Microbiological Status of Broiler Respiratory Tracts Before and During Catching for Transport to the Processing Plant

Microbiological Status of Broiler Respiratory Tracts Before and During Catching for Transport to... Abstract A significant point of entry for Salmonella into a processing plant is within the broilers to be processed. Prior to transport to the processing plant, feed (4 h) and water are withdrawn from the broilers on the farm before they are caught and cooped. During catching, an increased presence of dust in the house air is visible and may affect the presence of bacteria within the broiler's respiratory tract. The objective of this study was to examine the effect of catching on the levels of aerobic bacteria (aerobic plate count [APC]), levels and prevalence of Enterobacteriaceae (EB), and prevalence of Salmonella within broiler respiratory tracts. To determine flock Salmonella status 1 wk prior to catching, broiler carcasses were sampled for APC, EB, and Salmonella by respiratory tract flushing and ceca were sampled for Salmonella. At 1 d prior to catching and when half of the broilers in the house were caught, broilers were again collected, transported to the pilot plant, euthanized, and sampled. In Trial 1, there were no significant differences between sampling times for ceca Salmonella or respiratory EB and APC. However, Salmonella prevalence in the respiratory tract was significantly higher at 1 wk preharvest compared to during catching. In Trial 2, EB was significantly lower during catching compared to 1 wk preharvest. No significant differences were detected in Trial 3. Based on these results, the presence of aerosolized dust in the broiler house during catching does not appear to lead to increases in broiler respiratory tract microbial contamination. DESCRIPTION OF PROBLEM When present, Salmonella can be detected from multiple locations within a broiler growout house, including both litter and dust [1,2]. Previous work sampling turkey flocks has indicated that Salmonella can be found in dust with the same frequency (41 to 58%) as boot swabs or litter sampling [2]. Broiler house dust contains a mixture of dander, litter, feed, feces, and microbes, with the vast majority of dust containing microbial DNA [3]. Generally, catch crew workers will wear respiratory protective equipment during broiler harvest (catching) to minimize the amount of dust they inhale. However, the broilers do not have any external respiratory protection other than the increased mechanical exhaust ventilation during harvest. Therefore, it was anticipated that microbes in increased levels of dust in the broiler house air may be inhaled by the broilers during catching and accumulate in the respiratory tract. The broiler respiratory tract is a potential reservoir for Salmonella. Several studies have demonstrated that inoculating broilers within the trachea with Salmonella leads to further intestinal colonization [4–6]. Intratracheal inoculations of Salmonella led to colonization as well as or more effectively than oral inoculation [6] when broilers were full fed. In work comparing 5 inoculation routes (oral, intratracheal, subcutaneous, ocular, and cloacal), the intratracheal inoculation route led to intestinal tract colonization significantly more frequently than all other routes [4]. Salmonella present in the litter may be transported on increased dust in the air during catching and enter into broiler respiratory tracts. The transfer of Salmonella from the litter or house dust to the broiler respiratory tract (specifically the trachea) may potentially have further impact in the processing plant on broiler product Salmonella status. The objective of this study was to examine the effect of catching on the levels of aerobic bacteria (aerobic plate count [APC]), the levels and prevalence of Enterobacteriaceae (EB), and the prevalence of Salmonella within broiler respiratory tracts. MATERIALS AND METHODS Experimental Design The broilers and standard operating procedures used in this study were covered by an animal use proposal approved by the US National Poultry Research Center IACUC. To assess the effect of aerosolized dust during broiler catching on the levels APC, levels and prevalence of EB, and prevalence of Salmonella within broiler