23 Introduction Campylobacter jejuni and coli are the most important cause of bacterial gastroenteritis worldwide infecting humans mostly through consumption of contaminated poultry (Dingle et  al. 2002, Food et  al. 2014). Campylobacter species colonise the gastrointestinal tract of domestic and wild animals and their prevalence in food producing animals, such as cattle, swine and poultry, can exceed the 80% (Mughini Gras et  al. 2012, EFSA 2017). Campylobacter has a broad host range and has been detected everywhere, from farm and urban environments to slaughter plants, in wild birds and mammals, companion animals and farm production animals (Whiley et  al. 2013, Alter et  al. 2005, Pearce et  al. 2003). Campylobacter species are highly adapted to asymptomatically colonise the intestinal tract of most avian species, reaching high numbers (up to 1010 cfu/g caeca content) in chickens and turkeys (Newell et  al. 2008). Once Campylobacter is introduced into a flock, it spreads quickly. Indeed, it can reach a within‑flock prevalence ranging from 60% to 100% (Barrios et al. 2006). Higher prevalence of infection has been observed in many countries in warmer months, suggesting a seasonal pattern in the colonization of poultry flocks (Horroks et  al. 2009). The reason behind this seasonal effect is largely unknown, although a possible role of migratory birds or insects has been suggested (Jacobs‑Reitsma 1997). Campylobacter infections in colonized flocks could be transmitted horizontally within the farm via a variety of routes and vehicles. The possible primary infection sources and transmission routes of Campylobacter for poultry flocks have been investigated in numerous studies, but no definitive factors have been identified so far that explain the high levels of prevalence observed in commercial poultry flocks. Risk factors associated with the introduction and dissemination of Campylobacter within the flocks may include lack of biosecurity Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise 'G. Caporale', Campo Boario, 64100 Teramo, Italy *Corresponding author at: Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise 'G. Caporale', Campo Boario, 64100 Teramo, Italy. E‑mail: s.iannetti@izs.it Keywords Broiler carcasses, Campylobacter, Contamination levels, Production chain, PFGE. Summary A research was carried out in Italy with the aim of assessing Campylobacter contamination in broilers from breeding to slaughter, of defining the genetic diversity of isolates and their antibiotic resistance. Sampling was carried out in a slaughterhouse, and in farms representative of the most common broiler production in Italy. At farm, the 78.8% (95% C.I.: 74.5%‑82.5%) of cloacal samples tested positive for Campylobacter spp. C. jejuni showed higher prevalence in winter than in spring and summer (p < 0.00001, χ2 = 32.9), while C. coli showed an opposite trend (p < 0.00001, χ2= 41.1). At slaughterhouse, the 32.3% (95% C.I.: 30.2%‑35.2%) and the 23.9% (95% C.I.: 21.7%‑26.3%) of skin samples tested positive for C.  jejuni for C. coli, respectively. C. coli showed higher prevalence than C. jejuni at washing (p < 0.05, χ2 = 11.11) and at chilling (p < 0.05, χ2 = 9.26). PFGE revealed high heterogeneity among isolates. Some clones were identified within the same farm in more than one season, suggesting environmental conditions able to support their persistence; other clones resulted to be spatially distant, suggestive of cross‑contamination. Both Campylobacter species showed high resistance to nalidixic acid and ciprofloxacin, while resistance to erythromycin was more frequent in C. coli than C. jejuni (p < 0.05; χ2 test). Simona Iannetti*, Paolo Calistri, Gabriella Di Serafino, Francesca Marotta, Alessandra Alessiani, Salvatore Antoci, Diana Neri, Margherita Perilli, Giorgio Iannitto, Luigi Iannetti, Giacomo Migliorati and Elisabetta Di Giannatale Campylobacter jejuni and Campylobacter coli: prevalence, contamination levels, genetic diversity and antibiotic resistance in Italy Veterinaria Italiana 2020, 56 (1), 23‑34. doi: 10.12834/VetIt.1819.9596.2 Accepted: 11.11.2019 | Available on line: 24.04.2020 24 Veterinaria Italiana 2020, 56 (1), 23‑34. doi: 10.12834/VetIt.1819.9596.2 Campylobacter coli and jejuni in Italy Iannetti et al. carcasses contamination during slaughtering and Campylobacter colonisation at primary production level (EFSA 2010c). The aim of this research was to study the prevalence of C.  jejuni and C.  coli infection in chickens at farm and the contamination levels of carcasses in a typical Italian chicken production and slaughtering chain. Moreover, this study aimed at evaluating the antibiotic susceptibility profiles, the survival and genetic diversity of Campylobacter isolated at farm and at different stages of the slaughtering process using PFGE. Materials and methods Sample collection The sampling activities were carried out in one slaughterhouse (slaughter capacity about 110,000 chickens per day) and in three farms located in Abruzzi region (Central Italy). The farms (A, B, C) selected belonged to the same company and were representative of the most common intensive broiler farms in Italy, on the basis of the breeding management system, the animal housing system, feeding programs and sanitary protocols. In such farms, broilers are usually grown as mixed‑sex flocks in large sheds under intensive conditions; they are reared on ground on deep litter consisting in chopped straw and fed ad libitum with different feed formulas, depending on different stages of the animals’ growth. The temperatures (24‑33  °C), relative humidity (80‑100%) with natural daylight of 12 h and artificial lighting during 12 h of darkness, are differently settled in relation of animals’ age. Sampling was carried out twice for each season (with the exception of August and March for logistic reasons) to take into consideration possible seasonal variations in the infection rates: • Winter: from December to February; • Spring: from April to May; • Summer: from June to July; • Autumn: from September to November. At farm, sampling activities were performed during four breeding cycles in farms A, B and C. In particular, samples were collected at different stages of the breeding cycle: • fifteen days before day‑old chicks restocking (water trough, feed trough, fan blades, ground and insects of the species Alphitobius diaperinus, litter beetles commonly found in poultry houses); • during day‑old chicks restocking (shipping containers swabs, starter feed, manure); measures, contaminated water or feed, contacts with other infected animal species (wild birds, pets, mice, etc.) and mechanical transmission via insects (Barrios et al. 2006, Horroks et al. 