Curr Microbiol DOI 10.1007/s00284-014-0768-4
Effects of Fibrinopurulent Polyserositis in Broilers on Postharvest Microbiological Parameters Relevant to Public Health of Broiler Meat Wiebke Jansen • Felix Reich • Gu¨nter Klein
Received: 6 October 2014 / Accepted: 25 November 2014 Ó Springer Science+Business Media New York 2014
Abstract Fibrinopurulent polyserositis is of utmost importance in commercial broiler production worldwide. This multifactorial endemic disease is marked by severe clinical alterations post-mortem, yet its effects on food safety and processing hygiene criteria remain unclear. Current considerations presume that bacteraemia lead to meat being unfit for consumption. In the present study, we evaluated some microbiological criteria of affected broiler carcasses in comparison to unaffected control broiler carcasses. The results thereof indicated that the lesions did not result in higher bacterial counts or in an increased percentage of contaminated meat. The carry-over of associated zoonotic pathogens into the food chain seems to be not more prevalent in birds affected with non-systemic affections of polyserositis.
Introduction Endemic fibrinopurulent polyserositis (FP) in broilers is known as a final outcome of a multifactorial aetiology by predisposing non-infectious factors and infectious agents. The primary aetiological agents include Mycoplasma spp., Avibacterium (Haemophilus) paragallinarum, Ornithobacterium rhinotracheale and very frequently avian pathogenic Escherichia coli (APEC) [2]. Broilers that are exposed to faecal-contaminated dust are more susceptible of bacteria multiplying in the upper respiratory tract, and colonise the air sacs and the lungs. Followed by defined
W. Jansen F. Reich G. Klein (&) Institute of Food Quality and Food Safety, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, 30173 Hannover, Germany e-mail: [email protected]
exudations of serum, fibrin and inflammatory cells such as neutrophil granulocytes, these alterations can be found covering the intestine, air sacs, heart and liver [2, 26]. The terminology is inconsistent, similar post-mortem lesions in broilers at meat inspection are described as airsacculitis, aerosacculitis, salpingitis, peritonitis, pericarditis and perihepatitis. Due to the predominant influence of E. coli, these symptoms are commonly related to the term ‘‘colibacilliosis’’. This complex disease is of great importance in commercial broiler rearing but also for layer hens and turkey rearing [15]. The potentially zoonotic aetiology of E. coli and coagulase-positive staphylococci is widely known, yet scientific investigations on public health consequences and the carry-over effect in the food chain are scarce. To date, little is known about the occurrence and impact of indicator and pathogenic bacteria in the processed broiler meat of broilers affected by FP. It needs to be elucidated whether, and to which degree, FP and the associated zoonotic pathogens such as E. coli and coagulase-positive staphylococci harbour possible effects for public health. The study was performed to identify if bacteria spread to the meat of birds affected with FP, and that does not show obvious signs of systemic infection and if such meat would be acceptable for human consumption in comparison to established microbiological parameters for broiler meat. In this study, broilers affected with FP were tested for the percentage and mean counts of the aerobic growing bacteria (aerobic colony count, ACC) and Enterobacteriaceae in the meat of affected birds and of healthy control birds of the same flocks after slaughter. Additionally, E. coli and coagulase-positive staphylococci were assessed in the meat as indicator agents for the presence of bacteraemia associated with FP and posing a potential foodborne hazard. Affected giblets were examined exclusively for these two organisms.
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W. Jansen et al.: Post-harvest Microbiological Parameter of Broiler Meat
Materials and Methods Sampling Six conventional broiler flocks from six different integrated rearing farms were selected due to their history for fibrinopurulent polyserositis between June 2011 and November 2012. The flocks represented broilers, which were reared under conventional all in all out conditions, caught and delivered to the abattoir and processed in one batch each. These flocks at the abattoir were each classified as a single epidemiological unit. All flocks were slaughtered and processed in the same EU-licenced abattoir in Northern Germany. Flock sizes were around 40,000 birds. From each flock, 12 affected broilers and their corresponding giblets were selected due to similar pathomorphological alterations. Affected broilers and giblets fulfilled the selection criteria if they had alterations as follows: fibrinopurulent plaques larger than 1 cm in diameter, and visible, acute inflammatory serosal lesions and lesions in the body cavity. These criteria were the requirements for sample collection set by the slaughterhouse quality control in agreement with the local veterinarian meat control officials. Broilers showing signs of generalisation and chronic pericarditis, perihepatitis/hepatitis or septicaemia were not taken into account, because these signs already demand condemnation in accordance with Regulation EC 854/2004 [1]. The samples were chosen with the assistance of the local veterinary officials out of the processing line during the routine meat inspection. Each two non-affected healthy broilers without signs of lesions per flock were selected as control. Consequently, 12 affected carcasses, the 12 corresponding giblets and 2 control carcasses were examined per flock. This resulted for six flocks in 71 examined carcasses, 72 examined giblets and 12 control carcasses in total. Carcasses and corresponding giblets of affected birds were collected during the whole slaughter period representatively for each batch. The giblets were collected individually in labelled, sterile plastic bags and chilled immediately at 4 °C. Carcasses were tagged with coloured straps, labelled like corresponding giblets and put back in the processing line. The tagged carcasses were identified at the end of the processing line post-chilling but prior to cutting, where they were considered market ready broiler meat. They were stored in labelled, sterile plastic bags at 4 °C until further examination in the laboratory within 24–48 h. One carcass could not be identified after chilling, and a second carcass was identified after cutting, with legs already missing, and so one whole carcass and one leg sample could not be analysed. Sample Preparation Four different portions of each carcass were evaluated. Meat samples were taken under aseptic conditions from the breast,