respiratory tracts, broilers were sampled at 3 different time points. For all trials broilers and litter were first sampled at 1 wk preharvest to confirm the presence of Salmonella in the commercial broiler house and flock prior to further sampling. If the house litter and flock were confirmed to be Salmonella-positive at 1 wk preharvest, then at 1 d preharvest broilers were again sampled to determine the levels and/or prevalence of bacteria prior to catching. On the next day, broilers were sampled during catching after approximately half of the house had been caught and the last transport coop for an entire trailer (22 coops/trailer) was leaving the house. Results from the during catching sampling time were compared to the 1 d preharvest sampling results in order to determine if dust in the air during catching aerosolized from the litter and house would impact broiler respiratory tract microbiology. At all 3 sampling times, male broilers were caught individually with a leg hook and from the middle of the house to minimize exciting the flock and elevating dust levels in the air. Litter and Broiler Sampling For each of 3 trials, commercial broiler house litter (between the water and feed lines) was sampled using intermittently stepped on drag swabs 1 wk prior to catching for transport to a processing plant [7,8]. Following collection, drag swab samples were tested for the prevalence of Salmonella [9]. In addition to litter sampling, 20 male broilers per trial (60 total) were individually caught, cooped, and transported less than 1 h to the pilot processing plant for sampling. In Trial 1, the 20 broilers were collected from 4 commercial houses (5 broilers/house) at 5 wk of age for the 1-wk preharvest sampling time and at 6 wk of age for the 1-d preharvest and during catching sampling times. In Trials 2 and 3, 10 broilers were collected from each of 2 adjacent commercial houses. For Trials 2 and 3 broilers were 6 wk of age at the 1-wk preharvest sampling time and at 7 wk of age for the 1-d preharvest and during catching times. Each trial was conducted with different flocks on different commercial farms. Following arrival at the pilot processing plant, broilers were individually euthanized by electrocution prior to sampling within 2 h of catching. Respiratory tracts were sampled as previously described [10,11]. Briefly, the carcass neck feathers and skin were sprayed with ethanol and the neck skin cut about 10 cm (approximately mid-neck) to expose the underlying trachea. Then the trachea was partially cut for insertion of plastic tubing (toward the syrinx) attached to a syringe containing 60 mL of 1% Buffered Peptone Water (BPW). During sampling, the tubing was secured within the trachea with a small cable tie. The BPW was slowly introduced into the respiratory tract, the carcass inverted 30 times, the carcass was hung in a shackle by the feet, and then the rinse was collected back into the same syringe. Rinsates were analyzed for APCs, EB counts and prevalence, and Salmonella prevalence [11,12]. Following respiratory tract sampling, the abdominal feathers and skin were sprayed with ethanol and opened for aseptic removal of ceca for Salmonella prevalence analysis [13,14]. RESULTS AND DISCUSSION Only the broiler houses with litter positive for Salmonella were sampled at all 3 time points (1 wk prior to catching, 1 d prior to catching, and during catching) and are reported in this study. Results from microbiological sampling of broiler respiratory tracts and ceca are shown in Table 1. There were no significant differences between the sampling time points for cecal Salmonella prevalence (50 to 60% positive). In Trial 1, broilers collected during catching had a significantly lower prevalence (20%) of Salmonella in the respiratory tract rinsate than broilers collected 1 wk prior to slaughter (55%). Broilers collected 1 d prior to slaughter were intermediate (50%). This significant difference was not observed in Trials 2 and 3, or overall. In Trial 2, respiratory tract EB counts were significantly higher (2.17 log10) for broilers collected during catching than 1 wk prior to slaughter (0.58 log10); however, this difference was not seen in Trials 1, 3, or overall. Enterobacteriaceae prevalence and APCs were not significantly different