2009). Some authors have suggested that Campylobacter can spread from the parent flocks to the progeny (Cox et  al. 2002, Petersen et  al. 2001b, Sahin et  al. 2003, Shanker et  al. 1986) although some observations indicate that vertical transmission plays a minor role in Campylobacter flock colonization (Petersen et  al. 2001a, Callicott et  al. 2006). In the European Union (EU), enteric infections caused by Campylobacter are the most frequently reported zoonosis in humans; in 2016, 246,307 cases of campylobacteriosis have been reported in the EU, with an increase of 6.1% compared with 2015. (EFSA/ECDC 2017). The majority of Campylobacter infections in humans originate from the consumption and handling of raw or undercooked poultry meat products. In Italy, a recent study aimed at investigating an outbreak of campylobacteriosis, showed that in the 70% of cases of C.  jejuni infection, the MLST profiles associated with human disease were most similar to those associated with chicken source (Di Giannatale et  al. 2016). This result is in line with many other European countries, even if the percentage of human campylobacteriosis due to the chicken source vary among the studies (Wilson et al. 2008, Mullner et al. 2009, Sheppard et al. 2009). Therefore, the control of Campylobacter in poultry flocks and the reduction of poultry meat contamination are the cornerstones for any public health strategy aiming at reducing the incidence of campylobacteriosis in humans (EFSA 2008). In the EU, during 2008 a harmonised and standardised baseline survey on the prevalence of Campylobacter in broiler flocks and broiler carcasses was carried out, which assessed a prevalence of Campylobacter contamination in broiler carcasses in Italy equal to 49.6% (95% C.I. 39.5%‑59.7%) (EFSA 2010b). The results of this survey showed that a Campylobacter‑colonised broiler batch was about 30 times more likely to have the sampled carcass contaminated with Campylobacter, compared to a non‑colonised batch, and that the risk of carcasses contamination increases from July to September (EFSA 2010c). According to relevant studies, faecal contamination of carcasses during slaughtering represents the main source of Campylobacter in fresh poultry meat (Mahler et  al. 2011, Guerin et  al. 2011). However, the main factors responsible for Campylobacter presence on carcasses have not been identified yet. Moreover, previous studies comparing C.  jejuni and C.  coli concentrations at farm and along the slaughtering chain are few (Schets FM et  al. 2017). Given the results of the baseline survey, the European Food Safety Authority (EFSA) recommended the EU Member States to identify more clearly the risk factors of 25Veterinaria Italiana 2020, 56 (1), 23‑34. doi: 10.12834/VetIt.1819.9596.2 Iannetti et al. Campylobacter coli and jejuni in Italy Italy). Colonies were cultured on Columbia agar for 48 hours in micro‑aerobic atmosphere, inoculated in Mueller Hinton Broth supplemented with blood, and dispensed into Eucamp microtiter plates (TREK Diagnostic Systems, Biomedical Service, Italy). The plates contained known scalar concentrations of the following antimicrobial substances: gentamicin (Gm) (0.12‑16 µg/ml), streptomycin (S) (1‑16 µg/ml), ciprofloxacin (Cip) (0.06‑4 µg/ml), tetracycline (Te) (0.25‑16 µg/ml), erythromycin (ERY) (0‑5‑32 µg/ml), nalidixic acid (NA) (2‑64 µg/ml), and chloramphenic (CPL) (2‑32  µg/ml). The plates were then incubated at 42  °C in micro‑aerobic atmosphere for 48  hours. C.  jejuni strain NCTC 11351 was included for the quality control of the minimal inhibitory concentration (MIC) test. Antimicrobial resistance was interpreted according to CLSI breakpoints (CLSI Clinical and Laboratory Standards Institute). PFGE Pulsed‑field gel electrophoresis (PFGE) was performed according to the instructions of the 2013 U.S. Pulse Net protocol for Campylobacter (Pulse Net International 2013). Strains of C. jejuni and C. coli were sub cultured on Columbia agar at 42 °C for 2 days in micro‑aerobic atmosphere and embedded in agarose blocks (Seakem Gold agarose, Lonza, Rockland, USA). The blocks were then lysed, washed and digested with SmaI enzyme (Promega, Italy), 25  U at 25  °C for 4 hours. Salmonella serovar Branderup H9812 was used as standard molecular weight size. PFGE was performed using a Chef Mapper XA (Biorad Laboratories) with the following parameters: initial switch time of 6.75 s, final switch time of 35.38 s for 18 hours at 6 V and 14 °C in 0.5 X TBE buffer (Sigma). After electrophoresis, the gel was stained with Sybr Safe DNA gel stain (Invitrogen, USA) and photographed at transilluminator (Alpha Innotech, USA). Bionumerics v. 6.6 software (Applied Maths, Belgium) was used for the analysis of PFGE fingerprinting profiles. Level of similarity were calculated with the Dice correlation coefficient (position tolerance was set at 1%) and unweighted pair group mathematical average UPGMA clustering algorithm was used for cluster analysis of the PFGE pattern. PFGE‑clusters were defined at 100% similarity between macro restriction patterns (Grotheus et  al. 1991). Untypeable isolates were not included in the analysis. Data collection and analysis Sampling information was collected using specific sampling cards and recorded into Microsoft® Access database (MS‑Access 2010) for further analyses. Microsoft® Excel (MS‑Excel 2010) and XLStat©‑Pro (Version 7.5) were used for descriptive and statistical analysis of the data. • thirty days after restocking (water trough, feed trough, fan blades, growth feed and the water from the lake located inside the farm and used for chickens). One day before slaughtering, fifty broilers for each batch were identified before leaving the farm with numbered leg‑rings, and cloacal swabs were collected from each identified animal: the number of broilers identified was increased to take into account the possible lack of the leg‑ring during the slaughtering operations. The identification of the broilers allowed preserving the link between the animal sampled before and during the slaughtering process. At slaughterhouse, samples were collected from the neck skin of each identified carcass by excision after bleeding, defeathering, evisceration, washing, chilling. Caeca samples were also collected from the same carcasses after the evisceration stage. Culture conditions and PCR assays Campylobacter strains were recovered from skin after the enrichment according to ISO 10272‑1:2006 and to ISO 10272‑2:2006 methods. Caeca contents were directly plated on mCCD agar and incubated at 42 °C for 48 h under micro‑aerobic conditions. For enumeration, 1 ml of caeca contents was added to 9 ml of peptone water pH 7.0 (1:10 g/ml), log‑dilution were performed (until 10‑9 dilution), and the plates were incubated at same conditions. The suspected colonies were then examined according to ISO 10272 method and confirmed by multiplex PCR (Wang et al. 2002). Campylobacter isolates were cultured on Columbia blood agar in microaerophilic conditions at 42  °C for 48 hours. Species identification was performed using a multiplex PCR (Wang et al. 2002). DNA from Campylobacter strains was extracted using the Maxwell 16 tissue DNA purification kit (Promega Corporation, Madison, WI) according to the manufacturer's instructions. All isolates were stored at ‑ 80 °C. One thousand ml of water lake was filtered using membrane filter (pore size 0.45 micron, Millipore). After filtration, membrane filter was placed in Bolton enrichment broth for 48 h at 42  °C in microaerobic condition, later streaked on mCCD agar plate for isolation, and then incubated again for 48 h at 42 °C in microaerobic condition. The suspected colonies were identified by multiplex PCR (Wang et al. 2002). Antimicrobial susceptibility The susceptibility of Campylobacter isolates to seven antimicrobials was evaluated with a micro broth dilution method using the ‘Sensititre’ automated system (TREK Diagnostic Systems, Biomedical Service, 26 Veterinaria Italiana 2020, 56 (1), 23‑34. doi: 10.12834/VetIt.1819.9596.2 Campylobacter coli and jejuni in Italy Iannetti et al. Multiple comparisons showed a significantly higher prevalence of contamination in winter than in spring and summer (p < 0.00001, χ2 = 32.9; Multiple chi‑squared test) for C.  jejuni, while C.  coli showed a higher prevalence level in spring than in summer and autumn (p < 0.00001, χ2 = 41.1; Multiple Chi‑squared test). The environmental samples, the feed and insects tested all negative for Campylobacter  spp. by the detection method. The water collected from the lake located inside the farm 30 days after placement of the day‑old chicks in the shed, and used for chickens, tested positive for Campylobacter  spp. by filtration method and Campylobacter coli was identified by multiplex PCR. At slaughterhouse, 230 broilers out of 400 (57.5%) were sampled, for 1,333 samples (Table I). The rest of the broilers was not sampled due to the loss of the leg‑ring during transport. The 32.3% (95% C.I.: 30.2%‑35.2%) of skin samples tested positive for C. jejuni and the 23.9% (95% C.I.: 21.7%‑26.3) of samples tested positive for C.  coli. The 48.9% (95% C.I.: 42.9‑55.8) of caeca samples tested positive for C.  jejuni and the 28.9% (95% C.I.: 23.4‑35.1) tested positive for C.  coli. Figure  2 compares the prevalence of C.  coli and C.  jejuni contamination in samples taken at slaughterhouse. C.  jejuni showed higher prevalence levels than C.  coli in caeca samples, at bleeding, defeathering and evisceration, while after the evisceration stage Multiple Chi‑Squared test was used to verify if in samples taken at slaughterhouse there were significant differences in prevalence levels among sampling seasons and sampling stages. Chi‑Squared test was used to verify whether the prevalence levels observed for C.  jejuni were significantly different from prevalence levels observed for C. coli in samples taken at slaughterhouse. Contamination levels were checked for a normal distribution with the Kolmogorov‑Smirnov test, and then non‑parametric tests have been used because of non‑normality of the data. Kruskal‑Wallis test was used to verify significant differences among contamination levels of sampling seasons and significant differences among contamination levels of sampling stages in samples taken at slaughterhouse. Multiple comparisons among contamination levels of C. coli and of C. jejuni in skin and caeca samples were performed with Dunn Test. Friedman test was used to verify if there were significant differences among contamination levels of skin samples taken from the same animal after bleeding, defeathering, evisceration, washing and chilling and multiple comparisons were performed with Nemenyi test. Fisher F‑test was also used to verify whether the contamination levels observed for C.  jejuni were significantly different from contamination levels observed for C.  coli in samples taken at slaughterhouse. Results Campylobacter prevalence and contamination levels At farm, out of 400 cloacal samples, 315 tested positive for Campylobacter spp. (Prevalence = 78.8%, 95% C.I.: 74.5%‑82.5%). C.  jejuni showed higher prevalence levels than C. coli during each sampling season. A lower prevalence level was observed in spring than in the other seasons. C.  coli, showed a different seasonal pattern with higher prevalence levels in spring than in the other seasons (Figure 1). Table I. Number of samples taken at slaughterhouse. Month of sampling Season N. of animals N. of caeca N. of samples taken after the slaughtering stages Total n. of samplesBleeding Defeathering Evisceration Washing Chilling Dec-Feb Winter 46 46 46 46 46 46 45 275 Apr-May Spring 60 55 55 56 55 59 55 335 Jun-July Summer 68 68 68 63 63 63 62 387 Sep-Nov Autumn 56 56 56 56 56 56 56 336 Total 230 225 225 221 220 224 218 1,333 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Winter Spring Summer Autumn P re va le n ce (% ) Sampling season Figure 1. C. coli (blue dots) and C. jejuni (orange dots): prevalence (± C.L. 95%) of contamination of samples taken at farm (cloacal swabs). 27Veterinaria Italiana 2020, 56 (1), 23‑34. doi: 10.12834/VetIt.1819.9596.2 Iannetti et al. Campylobacter coli and jejuni in Italy Wallis test). Contamination levels of C.  jejuni were significantly higher at bleeding [Mean: 4.08 Log (CFU)/g; SD: 1.05] than at the other slaughtering stages (p  <  0.0001; Kruskal Wallis test). Details on C.  coli and C.  jejuni concentrations in samples of carcasses collected after the slaughter operations and on the results of the multiple comparisons performed are shown in Table II and Table III. As regards seasonality, concentration of C.  jejuni in skin samples was found significantly higher in summer [Mean: 3.89 Log (CFU)/g; SD: 1.20] than in other sampling seasons (p < 0.0001; Kruskal Wallis test), while no statistically significant difference was found for C. coli. In caeca samples no statistically significant difference was observed among contamination levels of seasons for C.  coli, while a significant lower contamination C.  coli showed significantly higher prevalence levels than of C.  jejuni at washing (p  <  0.05, χ2 = 11.11) and at chilling (p  <  0.05, χ2 =  9.26). Figure 3 shows contamination levels of C.  jejuni and C.  coli in samples taken at slaughterhouse at different slaughtering stages. Concentrations of C.  coli and C. jejuni on neck skin samples of carcasses collected after slaughter operations are shown in Table  II and Table  III, respectively. Statistically significant differences were found in Campylobacter concentration among the slaughtering stages (p  <  0.0001; Kruskal Wallis test). Results of multiple comparisons performed with Dunn test highlighted that contamination levels of C.  coli were significantly lower at defeathering [Mean: 1.54 Log (CFU)/g; SD: 0.91] and significantly higher at bleeding [Mean: 4.17 Log (CFU)/g; SD: 0.69] than at the other slaughtering stages (p < 0.0001; Kruskal 0% 10% 20% 30% 40% 50% 60% Caeca Bleeding Defeathering Evisceration Washing Chilling Figure 2. C. coli (blue dots) and C. jejuni (red dots): prevalence (± C.L. 95%) of contamination of samples taken at slaughterhouse. 