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Bacterial contamination of eggs and behaviour of poultry flocks in the free range environment
Accepted Manuscript Title: Bacterial contamination of eggs and behaviour of poultry flocks in the free range environment Author: Talia Moyle Kelly Drake Vaibhav Gole Kapil Chousalkar Susan Hazel PII: DOI: Reference:
S0147-9571(16)30101-1 http://dx.doi.org/doi:10.1016/j.cimid.2016.10.005 CIMID 1102
To appear in: Received date: Revised date: Accepted date:
14-2-2016 27-5-2016 20-10-2016
Please cite this article as: Moyle Talia, Drake Kelly, Gole Vaibhav, Chousalkar Kapil, Hazel Susan.Bacterial contamination of eggs and behaviour of poultry flocks in the free range environment.Comparative Immunology, Microbiology and Infectious Diseases http://dx.doi.org/10.1016/j.cimid.2016.10.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Bacterial contamination of eggs and behaviour of poultry flocks in the free range environment Talia Moylea, Kelly Drakeb, Vaibhav Golec, Kapil Chousalkara, Susan Hazela
a School of Animal and Veterinary Science, The University of Adelaide, Roseworthy, South Australia, 5371, Australia b South Australia Research and Development Institute (SARDI), JS Davies Building, Roseworthy, South Australia. 5371, Australia c Rivalea (Australia) Pty Ltd , PO Box 78 Corowa NSW 2646, Australia
Corresponding Author:
K. K. Chousalkar School of Animal and veterinary Science The University of Adelaide Roseworthy, 5371, SA Australia Telephone: +61 8 8313 1502 Facsimile: + 61 8 8303 7356 Email: [email protected]
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Highlights
Significant effect of hen age on total bacterial levels on the egg shell surface.
Hen age had a significant effect on the level of total bacteria in egg shell pores.
Escherichia coli were the most frequently isolated Enterobacteriaceae species.
Significant effect of hen age on feather condition score.
Significant effect of hen age on submissive/crouching behaviour.
Abstract The free range production system is becoming more common in Australia and is expected to increase. Free range hens are exposed to more stressors in comparison to hens from barn and cage systems and it is suggested that stress can increase bacterial shedding on eggs. The aims of this study were to examine the level of total bacteria and Enterobacteriaceae populations, as well as the presence of Salmonella and Campylobacter, in eggs collected from two free range flocks on two different farms and to conduct longitudinal observations of the behaviour and welfare of hens in the free range production system. Hen age (weeks) was shown to have a significant effect (increase) on the level of total bacteria on the egg shell surface and in shell pores, as well as having an effect on feather condition score. As the hens aged, the frequency of external visual egg characteristics increased, as did feather condition score (where feather condition was poorer). These observations indicate areas which should be investigated further to improve the food safety of eggs and optimise the welfare of free range hens.
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Key words: Eggs, bacteria, behavior, welfare, food safety, Free range poultry.
1. Introduction In 2014, the annual production of eggs in Australia was 397.4 million dozen and worth $1.7 billion. Approximately 38% of egg production is derived from the free range production system [1]. The definition of free range layer hens in Australia is; birds housed in sheds that also have access to an outdoor range [2]. Due to public perception of intensive farming systems, animal welfare and major retailer pressures, the free range production system is becoming more common in Australia and is expected to increase. Free range hens are exposed to more stressors (e.g. predators, extreme weather conditions, larger and more variable social hierarchies and potentially aggressive behaviours from other hens) in comparison to hens from barn and cage systems [3, 4]. These challenges can affect hen welfare and therefore the ability to produce eggs [5]. Welfare measures such as feather pecking, vent pecking, cannibalism, body weight and external visual egg characteristics are useful indicators of hen welfare. Results of these measures can be compared between different production systems (free range, barn, cage, aviary etc.) [3, 5]. Free range may be perceived by consumers and welfare advocates as the best option for egg production, but it has negative and positive welfare aspects for the hens [5]. The free range system increases the risk of disease transmission, injury, predation [3] and parasite occurrence [6]. The positive welfare aspects of free range systems enable the hens to exhibit a larger repertoire of behaviours, including dust bathing, foraging, experience natural light, and have an increased area outdoors in which to range, in comparison to hens in cage systems [5, 6]. Consumption of eggs and egg products is often linked to food poisoning outbreaks [7]. Total bacteria and Enterobacteriaceae populations can be used as an indicator of the food 3
safety of eggs [8]. Bacteria have the ability to move from the surface of the egg, through the cuticle, into the egg shell pores, through the egg shell membranes and into the egg internal contents (albumen and yolk). As this can occur, it is important to study bacterial count in all parts of the egg, not just the egg surface and internal contents. There is a lack of research regarding the level of egg shell pore contamination by Enterobacteriaceae. The Enterobacteriaceae family includes many genera of bacteria, one of which is Salmonella [9]. Food poisoning is often due to the bacteria Salmonella and Campylobacter. Campylobacter is commonly associated with broiler chickens and is the leading cause of bacterial food-borne illness [10], but little research has been conducted in relation to Campylobacter contamination of eggs [11]. Given its importance to food safety and the increase in free range production, it is necessary to study the prevalence of Campylobacter and Salmonella spp in eggs to potentially improve food safety [11]. The level of bacteria, when present, in the egg shell, pore and internal contents varies greatly due to many factors. Factors such as season, stress, stocking density, flock size, individual flock management, farm management, infection from pests and hygiene play major roles in the level of bacteria present in eggs [12]. Egg washing with sanitisers is a common method to minimise shell contamination however, in Australia, this practice is highly variable as it is not compulsory to wash eggs. Australia does have regulations preventing the sale of dirty and/or cracked eggs though [12]. Limited research has been conducted assessing bacterial contamination in free range eggs collected over time from the Australian commercial free range production system. The aims of this study were to; examine the level of total bacteria and Enterobacteriaceae populations, as well as the presence of Salmonella and Campylobacter, in eggs collected from two free range flocks from two farms over a period of time and to conduct longitudinal observations of the behaviour and welfare of hens in the free range production system over time. 4
2. Methods and materials 2.1 Bacterial isolation 2.1.1 Collection of eggs Eggs (n= 600) were collected from two free range flocks on two different farms. These farms were selected based on the willingness of the producers to participate in the study. Each flock was sampled five times at six week intervals from February to August (mid to late lay). Both flocks were Hy-Line Brown hens, with approximately 9,500 hens (aged 43 weeks) on Farm One and approximately 9,000 hens (aged 49 weeks) on Farm Two at the commencement of the study. Both farms complied with the stocking density allowance outlined in Model Code of Practice for the Welfare of Animals- Domestic poultry (1,500 hens per hectare) [2]. Sampling of at least 40 eggs is essential for statistically reliable determination of bacterial load in eggs [13]. Therefore, during each sampling visit, 60 visibly clean eggs, from the nest boxes, were randomly selected. 2.1.2 Shell wash For bacterial analysis, six eggs were pooled (10 samples/sampling visit) as one sample [14]. Samples were not duplicated. 2.1.2.1 Total bacterial count Six eggs were placed into one Whirl- Pak (Nasco) bag with 60 mL of Buffered Peptone Water (Oxoid Australia). Each egg was massaged by hand for 60 seconds. The eggs were removed from the bags and 100 µL of the Buffered Peptone Water egg shell wash solution was plated on nutrient agar (Oxoid Australia). The plates were incubated overnight at 37°C and colonies were counted and recorded [15]. Inoculated Buffered Peptone Water samples were stored in 80% glycerol for further characterisation of Enterobacteriaceae.