between sampling times for any trial or overall (EB 0.98 to 1.77 log10; APC 3.03 to 3.19 log10). Table 1. Broiler Respiratory Tract and Ceca Microbiology Prior to and During Broiler Catching. Respiratory tracts Sampling time Ceca Salmonella Prevalence Salmonella Prevalence EB1 Prevalence EB Counts3 APC2 Counts Trial 1  Preharvest 1 wk 17/20 11/20A 8/20 1.80 2.77  Preharvest 1 d 12/20 10/20A,B 8/20 0.68 2.83  During catching 11/20 4/20B 5/20 <0.01 3.11 Trial 2  Preharvest 1 wk 14/20 2/20 6/20 0.52A 2.77  Preharvest 1 d 13/20 0/20 2/20 0.58A,B 2.78  During catching 11/20 3/20 3/20 2.17B 2.49 Trial 3  Preharvest 1 wk 5/20 0/20 5/20 2.08 3.76  Preharvest 1 d 7/20 0/20 7/20 1.43 3.48  During catching 8/20 1/20 8/20 2.74 4.00 Overall  Preharvest 1 wk 36/60 13/60 19/60 1.47 3.10  Preharvest 1 d 32/60 10/60 17/60 0.98 3.03  During catching 30/60 8/60 16/60 1.77 3.19 Respiratory tracts Sampling time Ceca Salmonella Prevalence Salmonella Prevalence EB1 Prevalence EB Counts3 APC2 Counts Trial 1  Preharvest 1 wk 17/20 11/20A 8/20 1.80 2.77  Preharvest 1 d 12/20 10/20A,B 8/20 0.68 2.83  During catching 11/20 4/20B 5/20 <0.01 3.11 Trial 2  Preharvest 1 wk 14/20 2/20 6/20 0.52A 2.77  Preharvest 1 d 13/20 0/20 2/20 0.58A,B 2.78  During catching 11/20 3/20 3/20 2.17B 2.49 Trial 3  Preharvest 1 wk 5/20 0/20 5/20 2.08 3.76  Preharvest 1 d 7/20 0/20 7/20 1.43 3.48  During catching 8/20 1/20 8/20 2.74 4.00 Overall  Preharvest 1 wk 36/60 13/60 19/60 1.47 3.10  Preharvest 1 d 32/60 10/60 17/60 0.98 3.03  During catching 30/60 8/60 16/60 1.77 3.19 1 EB = Enterobacteriaceae. 2 APC = Aerobic plate count. 3 Counts are reported as Log10 CFU/mL. A,B Values within a Trial and within a column with differing superscripts are significantly different (P ≤ 0.05). View Large Table 1. Broiler Respiratory Tract and Ceca Microbiology Prior to and During Broiler Catching. Respiratory tracts Sampling time Ceca Salmonella Prevalence Salmonella Prevalence EB1 Prevalence EB Counts3 APC2 Counts Trial 1  Preharvest 1 wk 17/20 11/20A 8/20 1.80 2.77  Preharvest 1 d 12/20 10/20A,B 8/20 0.68 2.83  During catching 11/20 4/20B 5/20 <0.01 3.11 Trial 2  Preharvest 1 wk 14/20 2/20 6/20 0.52A 2.77  Preharvest 1 d 13/20 0/20 2/20 0.58A,B 2.78  During catching 11/20 3/20 3/20 2.17B 2.49 Trial 3  Preharvest 1 wk 5/20 0/20 5/20 2.08 3.76  Preharvest 1 d 7/20 0/20 7/20 1.43 3.48  During catching 8/20 1/20 8/20 2.74 4.00 Overall  Preharvest 1 wk 36/60 13/60 19/60 1.47 3.10  Preharvest 1 d 32/60 10/60 17/60 0.98 3.03  During catching 30/60 8/60 16/60 1.77 3.19 Respiratory tracts Sampling time Ceca Salmonella Prevalence Salmonella Prevalence EB1 Prevalence EB Counts3 APC2 Counts Trial 1  Preharvest 1 wk 17/20 11/20A 8/20 1.80 2.77  Preharvest 1 d 12/20 10/20A,B 8/20 0.68 2.83  During catching 11/20 4/20B 5/20 <0.01 3.11 Trial 2  Preharvest 1 wk 14/20 2/20 6/20 0.52A 2.77  Preharvest 1 d 13/20 0/20 2/20 0.58A,B 2.78  During catching 11/20 3/20 3/20 2.17B 2.49 Trial 3  Preharvest 1 wk 5/20 0/20 5/20 2.08 3.76  Preharvest 1 d 7/20 0/20 7/20 1.43 3.48  During catching 8/20 1/20 8/20 2.74 4.00 Overall  Preharvest 1 wk 36/60 13/60 19/60 1.47 3.10  Preharvest 1 d 32/60 10/60 17/60 0.98 3.03  During catching 30/60 8/60 16/60 1.77 3.19 1 EB = Enterobacteriaceae. 2 APC = Aerobic plate count. 3 Counts are reported as Log10 CFU/mL. A,B Values within a Trial and within a column with differing superscripts are significantly different (P ≤ 0.05). View Large Broiler litter Salmonella recovery has been shown to peak around 3 to 4 wk and diminish thereafter [15–17]. The lack of significant difference between ceca Salmonella prevalence was not unexpected because of the short time between sampling time points (6 or 7 d). However, the presence of Salmonella in 25 to 85% of the broiler ceca sampled indicated that Salmonella was likely continuously being shed into the litter. The respiratory tract results from Trial 1 in this study suggest that respiratory tract Salmonella prevalence may decrease with catching, but this result was not consistent in the later 2 trials. One possible explanation for the inconsistency between trials could be the lower combined prevalence of respiratory tract Salmonella detected in Trials 2 and 3 (8%, 5/60 and 2%, 1/60, respectively) compared to the higher combined prevalence