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leg, wing and carcass back, hereafter called back. Sample preparation was performed according to ISO 6887-2:2003 [12]. The surface of each broiler part was decontaminated by heat treatment until surface layers were denatured to a depth of about 2 mm. Samples of 10 g each were collected from the depth of the muscles. The four meat samples per carcass were enumerated for the aerobic colony count and Enterobacteriaceae representing general microbiological criteria. E. coli and coagulase-positive staphylococci were quantified representing indicator organisms associated with pathological affections generally found in broilers at slaughter and with relevance for food hygiene, in consultation with the clinic for poultry at the University of Veterinary Medicine, Foundation in Hannover, Germany (Glu¨nder, personal communication). From the corresponding giblets, the affected serosa and fibrinopurulent plaques were sampled and assessed for E. coli and coagulase-positive staphylococci counts. For six batches, this amounted to 283 meat samples and 72 giblet samples from affected broilers. Each two broiler carcasses from the same batch were estimated as being unaffected, and consequently a total of 48 control meat samples were analysed. Sample preparation was performed according to ISO 6887-2:2003 [12] with an initial dilution of 1:10 in diluent containing 0.85 % NaCl and 0.1 % peptone (Oxoid, Wesel, Germany) and 120 s homogenisation in a StomacherÒ 400 Circulator (Seward, Worthing, West Sussex, United Kingdom). Subsequent decadic dilutions steps followed according to ISO. The aerobic colony count was performed according to ISO 4833:2003 [13] on plate count agar (Oxoid) incubated aerobically for 72 ± 3 h at 30 ± 0.5 °C with an enumeration limit of 5.0 9 100 cfu/g. Enterobacteriaceae were analysed according to ISO 21528-2:2004 [14] on VRBD-agar (Oxoid), being incubated aerobically for 24 ± 2 h at 37 ± 0.5 °C with an enumeration limit of 5.0 9 100 cfu/g. E. coli was enumerated according to ISO 16649-2:2001 [11] on TBX-agar (VWR, Darmstadt, Germany) and incubated aerobically for 24 ± 2 h at 44 ± 0.5 °C, the limit of enumeration being 5.0 9 100 cfu/g. Coagulase-positive staphylococci were enumerated according to ISO 6888-1:1999 [10] on Baird Parker-agar (Oxoid) incubated aerobically for 48 ± 2 h at 37 ± 0.5 °C, with an enumeration limit of 5.0 9 101 cfu/g. Statistics The results of the affected broilers were compared to the control group in order to detect statistical differences. The analysis and calculations were divided into quantitative data for aerobic colony counts and the percentage values of the presence/absence for all assessed bacterial groups. Quantitative data of the aerobic colony count were tested for differences by means of the Mann–Whitney-U-test for independent samples. The percentage values of the presence versus absence of quantifiable bacteria between the
W. Jansen et al.: Post-harvest Microbiological Parameter of Broiler Meat
(Table 4). Countable E. coli was found in 8 wing and 12 back (each a total of n = 47) samples and in two leg samples from the affected group (total n = 46), while one wing sample and 4 back samples of the control group (total n = 12) tested positive (Table 2). The counts ranged between 0.7 and 3.6 log10 cfu/g of meat. Most samples ([80 %) yielded mean counts below the DGHM guiding limit of 2.7 log10 cfu/g. The higher values were found in the back samples of the affected group (Table 2). Samples positive for E. coli were found in both the groups affected and unaffected in batches 1–3, while in batches 4–6, only affected birds yielded countable E. coli (Table 4). The guiding limit was exceeded in one back sample from affected birds of batch 1 and batch 4 and in one back sample of batch 3 from unaffected birds, respectively. The warning limit was not exceeded in any sample (Table 5). Coagulase-positive staphylococci were found in 7 (2.5 %) meat samples of the affected broilers and in one (2.1 %) of the control group (Table 4). Single counts ranged between 1.7 and 3.2 log10 cfu/g meat. The counts were below the DGHM guiding limit of 2.7 log10 cfu/g, except for one wing sample and one leg sample from the affected group. Regarding the fibrinopurulent plaques of the affected broilers, E. coli was found in 95.8 % of the samples and coagulase-positive staphylococci in 25.0 % of the samples (n = 72). In addition, counts were higher for E. coli than for staphylococci (Table 3). The detection rates for the analysed microbiological criteria concerning all six broiler batches tested did not show statistically significant differences between the meat of affected broilers and the control group (Table 4).
two groups were tested for significance with the Chi square test for aerobic colony count, while Fishers’ exact test was performed on the data of E. coli, coagulase-positive staphylococci and Enterobacteriaceae. Tests were calculated with SAS software version 9.13 (Statistical Analysis Systems, Cary, NC, USA).
Results In total, 283 meat samples and the corresponding 72 fibrinopurulent plaque samples, altered serosa samples and 48 control meat samples were examined from six flocks. The results were evaluated according to the guiding and warning limits of the German Association for Hygiene and Microbiology (DGHM) for raw poultry meat. The DGHM defined microbiological criteria for fresh poultry meat which are summarised in Table 1. Hygiene Criteria Meat samples of the affected broilers yielded aerobic colony counts above the limit of enumeration in 57.7 % cases compared to 64.6 % cases in the control group (Table 4). Back samples were the most often contaminated samples, followed by wings, legs and breasts in descending order. No sample exceeded the guiding limit of 6.7 log10 cfu/g according to DGHM, yet counts up to 4.1/4.2 log10 cfu/g were detected for backs and wings (Table 2). There were neither statistically significant differences between the aerobic colony counts of affected broilers compared to the control (Table 2) nor for the percentage of detection (Table 4). Enterobacteriaceae were found in 9.9 % (mean count 1.6 log10 cfu/g, range 0.7–3.9) of the meat samples of the affected broilers and in 8.3 % (mean count 2.3 log10 cfu/g, range 0.7–3.3) of the control group (Table 4). The detection rate was higher in wings and backs.
Discussion This study focused on microbiological findings of the meat of processed broiler carcasses and corresponding affected giblets in six broiler flocks with endemic FP. Endemic FP has a multi-pathogenic aetiology, including zoonotic agents like E. coli and coagulase-positive staphylococci. The inflammatory process, however, depends on the host, its immunological status, presence and type of pathogens and
Indicator Bacteria In the meat samples of the affected broilers, E. coli was found in 7.8 % of these and in 10.4 % of the control birds
Table 1 Microbiological criteria for fresh poultry meat according to DGHM (2012) Guiding limita cfu/g (log10)
Warning limitb cfu/g (log10)
Aerobic colony count
5.0 9 106 (6.7)
No value defined
E. coli
5.0 9 102 (2.7)
5.0 9 103 (3.7)
Enterobacteriaceae
1.0 9 10 (4.0)
1.0 9 105 (5.0)
Coagulase-positive staphylococci
5.0 9 102 (2.7)
5.0 9 103 (3.7)
a
Guiding limit: borderline, equivalent to ‘‘m’’
b
Warning limit: unsatisfactory, equivalent to ‘‘M’’
4
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P-values for statistical comparison of ACC counts of the different broiler meat parts from affected and not affected birds: breast P = 0.94; leg P = 0.14; wing P = 0.40; back P = 0.12
n.d. not detected
a
n.d. 2.0 (n = 1) n.d. n.d. Control
1.6 (1.2; 0.7–3.1; n = 4)
1.5 (1.0; 0.7–3.6; n = 12)
1.7 (n = 1) 2.3 (0.6; 1.7–3.0; n = 5) 3.2 (n = 1) n.d. Affected Coagulase-positive staphylococci
2.1 (n = 1)
1.1 (0.4; 0.7–1.7; n = 8) 2.1 (0.8; 1.5–2.6; n = 2)
n.d.
n.d.
n.d.