0 1 2 3 4 5 6 7 8 Caeca Bleeding Defeathering Evisceration Washing Chilling Lo g (U FC )/ g Figure 3. C. coli (green dots) and C. jejuni (purple dots): levels of contamination (± C.L. 95%) of samples taken at slaughterhouse. Table II. The concentration of C. coli on samples of chicken carcasses collected after the slaughter operations. C. coli Caeca Bleeding* Defeathering# Evisceration Washing Chilling Mean 6.81505115 4.170834346 1.540735312 2.919966832 2.574238328 2.664002739 Median 7.173475592 4.170915028 0.995635195 3.146128036 2.579783597 2.676091259 SD 1.112598967 0.694096517 0.906040819 0.793316118 0.296979432 0.630023145 95° perc 7.830002887 5.313414732 3.151232096 4.097512676 3.009558145 3.491361694 5° perc 4.044136728 3.090273684 0.995635195 0.995635195 2.301029996 2 *Contamination levels significantly higher (p < 0.0001); #Contamination levels significantly lower (p < 0.0001). Table III. The concentration of C. jejuni on samples of chicken carcasses collected after the slaughter operations. C. jejuni Caeca Bleeding* Defeathering# Evisceration# Washing# Chilling# Mean 5.374044013 4.078751274 1.831685688 2.787108632 2.545574554 2.76650184 Median 5.627636253 3.954215699 0.995635195 2.903089987 2.544068044 3 SD 1.613333167 1.047516471 1.014431325 0.778875511 0.278445066 0.757715035 95° perc 7.661623156 5.946502352 3.538718726 3.678379134 3.004967555 3.614852317 5° perc 2.903089987 2.618264014 0.995635195 0.995635195 2.176091259 0.995635195 *Contamination levels significantly higher (p < 0.0001); #Contamination levels significantly lower (p < 0.0001). 28 Veterinaria Italiana 2020, 56 (1), 23‑34. doi: 10.12834/VetIt.1819.9596.2 Campylobacter coli and jejuni in Italy Iannetti et al. test). Campylobacter concentration was significantly lower at defeathering than at evisceration, washing and chilling, and significantly lower also at washing than at chilling stages (p  <  0.0001; Nemenyi test). Figure 4 shows mean and median contamination levels found in skin samples of the same animal along the slaughtering chain. PFGE PFGE analysis with SmaI restriction enzyme of 367 C.  jejuni isolates in caeca and in the phases of the slaughter process resulted in a total of 103 pulsotypes (with 100% similarity). PFGE types of C.  jejuni strains isolated at the various stages of the slaughter process vary from 1 to 6 pulsotypes per cycle. Three PFGE types (31, 35 and 75) were always present in different product of all batches and farms, while six PFGE types (41, 53, 75, 82, 85 and 86) were present in all slaughtering stages (Table IV). For each slaughter batch, up to four different C.  jejuni pulsotypes were found at slaughterhouse. Cluster analysis of 273 C.  coli isolates showed 70  distinct pulsotypes (with 100% similarity) with none of the PFGE types overlapping between the two farms. In particular, PFGE types of C. coli strains isolated at the different stages of the slaughter line (p < 0.05; Kruskal Wallis test) was detected in spring for C.  jejuni [Mean: 4.30 Log (CFU)/g; SD: 0.77]. Significant differences between contamination levels of C.  coli and C.  jejuni along the slaughtering stages were found only for caeca samples with higher level of C. jejuni (p < 0.05; Fisher F‑test). As regards Campylobacter concentration in the same animal during the slaughtering chain, Campylobacter concentration was found significantly different among each sampling point (p  <  0.0001; Friedman 2.745 1.214 2.322 1.916 2.330 3.079 0.996 2.653 2.301 2.602 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Bleeding Defeathering Evisceration Washing Chilling Lo g (C FU )/ g Mean Median Figure 4. Contamination levels of skin samples taken at slaughterhouse from the same animal. Table IV. PFGE pulsotypes of C. jejuni strains isolated at different stages of the slaughter process and at farm and sampling season. Major pulsotypes (100% similarity) Isolates (No.) Source Farm Sampling season 26 4 Washing, Evisceration B Autumn 29 14 Defeathering, Evisceration, Washing, Chilling B Autumn 30 6 Evisceration, Washing, Chilling B Autumn 31 15 Defeathering, Evisceration, Washing, Chilling B Autumn Washing C Spring 35 14 Chilling B Autumn Washing C Winter Slaughtering bleeding, Evisceration, Washing C Spring 41 10 Slaughtering bleeding, Washing, Chilling B Summer 43 4 Slaughtering bleeding, Evisceration C Spring 53 25 Slaughtering bleeding, Evisceration, Chilling A Spring 68 10 Slaughtering bleeding, Evisceration, Washing, Caeca A Winter 72 2 Slaughtering bleeding, Evisceration B Summer 75 24 Defeathering A Summer Slaughtering bleeding, Evisceration, Washing A Winter Slaughtering bleeding, Evisceration, Washing, Chilling B Summer 77 2 Chilling, Defeathering A Summer 82 16 Slaughtering bleeding, Evisceration, Washing, Chilling B Summer 85 10 Slaughtering bleeding, Defeathering, Evisceration, Washing Chilling C Winter 86 32 Slaughtering bleeding, Defeathering, Chilling C Winter 87 10 Defeathering, Evisceration C Winter 93 2 Slaughtering bleeding, Defeathering A Summer 103 9 Slaughtering bleeding, Defeathering B Autumn 29Veterinaria Italiana 2020, 56 (1), 23‑34. doi: 10.12834/VetIt.1819.9596.2 Iannetti et al. Campylobacter coli and jejuni in Italy for the reduction of campylobacteriosis in humans. To date, the mechanisms underlying Campylobacter colonization of farmed broiler flocks and contamination of carcasses during slaughtering have not been fully clarified yet. Investments in research are fundamental to improve the knowledge of the physiology, ecology, metabolism and colonisation mechanisms of C.  jejuni and C.  coli in poultry and their surviving capacity in the environment. In addition, although the role of C. jejuni contamination in broiler meat has been extensively studied in many European countries, the importance of C. coli in these food products has been not fully investigated yet. In particular, while the contribution of C.  jejuni to the burden of human illness, through the consumption of raw or undercooked broiler meat, is well