.ns.ac.rs Arhiv veterinarske medicine, Vol. 12, No. 2, 27 38, 2019 Petrović J. ... et al.: Th ermophilic Campylobacter spp. in...


INTRODUCTION
Campylobacter spp. are microaerophilic, Gram-negative, curved and motile rods, which commonly cannot grow below 30°C; however, some low metabolic activity is detectable at 40°C (EFSA, 2010). Th e most important foodborne Campylobacter species are thermophilic C. jejuni and C. coli. C. jejuni can be found in the intestines of a range of wild and domestic animals, especially birds. Its prevalence in birds could be attributed to the optimal growth temperature being 42°C. C. coli can oft en be identifi ed in the intestines of pigs. In the environment, the organism is isolated from the water, dust, soil, air and also in fi sh and vegetables (EFSA, 2010). Virulence factors of Campylobacter include motility, chemotaxis, adherence and invasion (Bolton 2015).
Th ermophilic non-foodborne Campylobacter species are common causative agents of abortions in sheep and cattle and, occasionally, in other animal species. Th ermophilic foodborne species can cause diarrhoea in animals and hepatitis in birds. Campylobacter jejuni/coli oft en colonize the alimentary tract of poultry and other domestic livestock, yet without development of diseases symptoms. Th e organisms can commonly be found in the caecum, colon and cloaca, colonizing the crypt and villus region, and are present in the mucus but not in the epithelium. Mucus is the environment that provides optimal oxygen level and improved motility of the bacteria (Mead, 2002). Campylobacter jejuni/coli are much more frequently detected in the caecum and rectum (70.0%) than in the reproductive system (uterus and magnum) (6.7%) of poultry (Stojanov et al., 2008).