in Trial 1 (42%, 25/60). Potential differing levels of Salmonella in the house litter, differing times of year (April, August, May), age (5 to 6 wk vs 6 to 7 wk), and differing housing construction and ventilation may all have contributed to the inconsistency among trials. There are also inconsistent results in previous studies evaluating dust concentrations in broiler houses ranging from 0.03 to 6.5 mg/m3 [18,19]. These differences were attributed to sampling time, bird activity, and house ventilation differences due to ambient temperature [19]. In caged turkeys exposed to Salmonella contaminated fecal dust for 2 or 4 h (105 cfu/g), Harbaugh [20] reported very low recovery (≥1/16 turkeys) from respiratory tract samples (nasal passages, infraorbital sinuses, trachea, or air sacs); and when the dust exposure level was elevated to 107 or 109 cfu/g then 0 to 6/8 turkeys had positive samples. In contrast, lungs (31 to 94% positive) and small intestines (44 to 88% positive) had very high recovery of Salmonella at all 3 dust exposure levels. Cross contamination from Salmonella-inoculated turkeys to non-inoculated turkeys by fans was less successful with the highest recovery of 14% for the respiratory tract samples but no recovery for the trachea samples. In the current study, dust levels prior to and during catching visually appeared to increase but were not directly measured. The levels of dust present during catching may depend on the outside ambient temperature. During hot weather, ventilation rates are at maximum levels to prevent bird heat stress; however, during cold weather ventilation rates are at lower levels to prevent bird chilling. This difference in ventilation could significantly impact levels of dust present during broiler catching. Ambient temperatures for Trials 1, 2, and 3 were 26°C, 31°C, and 29°C (79°F, 88°F, 84°F), respectively. This would indicate that ventilation rates may have been at higher levels than they would have been during the winter months. Although it would seem that increased ventilation would decrease dust [21], some authors have suggested that increased ventilation rates may cause resuspension of dust particles [22]. This is further demonstrated by Chinivasagam et al. when greater levels of Escherichia coli were detected in air from tunnel ventilated broiler houses during the summer in comparison to winter [23]. Broiler activity can also significantly impact the levels of dust present in the growout house. Calvet et al. [24] determined a direct cause-effect relationship between animal activity and dust concentration (r2 = 0.89) where an increase in activity led to an increase in dust concentrations. Although dust concentrations were not measured in the current study, it is anticipated that there were greater levels of dust during catching than the day prior to catching. It is interesting to note that broilers with Salmonella-positive respiratory tracts did not always have Salmonella-positive ceca. There were 26% (8/31) of broilers that had positive respiratory tracts that were ceca Salmonella-negative. Of these 8 carcasses that were respiratory tract Salmonella-positive/ceca Salmonella-negative, 5 were detected from broilers collected during catching. In comparison overall, 23% (23/98) of the broilers were Salmonella-positive for both the respiratory tracts and the ceca. Kallapura [6] also reported dissimilar recovery from trachea and ceca for the same broiler; from 100 commercial broilers there were 28 Salmonella-positive trachea and only 10 Salmonella-positive ceca (Trial 3), and in contrast from 150 broilers only 3 Salmonella-positive trachea and 27 Salmonella-positive ceca (Trial 5). This indicates that the presence of Salmonella within the respiratory tract but not the ceca may have been due to means other than systemic intestinal colonization, such as dust in the air. However, the route by which Salmonella became present in the respiratory tract or the duration of persistence was not examined within the scope of this work. It was expected that the increased dust in the air would lead to increased levels of EB and APC within the broiler respiratory tracts between 1 d prior to catching and during catching. However, this did not occur. Overall counts of EB at 1.41 log10 CFU/mL and APC at 3.11 