Affected
Control
E. coli
2.4 (1.4; 0.7–3.3; n = 3) 2.0 (n = 1) n.d. n.d. Control
1.7 (1.1: 0.7–4.0; n = 11)
1.5 (0.9; 0.7–3.9; n = 17) 2.4 (1.1; 1.6–3.2; n = 2) 1.5 (0.5; 0.7–2.2; n = 9) n.d. Affected Enterobacteriaceae
1.9 (0.81; 0.7–4.1; n = 57) 1.7 (0.78: 0.7–3.7; n = 50)
2.1 (1.1: 0.7–4.2; n = 8) 1.0 (0.6: 0.7–2.2; n = 6) 1.1 (0.38: 0.7–1.7; n = 6) Control
1.3 (0.70; 0.7–3.6; n = 30) 1.1 (0.49; 0.7–2.9; n = 27) Affected ACCa
Back Wing Leg Breast
Mean log10 cfu/g (SD; range) Bacteria
Table 2 Microbiological counts for hygiene criteria: aerobic colony count (ACC), Enterobacteriaceae and indicator bacteria: E. coli and coagulase-positive staphylococci; mean values and standard deviation (SD) in broiler meat samples of affected and control group
W. Jansen et al.: Post-harvest Microbiological Parameter of Broiler Meat
predisposing factors. Vaccinations, predominant infections, unfavourable nutrition, poor sanitary conditions and poor ventilation can be considered as contributing predisposing factors [25]. These immunosuppressive conditions are triggered by common respiratory viral infections [18]. Those predisposed flocks exposed to other non-infectious and infectious agents are especially susceptible to FP. Leading to severe, visible post-mortem lesions, the meat of affected birds may represent a public health threat. As E. coli is often associated with these lesions, the alterations might harbour considerable zoonotic potential [3, 20]. Enterobacteriaceae and coliform bacteria are common contaminations on broiler backs found up to 100 % in batches at slaughter [7, 24]. Contamination of broiler meat and meat products with other zoonotic Enterobacteriaceae such as Salmonella and Campylobacter may occur inevitably while processing at the abattoir due to faecal shedding [5, 19, 21]. Indicator organisms are therefore a crucial amendment for controlling meat and processing hygiene even when taking only a limited number of samples into account. The sample size allowed for statistical comparison of the quantitative and qualitative results. No statistically significant difference was shown between the two groups. The bacterial counts complied with microbiological standards in both the groups according to DGHM [4]. Internationally accepted limits for aerobic colony counts consider counts below 5.0 9 105 cfu/g for raw broiler meat. In general, these counts are feasible under current conditions in broiler processing and thus considered as satisfactory [8, 9]. In the present study, the detection rate of E. coli in 95.8 % of the plaques in affected chicken is considerably high, supporting previous findings, indicating E. coli as an important primary infectious agent in the aetiological complex of FP [6]. The results therefore indicate that E. coli seems to be associated with the aetiology of FP. Although the giblets were highly contaminated, the detection rates in the meat of the affected broilers in comparison to unaffected control remained alike. This implies a negligible risk of a carry-over effect from the highly contaminated giblets to the meat in the present study, as meat of affected chickens was not contaminated in a significant manner with the predisposing agent E. coli. Additionally, E. coli was found at similar levels in the meat of affected and control samples in the present study, suggesting instead cross-contamination during processing [17, 23]. Further strain analysis might show an epidemiological correlation between infection in the flock and contamination of the meat at the abattoir. Coagulase-positive staphylococci were detected less often in the affected meat samples and the corresponding plaques than E. coli, indicating a minor contribution to the complex aetiology. Yet, the DGHM warning limits for raw broiler meat were
W. Jansen et al.: Post-harvest Microbiological Parameter of Broiler Meat Table 3 Distribution of E. coli and coagulase-positive staphylococci colony count categories in % in fibrinopurulent plaques of broilers with fibrinopurulent polyserositis log10 cfu/g E. coli (n = 71) log10 cfu/g Staphylococci (n = 72)
\1.0 5.6 \1.7 75.0
C1.0 \ 2.0
C2.0 \ 3.0
C3.0 \ 4.0
C4.0 \ 5.0
C5.0
23.9
36.6
18.3
9.9
5.6
C1.7 \ 2.0
C2.0 \ 3.0
C3.0 \ 4.0
C4.0
5.6
15.3
2.8
1.4
Table 4 Detection rates of samples yielding counts for the four different microbiological criteria analysed shown per batch and in total for broilers affected by polyserositis and unaffected controls Group Broilers affected by polyserositis
Control broilers
Microbiological criteria
Batch 1
Batch 2
Batch 3
Batch 4
Batch 5
Totala
Batch 6
n
n = 44
n = 47
n = 48
n = 48
n = 48
n = 48
n = 283
Aerobic colony count (%)
54.5
42.6
50.0
56.3
77.1
66.7
58.0
Enterobacteriaceae (%) E. coli (%)
9.1 11.4
17.0 10.6
12.5 6.3
6.3 6.3
10.4 8.3
4.2 4.2
9.9 7.8
Coagulase-positive staphylococci (%)
2.3
4.3
0.0
2.1
4.2
2.1
2.5
n
n = 12
n = 12
n = 12
n = 12
n = 12
n = 12
n = 48
Aerobic mean counts (%)
37.5
25.0
62.5
87.5
100.0
75.0
64.6
Enterobacteriaceae (%)
0.0
12.5
37.5
0.0
0.0
0.0
8.3
E. coli (%)
12.5
12.5
37.5
0.0
0.0
0.0
10.4
Coagulase-positive staphylococci (%)
0.0
0.0
0.0
0.0
0.0
12.5
2.1
a
P-values of the statistical comparison of affected versus unaffected groups were aerobic colony count P = 0.388; Enterobacteriaceae P = 0.49; E. coli P = 0.35; coagulase-positive staphylococci P = 0.68
Table 5 Percentage of samples with contamination levels falling into the three different categories according to DGHM, (a) below ‘‘m’’, (b) between ‘‘m’’ and ‘‘M’’ and (c) above ‘‘M’’ Below m
Between m and M
Above M
Affected
Control
Affected
Control
Affected
Control
Aerobic colony count
100
100
0
0
No value defined
No value defined
E. coli
99.3
97.9
0.7
2.1
0
0
Enterobacteriaceae
100
100
0
0
0
0
Coagulase-positive staphylococci
99.3
100
0.7
0
0
0
neither exceeded in any meat sample of the affected nor in the control meat samples (Table 5). These findings indicate that current handling considerations of food business operators comply with microbiological criteria for fresh poultry meat. Additionally, the contamination of raw meat at the abattoir and management measures seem to be appropriate for controlling the risk. The carry-over of the associated zoonotic pathogens from the giblets to the meat and subsequently into the food chain seems to be avoidable. No correlation was found between E. coli counts in meat and the corresponding giblets and the control. However, the fundamental impact of proper processing hygiene to reduce the likelihood of cross-contamination should not be underestimated (Table 5).