CAMPYLOBACTER SPP. IN POULTRY FARMING
Campylobacter is present in farm surrounding environment, including the soil, water sources, dust, building surfaces, and the air (Ellis-Iversen et al., 2012). Major contamination sources include the environment, domestic and wild animals, water, partial depopulation (thinning) and carry over from previous fl ocks ( Barrios et al., 2006;Newell et al., 2011). Th e prevalence of Campylobacter positive fl ocks in Europe greatly diff ers between the countries and ranges between 5 and 90% depending on the geographic location, climatic diff erences, biosecurity measures as well as diff erent research methodologies (Berndtson et al., 1996a,b).
If Campylobacter is introduced into the fl ock at an early phase of breeding, the colonization will most likely persist in all birds until slaughter, and the fl ock prevalence can reach even 100%. Campylobacter infection spreads within the fl ock via bird-to-bird transmission route. Such a rapid spread is a result of high expression level (8-9 log cfu/g faeces), coprophagia as well as contamination of drinking water and feeders (Th ibodeau et al., 2015). Th e spread of Campylobacter is mediated also by the presence of fl ies, mice, and farm workers as the mechanical transmission vectors. Low fl ock prevalence of Campylobacter indicates recent colonization (Evans and Sayers, 2000).
Colonization occurs most commonly in birds at the age of 2-4 (van Gerwe et al., 2009). Despite extensive colonization, clinical symptoms in poultry are rare and unspecifi c and include watery diarrhoea oft en in the absence of pathoanatomical changes in the jejunal mucosa and the caecum. Th e intestinal bloating and distension consequent to the accumulation of watery and mucous contents are also diagnosed (Evans and Sayers, 2000). Macroscopic necrotic changes are visible in the liver of poultry (Lemos et al., 2015). Diarrhoea commonly occurs some 6 hours post infection, and can persist for up to 10 days. Clinical signs are greatly determined by the amount and virulence of the individual strain of C. jejuni, stress factors and immunosuppression (Evans and Sayers, 2000). Our research revealed that clinically manifest campylobacteriosis occurred in experimentally infected chickens with both C. jejuni and Salmonella Enteritidis or C. jejuni with simultaneous administration of live D78 Gumboro vaccine. Major clinical symptom was watery diarrhoea that varied in intensity between birds. Diarrhoea did not occur in chickens infected with only C. jejuni or only Salmonella Enteritidis. Th e administration of immunosuppressive agents induced clinically manifested signs associated with the occurrence of red or yellow spots on the liver (Stojanov et al., 2008). Immunosuppressive agents such as dexamethasone, ochratoxin A and secondary bacterial infections also promote clinical signs of campylobacteriosis (Stojanov et al., 2011).
In chicken categories older than three weeks, the infection with C.  Figure 1. aft er an initial growing tendency during fi rst fi ve days aft er infection, a continuous decrease has been observed during the period between day 11 and day 18 post-infection (from 4.97 to 3.02 log cfu/g) followed by an increase in C. jejuni count to 4.95 log cfu/g (day 28 post-infection). Campylobacter contamination at slaughterhouse is closely related with the colonization status of broilers during rearing. Colonized chickens carry large amounts of Campylobacter in the caecum (5-8 log cfu) and intestines. Such chickens shed Campylobacter in the faeces during transportation and spread it into the environment (Dogan et al., 2019). Flock prevalence rate and Campylobacter concentration in the caecum along with the hygienic standards applied during processing strongly infl uence the amount of the pathogens in fi nal poultry products and hence the exposure of the consumers to thermophilic Campylobacter species from poultry meat.
Major points of potential contamination in the slaughterhouse include picking, evisceration and some chilling processes. Leaking of faecal content during picking and evisceration process leads to carcass contamination; even very small amounts of the faeces can signifi cantly elevate the Campylobacter counts on the carcasses. Immersion chilling with chlorinated water reduces contamination but without chlorine the contamination increases ( Contamination of carcasses could be reduced by scalding, washing and cold storage. During scalding, a part of Campylobacter organisms is washed out of the carcass surface. However, scalding water easily becomes contaminated with faecal contents and dust, thus causing recontamination of the carcass with certain amount of pathogens if scalding technology is not properly applied. Th e temperature of scalding water does not signifi cantly infl uence the Campylobacter counts. Washing substantially reduces the concentration of Campylobacter (90%), while airfl ow-chilling reduces Campylobacter counts depending on the temperature and humidity (Guerin et al., 2010;Dogan et al., 2019). Campylobacter contamination can be detected on all parts of broiler carcass neck skin, visceral cavity, even the region under the skin. Th e number of Campylobacter on the carcass commonly ranges around 2-3 log cfu/cm 2 . Dry environment as well as the storage at 4°C or freezing result in the decrease of Campylobacter counts. Freezing at 18°C during 10 and 21 days will reduce the number of Campylobacter for 90% and 99%, respectively. However, there are reports on Campylobacter isolation from broiler carcasses even aft er 83 weeks of freezing. Even though current processing procedures reduce the Campylobacter count, the organism can oft en be found in fi nal products at the range 1-4 log/cm 2 . Cross-contamination at the slaughterhouse is considered possible yet limited, having in mind low concentration of Campylobacter on cross-contaminated carcasses as related to those originating from poultry that has previously been colonized with Campylobacter. Transmission of Campylobacter from cross-contaminated meat to the fi nal products during processing is very unlikely, as well as the probability for consequent human infection and disease (EFSA, 2010).
Th e rate of carcass contamination at the slaughterhouse varies among poultry originating from Campylobacter colonized fl ocks. At certain production stages, the count of Campylobacter on the carcass can increase or decrease, and there are reports about complete elimination of all Campylobacter so the carcasses were free from Campylobacter. Various technological procedures applied at the slaughterhouses can aff ect cross-contamination and contamination of carcasses of poultry originating from Campylobacter-free fl ocks (Rosenquist et al., 2003). Slaughtering of fl ocks with 100% of infected birds results in 50% contaminated carcasses (Petrović et al., 2008). Th e prevalence of contamination signifi cantly varies between individual slaughterhouses (Table 1).