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2.1.2.2 Total Enterobacteriaceae count Using a micropipette, 100 µL of the Buffered Peptone Water egg shell wash solution was plated on violet red bile glucose agar (Oxoid, Australia). The plates were incubated overnight at 37°C and colonies were counted and recorded [14]. 2.1.2.3 Campylobacter isolation Using the Buffered Peptone Water egg shell wash solution, 100 µL was plated onto Brilliance CampyCount agar (Oxoid, Australia). Plates were placed in Compact Plastic Pouches (Oxoid, Australia), with CampyGen Compact Sachets (Oxoid, Australia) and sealed to create microaerobic conditions. After incubation at 37°C for 48 hours, colony numbers were counted and recorded. The suspect cultures were tested by Gram staining and biochemical tests such as, Gram-lysis and L-ALA tests, to confirm potential Campylobacter species. 2.1.2.4 Salmonella isolation The Buffered Peptone Water egg shell wash solution was incubated overnight at 37°C. For Salmonella isolation, 100 µL of the incubated Buffered Peptone Water was added to 10 mL Rappaport Vassiliadis Soya Peptone broth (Oxoid, Australia) and incubated overnight at 42°C. Samples were streaked on xylose lysine deoxycholate agar (Oxoid, Australia) and incubated overnight at 37°C. Suspected Salmonella colonies were confirmed by streaking on Brilliance Salmonella Agar (Oxoid, Australia) and stored in 80% glycerol for serotyping [14]. 2.1.3 Shell crush After the shell wash procedure, eggs were dipped in 70% ethanol for 90 seconds to kill egg shell surface bacteria. After air drying, the eggs (six eggs pooled as one sample) were cracked open and internal contents placed in a Whirl-Pak bag (Nasco). The shells were washed with phosphate buffered saline to remove any albumen, placed in a new Whirl-Pak bag and crushed
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thoroughly. To each bag, 60 mL of Buffered Peptone Water was added [15]. The shell crush samples were processed for bacteriological examination as stated. 2.1.4 Internal contents The internal egg contents samples were mixed thoroughly by hand. Using a 5 mL syringe, 5 mL of egg internal contents was added to 45 mL of Buffered Peptone Water in a zip-lock bag [15]. The internal contents samples were plated on nutrient agar, violet red bile glucose agar and Brilliance CampyCount agar (Oxoid, Australia), incubated as mentioned above and the bacteria counted and recorded if present. 2.1.5 Characterisation of Enterobacteriaceae A loopful of the stored inoculated Buffered Peptone Water (Oxoid Australia) was plated on violet red bile glucose agar and incubated overnight at 37°C. An individual colony was selected to be characterised using API 20E strips (Biomerieux, Australia) as per the manufacturer's instructions and results were determined using apiweb software (Biomerieux, Australia) [14]. 2.2 Welfare and behavioural observation The following hen welfare and behavioural measures were observed at the same time as the egg collection between approximately 10:00am and 1:00pm. During each sampling visit, weather conditions were also recorded. 2.2.1 Feather condition score Eighty hens inside the shed were assessed for feather condition (20 hens in each quarter of the shed). Three areas on the hen were scored, including the head and neck, back and vent, using a score of 0-2 (Table 1) to obtain a total score for the hens. Assessment was performed by selecting a random hen, counting two hens to the left or right and scoring the feather condition of that hen. Scores were assessed by observing hens from a short distance [16].
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2.2.2 Submissive/crouching behaviour Submissive behaviour of the hens was assessed by walking slowly through the shed and counting the number of hens within one metre of the observer which crouched on approach of the observer. The number of hens displaying the behaviour was counted in each quarter of the shed. 2.2.3 External visual egg shell characteristics The external visual egg shell characteristics from the sample of 60 eggs were recorded using the terms: misshapen, light calcium dusting, heavy calcium dusting, blood stains, white bands and pink colouring (Table 2) [17]. 2.3 Statistical analysis Univariate general linear models using IBM SPSS Statistics version 22 [18] were used to determine the variation in the load of total bacteria and Enterobacteriaceae (log 10 transformed colony forming unit (CFU)) populations in and on eggs, to examine changes in external visual egg characteristics and changes in submissive/crouching behaviour, during the period of the longitudinal samplings. The same analysis was also used to examine feather condition data, which were transformed by square root to minimise variation in the data. Significance was tested at the P<0.05 level.
3. Results 3.1 Total bacteria- egg shell surface The average level of bacteria isolated from the egg shell surfaces from Farm One was 3.84 log CFU/egg shell (Figure 1). The average level of bacteria isolated from the egg shell surfaces from Farm Two was 3.69 log CFU/egg shell. Table 3 shows raw data for average number of bacterial colonies per egg shell. Statistical analysis indicated there was a significant effect of age (weeks) on the total bacteria count. For each week increase in age, the amount of total 8
bacteria on the egg shell surface increased by 0.0145 log CFU/egg shell for Farm One and 0.014 log CFU/egg shell for Farm Two (P=0.016). 3.2 Total bacteria- egg shell pores The level of total bacteria in the shell pores increased during the study (Figure 2). The average level of bacteria isolated from shell pores from Farm One was 0.52 log CFU/egg shell and 0.40 log CFU/egg shell from Farm Two. Statistical analysis indicated there was a significant effect of age (weeks) on the total bacteria in egg shell pores (P=0.002). For each week increase of age, the amount of bacteria in the shell pores increased by 0.032 log CFU/egg shell for Farm One and 0.038 for Farm Two. Eleven samples from Farm One and eight from Farm Two were positive. One positive total bacteria internal contents sample was isolated from Farm One and four from Farm Two. 3.3 Enterobacteriaceae- egg shell surface The level of Enterobacteriaceae in the shell wash was not significantly different (P=0.737) between samplings during the study. The average level of Enterobacteriaceae isolated from the egg shell surface from Farm One was 0.98 log CFU/egg shell and 0.68 log CFU/egg shell from Farm Two. Statistical analysis indicated that there was no significant effect of age on the level of bacteria present on the egg shells. The level of Enterobacteriaceae on eggs decreased on Farm One at a rate of 0.021 log CFU/egg shell per week and slowly increased each week at 0.011 log CFU/egg shell on eggs from Farm Two. 3.4 Isolated Enterobacteriaceae species Seven different species of Enterobacteriaceae were identified on the two farms. The apiweb software (Biomerieux, Australia) was unable to identify two samples from each farm and these were classed as inconclusive samples. Escherichia coli were the most frequently isolated species of Enterobacteriaceae from both Farm One and Farm Two. Escherichia hermannii and 9
Kluyvera spp were isolated from Farm One only, whilst Proteus mirabilis and Klebsiella pneumoniae were isolated from Farm Two only. Two positive Enterobacteriaceae shell crush samples were isolated from Farm One and three from Farm Two. These were all Escherichia coli. Campylobacter spp was not isolated from shell wash, shell crush or internal contents samples. Salmonella Mbandaka was isolated from one shell crush sample from Farm Two. Between the two farms, 90% (n=100) of the pooled egg shell wash samples and 5% (n=100) of the pooled egg shell crush samples were contaminated with Enterobacteriaceae. 3.5 External visual egg shell characteristics There was no significant effect of age (weeks) on the external visual egg characteristics from Farm One and Two. However, the percentage of lightly calcium dusted eggs increased, as did the presence of heavily calcium dusted eggs in late lay on both farms. Egg characteristics, such as blood stains, misshapen and pink eggs were also observed more often towards late lay, however the occurrence of these characteristics were not found to be statistically significant. 3.6 Feather condition score The feather condition scores of both flocks increased over time indicating poorer feather condition. For each week increase in hen age, the feather condition score increased by 0.031 for Farm One and 0.014 for Farm Two (P<0.001). 3.7 Submissive/crouching behaviour The percentage of both flocks exhibiting submissive behaviour decreased during the study (Figure 3). For each week increase of age, the percentage of the flock exhibiting submissive/crouching behaviour decreased by 0.011% for Farm One and 0.007% for Farm Two (P=0.005). 4. Discussion
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The aims of this study were to examine the level of total bacteria and Enterobacteriaceae populations in eggs collected from two commercial free range flocks from two farms over time and to conduct longitudinal observations of the behaviour and welfare of hens in the free range production system. The presence of Salmonella and Campylobacter in the eggs was also examined. Campylobacter was not isolated and only one positive Salmonella sample (Salmonella Mbandaka) was identified. Hen age (weeks) was shown to have a significant effect on the level of total bacteria on the egg shell surface and in shell pores, as well as having an effect on feather condition score and submissive/crouching behaviour. On both farms feather condition score increased (feather condition was poorer) and the percentage of the flock exhibiting submissive/crouching behaviour decreased. Table 3 showing average number of bacterial colonies per egg provides an indicator of the level of which bacteria was present on and in eggs used in this study and is useful information in regards to food safety. 4.1 Total bacteria- egg shell surface A larger population of bacteria on the surface of the shell can increase the likelihood of penetration of bacteria into egg shell pores and internal contents if eggs are not handled correctly [14, 19]. Total bacteria and Enterobacteriaceae populations can also be used as an indicator of food safety of eggs. Bacteria on the surface of egg shells are commonly found in the air, water and soil, so the types vary due to the environment of the egg production facility [20]. In previous studies, there are conflicting results in the level of total bacteria contamination in both alternative (barn and free range) and conventional (cage) egg production systems [21]. This study found a statistically significant age (weeks) effect on the level of total bacteria found on the egg shell surface as was reported earlier [15]. The average level of bacteria isolated from the surface of the egg shells from Farm One during the study was 3.84 log CFU/egg shell and 3.69 log CFU/egg shell for Farm Two. These results are similar to previous studies [15], who reported an average of 3.48 log CFU/egg shell for total bacteria on free range eggs. Another 11