Yet, proper flock management and complementary conditions are therefore crucial to avoid high condemnation rates at abattoir [22]. Prevention strategies avoiding immunosuppression should include adequate temperature, air quality, rodent control and waste management. Complementary nutrition such as prebiotics and probiotics showed promising results [6]. Future approaches could include optional spray disinfection of flocks and permanent supplementation of in-feed and water additives based on organic acids, medium chain fatty acids (MCFA) or electrolysed water during the rearing periods [26]. Future developments might be subunit vaccines, multivalent bacteriocins and bacteriophages as therapeutic agents and preventive measures [16].
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W. Jansen et al.: Post-harvest Microbiological Parameter of Broiler Meat Acknowledgments We thank Dr. Glu¨nder of the Clinic for Poultry at the University of Veterinary Medicine, Foundation, Hannover, Germany for scientific advice.
References 1. Anon (2004) Regulation (EC) No 854/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific rules for the organisation of official controls on products of animal origin intended for human consumption 2. Beckman DB (2006) Current perspectives on peritonitis. In: Midwest Poultry Federation Convention Proceedings Dallas Center, St. Paul, Minnesota, 2006. pp http://www.zootecnicainternational.com/arti cle-archive/veterinary/909-currentperspectives-on-peritonitis.html 3. Cox JM, Pavic A (2010) Advances in enteropathogen control in poultry production. J Appl Microbiol 108(3):745–755. doi:10. 1111/j.1365-2672.2009.04456.x 4. DGHM (2012) Microbiological guiding and warning limits for food stuffs. German Association for Hygiene and Microbiology, Hannover 5. EFSA (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, 2008, Part A: Campylobacter and Salmonella prevalence estimates. EFSA J 8(03):1503 6. Ferreira TZ, Sesterhenn R, Kindlein L (2012) Economic losses of main causes of condemnation of the broiler carcass in a slaughterhouse under federal inspection on Rio Grande do Sul, Brazil. Acta Sci Vet 40(1):1021 7. Huezo R, Northcutt JK, Smith DP, Fletcher DL, Ingram KD (2007) Effect of dry air or immersion chilling on recovery of bacteria from broiler carcasses. J Food Prot 70(8):1829–1834 8. ICMSF (1986) Microorganisms in Foods 2. Sampling for microbiological analysis: Principles and specific applications. 2nd Ed. International Commission on Microbiological Specifications for Foods. Blackwell Scientific, Oxford 9. IFST (1999) Development and use of microbiological criteria for foods. Institute for food safety and technology, London 10. ISO (1999) ISO 6888-1:1999 Microbiology of food and animal feeding stuffs – Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) – Part 1: Technique using Baird-Parker agar medium 11. ISO (2001) ISO 16649-2:2001 Microbiology of food and animal feeding stuffs – Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli – Part 2: Colony-count technique at 44 degrees C using 5-bromo-4-chloro-3-indolyl beta-D-glucuronide 12. ISO (2003) ISO 6887-2:2003 Microbiology of food and animal feeding stuffs – Preparation of test samples, initial suspension and decimal dilutions for microbiological examination – Part 2: Specific rules for the preparation of meat and meat products 13. ISO (2003) ISO 4833-1:2003 Microbiology of food and animal feeding stuffs – Horizontal method for the enumeration of microorganisms – Colony-count technique at 30 degrees C
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14. ISO (2004) ISO 21528-2:2004 Microbiology of food and animal feeding stuffs – Horizontal methods for the detection and enumeration of Enterobacteriaceae – Part 2: Colony-count method 15. Kabir SML (2010) Avian Colibacillosis and Salmonellosis: a closer look at epidemiology, pathogenesis, diagnosis, control and public health concerns. Int J Environ Res Public Health 7(1):89–114. doi:10.3390/ijerph7010089 16. Kittler S, Fischer S, Abdulmawjood A, Glu¨nder G, Klein G (2013) Effect of bacteriophage application on Campylobacter jejuni loads in commercial broiler flocks. Appl Environ Microbiol 79(23):7525–7533 17. Lupo C, Le Bouquin S, Balaine L, Michel V, Peraste J, Petetin I, Colin P, Chauvin C (2009) Feasibility of screening broiler chicken flocks for risk markers as an aid for meat inspection. Epidemiol Infect 137(8):1086–1098. doi:10.1017/s095026880900209x 18. Marien M (2007) Mixed respiratory infections in turkeys, with emphasis on avian metapneumovirus, Ornithobacterium rhinotracheale, Escherichia coli and Mycoplasma gallisepticum. University of Ghent, Ghent 19. Milnes AS, Sayers AR, Stewart I, Clifton-Hadley FA, Davies RH, Newell DG, Cook AJC, Evans SJ, Smith RP, Paiba GA (2009) Factors related to the carriage of Verocytotoxigenic E. coli, Salmonella, thermophilic Campylobacter and Yersinia enterocolitica in cattle, sheep and pigs at slaughter. Epidemiol Infect 137(8):1135–1148. doi:10.1017/s095026880900199x 20. Reich F, Atanassova V, Klein G (2013) Extended-spectrum betalactamase- and AmpC-producing enterobacteria in healthy broiler chickens, Germany. Emerg Infect Dis 19(8):1253–1259. doi:10. 3201/eid1908.120879 21. Reich F, Atanassova V, Haunhorst E, Klein G (2008) The effects of Campylobacter numbers in caeca on the contamination of broiler carcasses with Campylobacter. Int J Food Microbiol 127(1–2):116–120 22. Shane SM (2009) Reducing pathogenic E. coli infection by vaccination. World Poult 25(5):21–23 23. Silva MCA, Melo DB, Costa WLR, Fernandes LMB, Pinna MH, Vieira Neto J (2011) Macroscopic and microscopic aspects of airsacculitis in slaughtered broilers in Brazil. In: Animal hygiene and sustainable livestock production. Proceedings of the XVth International Congress of the International Society for Animal Hygiene, pp. 603–605 Vienna, Austria. Tribun EU, pp 603–605 24. Smith DP, Cason JA, Berrang ME (2005) Effect of fecal contamination and cross-contamination on numbers of coliform, Escherichia coli, Campylobacter, and Salmonella on immersionchilled broiler carcasses. J Food Prot 68(7):1340–1345 25. Tessier M, Fredette MA, Beauchamp G, Boulianne M (2001) Cellulitis in broiler chickens: a one-year retrospective study in four Quebec abattoirs. Avian Dis 45(1):191–194. doi:10.2307/ 1593027 26. USDA (2012) E. coli peritonitis on breeder chicken farms in the United States, a study of US breeder-chicken farms in 2010. National Animal Health Monitoring System (NAHMS), Veterinary Service Washington
Effects of Fibrinopurulent Polyserositis in Broilers on Postharvest Microbiological Parameters Relevant to Public Health of Broiler Meat Wiebke Jansen • Felix Reich • Gu¨nter Klein
Received: 6 October 2014 / Accepted: 25 November 2014 Ó Springer Science+Business Media New York 2014
Abstract Fibrinopurulent polyserositis is of utmost importance in commercial broiler production worldwide. This multifactorial endemic disease is marked by severe clinical alterations post-mortem, yet its effects on food safety and processing hygiene criteria remain unclear. Current considerations presume that bacteraemia lead to meat being unfit for consumption. In the present study, we evaluated some microbiological criteria of affected broiler carcasses in comparison to unaffected control broiler carcasses. The results thereof indicated that the lesions did not result in higher bacterial counts or in an increased percentage of contaminated meat. The carry-over of associated zoonotic pathogens into the food chain seems to be not more prevalent in birds affected with non-systemic affections of polyserositis.