known, the same cannot be stated for C. coli. Currently, in the EU it is generally considered that, given food regulations (EU 2017) precluding the use of antimicrobial treatments on carcasses (such as hyper chlorination), the most effective intervention strategy is to prevent or reduce flock colonisation at the farm level. The results obtained at farm confirm the high level of prevalence already detected by other previous studies (Allen et  al. 2007, Di Giannatale et  al. 2010, Hadžiabdić et  al. 2013, Henry et  al. 2011, Hue et  al. 2010, Rosenquist et  al. 2006, Thakur et  al. 2013). Regarding environmental samples, feed, water and pests, our results, however, are not in line with the findings of other studies (Evans et  al. 2000, Bull et  al. 2006) in which the isolation of Campylobacter was frequently obtained from feed, water in the drinkers and litter samples. In our research, the environmental samples and those taken from the feed, water and pests resulted all negative for Campylobacter detection, with the exception of one sample of water lake, which could suggest a possible introduction of contamination through the use of this water source for animal drinking. vary from one to three pulsotypes for each batch and only three PFGE types (18, 44 and 70) were isolated at all slaughtering stages (Table V). Besides, for C. coli slaughter batches showed a multiple number of PFGE types, up to three, with the exception of four carcasses from farm C having a single pulsotype identified in all slaughtering stages. Antimicrobial resistance The results of MIC and antimicrobial resistance revealed that 92.0% and 93.8% of the isolates from caeca were resistant to quinolones and fluoroquinolones (NAL and Cip), respectively. The 39.3% of the strains showed resistance to tetracycline, the 13.4% to erythromycin, and few strains resulted resistant to other antimicrobials such as chloramphenicol (1.8%) and streptomycin (0.9%). None of the isolates tested was resistant or sensitive to gentamicin. Moreover, resistance to erythromycin was more frequent in C.  coli (27.8%) compared to C. jejuni (6.6%) isolates (p < 0.05; χ2 test), whereas these differences were not observed for the remaining antimicrobial substances. The highest level of resistance was observed to NAL and Cip, for Campylobacter isolates from carcasses. In detail, the 90.0% and 90.6% of the strains were resistant to fluoroquinolones and quinolones, the 64.7% were resistant to tetracycline, and the 31.9% were resistant to erythromycin. The 99% of strains were susceptible to chloramphenicol, streptomycin gentamicin antimicrobials. Also for the strains isolated from carcasses, resistance to erythromycin was more frequent in C.  coli isolates (44.0%) compared to C. jejuni (13.2%) (p < 0.05; χ2 test). Discussion The prevention and control of Campylobacter colonisation in broiler flocks is an important goal Table V. PFGE pulsotypes of C. coli strains isolated at different stages of the slaughter process and at farm and sampling season. Major pulsotypes (100% similarity) Isolates (No.) Source Farm Sampling season 9 6 Defeathering, Washing, Chilling A Spring 10 2 Chilling, Evisceration A Spring 15 3 Defeathering, Washing A Autumn 17 7 Evisceration, Chilling A Autumn 18 18 Slaughtering bleeding, Washing, chilling A Autumn 24 18 Defeathering, Evisceration, Washing, Chilling A Winter 33 4 Evisceration, Washing, Chilling C Spring 41 6 Evisceration, Washing A Summer 43 3 Evisceration, Washing A Summer 44 36 Slaughtering bleeding, Evisceration, Washing, Chilling A Summer 70 42 Slaughtering bleeding, Defeathering, Evisceration, Washing, Chilling A Spring 30 Veterinaria Italiana 2020, 56 (1), 23‑34. doi: 10.12834/VetIt.1819.9596.2 Campylobacter coli and jejuni in Italy Iannetti et al. genomic relationships among bacterial isolates, with the ability to correlate isolated microorganisms from different sites and samples (Frasao et  al. 2017). Typing of Campylobacter strains isolated from pigs, poultry, turkey, sheep, and lambs by PFGE has been widely described (Silva et  al. 2016, Lahti et  al. 2017). In particular, Silva and colleagues found that Campylobacter clones belong to poultry flocks to indicate endemic strains with horizontal transmission among birds, and that the genetic profile associated with different farms suggested different sources of contamination (Silva et al. 2016). PFGE results showed a high genetic heterogeneity of Campylobacter population: the same flocks were colonized by more genotypes. Some clones recovered at the early stages of the production chain were not recovered at the later stages, while other clones were predominant in individual breeding cycles and were present in all production chain stages (pulsotype 85 for C. jejuni or pulsotype 70 for C.  coli), confirming the traceability of flock specific strains along the entire processing chain. Every season would seem characterised by different genetic sub‑populations of strains of Campylobacter. However, other clones were identified within the same farm in more than one season (pulsotype 35 or 75 for C.  jejuni), suggesting the persistence of these genotypes in the environment. These results are consistent with a study of Peyrat and colleagues showing the existence of C.  jejuni and C.  coli clones particularly able to adapt and survive overnight on the surfaces of slaughterhouse equipment after cleaning and disinfection (Peyrat et al. 2009). On the contrary, the presence of the same clones of C. jejuni and C. coli in different herds (pulsotypes 31 for C.  jejuni), spatially distant, might suggest a cross‑contamination linked to the operators or the means used, favouring the recirculation of these strains among farms. Cross‑contamination of carcasses from poultry coming from different flocks but slaughtered at same slaughterhouse seems, therefore, to be unavoidable. The prevalence and contamination levels observed in carcasses confirm the deep influence of slaughtering operations to the final contamination levels. After a clear decrease of prevalence and contamination levels between bleeding and defeathering, a significant increase was observed after the evisceration stage. This trend may be related to the contamination occurred in case of intestine ruptures at the evisceration stage and supported by cross‑contamination due to the strict contact among the carcasses during the slaughtering chain. The reduction of prevalence and contamination levels in skin samples taken after defeathering may be due to the effect of heat treatment after scalding (around 55  °C) (Bolder 2002), and to the abrasive Many studies are available on the different behaviour of Campylobacter species in farms and environment suggesting