log10 CFU/mL were similar to a previous study where respiratory tracts were sampled for E. coli, coliforms, and aerobes following slaughter and prior to scalding [11]. A lack of significant difference between bacterial levels prior to and following catching may have been due to the length of time (1 to 2 h) between collecting the broilers at the farm and respiratory tract sampling. The mucus and cilia lining the trachea are continuously removing dust particles, bacteria, and toxins from the air within the trachea into the pharynx where they are swallowed and enter the esophagus. This mucus coating may facilitate the passage through the acid environments of the proventriculus and gizzard (pH 2) of young chicks and result in intestinal colonization [4,5,16]. An in vitro tracheal clearance assay reported a clearance velocity of 2.39 mm/min for 19- to 20-d chicken embryos [25]. Using this clearance rate, the entire broiler trachea (15 cm glottis to syrinx) would clear every 1.05 min. In humans for particles ≤6 μm, a certain fraction was retained in the trachea for more than 24 h [26]. When anesthetized White Leghorn roosters were exposed to airborne particles for 30 min and immediately euthanized, the largest particles (3.7 to 7 μm) were captured in the head and anterior trachea [27]. Tracheal clearance of dust inhaled during catching may contribute to downstream contamination during transport and processing. In future work, direct comparisons of dust levels, respiratory tract bacteria, and processed carcasses should be examined to determine if there is any significant relationship. CONCLUSION AND APPLICATIONS The aerosolized dust that occurred during catching broiler chickens did not significantly impact Salmonella prevalence, EB prevalence and counts, or aerobic bacterial counts when compared to broilers collected 1 d prior to harvest. Twenty-six percent of broilers with Salmonella-positive respiratory tracts did not have cecal Salmonella colonization. There is the potential for downstream poultry product contamination from the presence of Salmonella within broiler respiratory tracts; however, this relationship needs to be demonstrated in future research. Note Primary Audience: Researchers, Veterinarians, Poultry Farm Managers, Quality Assurance Personnel REFERENCES AND NOTES 1. Marin C. , Balasch S. , Vega S. , Lainez M. . 2011 . Sources of Salmonella contamination during broiler production in Eastern Spain . Prev. Vet. Med. 98 : 39 – 45 . Google Scholar CrossRef Search ADS PubMed 2. Mueller-Doblies D. , Sayers A. R. , Carrique-Mas J. J. , Davies R. H. . 2009 . Comparison of sampling methods to detect Salmonella infection of turkey flocks . J. Appl. Microbiol. 107 : 635 – 645 . Google Scholar CrossRef Search ADS PubMed 3. O’Brien K. M. , Chimenti M. S. , Farnell M. , Tabler T. , Bair T. , Bray J. L. , Nonnenmann M. 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Evaluation of the respiratory route as a viable portal of entry for Salmonella in poultry via intratracheal challenge of Salmonella Enteritidis and Salmonella Typhimurium . Poult. Sci. 93 : 340 – 346 . Google Scholar CrossRef Search ADS PubMed 7. Buhr R. J. , Richardson L. J. , Cason J. A. , Cox N. A. , Fairchild B. D. . 2007 . Comparison of four sampling methods for the detection of Salmonella in broiler litter . Poult. Sci. 86 : 21 – 25 . Google Scholar CrossRef Search ADS PubMed 8. ISODS (intermittently stepped on drags swabs) were used to sample commercial broiler house litter . For each house, four drag swabs (DS-001, Solar Biologicals Inc., Ogdensburg, NY) presoaked in skim milk were unwound and dragged between the feeder and water lines on top of the litter while intermittently being stepped on to increase swab-litter contact. Two swabs were used to sample each half of the house, then pooled yielding two samples per house . 