Introduction Endemic fibrinopurulent polyserositis (FP) in broilers is known as a final outcome of a multifactorial aetiology by predisposing non-infectious factors and infectious agents. The primary aetiological agents include Mycoplasma spp., Avibacterium (Haemophilus) paragallinarum, Ornithobacterium rhinotracheale and very frequently avian pathogenic Escherichia coli (APEC) [2]. Broilers that are exposed to faecal-contaminated dust are more susceptible of bacteria multiplying in the upper respiratory tract, and colonise the air sacs and the lungs. Followed by defined
W. Jansen F. Reich G. Klein (&) Institute of Food Quality and Food Safety, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, 30173 Hannover, Germany e-mail: [email protected]
exudations of serum, fibrin and inflammatory cells such as neutrophil granulocytes, these alterations can be found covering the intestine, air sacs, heart and liver [2, 26]. The terminology is inconsistent, similar post-mortem lesions in broilers at meat inspection are described as airsacculitis, aerosacculitis, salpingitis, peritonitis, pericarditis and perihepatitis. Due to the predominant influence of E. coli, these symptoms are commonly related to the term ‘‘colibacilliosis’’. This complex disease is of great importance in commercial broiler rearing but also for layer hens and turkey rearing [15]. The potentially zoonotic aetiology of E. coli and coagulase-positive staphylococci is widely known, yet scientific investigations on public health consequences and the carry-over effect in the food chain are scarce. To date, little is known about the occurrence and impact of indicator and pathogenic bacteria in the processed broiler meat of broilers affected by FP. It needs to be elucidated whether, and to which degree, FP and the associated zoonotic pathogens such as E. coli and coagulase-positive staphylococci harbour possible effects for public health. The study was performed to identify if bacteria spread to the meat of birds affected with FP, and that does not show obvious signs of systemic infection and if such meat would be acceptable for human consumption in comparison to established microbiological parameters for broiler meat. In this study, broilers affected with FP were tested for the percentage and mean counts of the aerobic growing bacteria (aerobic colony count, ACC) and Enterobacteriaceae in the meat of affected birds and of healthy control birds of the same flocks after slaughter. Additionally, E. coli and coagulase-positive staphylococci were assessed in the meat as indicator agents for the presence of bacteraemia associated with FP and posing a potential foodborne hazard. Affected giblets were examined exclusively for these two organisms.
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Materials and Methods Sampling Six conventional broiler flocks from six different integrated rearing farms were selected due to their history for fibrinopurulent polyserositis between June 2011 and November 2012. The flocks represented broilers, which were reared under conventional all in all out conditions, caught and delivered to the abattoir and processed in one batch each. These flocks at the abattoir were each classified as a single epidemiological unit. All flocks were slaughtered and processed in the same EU-licenced abattoir in Northern Germany. Flock sizes were around 40,000 birds. From each flock, 12 affected broilers and their corresponding giblets were selected due to similar pathomorphological alterations. Affected broilers and giblets fulfilled the selection criteria if they had alterations as follows: fibrinopurulent plaques larger than 1 cm in diameter, and visible, acute inflammatory serosal lesions and lesions in the body cavity. These criteria were the requirements for sample collection set by the slaughterhouse quality control in agreement with the local veterinarian meat control officials. Broilers showing signs of generalisation and chronic pericarditis, perihepatitis/hepatitis or septicaemia were not taken into account, because these signs already demand condemnation in accordance with Regulation EC 854/2004 [1]. The samples were chosen with the assistance of the local veterinary officials out of the processing line during the routine meat inspection. Each two non-affected healthy broilers without signs of lesions per flock were selected as control. Consequently, 12 affected carcasses, the 12 corresponding giblets and 2 control carcasses were examined per flock. This resulted for six flocks in 71 examined carcasses, 72 examined giblets and 12 control carcasses in total. Carcasses and corresponding giblets of affected birds were collected during the whole slaughter period representatively for each batch. The giblets were collected individually in labelled, sterile plastic bags and chilled immediately at 4 °C. Carcasses were tagged with coloured straps, labelled like corresponding giblets and put back in the processing line. The tagged carcasses were identified at the end of the processing line post-chilling but prior to cutting, where they were considered market ready broiler meat. They were stored in labelled, sterile plastic bags at 4 °C until further examination in the laboratory within 24–48 h. One carcass could not be identified after chilling, and a second carcass was identified after cutting, with legs already missing, and so one whole carcass and one leg sample could not be analysed. Sample Preparation Four different portions of each carcass were evaluated. Meat samples were taken under aseptic conditions from the breast,
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leg, wing and carcass back, hereafter called back. Sample preparation was performed according to ISO 6887-2:2003 [12]. The surface of each broiler part was decontaminated by heat treatment until surface layers were denatured to a depth of about 2 mm. Samples of 10 g each were collected from the depth of the muscles. The four meat samples per carcass were enumerated for the aerobic colony count and Enterobacteriaceae representing general microbiological criteria. E. coli and coagulase-positive staphylococci were quantified representing indicator organisms associated with pathological affections generally found in broilers at slaughter and with relevance for food hygiene, in consultation with the clinic for poultry at the University of Veterinary Medicine, Foundation in Hannover, Germany (Glu¨nder, personal communication). From the corresponding giblets, the affected serosa and fibrinopurulent plaques were sampled and assessed for E. coli and coagulase-positive staphylococci counts. For six batches, this amounted to 283 meat samples and 72 giblet samples from affected broilers. Each two broiler