study investigating free range and cage production system eggs collected during the same seasons (autumn and winter) found total bacteria levels on the surface of free range eggs of 3.2 log CFU/mL for free range eggs and 3.6 log CFU/mL for caged eggs [22]. These results are lower in comparison to a previous study [21], but comparable to the present study. Another study investigating bacterial contamination of free range eggs and caged eggs found very high levels of total bacteria, 5.55 log CFU/mL for free range eggs and 5.23 log CFU/mL for cage eggs [21]. Results may differ between studies due to differing isolation techniques, age and breed of hens, climate and country, hygiene and farm management. 4.2 Total bacteria- egg shell pores There was a significant age (weeks) effect on the level of total bacteria in egg shell pores, consistent with previous findings [15]. The results of this study (Farm One 0.40 log CFU/egg shell and Farm Two 0.52 log CFU/egg shell) are lower than results reported previously [15], who observed bacterial levels in egg shell pores of 1.27 log CFU/egg shell in free range eggs and 1.21 log CFU/egg shell in caged eggs. It is likely these results differ due to the quality of the egg shell and the cuticle, which aids in preventing bacteria entering the egg shell pores [7], however results may also differ due to egg handling practices, isolation techniques, farm practices and hygiene. 4.3 Enterobacteriaceae- egg shell surface The average level of Enterobacteriaceae isolated from the egg shell surface from Farm One was 0.98 log CFU/egg shell and 0.68 log CFU/egg shell from Farm Two. There was no significant effect of age on the level of bacteria present on the egg shells. However, previous study has reported a significant effect of age on increasing levels of Enterobacteriaceae on the egg shell surface [15]. The level of Enterobacteriaceae isolated in this study was low in comparison to other studies. Previous study by Gole et al. [14] found an average count of 1.46
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log CFU/egg shell in caged eggs was in agreement with De Reu et al. [23] (1.51 log CFU/egg shell). De Reu et al. [23] also reported an average log CFU/egg shell of 1.54 present in noncage eggs. Another study found the average level of Enterobacteriaceae on the egg shell surface of free range eggs to be 1.5 log CFU/egg shell [15]. All of these results differ from those observed previously [21], who showed an average of 4.38 log CFU/mL for free range eggs and 3.05 log CFU/mL for caged eggs. Violet red bile glucose agar (Oxoid, Australia) was used for detection and enumeration of Enterobacteriaceae in this study. Results may have differed from other studies due to the isolation technique used. Bacteria present may not have recovered sufficiently to grow proficiently on the selective media. 4.4 Enterobacteriaceae species Escherichia coli, Enterobacter cloacae, Escherichia fergusonii, Kluyvera spp. and Klebsiella pneumoniae have been isolated during previous studies investigating Enterobacteriaceae contamination of eggs [9, 13, 23] and many others have also been isolated from egg production facilities [20]. Proteus mirabilis and Escherichia hermannii have previously been isolated from the surface of egg shells [24]. Escherichia coli were the most commonly isolated bacterial species from both Farm One and Two (60.0% and 82.22%). A caged layer hen study also reported Escherichia genus most frequently (60.8%) [14]. In that study, Enterobacter was the third most isolated genus and in this study, Enterobacter cloacae (24%) was the second most frequently isolated bacteria for Farm One followed by Escherichia hermannii (8%). All other species for both farms were identified less frequently (<5%), as was the case with Gole et al. [14]. The Escherichia genus was also the most commonly isolated bacteria in a study by Musgrove et al. [9]. Escherichia coli, the most frequently isolated Enterobacteriaceae in this study, is commonly a source of food borne illness and is recognised as an important human pathogen [25, 26]. Salmonella Mbandaka was identified from one shell crush sample from Farm Two. Salmonella belong to the Enterobacteriaceae family and 13
initially, when testing for Enterobacteriaceae, this sample was negative. As Salmonella isolation occurs after enrichment of the Buffered Peptone Water solution, the bacteria were able to recover and grow. Therefore, when the sample was positive when tested for Salmonella presence. 4.5 External visual egg shell characteristics Stress can cause deformities in eggs. For example, hens may delay laying an egg if stressed, which can result in extra calcium carbonate forming on the egg shell or other shell deformities. Blood stains may also be present on egg shells due to prolapse or tearing of the vent due to laying an overly large egg [5]. Viral infections also cause shell deformities and affect egg quality therefore; hens in this study were vaccinated against infectious bronchitis virus and egg drop syndrome. There was no significant effect of age (weeks) on the external visual egg characteristics for either farm. However, the percentage of lightly calcium dusted eggs increased and observations of heavily calcium dusted eggs occurred in late lay on both farms. Other external visual egg characteristics, including blood stains, misshapen and pink eggs, were also observed more often towards late lay. A study of 25 free range farms combined pink and calcium dusted egg data, classed as “off colour” and found an occurrence of 0.44%, lower than that observed in this study [27]. Sherwin et al. [5] reported 1.7% calcium spots and 1.4% blood stains on free range eggs in their study. In Sherwin et al. [5], producers were asked to examine and record external visual egg characteristics. This may have contributed to the low averages, as farmers may have been biased when reporting the findings. 4.6 Feather condition Feather pecking has been commonly observed in larger free range flocks, when a large percentage of the flock does not range [28]. Feather pecking is worse in sparse environments that are void of stimuli that can distract hens from extreme feather pecking [29, 30]. During
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this study, the feather condition of the hens deteriorated, with a significant effect of age (weeks) on feather condition score for both farms. On farms where feather pecking occurs, it generally increases with age, as it is a learnt behaviour. Feather pecking can be hard to control once it has begun, therefore, the feather condition of the hens worsens [31], as was the case in the present study. Whay et al. [27] also observed an increase in the degeneration of feather condition over time, as well as an increase in the number of hens subject to feather loss, as was observed in this study. On each of the farms, more hens with feather damage and higher feather condition scores were observed over time. While it is the most common reason for poor feather condition, feather pecking is not the only cause. It may also be due to moulting or abrasive surfaces in the environment [29]. As a result, it cannot be concluded feather pecking is the sole cause of feather degeneration in this study. 4.7 Submissive/crouching behaviour The submissive crouching behaviour exhibited by hens is a sign of sexual maturity, sexual receptivity, submission to dominant hens and fear [31]. Submissive behaviour is a novel measure of welfare, which has not been investigated in depth. Hens can often be observed exhibiting this crouching behaviour due to the approach of a person. A person can be perceived as a predator and assuming this crouching submissive posture or freezing could potentially reduce the likelihood of being seen by a predator (person) [32]. There was a significant effect of age on the percentage of hens on both farms exhibiting submissive behaviour, resulting in a decrease of the behaviour being observed. The decrease is most likely due to hens habituating to the presence of an observer. The free range production system is becoming a major source of egg production in Australia and other parts of world. Longitudinal studies are helpful to understand the level of bacterial shedding. Hens raised in a free range production system could be exposed to environmental
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stressors, which could induce bacterial shedding. In Australia, USA and Japan, commercial table eggs are washed [12] to reduce bacterial load. However, the presence of bacteria inside shell pores could still pose a challenge if eggs are not handled correctly. The presence of Escherichia coli and Enterobacteriaceae have been used as quality and safety indicators in food chains [8]. There are no established levels or regulatory thresholds of such indicator organisms in the egg industry in Australia. Findings from this study could be helpful for establishing base levels of indicator organisms, but large scale longitudinal studies are required in the future. Significant associations could not be made between behaviour and bacterial shedding due to limited sample size. Further studies could be conducted for objective measurement of stress (such as measuring level of corticosterone) along with hen behaviour and bacterial shedding. 5. Conclusion Hen age was shown to have a significant effect on the level of total bacteria on the egg shell surface and in shell pores, as well as having an effect on feather condition score and submissive/crouching behaviour. Escherichia coli were the most frequently isolated Enterobacteriaceae species from both farms. Limited research has been conducted examining bacterial load in eggs from commercial free range farms in Australia therefore; these observations are important to the industry as they may indicate areas which should be investigated further to improve food safety of eggs and optimise the welfare of free range hens in Australia. As there were only two farms in this study, no significant associations between levels of bacteria, hen behaviour and welfare were able to be made. The bacteria and behavioural observations need to be further investigated on a larger scale to obtain substantial results. Two farms were not enough to obtain significant results, from which conclusions could be drawn.
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Sampling farms more often (monthly/weekly) would be preferable, as this would provide more in depth data in regards to the bacteria, hen behaviours and welfare. Further research also needs to be conducted to demonstrate the link between behaviours and/or welfare indicators and stress in free range hens. This could be achieved by using additional physiological measures of stress such as corticosterone, and heterophil to lymphocyte ratio [28]. Acknowledgements This research was funded by the Poultry Cooperative Research Centre and The University of Adelaide. Thanks to Dr Michelle Hebart for her assistance with statistical analysis.