9. Following ISODS sampling, 120 mL of 1% BPW (Difco, Becton, Dickinson and Co., Sparks, MD) was added to each bag and manually mixed . Then samples were incubated 24 h at 37°C. Following enrichment, 0.5 ml of each rinsate was transferred into 9.5 ml of tetrathionate broth Hajna (TT; BD), and an additional 0.1 mL was transferred into 9.9 mL of Rappaport-Vassiliadis R10 broth (RV; Acumedia, Lansing, MI). These broths were then incubated at 42°C for 24 h. Two 10-μL loops from each of the two broths were streaked for isolation onto brilliant green sulfur (BGS; Acumedia) and xylose lysine Tergitol 4 agar (XLT4; BD) plates, and the plates were incubated for 24 h at 37°C. Presumptive colonies were selected, stabbed individually into triple sugar iron and lysine iron agar slants (Acumedia), and then incubated at 37°C for 24 h. After incubation, the presumptive-positive colonies were subjected to Salmonella O antiserum Poly A-I & Vi agglutination assay . 10. Berrang M. E. , Meinersmann R. J. , Buhr R. J. , Reimer N. A. , Phillips R. W. , Harrison M. A. . 2003 . Presence of Campylobacter in the respiratory tract of broiler carcasses before and after commercial scalding . Poult. Sci. 82 : 1995 – 1999 . Google Scholar CrossRef Search ADS PubMed 11. Buhr R. J. , Berrang M. E. , Cason J. A. , Bourassa D. V. . 2005 . Recovery of bacteria from broiler carcass respiratory tracts before and after immersion scalding . Poult. Sci. 84 : 1769 – 1773 . Google Scholar CrossRef Search ADS PubMed 12. Respiratory tract rinsates were diluted in sterile saline and 1 mL plated in duplicate onto both aerobic plate count (APC) petrifilm and Enterobacteriaceae (EB) petrifilm (3 M Food Safety). APC petrifilm were incubated at 37°C for 48 h and EB were incubated at 37°C for 24 h. Following incubation, colony forming units were recorded. For each respiratory tract sample, 20 mL of rinsate was transferred to a sterile tube for Salmonella prevalence testing. Tubes were enriched by incubating 24 h at 37°C then analyzed for Salmonella as described previously [9] . 13. Bourassa D. V. , Holmes J. M. , Cason J. A. , Cox N. A. , Rigsby L. L. , Buhr R. J. . 2015 . Prevalence and serogroup diversity of Salmonella for broiler neck skin, whole carcass rinse, and whole carcass enrichment sampling methodologies following air or immersion chilling . J. Food Prot. 78 : 1938 – 1944 . Google Scholar CrossRef Search ADS PubMed 14. Ceca were bagged and stored on ice prior to sampling within 2 h. For each ceca sample, a mL volume of 1% BPW at 3 times the average ceca weight was added to each sample bag, macerated to release internal contents, and stomached. Stomached samples were enriched by incubating 24 h at 37°C then analyzed for Salmonella as described previously [4] . 15. Gradel K. O. , Andersen J. , Madsen M. . 2002 . 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Improving In-House air quality in broiler production facilities using an electrostatic space charge system . J. Appl. Poult. Res. 15 : 333 – 340 . Google Scholar CrossRef Search ADS 23. Chinivasagam H. N. , Tran T. , Maddock L. , Gale A. , Blackall P. J. . 2009 . Mechanically ventilated broiler sheds: a possible source of aerosolized Salmonella, Campylobacter, and Escherichia coli . Appl. Environ. Microbiol. 75 : 7417 – 7425 . Google Scholar CrossRef Search ADS PubMed 24. Calvet S. , Van den Weghe H. , Kosch R. , Estellés F. . 2009 . The influence of the lighting program on broiler activity and dust production . Poult. Sci. 88 : 2504 – 2511 . Google Scholar CrossRef Search ADS PubMed 25. Henning A. , Schneider M. , Bur M. , Blank F. , Gehr P. , Lehr C. M. . 2008 . Embryonic chicken trachea as a new in vitro model for the investigation of mucociliary particle clearance in the airways . AAPS PharmSciTech 9 : 521 – 527 . Google Scholar CrossRef Search ADS PubMed 26. Scheuch G. , Stahlhofen W. . 1987 . Particle deposition of inhaled aerosol boluses in the upper human airways . J. Aerosol. Sci. 18 : 725 – 727 . Google Scholar CrossRef Search ADS 27. Hayter R. B. , Besch E. L. . 1974 . Airborne-particle deposition in the respiratory tract of chickens . Poult. Sci. 53 : 1507 – 1511 . Google Scholar CrossRef Search ADS PubMed © 2018 Poultry Science Association Inc. This article is published and distributed under the term of oxford University Press, standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Applied Poultry Research Oxford University Press

Microbiological Status of Broiler Respiratory Tracts Before and During Catching for Transport to the Processing Plant

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Applied Poultry Science, Inc.
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© 2018 Poultry Science Association Inc.