carcasses from the same batch were estimated as being unaffected, and consequently a total of 48 control meat samples were analysed. Sample preparation was performed according to ISO 6887-2:2003 [12] with an initial dilution of 1:10 in diluent containing 0.85 % NaCl and 0.1 % peptone (Oxoid, Wesel, Germany) and 120 s homogenisation in a StomacherÒ 400 Circulator (Seward, Worthing, West Sussex, United Kingdom). Subsequent decadic dilutions steps followed according to ISO. The aerobic colony count was performed according to ISO 4833:2003 [13] on plate count agar (Oxoid) incubated aerobically for 72 ± 3 h at 30 ± 0.5 °C with an enumeration limit of 5.0 9 100 cfu/g. Enterobacteriaceae were analysed according to ISO 21528-2:2004 [14] on VRBD-agar (Oxoid), being incubated aerobically for 24 ± 2 h at 37 ± 0.5 °C with an enumeration limit of 5.0 9 100 cfu/g. E. coli was enumerated according to ISO 16649-2:2001 [11] on TBX-agar (VWR, Darmstadt, Germany) and incubated aerobically for 24 ± 2 h at 44 ± 0.5 °C, the limit of enumeration being 5.0 9 100 cfu/g. Coagulase-positive staphylococci were enumerated according to ISO 6888-1:1999 [10] on Baird Parker-agar (Oxoid) incubated aerobically for 48 ± 2 h at 37 ± 0.5 °C, with an enumeration limit of 5.0 9 101 cfu/g. Statistics The results of the affected broilers were compared to the control group in order to detect statistical differences. The analysis and calculations were divided into quantitative data for aerobic colony counts and the percentage values of the presence/absence for all assessed bacterial groups. Quantitative data of the aerobic colony count were tested for differences by means of the Mann–Whitney-U-test for independent samples. The percentage values of the presence versus absence of quantifiable bacteria between the
W. Jansen et al.: Post-harvest Microbiological Parameter of Broiler Meat
(Table 4). Countable E. coli was found in 8 wing and 12 back (each a total of n = 47) samples and in two leg samples from the affected group (total n = 46), while one wing sample and 4 back samples of the control group (total n = 12) tested positive (Table 2). The counts ranged between 0.7 and 3.6 log10 cfu/g of meat. Most samples ([80 %) yielded mean counts below the DGHM guiding limit of 2.7 log10 cfu/g. The higher values were found in the back samples of the affected group (Table 2). Samples positive for E. coli were found in both the groups affected and unaffected in batches 1–3, while in batches 4–6, only affected birds yielded countable E. coli (Table 4). The guiding limit was exceeded in one back sample from affected birds of batch 1 and batch 4 and in one back sample of batch 3 from unaffected birds, respectively. The warning limit was not exceeded in any sample (Table 5). Coagulase-positive staphylococci were found in 7 (2.5 %) meat samples of the affected broilers and in one (2.1 %) of the control group (Table 4). Single counts ranged between 1.7 and 3.2 log10 cfu/g meat. The counts were below the DGHM guiding limit of 2.7 log10 cfu/g, except for one wing sample and one leg sample from the affected group. Regarding the fibrinopurulent plaques of the affected broilers, E. coli was found in 95.8 % of the samples and coagulase-positive staphylococci in 25.0 % of the samples (n = 72). In addition, counts were higher for E. coli than for staphylococci (Table 3). The detection rates for the analysed microbiological criteria concerning all six broiler batches tested did not show statistically significant differences between the meat of affected broilers and the control group (Table 4).
two groups were tested for significance with the Chi square test for aerobic colony count, while Fishers’ exact test was performed on the data of E. coli, coagulase-positive staphylococci and Enterobacteriaceae. Tests were calculated with SAS software version 9.13 (Statistical Analysis Systems, Cary, NC, USA).
Results In total, 283 meat samples and the corresponding 72 fibrinopurulent plaque samples, altered serosa samples and 48 control meat samples were examined from six flocks. The results were evaluated according to the guiding and warning limits of the German Association for Hygiene and Microbiology (DGHM) for raw poultry meat. The DGHM defined microbiological criteria for fresh poultry meat which are summarised in Table 1. Hygiene Criteria Meat samples of the affected broilers yielded aerobic colony counts above the limit of enumeration in 57.7 % cases compared to 64.6 % cases in the control group (Table 4). Back samples were the most often contaminated samples, followed by wings, legs and breasts in descending order. No sample exceeded the guiding limit of 6.7 log10 cfu/g according to DGHM, yet counts up to 4.1/4.2 log10 cfu/g were detected for backs and wings (Table 2). There were neither statistically significant differences between the aerobic colony counts of affected broilers compared to the control (Table 2) nor for the percentage of detection (Table 4). Enterobacteriaceae were found in 9.9 % (mean count 1.6 log10 cfu/g, range 0.7–3.9) of the meat samples of the affected broilers and in 8.3 % (mean count 2.3 log10 cfu/g, range 0.7–3.3) of the control group (Table 4). The detection rate was higher in wings and backs.
Discussion This study focused on microbiological findings of the meat of processed broiler carcasses and corresponding affected giblets in six broiler flocks with endemic FP. Endemic FP has a multi-pathogenic aetiology, including zoonotic agents like E. coli and coagulase-positive staphylococci. The inflammatory process, however, depends on the host, its immunological status, presence and type of pathogens and
Indicator Bacteria In the meat samples of the affected broilers, E. coli was found in 7.8 % of these and in 10.4 % of the control birds
Table 1 Microbiological criteria for fresh poultry meat according to DGHM (2012) Guiding limita cfu/g (log10)
Warning limitb cfu/g (log10)
Aerobic colony count
5.0 9 106 (6.7)
No value defined
E. coli
5.0 9 102 (2.7)
5.0 9 103 (3.7)
Enterobacteriaceae
1.0 9 10 (4.0)
1.0 9 105 (5.0)
Coagulase-positive staphylococci
5.0 9 102 (2.7)
5.0 9 103 (3.7)
a
Guiding limit: borderline, equivalent to ‘‘m’’
b
Warning limit: unsatisfactory, equivalent to ‘‘M’’
4
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P-values for statistical comparison of ACC counts of the different broiler meat parts from affected and not affected birds: breast P = 0.94; leg P = 0.14; wing P = 0.40; back P = 0.12
n.d. not detected
a
n.d. 2.0 (n = 1) n.d. n.d. Control
1.6 (1.2; 0.7–3.1; n = 4)
1.5 (1.0; 0.7–3.6; n = 12)
1.7 (n = 1) 2.3 (0.6; 1.7–3.0; n = 5) 3.2 (n = 1) n.d. Affected Coagulase-positive staphylococci
2.1 (n = 1)
1.1 (0.4; 0.7–1.7; n = 8) 2.1 (0.8; 1.5–2.6; n = 2)
n.d.
n.d.
n.d.