CAMPYLOBACTER SPP. AT RETAIL
Th e presence of thermophilic Campylobacter in meat on the market varies depending on the product type the highest risk is associated with whole poultry carcass, somewhat lower prevalence is evident in skin-off whole pieces. Minced meat is considered low-risk product because of benefi cial eff ects of spices and oxidative stress as well as the range of poultry meat products (such as sausages, etc.) (Simone et al., 2017). In Serbian market, poultry meat is available as chilled (+4°C) or frozen (-20°C) product. Chilling does not signifi cantly aff ect the survival of Campylobacter, whereas freezing reduces contamination (Reiersen et al., 2001). Campylobacter has been identifi ed in 18.8% poultry meat samples and 10.0% of samples of other meat samples in a retail chain (Trajković et al., 2007).
Campylobacteriosis is the most commonly reported gastrointestinal human disease in the EU/EEA (EFSA and ECDC, 2018). Most common sources of human disease are undercooked or improperly heat treated meat, unpasteurized milk, dairy products and contaminated water. Some half of all cases of human campylobacteriosis in the USA is considered to be associated with consumption of poultry meat. Aft er the incident with dioxin contamination in Belgium in 1999, poultry meat and relevant products were withdrawn from the market, which resulted in a 40% drop of the incidence of human campylobacteriosis. Grilling meat is at particular risk of infection development due to easy transmission of bacteria from raw meat to the hands and thus to other food while handling (Jorgensen, 2002). Th e risk of campylobacteriosis associated with consumption of pork and beef meat is considered relatively law; however, undertreated off al products still pose substantial risk. Important sources of human campylobacteriosis include drinking water and fresh products contaminated with campylobacteria from irrigation water. Th e presence of Campylobacter spp. in drinking and irrigation water is due to faecal contamination (EFSA, 2010).

CONTROL MEASURES
Qualitative risk analysis revealed that decrease in fl ock contamination level by 3 log cfu does not lead to signifi cant decrease in the number of contaminated carcasses processed at the slaughterhouse; however, the Campylobacter count on the carcasses was signifi cantly reduced (Reiersen et al., 2001). Quantitative risk analysis proved that even law reduction of Campylobacter spp. count in poultry faeces (2 log) decreases the incidence of human infection 30 times (Rosenquist et al., 2018) suggesting that control, that is, reduction of Campylobacter in poultry faeces at the farms is a crucial measure for the prevention of human infections. Quantitative risk analysis also revealed a linear relationship between fl ock prevalence and number of contaminated carcasses aft er processing in the slaughterhouse, thus, compliance to strict hygienic barriers is the only eff ective approach to reducing the fl ock prevalence of Campylobacter (Reiersen et al., 2001).
Reduction of Campylobacter count on carcass surface can be accomplished by increasing scalding temperature, improving evisceration techniques, usage of large amounts of water across the entire production line, increasing the airfl ow at chilling and introducing relevant disinfection practices. Prevention of cross-contamination during transport of live chickens is of vital importance, also. However, all the aforementioned measures are of lesser infl uence on the decrease in rate of human infections as compared to the reduction of the incidence of Campylobacter in the fl ock.

CONCLUSION
Campylobacteriosis is one of the most important foodborne diseases mostly associated with consumption of contaminated poultry meat. In order to reduce the number of disease episodes, relevant measures should be applied across the entire poultry meat production chain and especially at poultry farms. Strict compliance with relevant biosecurity measures prevents the introduction of the pathogen into the production process at the farm. Adequate hygiene and sanitation practices in the slaughterhouses can prevent cross-contamination and decrease the contamination level. Ensuring of cold chain supply of poultry meat also contributes to an eff ective disease control. Successful protection of human population from alimentary diseases is closely associated with a range of preventive activities across the entire meat production chain and responsibility of each individual participant of this production chain.
teriosis. JP contributed with data about Campylobacter dynamics in artifi cially infected poultry and data about slaughterhouse prevalence. VG and JL contributed with data about Campylobacter prevalence at retail level. SM revised the manuscript critically and together with JP prepared the fi nal draft of the manuscript. All authors read and approved the fi nal manuscript.