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References [1] Australian Egg Corporation Limited (AECL), Australian Egg Corporation Annual Report. http://www.aecl.org/assets/Uploads/Annual-Reports/AECL-Annual-Report-2014.pdf,
2014
(accessed 02.03015). [2] CSIRO. Primary Industries Standing Committee Model Code of Practice for the Welfare of Animals Domestic Poultry 4th Edition. Appendix 2. Collingwood, VIC, AUS: CSIRO Publishing; 2002. p. 27-8. [3] Daigle C, Siegford J. Welfare Quality® parameters do not always reflect hen behaviour across the lay cycle in non-cage laying hens. Anim. Welf. 2014;23:423-434. [4] Walker A, Hughes B. Egg shell colour is affected by laying cage design. Br. Poult Sci. 1998;39:696-699. [5] Sherwin C, Richards G, Nicol C. Comparison of the welfare of layer hens in 4 housing systems in the UK. Br. Poult. Sci. 2010;51:488-499. [6] Tauson R. Management and housing systems for layers–effects on welfare and production. World's Poult. Sci. J. 2005;61:477-490. [7] De Reu K. Bacteriological contamination and infection of shell eggs in the production chain: University of Ghent, Belgium; 2006. [8] Kornacki JL, Johnson JL. Enterobacteriaceae, coliforms, and Escherichia coli as quality and safety indicators. In: Downes FP, Ito K, (Eds.), Compendium of Methods for the Microbiological Examination of Foods. 4th ed. American Public Health Association, Washington, DC, 2001, p. 69-82. [9] Musgrove MT, Jones DR, Northcutt JK, Cox NA, Harrison MA. Identification of Enterobacteriaceae from washed and unwashed commercial shell eggs. J. of Food Protection®. 2004;67:2613-2616.
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[10] Sahin O, Kobalka P, Zhang Q. Detection and survival of Campylobacter in chicken eggs. J. of Appl. Microbiol. 2003;95:1070-1079. [11] Messelhäusser U, Thärigen D, Elmer-Englhard D, Bauer H, Schreiner H, Höller C. Occurrence of thermotolerant Campylobacter spp. on eggshells: a missing link for food-borne infections? Appl. and Environ. Microbiol. 2011;77:3896-3897. [12] Chousalkar K, Sexton M, McWhorter A, Hewson K, Martin G, Shadbolt C, Paul Goldsmith. Salmonella Typhimurium in the Australian Egg Industry: Multidisciplinary Approach to Addressing the Public Health Challenge and Future Directions. Crit. Rev. Food Sci. Nutr. 2015. [13] De Reu K, Grijspeerdt K, Heyndrickx M, Zoons J, De Baere K, Uyttendaele M, Debevere J, Herman L. Bacterial eggshell contamination in conventional cages, furnished cages and aviary housing systems for laying hens. Br. Poult. Sci. 2005;46:149-155. [14] Gole V, Chousalkar K, Roberts J. Survey of Enterobacteriaceae contamination of table eggs collected from layer flocks in Australia. Int. J. of Food Microbiol. 2013;164:161-165. [15] Samiullah S, Roberts J, Chousalkar K. Effect of production system and flock age on egg quality and total bacterial load in commercial laying hens. The J. of Appl. Poult. Res. 2014;23:59-70. [16] Bristol University, RSPCA, Soil Association, Laying hens assessment protocol. http://www.assurewel.org/layinghens, 2013 (accessed 24.11.15). [17] Hughes B, Gilbert A, Brown MF. Categorisation and causes of abnormal egg shells: relationship with stress. Br. Poult. Sci. 1986;27:325-337. [18] IBM Corporporation . IBM SPSS Statistics for Windows. 22.0 ed. Armonk, NY: IBM Corp; 2013.
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[19] Smith A, Rose S, Wells R, Pirgozliev V. The effect of changing the excreta moisture of caged laying hens on the excreta and microbial contamination of their egg shells. Br. Poult. Sci. 2000;41:168-173. [20] Musgrove M, Jones D, Shaw J, Sheppard M, Harrison M. Enterobacteriaceae and related organisms isolated from nest run cart shelves in commercial shell egg processing facilities. Poult. Sci. 2009;88:2113-2117. [21] Parisi M, Northcutt J, Smith D, Steinberg E, Dawson P. Microbiological contamination of shell eggs produced in conventional and free-range housing systems. Food Control. 2015;47:161-165. [22] Jones D, Anderson K, Musgrove M. Comparison of environmental and egg microbiology associated with conventional and free-range laying hen management. Poult. Sci. 2011;90:20632068. [23] De Reu K, Rodenburg T, Grijspeerdt K, Messens W, Heyndrickx M, Tuyttens F, et al. Bacteriological contamination, dirt, and cracks of eggshells in furnished cages and noncage systems for laying hens: An international on-farm comparison. Poult. Sci. 2009;88:2442-2448. [24] Stępień-Pyśniak D. Occurrence of Gram-negative bacteria in hens' eggs depending on their source and storage conditions. Pol. J. of Vet. Sci. 2010;13:507-513. [25] Gorman R, Bloomfield S, Adley CC. A study of cross-contamination of food-borne pathogens in the domestic kitchen in the Republic of Ireland. Int. J of Food Microbiol. 2002;76:143-150. [26] Tauxe RV. Emerging foodborne diseases: an evolving public health challenge. Emerg. Infect. Dis. 1997;3:425. [27] Whay H, Main D, Green LE, Heaven G, Howell H, Morgan M, et al. Assessment of the behaviour and welfare of laying hens on free-range units. The Vet. Rec. 2007;161:119-128.
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[28] Lay D, Fulton R, Hester P, Karcher D, Kjaer J, Mench J, et al. Hen welfare in different housing systems. Poult. Sci. 2011;90:278-294. [29] Appleby MC, Hughes BO. Welfare of laying hens in cages and alternative systems: environmental, physical and behavioural aspects. World's Poult. Sci. J. 1991;47:109-128. [30] Blokhuis H. The effect of a sudden change in floor type on pecking behaviour in chicks. Appl. Anim. Behav. Sci. 1989;22:65-73. [31] Nicol C. Behavioural contributions to welfare assessment. In: Nicol C (Eds.), The behavioural biology of chickens. CABI, Wallingford, UK, 2015, p. 58-78. [32] Jones RB. Fear and adaptability in poultry: insights, implications and imperatives. World's Poult. Sci. J. 1996;52:131-174.
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5 4.5
log CFU
4 3.5 3 2.5 2 40
45
50
55
60
65
70
75
Age (weeks) Farm 1
Farm 2
Figure 1. Average level of total bacteria (log CFU/egg shell) in egg shell wash from two free range farms, Farm One and Farm Two over the period of the study (age-weeks). CFU=colony forming unit. Farm One n=300 eggs and Farm Two n=300 eggs.
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1.8 1.6 1.4
log CFU
1.2 1 0.8 0.6 0.4 0.2 0 40
45
50
55
60
65
70
75
Age (weeks) Farm 1
Farm 2
Figure 2. Average level of total bacteria (log CFU/egg shell) in egg shell pores from two free range farms, Farm One and Farm Two, over the period of the study (age-weeks). CFU=colony forming unit. Farm One n=300 eggs and Farm Two n=300 eggs.
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0.6
Percentage %
0.5 0.4 0.3
Farm 1 Farm 2
0.2 0.1 0 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 Age (weeks)
Figure 3. Percentage of the flock from two free range farms, Farm One and Farm Two, exhibiting submissive/crouching behaviour over the period of the study (age-weeks).
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Table 1. Description of the feather scoring system used to assess the head and neck, back and vent area of the hens observed on two free range farms, Farm One and Farm Two. Feather score 0 1 2
Description No or minimal feather loss (no bare skin visible, no or slight feather wear, only single feathers missing). Moderate feather loss (moderate feather wear, i.e. damaged feather or one or more featherless areas, bare skin visible <5 cm maximum dimension). Severe feather loss (at least one area with bare skin visible ≥5 cm maximum dimension).
Table 2. Description of external visual egg characteristics observed on eggs collected from two free range farms, Farm One and Farm Two, over the period of the study (age-weeks) [17]. Egg shell Description characteristic An egg that is not the normal shape and size (It may be too small or large or Misshapen round instead of oval). They generally look round at one end of the egg and pointy at the other end. Light calcium Small, irregular shaped white spots deposited on the external surface of the shell, in small numbers covering a small proportion of the shell. dusting Larger, irregular shaped white spots deposited on the external surface of the Heaving calcium dusting shell, in great numbers covering a large proportion of the shell Smears of blood on the external surface of the shell. Blood stains The egg appears to be a pink colour due to a very fine calcium layer. Pink
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Table 3: Raw total bacterial and Enterobacteriaceae data. Average number of colonies per egg of total bacterial shell wash (TB SW), shell crush (TB SC) and internal contents (TB IC) and Enterobacteriaceae shell wash (E SW) and shell crush (E SC), for two free range farms, Farm One and Farm Two. Standard error in brackets. Age (wks) Farm ID
43
49
50
54
56
60
62
66
69
71
TB SW (n=60)
1 10890 (3197)
2 8660 (1325)
1 4200 (2890)
2 3570 (1134)
1 11840 (3144)
2 9620 (2918)
1 25510 (1992)
2 1210 (222)
1 9190 (2608)
2 29490 (510)
E SW (n=60) TB SC (n=60) E SC (n=60) TB IC (n=60)
3470 (2777) 0 (0) 0 (0) 0 (0)
1630 (1619) 0 (0) 0 (0) 0 (0)
3000 (3000) 0 (0) 0 (0) 0 (0)
200 (13) 10 (10) 0 (0) 0 (0)
600 (34) 170 (68) 10 (10) 20 (20)
220 (100) 60 (43) 40 (40) 30 (30)
2550 (1122) 50 (85) 0 (0) 0 (0)
0 (0) 20 (20) 0 (0) 10 (10)
0 (0) 90 (55) 10 (10) 0 (0)
120 (53) 230 (123) 60 (40) 3020 (2998)
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S0147-9571(16)30101-1 http://dx.doi.org/doi:10.1016/j.cimid.2016.10.005 CIMID 1102
To appear in: Received date: Revised date: Accepted date:
14-2-2016 27-5-2016 20-10-2016
Please cite this article as: Moyle Talia, Drake Kelly, Gole Vaibhav, Chousalkar Kapil, Hazel Susan.Bacterial contamination of eggs and behaviour of poultry flocks in the free range environment.Comparative Immunology, Microbiology and Infectious Diseases http://dx.doi.org/10.1016/j.cimid.2016.10.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Bacterial contamination of eggs and behaviour of poultry flocks in the free range environment Talia Moylea, Kelly Drakeb, Vaibhav Golec, Kapil Chousalkara, Susan Hazela
a School of Animal and Veterinary Science, The University of Adelaide, Roseworthy, South Australia, 5371, Australia b South Australia Research and Development Institute (SARDI), JS Davies Building, Roseworthy, South Australia. 5371, Australia c Rivalea (Australia) Pty Ltd , PO Box 78 Corowa NSW 2646, Australia
Corresponding Author:
K. K. Chousalkar School of Animal and veterinary Science The University of Adelaide Roseworthy, 5371, SA Australia Telephone: +61 8 8313 1502 Facsimile: + 61 8 8303 7356 Email: [email protected]
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Highlights
Significant effect of hen age on total bacterial levels on the egg shell surface.