Affected
Control
E. coli
2.4 (1.4; 0.7–3.3; n = 3) 2.0 (n = 1) n.d. n.d. Control
1.7 (1.1: 0.7–4.0; n = 11)
1.5 (0.9; 0.7–3.9; n = 17) 2.4 (1.1; 1.6–3.2; n = 2) 1.5 (0.5; 0.7–2.2; n = 9) n.d. Affected Enterobacteriaceae
1.9 (0.81; 0.7–4.1; n = 57) 1.7 (0.78: 0.7–3.7; n = 50)
2.1 (1.1: 0.7–4.2; n = 8) 1.0 (0.6: 0.7–2.2; n = 6) 1.1 (0.38: 0.7–1.7; n = 6) Control
1.3 (0.70; 0.7–3.6; n = 30) 1.1 (0.49; 0.7–2.9; n = 27) Affected ACCa
Back Wing Leg Breast
Mean log10 cfu/g (SD; range) Bacteria
Table 2 Microbiological counts for hygiene criteria: aerobic colony count (ACC), Enterobacteriaceae and indicator bacteria: E. coli and coagulase-positive staphylococci; mean values and standard deviation (SD) in broiler meat samples of affected and control group
W. Jansen et al.: Post-harvest Microbiological Parameter of Broiler Meat
predisposing factors. Vaccinations, predominant infections, unfavourable nutrition, poor sanitary conditions and poor ventilation can be considered as contributing predisposing factors [25]. These immunosuppressive conditions are triggered by common respiratory viral infections [18]. Those predisposed flocks exposed to other non-infectious and infectious agents are especially susceptible to FP. Leading to severe, visible post-mortem lesions, the meat of affected birds may represent a public health threat. As E. coli is often associated with these lesions, the alterations might harbour considerable zoonotic potential [3, 20]. Enterobacteriaceae and coliform bacteria are common contaminations on broiler backs found up to 100 % in batches at slaughter [7, 24]. Contamination of broiler meat and meat products with other zoonotic Enterobacteriaceae such as Salmonella and Campylobacter may occur inevitably while processing at the abattoir due to faecal shedding [5, 19, 21]. Indicator organisms are therefore a crucial amendment for controlling meat and processing hygiene even when taking only a limited number of samples into account. The sample size allowed for statistical comparison of the quantitative and qualitative results. No statistically significant difference was shown between the two groups. The bacterial counts complied with microbiological standards in both the groups according to DGHM [4]. Internationally accepted limits for aerobic colony counts consider counts below 5.0 9 105 cfu/g for raw broiler meat. In general, these counts are feasible under current conditions in broiler processing and thus considered as satisfactory [8, 9]. In the present study, the detection rate of E. coli in 95.8 % of the plaques in affected chicken is considerably high, supporting previous findings, indicating E. coli as an important primary infectious agent in the aetiological complex of FP [6]. The results therefore indicate that E. coli seems to be associated with the aetiology of FP. Although the giblets were highly contaminated, the detection rates in the meat of the affected broilers in comparison to unaffected control remained alike. This implies a negligible risk of a carry-over effect from the highly contaminated giblets to the meat in the present study, as meat of affected chickens was not contaminated in a significant manner with the predisposing agent E. coli. Additionally, E. coli was found at similar levels in the meat of affected and control samples in the present study, suggesting instead cross-contamination during processing [17, 23]. Further strain analysis might show an epidemiological correlation between infection in the flock and contamination of the meat at the abattoir. Coagulase-positive staphylococci were detected less often in the affected meat samples and the corresponding plaques than E. coli, indicating a minor contribution to the complex aetiology. Yet, the DGHM warning limits for raw broiler meat were
W. Jansen et al.: Post-harvest Microbiological Parameter of Broiler Meat Table 3 Distribution of E. coli and coagulase-positive staphylococci colony count categories in % in fibrinopurulent plaques of broilers with fibrinopurulent polyserositis log10 cfu/g E. coli (n = 71) log10 cfu/g Staphylococci (n = 72)
\1.0 5.6 \1.7 75.0
C1.0 \ 2.0
C2.0 \ 3.0
C3.0 \ 4.0
C4.0 \ 5.0
C5.0
23.9
36.6
18.3
9.9
5.6
C1.7 \ 2.0
C2.0 \ 3.0
C3.0 \ 4.0
C4.0
5.6
15.3
2.8
1.4
Table 4 Detection rates of samples yielding counts for the four different microbiological criteria analysed shown per batch and in total for broilers affected by polyserositis and unaffected controls Group Broilers affected by polyserositis
Control broilers
Microbiological criteria
Batch 1
Batch 2
Batch 3
Batch 4
Batch 5
Totala
Batch 6
n
n = 44
n = 47
n = 48
n = 48
n = 48
n = 48
n = 283
Aerobic colony count (%)
54.5
42.6
50.0
56.3
77.1
66.7
58.0
Enterobacteriaceae (%) E. coli (%)
9.1 11.4
17.0 10.6
12.5 6.3
6.3 6.3
10.4 8.3
4.2 4.2
9.9 7.8
Coagulase-positive staphylococci (%)
2.3
4.3
0.0
2.1
4.2
2.1
2.5
n
n = 12
n = 12
n = 12
n = 12
n = 12
n = 12
n = 48
Aerobic mean counts (%)
37.5
25.0
62.5
87.5
100.0
75.0
64.6
Enterobacteriaceae (%)
0.0
12.5
37.5
0.0
0.0
0.0
8.3
E. coli (%)
12.5
12.5
37.5
0.0
0.0
0.0
10.4
Coagulase-positive staphylococci (%)
0.0
0.0
0.0
0.0
0.0
12.5
2.1
a
P-values of the statistical comparison of affected versus unaffected groups were aerobic colony count P = 0.388; Enterobacteriaceae P = 0.49; E. coli P = 0.35; coagulase-positive staphylococci P = 0.68
Table 5 Percentage of samples with contamination levels falling into the three different categories according to DGHM, (a) below ‘‘m’’, (b) between ‘‘m’’ and ‘‘M’’ and (c) above ‘‘M’’ Below m
Between m and M
Above M
Affected
Control
Affected
Control
Affected
Control
Aerobic colony count
100
100
0
0
No value defined
No value defined
E. coli
99.3
97.9
0.7
2.1
0
0
Enterobacteriaceae
100
100
0
0
0
0
Coagulase-positive staphylococci
99.3
100
0.7
0
0
0
neither exceeded in any meat sample of the affected nor in the control meat samples (Table 5). These findings indicate that current handling considerations of food business operators comply with microbiological criteria for fresh poultry meat. Additionally, the contamination of raw meat at the abattoir and management measures seem to be appropriate for controlling the risk. The carry-over of the associated zoonotic pathogens from the giblets to the meat and subsequently into the food chain seems to be avoidable. No correlation was found between E. coli counts in meat and the corresponding giblets and the control. However, the fundamental impact of proper processing hygiene to reduce the likelihood of cross-contamination should not be underestimated (Table 5).