also a possible contribution of broiler farms to the aquatic environmental Campylobacter load (Schets et al. 2017). In our study, the comparison among C.  jejuni and C.  coli seems suggesting a different seasonal behaviour of C. jejuni with respect to C. coli in chickens, as shown in Figure 1. Moreover, it is noteworthy that whereas a clear seasonality of Campylobacter spp. contamination, with a highest risk from July to September, was observed in many EU  member countries, this pattern was not recognised in Italy (EFSA 2010c). The increase of Campylobacter spp. contamination in broiler flocks during summer was frequently associated with the possible role of flies in spreading the infection within and between farms, especially in northern countries (Hald 2004). At southern latitudes, like in Italy, the temperatures conditions could be favourable for the presence of flies and other insects all around the year, thus lacking a clear seasonality in Campylobacter spp. contamination. A recent report comparing different types of samples from broiler flocks at farm, showed a greater level of genetic diversity in strains isolated from neck skin and caeca samples than in chicken meat (Ugarte et  al. 2015). Other studies investigating the genetic diversity of Campylobacter concluded that Campylobacter concentration increases from farm to slaughter, suggesting also that the full diversity of Campylobacter genotypes found at slaughter could be also the result of cross‑contamination during the slaughtering process (Colles et al. 2010). Commercial broiler farms are an important ecological niche for a wide variety of Campylobacter genotypes, thus confirming the complexity of the population structure of these organisms in broiler production and in the chicken food chain. Therefore, it is very important to improve sampling strategies with the aim of investigating Campylobacter structure population in broiler production (Vidal et al. 2016). To our knowledge, this is the first extended study in which the sources of cross‑contamination in a poultry slaughterhouse were studied by PFGE in Italy: we used PFGE to check the traceability of flock specific strains along the entire processing chain. With regard to species difficult to quantify, isolate, or distinguish, such as Campylobacter spp., PFGE is an important technique enabling research on the entire food supply chain as well as the tracing and estimation of the same strain responsible for contamination, from the raising of the animal to the foodborne illness in a human (Frasao et  al. 2017). Even if PFGE is not the method of choice for molecular typing of Campylobacter, this technique is widely used for obtaining a clear comparison of 31Veterinaria Italiana 2020, 56 (1), 23‑34. doi: 10.12834/VetIt.1819.9596.2 Iannetti et al. Campylobacter coli and jejuni in Italy nalidixic acid and tetracycline has been observed in this study. Erythromycin resistance is significantly more frequent in Campylobacter coli (27.8%) compared to Campylobacter jejuni (6.6%) in caeca isolates (p < 0.05; χ2 test), and in Campylobacter coli isolates from chicken carcasses (44.0%) compared to Campylobacter jejuni (13.2%) (p < 0.05; χ2  test). According to the other studies, high levels of resistance to tetracycline and ciprofloxacin are frequently reported in both the species (Ge et  al. 2013, Rozynek et  al. 2008). C.  coli is usually more resistant to erythromycin than C.  jejuni, although resistance in C.  jejuni has been increasing (Wang et al. 2013). Our study demonstrates the significant diffusion of Campylobacter infection in broilers in a typical breeding and slaughtering production context and the high genetic variability of the bacterium. To reduce cross contamination of Campylobacter flocks with persistent clones during the slaughtering process, efficient hygiene measures are needed. The results of this study provide more information for the definition of proper control options at slaughterhouses and new insight about the possible different behaviour of C.  coli in comparison of C. jejuni. Grant This project was funded by the Italian Ministry of Health in the framework of the national research program and carried out by the Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’ (IZSAM), which is the National Reference Laboratory for Campylobacter. action caused by machines that remove microbial slide on the chicken skin. The favourable effect in reducing the level of contamination by the exposure to low temperatures and the dehydration of the carcasses surface during the transit through the chilling tunnel was not confirmed in our study unlike other researches (Guerin et al. 2011). The comparison among C.  jejuni and C.  coli concentrations in skin samples taken at slaughterhouse during seasons suggested a different behaviour of these organisms: C.  jejuni seems to be significantly higher in summer, while C.  coli concentration was not significantly different among seasons. An opposite prevalence trend among C.  jejuni and C.  coli was found after evisceration stage, as shown (Figure 2). C. coli showed significantly higher prevalence of contamination than of C.  jejuni at washing and at chilling. This finding could suggest a greater resistance of C.  coli than C.  jejuni at lower temperatures, although no statistically significant difference was found among contamination levels of C.  coli and of C.  jejuni at washing and at chilling (Figure 3). In general, it is rather difficult to interpret these apparent differences, especially considering the microclimatic conditions in slaughterhouses, which are roughly constant and standardised. However, all these differences might suggest different mechanisms of persistence or survival capacities between C.  jejuni and C.  coli in the slaughtering environment, which should be more in depth investigated. As regards antimicrobial resistance results, a significant increase in resistance to ciprofloxacin, 32 Veterinaria Italiana 2020, 56 (1), 23‑34. doi: 10.12834/VetIt.1819.9596.2 Campylobacter coli and jejuni in Italy Iannetti et al. Allen V.M., Bull S.A., Corry J.E.L., Domingue G., Jørgensen F., Frost J.A., Whyte R., Gonzalez A., Elviss N. & Humphrey T.J. 2007. Campylobacter spp. contamination of chicken carcasses during processing in relation to flock colonisation. Int J Food Microbiol, 113, 54‑61. Alter T., Gall F., Froeb A. & Fehlhaber K. 2005. Distribution of Campylobacter jejuni strains at different stages of a turkey slaughter line. Food Microbiol, 22, 345‑351. Barrios P.R., Reiersen J., Lowman R., Bisaillon J‑R., Michel P., Fridriksdóttir V., Gunnarsson E., Stern N., Berke O., McEwen S. & Martin W. 2006. Risk factors for Campylobacter spp. colonization in broiler