Hen age had a significant effect on the level of total bacteria in egg shell pores.
Escherichia coli were the most frequently isolated Enterobacteriaceae species.
Significant effect of hen age on feather condition score.
Significant effect of hen age on submissive/crouching behaviour.
Abstract The free range production system is becoming more common in Australia and is expected to increase. Free range hens are exposed to more stressors in comparison to hens from barn and cage systems and it is suggested that stress can increase bacterial shedding on eggs. The aims of this study were to examine the level of total bacteria and Enterobacteriaceae populations, as well as the presence of Salmonella and Campylobacter, in eggs collected from two free range flocks on two different farms and to conduct longitudinal observations of the behaviour and welfare of hens in the free range production system. Hen age (weeks) was shown to have a significant effect (increase) on the level of total bacteria on the egg shell surface and in shell pores, as well as having an effect on feather condition score. As the hens aged, the frequency of external visual egg characteristics increased, as did feather condition score (where feather condition was poorer). These observations indicate areas which should be investigated further to improve the food safety of eggs and optimise the welfare of free range hens.
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Key words: Eggs, bacteria, behavior, welfare, food safety, Free range poultry.
1. Introduction In 2014, the annual production of eggs in Australia was 397.4 million dozen and worth $1.7 billion. Approximately 38% of egg production is derived from the free range production system [1]. The definition of free range layer hens in Australia is; birds housed in sheds that also have access to an outdoor range [2]. Due to public perception of intensive farming systems, animal welfare and major retailer pressures, the free range production system is becoming more common in Australia and is expected to increase. Free range hens are exposed to more stressors (e.g. predators, extreme weather conditions, larger and more variable social hierarchies and potentially aggressive behaviours from other hens) in comparison to hens from barn and cage systems [3, 4]. These challenges can affect hen welfare and therefore the ability to produce eggs [5]. Welfare measures such as feather pecking, vent pecking, cannibalism, body weight and external visual egg characteristics are useful indicators of hen welfare. Results of these measures can be compared between different production systems (free range, barn, cage, aviary etc.) [3, 5]. Free range may be perceived by consumers and welfare advocates as the best option for egg production, but it has negative and positive welfare aspects for the hens [5]. The free range system increases the risk of disease transmission, injury, predation [3] and parasite occurrence [6]. The positive welfare aspects of free range systems enable the hens to exhibit a larger repertoire of behaviours, including dust bathing, foraging, experience natural light, and have an increased area outdoors in which to range, in comparison to hens in cage systems [5, 6]. Consumption of eggs and egg products is often linked to food poisoning outbreaks [7]. Total bacteria and Enterobacteriaceae populations can be used as an indicator of the food 3
safety of eggs [8]. Bacteria have the ability to move from the surface of the egg, through the cuticle, into the egg shell pores, through the egg shell membranes and into the egg internal contents (albumen and yolk). As this can occur, it is important to study bacterial count in all parts of the egg, not just the egg surface and internal contents. There is a lack of research regarding the level of egg shell pore contamination by Enterobacteriaceae. The Enterobacteriaceae family includes many genera of bacteria, one of which is Salmonella [9]. Food poisoning is often due to the bacteria Salmonella and Campylobacter. Campylobacter is commonly associated with broiler chickens and is the leading cause of bacterial food-borne illness [10], but little research has been conducted in relation to Campylobacter contamination of eggs [11]. Given its importance to food safety and the increase in free range production, it is necessary to study the prevalence of Campylobacter and Salmonella spp in eggs to potentially improve food safety [11]. The level of bacteria, when present, in the egg shell, pore and internal contents varies greatly due to many factors. Factors such as season, stress, stocking density, flock size, individual flock management, farm management, infection from pests and hygiene play major roles in the level of bacteria present in eggs [12]. Egg washing with sanitisers is a common method to minimise shell contamination however, in Australia, this practice is highly variable as it is not compulsory to wash eggs. Australia does have regulations preventing the sale of dirty and/or cracked eggs though [12]. Limited research has been conducted assessing bacterial contamination in free range eggs collected over time from the Australian commercial free range production system. The aims of this study were to; examine the level of total bacteria and Enterobacteriaceae populations, as well as the presence of Salmonella and Campylobacter, in eggs collected from two free range flocks from two farms over a period of time and to conduct longitudinal observations of the behaviour and welfare of hens in the free range production system over time. 4
2. Methods and materials 2.1 Bacterial isolation 2.1.1 Collection of eggs Eggs (n= 600) were collected from two free range flocks on two different farms. These farms were selected based on the willingness of the producers to participate in the study. Each flock was sampled five times at six week intervals from February to August (mid to late lay). Both flocks were Hy-Line Brown hens, with approximately 9,500 hens (aged 43 weeks) on Farm One and approximately 9,000 hens (aged 49 weeks) on Farm Two at the commencement of the study. Both farms complied with the stocking density allowance outlined in Model Code of Practice for the Welfare of Animals- Domestic poultry (1,500 hens per hectare) [2]. Sampling of at least 40 eggs is essential for statistically reliable determination of bacterial load in eggs [13]. Therefore, during each sampling visit, 60 visibly clean eggs, from the nest boxes, were randomly selected. 2.1.2 Shell wash For bacterial analysis, six eggs were pooled (10 samples/sampling visit) as one sample [14]. Samples were not duplicated. 2.1.2.1 Total bacterial count Six eggs were placed into one Whirl- Pak (Nasco) bag with 60 mL of Buffered Peptone Water (Oxoid Australia). Each egg was massaged by hand for 60 seconds. The eggs were removed from the bags and 100 µL of the Buffered Peptone Water egg shell wash solution was plated on nutrient agar (Oxoid Australia). The plates were incubated overnight at 37°C and colonies were counted and recorded [15]. Inoculated Buffered Peptone Water samples were stored in 80% glycerol for further characterisation of Enterobacteriaceae.