Yet, proper flock management and complementary conditions are therefore crucial to avoid high condemnation rates at abattoir [22]. Prevention strategies avoiding immunosuppression should include adequate temperature, air quality, rodent control and waste management. Complementary nutrition such as prebiotics and probiotics showed promising results [6]. Future approaches could include optional spray disinfection of flocks and permanent supplementation of in-feed and water additives based on organic acids, medium chain fatty acids (MCFA) or electrolysed water during the rearing periods [26]. Future developments might be subunit vaccines, multivalent bacteriocins and bacteriophages as therapeutic agents and preventive measures [16].
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W. Jansen et al.: Post-harvest Microbiological Parameter of Broiler Meat Acknowledgments We thank Dr. Glu¨nder of the Clinic for Poultry at the University of Veterinary Medicine, Foundation, Hannover, Germany for scientific advice.
References 1. Anon (2004) Regulation (EC) No 854/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific rules for the organisation of official controls on products of animal origin intended for human consumption 2. Beckman DB (2006) Current perspectives on peritonitis. In: Midwest Poultry Federation Convention Proceedings Dallas Center, St. Paul, Minnesota, 2006. pp http://www.zootecnicainternational.com/arti cle-archive/veterinary/909-currentperspectives-on-peritonitis.html 3. Cox JM, Pavic A (2010) Advances in enteropathogen control in poultry production. J Appl Microbiol 108(3):745–755. doi:10. 1111/j.1365-2672.2009.04456.x 4. DGHM (2012) Microbiological guiding and warning limits for food stuffs. German Association for Hygiene and Microbiology, Hannover 5. EFSA (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, 2008, Part A: Campylobacter and Salmonella prevalence estimates. EFSA J 8(03):1503 6. Ferreira TZ, Sesterhenn R, Kindlein L (2012) Economic losses of main causes of condemnation of the broiler carcass in a slaughterhouse under federal inspection on Rio Grande do Sul, Brazil. Acta Sci Vet 40(1):1021 7. Huezo R, Northcutt JK, Smith DP, Fletcher DL, Ingram KD (2007) Effect of dry air or immersion chilling on recovery of bacteria from broiler carcasses. J Food Prot 70(8):1829–1834 8. ICMSF (1986) Microorganisms in Foods 2. Sampling for microbiological analysis: Principles and specific applications. 2nd Ed. International Commission on Microbiological Specifications for Foods. Blackwell Scientific, Oxford 9. IFST (1999) Development and use of microbiological criteria for foods. Institute for food safety and technology, London 10. ISO (1999) ISO 6888-1:1999 Microbiology of food and animal feeding stuffs – Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) – Part 1: Technique using Baird-Parker agar medium 11. ISO (2001) ISO 16649-2:2001 Microbiology of food and animal feeding stuffs – Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli – Part 2: Colony-count technique at 44 degrees C using 5-bromo-4-chloro-3-indolyl beta-D-glucuronide 12. ISO (2003) ISO 6887-2:2003 Microbiology of food and animal feeding stuffs – Preparation of test samples, initial suspension and decimal dilutions for microbiological examination – Part 2: Specific rules for the preparation of meat and meat products 13. ISO (2003) ISO 4833-1:2003 Microbiology of food and animal feeding stuffs – Horizontal method for the enumeration of microorganisms – Colony-count technique at 30 degrees C
123
14. ISO (2004) ISO 21528-2:2004 Microbiology of food and animal feeding stuffs – Horizontal methods for the detection and enumeration of Enterobacteriaceae – Part 2: Colony-count method 15. Kabir SML (2010) Avian Colibacillosis and Salmonellosis: a closer look at epidemiology, pathogenesis, diagnosis, control and public health concerns. Int J Environ Res Public Health 7(1):89–114. doi:10.3390/ijerph7010089 16. Kittler S, Fischer S, Abdulmawjood A, Glu¨nder G, Klein G (2013) Effect of bacteriophage application on Campylobacter jejuni loads in commercial broiler flocks. Appl Environ Microbiol 79(23):7525–7533 17. Lupo C, Le Bouquin S, Balaine L, Michel V, Peraste J, Petetin I, Colin P, Chauvin C (2009) Feasibility of screening broiler chicken flocks for risk markers as an aid for meat inspection. Epidemiol Infect 137(8):1086–1098. doi:10.1017/s095026880900209x 18. Marien M (2007) Mixed respiratory infections in turkeys, with emphasis on avian metapneumovirus, Ornithobacterium rhinotracheale, Escherichia coli and Mycoplasma gallisepticum. University of Ghent, Ghent 19. Milnes AS, Sayers AR, Stewart I, Clifton-Hadley FA, Davies RH, Newell DG, Cook AJC, Evans SJ, Smith RP, Paiba GA (2009) Factors related to the carriage of Verocytotoxigenic E. coli, Salmonella, thermophilic Campylobacter and Yersinia enterocolitica in cattle, sheep and pigs at slaughter. Epidemiol Infect 137(8):1135–1148. doi:10.1017/s095026880900199x 20. Reich F, Atanassova V, Klein G (2013) Extended-spectrum betalactamase- and AmpC-producing enterobacteria in healthy broiler chickens, Germany. Emerg Infect Dis 19(8):1253–1259. doi:10. 3201/eid1908.120879 21. Reich F, Atanassova V, Haunhorst E, Klein G (2008) The effects of Campylobacter numbers in caeca on the contamination of broiler carcasses with Campylobacter. Int J Food Microbiol 127(1–2):116–120 22. Shane SM (2009) Reducing pathogenic E. coli infection by vaccination. World Poult 25(5):21–23 23. Silva MCA, Melo DB, Costa WLR, Fernandes LMB, Pinna MH, Vieira Neto J (2011) Macroscopic and microscopic aspects of airsacculitis in slaughtered broilers in Brazil. In: Animal hygiene and sustainable livestock production. Proceedings of the XVth International Congress of the International Society for Animal Hygiene, pp. 603–605 Vienna, Austria. Tribun EU, pp 603–605 24. Smith DP, Cason JA, Berrang ME (2005) Effect of fecal contamination and cross-contamination on numbers of coliform, Escherichia coli, Campylobacter, and Salmonella on immersionchilled broiler carcasses. J Food Prot 68(7):1340–1345 25. Tessier M, Fredette MA, Beauchamp G, Boulianne M (2001) Cellulitis in broiler chickens: a one-year retrospective study in four Quebec abattoirs. Avian Dis 45(1):191–194. doi:10.2307/ 1593027 26. USDA (2012) E. coli peritonitis on breeder chicken farms in the United States, a study of US breeder-chicken farms in 2010. National Animal Health Monitoring System (NAHMS), Veterinary Service Washington