flocks in Iceland. Prev Vet Med, 74, 264‑278. Bolder N.M. 2002. The microbiology of the slaughter and processing in poultry. In The microbiology of meat and poultry (Davies and Board eds.). Blackie Academic & Professional, London,158‑173. Boysen L., Rosenquist H., Larsson J.T., Nielsen E.M. & Sørensen G. 2014. Source attribution of human Campylobacteriosis in Denmark. Epidemiol Infect, 142, 1599‑1608. Bronowski C., James C.E. & Winstanley C. 2014. Role of environmental survival in transmission of Campylobacter jejuni. FEMS Microbiol Lett, 356, 8‑19. Bull S.A., Allen V.M., Domingue G., Jørgensen F., Fros J.A., Ure R., Whyte R., Tinker D., Corry J.E., Gillard‑King J. & Humprey T.J. 2006. Sources of Campylobacter spp. colonizing housed broilers flocks during rearing. Appl Environ Microbiol, 72, 645‑652. Cahill S.M. 2004. Risk assessment and Campylobacteriosis. In Risk management strategies: monitoring and surveillance (Smulders F.J.M. and Collins J.D., eds). Wageningen Academic Publishers, Wageningen, 151‑172. Callicott K.A., Fridriksdóttir V., Reiersen J., Lowman R., Bisaillon J‑R. & Gunnarsson E. 2006. Lack of evidence for vertical transmission of Campylobacter spp. in chickens. Appl Environ Microbiol, 72, 5794‑5798. Clinical and Laboratory Standards Institute. 2012. Performance standards for antimicrobial susceptibility testing. Twenty Second Informational supplement, 32 (3), M100‑S22. Cox N.A., Stern N.J., Hiett K.L. & Berrang M.E. 2002. Identification of a new source of Campylobacter contamination in poultry: transmission from breeder hens to broiler chickens. Avian Dis, 46, 535‑541. Di Giannatale E., Prencipe V., Colangeli P., Alessiani A., Barco L., Staffolani M., Tagliabue S., Grattarola C., Cerrone A., Costa A., Pisanu M., Santucci U., Iannitto G. & Migliorati G. 2010. Prevalenza di Campylobacter termotolleranti nel pollo da ingrasso in Italia. Vet Ital, 46 (4), 405‑414. Di Giannatale E., Garofolo G., Alessiani A., Di Donato G., Candeloro L., Vencia W., Decastelli L. & Marotta F. 2016. Tracing back clinical Campylobacter jejuni in the Northwest of Italy and assessing their potential source. Front Microbiol, 7, 887. Dingle K.E., Colles F.M., Ure R., Wagenaar J.A., Duim B., References Bolton F.J., Fox A.J., Wareing D.R. & Maiden C. 2002. Molecular characterization of Campylobacter jejuni clones: a basis for epidemiologic investigation. Emerg Infect Dis, 8, 949‑955. Dingle K.E., Colles F.M., Falush D. & Maiden M.C. 2005. Sequence typing and comparison of population biology of Campylobacter coli and Campylobacter jejuni. J Clin Microbiol, 43, 340‑347. European Food Safety Authority (EFSA) 2008. Assessing health benefits of controlling Campylobacter in the food chain. EFSA Scientific Colloquium Summary Report 12, 4‑5 December 2008, Rome, Italy. European Food Safety Authority (EFSA). 2010a. EFSA Panel on biological hazards (BIOHAZ); Scientific opinion on quantification of the risk posed by broiler meat to human Campylobacteriosis in the EU. EFSA Journal, 8 (1), 1437. European Food Safety Authority (EFSA). 2010b. Analysis of the baseline survey on the prevalence of Campylobacter in broiler batches and of Campylobacter and Salmonella on broiler carcasses in the EU, 2008, part A: Campylobacter and Salmonella prevalence estimates. EFSA Journal, 8 (3), 1503. European Food Safety Authority (EFSA) 2010c. Analysis of the baseline survey on the prevalence of Campylobacter in broiler batches and of Campylobacter and Salmonella on broiler carcasses in the EU, 2008, part B: Analysis of factors associated with Campylobacter colonisation of broiler batches and with Campylobacter contamination of broiler carcasses; and investigation of the culture method diagnostic characteristics used to analyse broiler carcass samples. EFSA Journal, 8 (8), 1522. European Food Safety Authority (EFSA) 2011. Scientific opinion on Campylobacter in broiler meat production: control options and performance objectives and/ or targets at different stages of the food chain. EFSA Journal, 9 (4), 2105. European Food Safety Authority and European Centre for Disease Prevention and Control (EFSA/ECDC). 2017. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food‑borne outbreaks in 2016. EFSA Journal, 15, 5077. European Union (EU). 2017. Commission Regulation (EU) 2017/1495 of 23 August 2017 amending Regulation (EC) No 2073/2005 as regards Campylobacter in broiler carcases. OJ, L 218, 24.08.2017. Evans S.J. & Sayers A.R., 2000. A longitudinal study of Campylobacter infection of broiler flocks in Great Britain. Prev Vet Med, 46, 209‑223. Evers E.G., Van Der Fels‑Klerx H.J., Nauta M.J., Schijven J.F. & Havelaar A.H. 2008. Campylobacter source attribution by exposure assessment. Int J Risk Ass Manag, 8, 174‑190. European Food Safety Authority (EFSA). 2014. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food‑borne outbreaks in 2012. EFSA Journal, 12 (2), 3547. Frasao B.S., Marin V.A. & Conte J.C.A. 2017. Molecular 33Veterinaria Italiana 2020, 56 (1), 23‑34. doi: 10.12834/VetIt.1819.9596.2 Iannetti et al. Campylobacter coli and jejuni in Italy Malher X., Simon M., Charnay V., Déserts R.D., Lehébel A. & Belloc C. 2011. Factors associated with carcass contamination by Campylobacter at slaughterhouse in cecal‑carrier broilers. Int J Food Microbiol, 150, 8‑13. Mughini Gras L., Smid J.H., Wagenaar J.A., de Boer A.G., Havelaar A.H., Friesema I.H., French N.P., Busani L. & van Pelt W. 2012. Risk factors for campylobacteriosis of chicken, ruminant, and environmental origin: a combined case‑control and source attribution analysis. PLoS One, 7, e42599. Mullner P., Spencer S.E., Wilson D.J., Jones G., Noble A.D., Midwinter A.C., Collins‑Emerson J.M., Carter P., Hathaway S. & French N.P. 2009. Assigning the source of human campylobacteriosis in New Zealand: a comparative genetic and epidemiological approach. Infect Genet Evol, 9, 1311‑1319. National Advisory Committee on Microbiological Criteria for Foods (NACMF). 1994. Campylobacter jejuni/coli. J Food Prot, 57, 1101‑1121. Newell D.G. , Allen V., Elvers K., Dorfper D., Hanssen I., Jones P., James S., Gittins J., Stern N., Davies R., Connerton I., Pearson D. & Salvat G. 2008. B15025: a critical review of interventions and strategies (both biosecurity and non‑biosecurity) to reduce Campylobacter on the poultry farm. Final Report. https://lwecext.rl.ac.uk/PDF/ RES3005_final_report.pdf. Peyrat M.B., Soumet C., Maris P. & Sanders P. 2009. Phenotypes and genotypes of Campylobacter strains isolated after cleaning and disinfection in poultry slaughterhouses. Vet Microbiol, 128, 313‑326. Pearce R.A., Wallace F.M., Call J.E., Dudley R.L., Oser A. & Yoder L. 2003. Prevalence of Campylobacter within a swine slaughter and processing facility. J Food Prot, 66, 1550‑1556. Petersen L., Nielsen E.M., On S.L.W. 2001a. Serotype and genotype diversity and hatchery transmission of Campylobacter jejuni in commercial poultry flocks. Vet Microbiol, 82, 141‑144. Petersen L. & Wedderkopp A. 2001b. Evidence that certain clones of Campylobacter jejuni persist during successive broiler flock rotations. Appl Environ Microbiol, 67, 2739‑2745. PulseNet International. 