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2.1.2.2 Total Enterobacteriaceae count Using a micropipette, 100 µL of the Buffered Peptone Water egg shell wash solution was plated on violet red bile glucose agar (Oxoid, Australia). The plates were incubated overnight at 37°C and colonies were counted and recorded [14]. 2.1.2.3 Campylobacter isolation Using the Buffered Peptone Water egg shell wash solution, 100 µL was plated onto Brilliance CampyCount agar (Oxoid, Australia). Plates were placed in Compact Plastic Pouches (Oxoid, Australia), with CampyGen Compact Sachets (Oxoid, Australia) and sealed to create microaerobic conditions. After incubation at 37°C for 48 hours, colony numbers were counted and recorded. The suspect cultures were tested by Gram staining and biochemical tests such as, Gram-lysis and L-ALA tests, to confirm potential Campylobacter species. 2.1.2.4 Salmonella isolation The Buffered Peptone Water egg shell wash solution was incubated overnight at 37°C. For Salmonella isolation, 100 µL of the incubated Buffered Peptone Water was added to 10 mL Rappaport Vassiliadis Soya Peptone broth (Oxoid, Australia) and incubated overnight at 42°C. Samples were streaked on xylose lysine deoxycholate agar (Oxoid, Australia) and incubated overnight at 37°C. Suspected Salmonella colonies were confirmed by streaking on Brilliance Salmonella Agar (Oxoid, Australia) and stored in 80% glycerol for serotyping [14]. 2.1.3 Shell crush After the shell wash procedure, eggs were dipped in 70% ethanol for 90 seconds to kill egg shell surface bacteria. After air drying, the eggs (six eggs pooled as one sample) were cracked open and internal contents placed in a Whirl-Pak bag (Nasco). The shells were washed with phosphate buffered saline to remove any albumen, placed in a new Whirl-Pak bag and crushed
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thoroughly. To each bag, 60 mL of Buffered Peptone Water was added [15]. The shell crush samples were processed for bacteriological examination as stated. 2.1.4 Internal contents The internal egg contents samples were mixed thoroughly by hand. Using a 5 mL syringe, 5 mL of egg internal contents was added to 45 mL of Buffered Peptone Water in a zip-lock bag [15]. The internal contents samples were plated on nutrient agar, violet red bile glucose agar and Brilliance CampyCount agar (Oxoid, Australia), incubated as mentioned above and the bacteria counted and recorded if present. 2.1.5 Characterisation of Enterobacteriaceae A loopful of the stored inoculated Buffered Peptone Water (Oxoid Australia) was plated on violet red bile glucose agar and incubated overnight at 37°C. An individual colony was selected to be characterised using API 20E strips (Biomerieux, Australia) as per the manufacturer's instructions and results were determined using apiweb software (Biomerieux, Australia) [14]. 2.2 Welfare and behavioural observation The following hen welfare and behavioural measures were observed at the same time as the egg collection between approximately 10:00am and 1:00pm. During each sampling visit, weather conditions were also recorded. 2.2.1 Feather condition score Eighty hens inside the shed were assessed for feather condition (20 hens in each quarter of the shed). Three areas on the hen were scored, including the head and neck, back and vent, using a score of 0-2 (Table 1) to obtain a total score for the hens. Assessment was performed by selecting a random hen, counting two hens to the left or right and scoring the feather condition of that hen. Scores were assessed by observing hens from a short distance [16].
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2.2.2 Submissive/crouching behaviour Submissive behaviour of the hens was assessed by walking slowly through the shed and counting the number of hens within one metre of the observer which crouched on approach of the observer. The number of hens displaying the behaviour was counted in each quarter of the shed. 2.2.3 External visual egg shell characteristics The external visual egg shell characteristics from the sample of 60 eggs were recorded using the terms: misshapen, light calcium dusting, heavy calcium dusting, blood stains, white bands and pink colouring (Table 2) [17]. 2.3 Statistical analysis Univariate general linear models using IBM SPSS Statistics version 22 [18] were used to determine the variation in the load of total bacteria and Enterobacteriaceae (log 10 transformed colony forming unit (CFU)) populations in and on eggs, to examine changes in external visual egg characteristics and changes in submissive/crouching behaviour, during the period of the longitudinal samplings. The same analysis was also used to examine feather condition data, which were transformed by square root to minimise variation in the data. Significance was tested at the P<0.05 level.
3. Results 3.1 Total bacteria- egg shell surface The average level of bacteria isolated from the egg shell surfaces from Farm One was 3.84 log CFU/egg shell (Figure 1). The average level of bacteria isolated from the egg shell surfaces from Farm Two was 3.69 log CFU/egg shell. Table 3 shows raw data for average number of bacterial colonies per egg shell. Statistical analysis indicated there was a significant effect of age (weeks) on the total bacteria count. For each week increase in age, the amount of total 8
bacteria on the egg shell surface increased by 0.0145 log CFU/egg shell for Farm One and 0.014 log CFU/egg shell for Farm Two (P=0.016). 3.2 Total bacteria- egg shell pores The level of total bacteria in the shell pores increased during the study (Figure 2). The average level of bacteria isolated from shell pores from Farm One was 0.52 log CFU/egg shell and 0.40 log CFU/egg shell from Farm Two. Statistical analysis indicated there was a significant effect of age (weeks) on the total bacteria in egg shell pores (P=0.002). For each week increase of age, the amount of bacteria in the shell pores increased by 0.032 log CFU/egg shell for Farm One and 0.038 for Farm Two. Eleven samples from Farm One and eight from Farm Two were positive. One positive total bacteria internal contents sample was isolated from Farm One and four from Farm Two. 3.3 Enterobacteriaceae- egg shell surface The level of Enterobacteriaceae in the shell wash was not significantly different (P=0.737) between samplings during the study. The average level of Enterobacteriaceae isolated from the egg shell surface from Farm One was 0.98 log CFU/egg shell and 0.68 log CFU/egg shell from Farm Two. Statistical analysis indicated that there was no significant effect of age on the level of bacteria present on the egg shells. The level of Enterobacteriaceae on eggs decreased on Farm One at a rate of 0.021 log CFU/egg shell per week and slowly increased each week at 0.011 log CFU/egg shell on eggs from Farm Two. 3.4 Isolated Enterobacteriaceae species Seven different species of Enterobacteriaceae were identified on the two farms. The apiweb software (Biomerieux, Australia) was unable to identify two samples from each farm and these were classed as inconclusive samples. Escherichia coli were the most frequently isolated species of Enterobacteriaceae from both Farm One and Farm Two. Escherichia hermannii and 9
Kluyvera spp were isolated from Farm One only, whilst Proteus mirabilis and Klebsiella pneumoniae were isolated from Farm Two only. Two positive Enterobacteriaceae shell crush samples were isolated from Farm One and three from Farm Two. These were all Escherichia coli. Campylobacter spp was not isolated from shell wash, shell crush or internal contents samples. Salmonella Mbandaka was isolated from one shell crush sample from Farm Two. Between the two farms, 90% (n=100) of the pooled egg shell wash samples and 5% (n=100) of the pooled egg shell crush samples were contaminated with Enterobacteriaceae. 3.5 External visual egg shell characteristics There was no significant effect of age (weeks) on the external visual egg characteristics from Farm One and Two. However, the percentage of lightly calcium dusted eggs increased, as did the presence of heavily calcium dusted eggs in late lay on both farms. Egg characteristics, such as blood stains, misshapen and pink eggs were also observed more often towards late lay, however the occurrence of these characteristics were not found to be statistically significant. 3.6 Feather condition score The feather condition scores of both flocks increased over time indicating poorer feather condition. For each week increase in hen age, the feather condition score increased by 0.031 for Farm One and 0.014 for Farm Two (P<0.001). 3.7 Submissive/crouching behaviour The percentage of both flocks exhibiting submissive behaviour decreased during the study (Figure 3). For each week increase of age, the percentage of the flock exhibiting submissive/crouching behaviour decreased by 0.011% for Farm One and 0.007% for Farm Two (P=0.005). 4. Discussion
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The aims of this study were to examine the level of total bacteria and Enterobacteriaceae populations in eggs collected from two commercial free range flocks from two farms over time and to conduct longitudinal observations of the behaviour and welfare of hens in the free range production system. The presence of Salmonella and Campylobacter in the eggs was also examined. Campylobacter was not isolated and only one positive Salmonella sample (Salmonella Mbandaka) was identified. Hen age (weeks) was shown to have a significant effect on the level of total bacteria on the egg shell surface and in shell pores, as well as having an effect on feather condition score and submissive/crouching behaviour. On both farms feather condition score increased (feather condition was poorer) and the percentage of the flock exhibiting submissive/crouching behaviour decreased. Table 3 showing average number of bacterial colonies per egg provides an indicator of the level of which bacteria was present on and in eggs used in this study and is useful information in regards to food safety. 4.1 Total bacteria- egg shell surface A larger population of bacteria on the surface of the shell can increase the likelihood of penetration of bacteria into egg shell pores and internal contents if eggs are not handled correctly [14, 19]. Total bacteria and Enterobacteriaceae populations can also be used as an indicator of food safety of eggs. Bacteria on the surface of egg shells are commonly found in the air, water and soil, so the types vary due to the environment of the egg production facility [20]. In previous studies, there are conflicting results in the level of total bacteria contamination in both alternative (barn and free range) and conventional (cage) egg production systems [21]. This study found a statistically significant age (weeks) effect on the level of total bacteria found on the egg shell surface as was reported earlier [15]. The average level of bacteria isolated from the surface of the egg shells from Farm One during the study was 3.84 log CFU/egg shell and 3.69 log CFU/egg shell for Farm Two. These results are similar to previous studies [15], who reported an average of 3.48 log CFU/egg shell for total bacteria on free range eggs. Another 11