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1056-6171
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1537-0437
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10.3382/japr/pfy029
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Abstract

Abstract A significant point of entry for Salmonella into a processing plant is within the broilers to be processed. Prior to transport to the processing plant, feed (4 h) and water are withdrawn from the broilers on the farm before they are caught and cooped. During catching, an increased presence of dust in the house air is visible and may affect the presence of bacteria within the broiler's respiratory tract. The objective of this study was to examine the effect of catching on the levels of aerobic bacteria (aerobic plate count [APC]), levels and prevalence of Enterobacteriaceae (EB), and prevalence of Salmonella within broiler respiratory tracts. To determine flock Salmonella status 1 wk prior to catching, broiler carcasses were sampled for APC, EB, and Salmonella by respiratory tract flushing and ceca were sampled for Salmonella. At 1 d prior to catching and when half of the broilers in the house were caught, broilers were again collected, transported to the pilot plant, euthanized, and sampled. In Trial 1, there were no significant differences between sampling times for ceca Salmonella or respiratory EB and APC. However, Salmonella prevalence in the respiratory tract was significantly higher at 1 wk preharvest compared to during catching. In Trial 2, EB was significantly lower during catching compared to 1 wk preharvest. No significant differences were detected in Trial 3. Based on these results, the presence of aerosolized dust in the broiler house during catching does not appear to lead to increases in broiler respiratory tract microbial contamination. DESCRIPTION OF PROBLEM When present, Salmonella can be detected from multiple locations within a broiler growout house, including both litter and dust [1,2]. Previous work sampling turkey flocks has indicated that Salmonella can be found in dust with the same frequency (41 to 58%) as boot swabs or litter sampling [2]. Broiler house dust contains a mixture of dander, litter, feed, feces, and microbes, with the vast majority of dust containing microbial DNA [3]. Generally, catch crew workers will wear respiratory protective equipment during broiler harvest (catching) to minimize the amount of dust they inhale. However, the broilers do not have any external respiratory protection other than the increased mechanical exhaust ventilation during harvest. Therefore, it was anticipated that microbes in increased levels of dust in the broiler house air may be inhaled by the broilers during catching and accumulate in the respiratory tract. The broiler respiratory tract is a potential reservoir for Salmonella. Several studies have demonstrated that inoculating broilers within the trachea with Salmonella leads to further intestinal colonization [4–6]. Intratracheal inoculations of Salmonella led to colonization as well as or more effectively than oral inoculation [6] when broilers were full fed. In work comparing 5 inoculation routes (oral, intratracheal, subcutaneous, ocular, and cloacal), the intratracheal inoculation route led to intestinal tract colonization significantly more frequently than all other routes [4]. Salmonella present in the litter may be transported on increased dust in the air during catching and enter into broiler respiratory tracts. The transfer of Salmonella from the litter or house dust to the broiler respiratory tract (specifically the trachea) may potentially have further impact in the processing plant on broiler product Salmonella status. The objective of this study was to examine the effect of catching on the levels of aerobic bacteria (aerobic plate count [APC]), the levels and prevalence of Enterobacteriaceae (EB), and the prevalence of Salmonella within broiler respiratory tracts. MATERIALS AND METHODS Experimental Design The broilers and standard operating procedures used in this study were covered by an animal use proposal approved by the US National Poultry Research Center IACUC. To assess the effect of aerosolized dust during broiler catching on the levels APC, levels and prevalence of EB, and prevalence of Salmonella within broiler respiratory tracts, broilers were sampled at 3 different time points. For all trials broilers and litter were first sampled at 1 wk preharvest to confirm the presence of Salmonella in the commercial broiler house and flock prior to further sampling. If the house litter and flock were confirmed to be Salmonella-positive at 1 wk preharvest, then at 1 d preharvest broilers were again sampled to determine the levels and/or prevalence of bacteria prior to catching. On the next day, broilers were sampled during catching after approximately half of the house had been caught and the last transport