2013. Standard operating procedure for PulseNet PFGE of Campylobacter jejuni. http://www.pulsenetinternational.org/assets/PulseNet/ uploads/pfge/PNL03CampyPFGEprotocol.pdf. Rosenquist H., Sommer H.M., Nielsen N.L. & Christensen B.B. 2006. The effect of slaughter operations on the contamination of chicken carcasses with thermotolerant Campylobacter. Int J Food Microbiol, 108, 226‑232. Sahin O., Kolbalka P. & Zhang Q. 2003. Detection and survival of Campylobacter in chicken eggs. J Appl Microbiol, 95, 1070‑1079. Shanker S., Lee A. & Sorrell T.C. 1986. Campylobacter jejuni in broilers: the role of vertical transmission. J Hyg (Lond), 96, 153‑159. Schets F.M., Jacobs‑Reitsma W.F., van der Plaats R.Q.J., Heer L.K., van Hoek A.H.A.M., Hamidjaja R.A., de Roda Husman A.M. & Blaak H. 2017. Prevalence and types detection, typing, and quantification of Campylobacter spp. Comprehensive Reviews in Food Science and Food Safety, 16 (4), 721‑734. Ge B., Wang F., Sjölund‑Karlsson M. & McDermott P.F. 2013. Antimicrobial resistance in campylobacter: susceptibility testing methods and resistance trends. J Microbiol Methods, 95, 57‑67. Grotheus D. & Tummler B. 1991. New approaches in genome analysis by pulsed‑field electrophoresis: application to the analysis of Pseudomonas species. Mol Microbiol, 5, 2763‑2776. Guerin M.T., Sir C., Sargeant J.M., Waddell L., O'Connor A.M., Wills R.W., Bailey R.H. & Byrd J.A. 2010. The change in prevalence of Campylobacter on chicken carcasses during processing: a systematic review. Poult Sci, 89, 1070‑1084. Hadžiabdić S., Rešidbegović E., Gruntar I., Kušar D., Pate M., Zahirović L., Kustura A., Gagić A. Goletić T. & Ocepek M. 2013. Campylobacters in broiler flocks in Bosnia and Herzegovina: prevalence and genetic diversity. Slov Vet Res, 50, 45‑55. Hald B., Skovgård H., Bang D.D., Pedersen K., Dybdahl J., Jespersen J.B. & Madsen M. 2004. Flies and Campylobacter infection of broiler flocks. Emerg Infect Dis, 10, 1490‑1492. Havelaar A.H., Galindo A.V., Kurowicka D. & Cooke R.M. 2008. Attribution of foodborne pathogens using structured expert elicitation. Foodborne Pathogens Dis, 5, 649‑659. Henry I., Reichardt J., Denisc M. & Cardinaled E. 2011. Prevalence and risk factors for Campylobacter spp. in chicken broiler flocks in Reunion Island (Indian Ocean). Prev Vet Med, 100, 64‑70. Horrocks S.M., Anderson R.C., Nisbet D.J. & Ricke S.C. 2009. Incidence and ecology of Campylobacter jejuni and coli in animals. Anaerobe, 15, 18‑25. Hue O., Le Bouquin S., Laisney M.‑J., Allain V., Lalande F., Petetin I., Rouxel S., Quesne S., Gloaguen P.‑Y., Picherot M., Santolini J., Salvat G., Bougeard S., Chemaly M. 2010. Prevalence of and risk factors for Campylobacter spp. contamination of broiler chicken carcasses at the slaughterhouse. Food Microbiol, 27, 992‑999. Jacobs‑Reitsma W.F. 1997. Aspects of epidemiology of Campylobacter in poultry. Vet Q, 19, 113‑117. Jones K. 2001. Campylobacters in water, sewage and the environment. J Appl Microbiol, 90, 68S‑79S. Jonsson M.E., Chriél M., Norström M. & Hofshagen M. 2012. Effect of cli‑mate and farm environment on Campylobacter spp. colonization in Norwegian broiler flocks. Prev Vet Med, 107, 95‑104. Kapperud G., Skjerve E., Vik L., Hauge K., Lysaker A., Aalmen I., Ostroff S.M. & Potter M. 1993. Epidemiological investigation of risk factors for Campylobacter colonization in Norwegian broiler flocks. Epidemiol Infect, 111 (2), 245‑255. Lahti E., Lofdahl M., Agren J., Hansson I. & Engvall E.O. 2017. Confirmation of a campylobacteriosis outbreak associated with chicken liver pate using PFGE and WGS. Zoonoses Public Health, 64,14‑20. 34 Veterinaria Italiana 2020, 56 (1), 23‑34. doi: 10.12834/VetIt.1819.9596.2 Campylobacter coli and jejuni in Italy Iannetti et al. M.C., Navarro‑Gonzalez N. & Dominguez L. 2013. The effect of different isolation protocols on detection and molecular characterization of Campylobacter from poultry. Lett Appl Microbiol, 57, 427‑443. Vidal A.B., Colles F.M., Rodgers J.D., McCarthy N.D., Davies R.H., Maiden M.C.J. & Clifton‑Hadley F.A. 2016. Genetic diversity of Campylobacter jejuni and Campylobacter coli isolates from conventional broiler flocks and the impacts of sampling strategy and laboratory method. Appl Environ Microbiol, 82, 2347‑2355. Wang G., Clark C.G., Taylor T.M., Pucknell C., Barton C., Price L., Woodward D.L. & Rodgers F.G. 2002. Colony multiplex PCR assay for identification and differentiation of Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus subsp. fetus. J Clin Microbiol, 40, 4744‑4747. Whelan C.D., Monaghan P., Girdwood R.W. & Fricker C.R. 1988. The significance of wild birds (Larus sp.) in the epidemiology of Campylobacter infections in humans. Epidemiol Infect, 101, 259‑267. Whiley H., van den Akker B., Giglio S. & Bentham R. 2013. The role of environmental reservoirs in human campylobacteriosis. Int J Environ Res Public Health, 10, 5886‑5907. Wilson D.J., Gabriel E., Leatherbarrow A.J.H., Cheesbrough J. & Gee S. 2008. Tracing the source of campylobacteriosis. PLoS Genet, 4 (9), e1000203. of Campylobacter on poultry farms and in their direct environment. J Water Health, 15, 849‑862. Sheppard S.K., Dallas J.F., Strachan N.J., MacRae M., McCarthy N.D., Wilson D.J., Gormley F.J., Falush D., Ogden I.D., Maiden M.C. & Forbes K.J. 2009. Campylobacter genotyping to determine the source of human infection. Clin Infect Dis, 48, 1072‑1078. Silva D.T., Tejada T.S., Blum‑Menezes D., Dias P.A. & Timm C.D. 2016. Campylobacter species isolated from poultry and humans, and their analysis using PFGE in southern Brazil. Intl J Food Microbiol, 217, 189‑194. Skirrow M.B. & Blaser M.J. 2000. Clinical aspects of Campylobacter infection. In Campylobacter (Nachamkin I. and Blaser M.J., eds). 2nd Edition. ASM Press, Washington, 69‑88. Stern N.J. & Robach M.C. 2003. Enumeration of Campylobacter spp. in broiler feces and in corresponding processed carcasses. J Food Prot, 66, 1557‑1563. Thakur S., Brake J., Keelara S., Zou M. & Susick E. 2013. Farm and environmental distribution of Campylobacter and Salmonella in broiler flocks. Res Vet Sci, 94, 33‑42. Thomas C., Gibson H., Hill D.J. & Mabey M.J. 1998. Campylobacter epidemiology: an aquatic perspective. Appl Microbiol, 85, 168S‑177S. Ugarte‑Ruiz M., Wassenaar T.M., Gomez‑Barrero S., Porrero