study investigating free range and cage production system eggs collected during the same seasons (autumn and winter) found total bacteria levels on the surface of free range eggs of 3.2 log CFU/mL for free range eggs and 3.6 log CFU/mL for caged eggs [22]. These results are lower in comparison to a previous study [21], but comparable to the present study. Another study investigating bacterial contamination of free range eggs and caged eggs found very high levels of total bacteria, 5.55 log CFU/mL for free range eggs and 5.23 log CFU/mL for cage eggs [21]. Results may differ between studies due to differing isolation techniques, age and breed of hens, climate and country, hygiene and farm management. 4.2 Total bacteria- egg shell pores There was a significant age (weeks) effect on the level of total bacteria in egg shell pores, consistent with previous findings [15]. The results of this study (Farm One 0.40 log CFU/egg shell and Farm Two 0.52 log CFU/egg shell) are lower than results reported previously [15], who observed bacterial levels in egg shell pores of 1.27 log CFU/egg shell in free range eggs and 1.21 log CFU/egg shell in caged eggs. It is likely these results differ due to the quality of the egg shell and the cuticle, which aids in preventing bacteria entering the egg shell pores [7], however results may also differ due to egg handling practices, isolation techniques, farm practices and hygiene. 4.3 Enterobacteriaceae- egg shell surface The average level of Enterobacteriaceae isolated from the egg shell surface from Farm One was 0.98 log CFU/egg shell and 0.68 log CFU/egg shell from Farm Two. There was no significant effect of age on the level of bacteria present on the egg shells. However, previous study has reported a significant effect of age on increasing levels of Enterobacteriaceae on the egg shell surface [15]. The level of Enterobacteriaceae isolated in this study was low in comparison to other studies. Previous study by Gole et al. [14] found an average count of 1.46
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log CFU/egg shell in caged eggs was in agreement with De Reu et al. [23] (1.51 log CFU/egg shell). De Reu et al. [23] also reported an average log CFU/egg shell of 1.54 present in noncage eggs. Another study found the average level of Enterobacteriaceae on the egg shell surface of free range eggs to be 1.5 log CFU/egg shell [15]. All of these results differ from those observed previously [21], who showed an average of 4.38 log CFU/mL for free range eggs and 3.05 log CFU/mL for caged eggs. Violet red bile glucose agar (Oxoid, Australia) was used for detection and enumeration of Enterobacteriaceae in this study. Results may have differed from other studies due to the isolation technique used. Bacteria present may not have recovered sufficiently to grow proficiently on the selective media. 4.4 Enterobacteriaceae species Escherichia coli, Enterobacter cloacae, Escherichia fergusonii, Kluyvera spp. and Klebsiella pneumoniae have been isolated during previous studies investigating Enterobacteriaceae contamination of eggs [9, 13, 23] and many others have also been isolated from egg production facilities [20]. Proteus mirabilis and Escherichia hermannii have previously been isolated from the surface of egg shells [24]. Escherichia coli were the most commonly isolated bacterial species from both Farm One and Two (60.0% and 82.22%). A caged layer hen study also reported Escherichia genus most frequently (60.8%) [14]. In that study, Enterobacter was the third most isolated genus and in this study, Enterobacter cloacae (24%) was the second most frequently isolated bacteria for Farm One followed by Escherichia hermannii (8%). All other species for both farms were identified less frequently (<5%), as was the case with Gole et al. [14]. The Escherichia genus was also the most commonly isolated bacteria in a study by Musgrove et al. [9]. Escherichia coli, the most frequently isolated Enterobacteriaceae in this study, is commonly a source of food borne illness and is recognised as an important human pathogen [25, 26]. Salmonella Mbandaka was identified from one shell crush sample from Farm Two. Salmonella belong to the Enterobacteriaceae family and 13
initially, when testing for Enterobacteriaceae, this sample was negative. As Salmonella isolation occurs after enrichment of the Buffered Peptone Water solution, the bacteria were able to recover and grow. Therefore, when the sample was positive when tested for Salmonella presence. 4.5 External visual egg shell characteristics Stress can cause deformities in eggs. For example, hens may delay laying an egg if stressed, which can result in extra calcium carbonate forming on the egg shell or other shell deformities. Blood stains may also be present on egg shells due to prolapse or tearing of the vent due to laying an overly large egg [5]. Viral infections also cause shell deformities and affect egg quality therefore; hens in this study were vaccinated against infectious bronchitis virus and egg drop syndrome. There was no significant effect of age (weeks) on the external visual egg characteristics for either farm. However, the percentage of lightly calcium dusted eggs increased and observations of heavily calcium dusted eggs occurred in late lay on both farms. Other external visual egg characteristics, including blood stains, misshapen and pink eggs, were also observed more often towards late lay. A study of 25 free range farms combined pink and calcium dusted egg data, classed as “off colour” and found an occurrence of 0.44%, lower than that observed in this study [27]. Sherwin et al. [5] reported 1.7% calcium spots and 1.4% blood stains on free range eggs in their study. In Sherwin et al. [5], producers were asked to examine and record external visual egg characteristics. This may have contributed to the low averages, as farmers may have been biased when reporting the findings. 4.6 Feather condition Feather pecking has been commonly observed in larger free range flocks, when a large percentage of the flock does not range [28]. Feather pecking is worse in sparse environments that are void of stimuli that can distract hens from extreme feather pecking [29, 30]. During
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this study, the feather condition of the hens deteriorated, with a significant effect of age (weeks) on feather condition score for both farms. On farms where feather pecking occurs, it generally increases with age, as it is a learnt behaviour. Feather pecking can be hard to control once it has begun, therefore, the feather condition of the hens worsens [31], as was the case in the present study. Whay et al. [27] also observed an increase in the degeneration of feather condition over time, as well as an increase in the number of hens subject to feather loss, as was observed in this study. On each of the farms, more hens with feather damage and higher feather condition scores were observed over time. While it is the most common reason for poor feather condition, feather pecking is not the only cause. It may also be due to moulting or abrasive surfaces in the environment [29]. As a result, it cannot be concluded feather pecking is the sole cause of feather degeneration in this study. 4.7 Submissive/crouching behaviour The submissive crouching behaviour exhibited by hens is a sign of sexual maturity, sexual receptivity, submission to dominant hens and fear [31]. Submissive behaviour is a novel measure of welfare, which has not been investigated in depth. Hens can often be observed exhibiting this crouching behaviour due to the approach of a person. A person can be perceived as a predator and assuming this crouching submissive posture or freezing could potentially reduce the likelihood of being seen by a predator (person) [32]. There was a significant effect of age on the percentage of hens on both farms exhibiting submissive behaviour, resulting in a decrease of the behaviour being observed. The decrease is most likely due to hens habituating to the presence of an observer. The free range production system is becoming a major source of egg production in Australia and other parts of world. Longitudinal studies are helpful to understand the level of bacterial shedding. Hens raised in a free range production system could be exposed to environmental
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stressors, which could induce bacterial shedding. In Australia, USA and Japan, commercial table eggs are washed [12] to reduce bacterial load. However, the presence of bacteria inside shell pores could still pose a challenge if eggs are not handled correctly. The presence of Escherichia coli and Enterobacteriaceae have been used as quality and safety indicators in food chains [8]. There are no established levels or regulatory thresholds of such indicator organisms in the egg industry in Australia. Findings from this study could be helpful for establishing base levels of indicator organisms, but large scale longitudinal studies are required in the future. Significant associations could not be made between behaviour and bacterial shedding due to limited sample size. Further studies could be conducted for objective measurement of stress (such as measuring level of corticosterone) along with hen behaviour and bacterial shedding. 5. Conclusion Hen age was shown to have a significant effect on the level of total bacteria on the egg shell surface and in shell pores, as well as having an effect on feather condition score and submissive/crouching behaviour. Escherichia coli were the most frequently isolated Enterobacteriaceae species from both farms. Limited research has been conducted examining bacterial load in eggs from commercial free range farms in Australia therefore; these observations are important to the industry as they may indicate areas which should be investigated further to improve food safety of eggs and optimise the welfare of free range hens in Australia. As there were only two farms in this study, no significant associations between levels of bacteria, hen behaviour and welfare were able to be made. The bacteria and behavioural observations need to be further investigated on a larger scale to obtain substantial results. Two farms were not enough to obtain significant results, from which conclusions could be drawn.
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Sampling farms more often (monthly/weekly) would be preferable, as this would provide more in depth data in regards to the bacteria, hen behaviours and welfare. Further research also needs to be conducted to demonstrate the link between behaviours and/or welfare indicators and stress in free range hens. This could be achieved by using additional physiological measures of stress such as corticosterone, and heterophil to lymphocyte ratio [28]. Acknowledgements This research was funded by the Poultry Cooperative Research Centre and The University of Adelaide. Thanks to Dr Michelle Hebart for her assistance with statistical analysis.
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References [1] Australian Egg Corporation Limited (AECL), Australian Egg Corporation Annual Report. http://www.aecl.org/assets/Uploads/Annual-Reports/AECL-Annual-Report-2014.pdf,
2014
(accessed 02.03015). [2] CSIRO. Primary Industries Standing Committee Model Code of Practice for the Welfare of Animals Domestic Poultry 4th Edition. Appendix 2. Collingwood, VIC, AUS: CSIRO Publishing; 2002. p. 27-8. [3] Daigle C, Siegford J. Welfare Quality® parameters do not always reflect hen behaviour across the lay cycle in non-cage laying hens. Anim. Welf. 2014;23:423-434. [4] Walker A, Hughes B. Egg shell colour is affected by laying cage design. Br. Poult Sci. 1998;39:696-699. [5] Sherwin C, Richards G, Nicol C. Comparison of the welfare of layer hens in 4 housing systems in the UK. Br. Poult. Sci. 2010;51:488-499. [6] Tauson R. Management and housing systems for layers–effects on welfare and production. World's Poult. Sci. J. 2005;61:477-490. [7] De Reu K. Bacteriological contamination and infection of shell eggs in the production chain: University of Ghent, Belgium; 2006. [8] Kornacki JL, Johnson JL. Enterobacteriaceae, coliforms, and Escherichia coli as quality and safety indicators. In: Downes FP, Ito K, (Eds.), Compendium of Methods for the Microbiological Examination of Foods. 4th ed. American Public Health Association, Washington, DC, 2001, p. 69-82. [9] Musgrove MT, Jones DR, Northcutt JK, Cox NA, Harrison MA. Identification of Enterobacteriaceae from washed and unwashed commercial shell eggs. J. of Food Protection®. 2004;67:2613-2616.
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[10] Sahin O, Kobalka P, Zhang Q. Detection and survival of Campylobacter in chicken eggs. J. of Appl. Microbiol. 2003;95:1070-1079. [11] Messelhäusser U, Thärigen D, Elmer-Englhard D, Bauer H, Schreiner H, Höller C. Occurrence of thermotolerant Campylobacter spp. on eggshells: a missing link for food-borne infections? Appl. and Environ. Microbiol. 2011;77:3896-3897. [12] Chousalkar K, Sexton M, McWhorter A, Hewson K, Martin G, Shadbolt C, Paul Goldsmith. Salmonella Typhimurium in the Australian Egg Industry: Multidisciplinary Approach to Addressing the Public Health Challenge and Future Directions. Crit. Rev. Food Sci. Nutr. 2015. [13] De Reu K, Grijspeerdt K, Heyndrickx M, Zoons J, De Baere K, Uyttendaele M, Debevere J, Herman L. Bacterial eggshell contamination in conventional cages, furnished cages and aviary housing systems for laying hens. Br. Poult. Sci. 2005;46:149-155. [14] Gole V, Chousalkar K, Roberts J. Survey of Enterobacteriaceae contamination of table eggs collected from layer flocks in Australia. Int. J. of Food Microbiol. 2013;164:161-165. [15] Samiullah S, Roberts J, Chousalkar K. Effect of production system and flock age on egg quality and total bacterial load in commercial laying hens. The J. of Appl. Poult. Res. 2014;23:59-70. [16] Bristol University, RSPCA, Soil Association, Laying hens assessment protocol. http://www.assurewel.org/layinghens, 2013 (accessed 24.11.15). [17] Hughes B, Gilbert A, Brown MF. Categorisation and causes of abnormal egg shells: relationship with stress. Br. Poult. Sci. 1986;27:325-337. [18] IBM Corporporation . IBM SPSS Statistics for Windows. 22.0 ed. Armonk, NY: IBM Corp; 2013.
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[19] Smith A, Rose S, Wells R, Pirgozliev V. The effect of changing the excreta moisture of caged laying hens on the excreta and microbial contamination of their egg shells. Br. Poult. Sci. 2000;41:168-173. [20] Musgrove M, Jones D, Shaw J, Sheppard M, Harrison M. Enterobacteriaceae and related organisms isolated from nest run cart shelves in commercial shell egg processing facilities. Poult. Sci. 2009;88:2113-2117. [21] Parisi M, Northcutt J, Smith D, Steinberg E, Dawson P. Microbiological contamination of shell eggs produced in conventional and free-range housing systems. Food Control. 2015;47:161-165. [22] Jones D, Anderson K, Musgrove M. Comparison of environmental and egg microbiology associated with conventional and free-range laying hen management. Poult. Sci. 2011;90:20632068. [23] De Reu K, Rodenburg T, Grijspeerdt K, Messens W, Heyndrickx M, Tuyttens F, et al. Bacteriological contamination, dirt, and cracks of eggshells in furnished cages and noncage systems for laying hens: An international on-farm comparison. Poult. Sci. 2009;88:2442-2448. [24] Stępień-Pyśniak D. Occurrence of Gram-negative bacteria in hens' eggs depending on their source and storage conditions. Pol. J. of Vet. Sci. 2010;13:507-513. [25] Gorman R, Bloomfield S, Adley CC. A study of cross-contamination of food-borne pathogens in the domestic kitchen in the Republic of Ireland. Int. J of Food Microbiol. 2002;76:143-150. [26] Tauxe RV. Emerging foodborne diseases: an evolving public health challenge. Emerg. Infect. Dis. 1997;3:425. [27] Whay H, Main D, Green LE, Heaven G, Howell H, Morgan M, et al. Assessment of the behaviour and welfare of laying hens on free-range units. The Vet. Rec. 2007;161:119-128.
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[28] Lay D, Fulton R, Hester P, Karcher D, Kjaer J, Mench J, et al. Hen welfare in different housing systems. Poult. Sci. 2011;90:278-294. [29] Appleby MC, Hughes BO. Welfare of laying hens in cages and alternative systems: environmental, physical and behavioural aspects. World's Poult. Sci. J. 1991;47:109-128. [30] Blokhuis H. The effect of a sudden change in floor type on pecking behaviour in chicks. Appl. Anim. Behav. Sci. 1989;22:65-73. [31] Nicol C. Behavioural contributions to welfare assessment. In: Nicol C (Eds.), The behavioural biology of chickens. CABI, Wallingford, UK, 2015, p. 58-78. [32] Jones RB. Fear and adaptability in poultry: insights, implications and imperatives. World's Poult. Sci. J. 1996;52:131-174.
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5 4.5
log CFU
4 3.5 3 2.5 2 40
45
50
55
60
65
70
75
Age (weeks) Farm 1
Farm 2
Figure 1. Average level of total bacteria (log CFU/egg shell) in egg shell wash from two free range farms, Farm One and Farm Two over the period of the study (age-weeks). CFU=colony forming unit. Farm One n=300 eggs and Farm Two n=300 eggs.
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1.8 1.6 1.4
log CFU
1.2 1 0.8 0.6 0.4 0.2 0 40
45
50
55
60
65
70
75
Age (weeks) Farm 1
Farm 2
Figure 2. Average level of total bacteria (log CFU/egg shell) in egg shell pores from two free range farms, Farm One and Farm Two, over the period of the study (age-weeks). CFU=colony forming unit. Farm One n=300 eggs and Farm Two n=300 eggs.
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0.6
Percentage %
0.5 0.4 0.3
Farm 1 Farm 2
0.2 0.1 0 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 Age (weeks)
Figure 3. Percentage of the flock from two free range farms, Farm One and Farm Two, exhibiting submissive/crouching behaviour over the period of the study (age-weeks).
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Table 1. Description of the feather scoring system used to assess the head and neck, back and vent area of the hens observed on two free range farms, Farm One and Farm Two. Feather score 0 1 2
Description No or minimal feather loss (no bare skin visible, no or slight feather wear, only single feathers missing). Moderate feather loss (moderate feather wear, i.e. damaged feather or one or more featherless areas, bare skin visible <5 cm maximum dimension). Severe feather loss (at least one area with bare skin visible ≥5 cm maximum dimension).
Table 2. Description of external visual egg characteristics observed on eggs collected from two free range farms, Farm One and Farm Two, over the period of the study (age-weeks) [17]. Egg shell Description characteristic An egg that is not the normal shape and size (It may be too small or large or Misshapen round instead of oval). They generally look round at one end of the egg and pointy at the other end. Light calcium Small, irregular shaped white spots deposited on the external surface of the shell, in small numbers covering a small proportion of the shell. dusting Larger, irregular shaped white spots deposited on the external surface of the Heaving calcium dusting shell, in great numbers covering a large proportion of the shell Smears of blood on the external surface of the shell. Blood stains The egg appears to be a pink colour due to a very fine calcium layer. Pink
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Table 3: Raw total bacterial and Enterobacteriaceae data. Average number of colonies per egg of total bacterial shell wash (TB SW), shell crush (TB SC) and internal contents (TB IC) and Enterobacteriaceae shell wash (E SW) and shell crush (E SC), for two free range farms, Farm One and Farm Two. Standard error in brackets. Age (wks) Farm ID
43
49
50
54
56
60
62
66
69
71
TB SW (n=60)
1 10890 (3197)
2 8660 (1325)
1 4200 (2890)
2 3570 (1134)
1 11840 (3144)
2 9620 (2918)
1 25510 (1992)
2 1210 (222)
1 9190 (2608)
2 29490 (510)
E SW (n=60) TB SC (n=60) E SC (n=60) TB IC (n=60)
3470 (2777) 0 (0) 0 (0) 0 (0)
1630 (1619) 0 (0) 0 (0) 0 (0)
3000 (3000) 0 (0) 0 (0) 0 (0)
200 (13) 10 (10) 0 (0) 0 (0)
600 (34) 170 (68) 10 (10) 20 (20)
220 (100) 60 (43) 40 (40) 30 (30)
2550 (1122) 50 (85) 0 (0) 0 (0)
0 (0) 20 (20) 0 (0) 10 (10)
0 (0) 90 (55) 10 (10) 0 (0)
120 (53) 230 (123) 60 (40) 3020 (2998)
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