coop for an entire trailer (22 coops/trailer) was leaving the house. Results from the during catching sampling time were compared to the 1 d preharvest sampling results in order to determine if dust in the air during catching aerosolized from the litter and house would impact broiler respiratory tract microbiology. At all 3 sampling times, male broilers were caught individually with a leg hook and from the middle of the house to minimize exciting the flock and elevating dust levels in the air. Litter and Broiler Sampling For each of 3 trials, commercial broiler house litter (between the water and feed lines) was sampled using intermittently stepped on drag swabs 1 wk prior to catching for transport to a processing plant [7,8]. Following collection, drag swab samples were tested for the prevalence of Salmonella [9]. In addition to litter sampling, 20 male broilers per trial (60 total) were individually caught, cooped, and transported less than 1 h to the pilot processing plant for sampling. In Trial 1, the 20 broilers were collected from 4 commercial houses (5 broilers/house) at 5 wk of age for the 1-wk preharvest sampling time and at 6 wk of age for the 1-d preharvest and during catching sampling times. In Trials 2 and 3, 10 broilers were collected from each of 2 adjacent commercial houses. For Trials 2 and 3 broilers were 6 wk of age at the 1-wk preharvest sampling time and at 7 wk of age for the 1-d preharvest and during catching times. Each trial was conducted with different flocks on different commercial farms. Following arrival at the pilot processing plant, broilers were individually euthanized by electrocution prior to sampling within 2 h of catching. Respiratory tracts were sampled as previously described [10,11]. Briefly, the carcass neck feathers and skin were sprayed with ethanol and the neck skin cut about 10 cm (approximately mid-neck) to expose the underlying trachea. Then the trachea was partially cut for insertion of plastic tubing (toward the syrinx) attached to a syringe containing 60 mL of 1% Buffered Peptone Water (BPW). During sampling, the tubing was secured within the trachea with a small cable tie. The BPW was slowly introduced into the respiratory tract, the carcass inverted 30 times, the carcass was hung in a shackle by the feet, and then the rinse was collected back into the same syringe. Rinsates were analyzed for APCs, EB counts and prevalence, and Salmonella prevalence [11,12]. Following respiratory tract sampling, the abdominal feathers and skin were sprayed with ethanol and opened for aseptic removal of ceca for Salmonella prevalence analysis [13,14]. RESULTS AND DISCUSSION Only the broiler houses with litter positive for Salmonella were sampled at all 3 time points (1 wk prior to catching, 1 d prior to catching, and during catching) and are reported in this study. Results from microbiological sampling of broiler respiratory tracts and ceca are shown in Table 1. There were no significant differences between the sampling time points for cecal Salmonella prevalence (50 to 60% positive). In Trial 1, broilers collected during catching had a significantly lower prevalence (20%) of Salmonella in the respiratory tract rinsate than broilers collected 1 wk prior to slaughter (55%). Broilers collected 1 d prior to slaughter were intermediate (50%). This significant difference was not observed in Trials 2 and 3, or overall. In Trial 2, respiratory tract EB counts were significantly higher (2.17 log10) for broilers collected during catching than 1 wk prior to slaughter (0.58 log10); however, this difference was not seen in Trials 1, 3, or overall. Enterobacteriaceae prevalence and APCs were not significantly different between sampling times for any trial or overall (EB 0.98 to 1.77 log10; APC 3.03 to 3.19 log10). Table 1. Broiler Respiratory Tract and Ceca Microbiology Prior to and During Broiler Catching. Respiratory tracts Sampling time Ceca Salmonella Prevalence Salmonella Prevalence EB1 Prevalence EB Counts3 APC2 Counts Trial 1  Preharvest 1 wk 17/20 11/20A 8/20 1.80 2.77  Preharvest 1 d 12/20 10/20A,B 8/20 0.68 2.83  During catching 11/20 4/20B 5/20 <0.01 3.11 Trial 2  Preharvest 1 wk 14/20 2/20 6/20 0.52A 2.77  Preharvest 1 d 13/20 0/20 2/20 0.58A,B 2.78  During catching 11/20 3/20 3/20 2.17B 2.49 Trial 3  Preharvest 1 wk 5/20 0/20 5/20 2.08 3.76  Preharvest 1 d 7/20 0/20 7/20 1.43 3.48  During catching 8/20 1/20 8/20 2.74 4.00 Overall  Preharvest 1 wk 36/60 13/60 19/60 1.47 3.10  Preharvest 1 d 32/60 10/60 17/60 0.98 3.03  During catching 30/60 8/60 16/60 1.77 3.19 Respiratory tracts Sampling time Ceca Salmonella Prevalence Salmonella Prevalence EB1 Prevalence EB Counts3 APC2 Counts Trial 1  Preharvest 1 wk 17/20 11/20A 8/20 1.80 2.77  Preharvest 1 d 12/20 10/20A,B 8/20 0.68 2.83  During catching 11/20 4/20B 5/20 <0.01 3.11 Trial 2  Preharvest 1 wk 14/20 2/20 6/20 0.52A 2.77  Preharvest 1 d 13/20 0/20 2/20 0.58A,B 2.78  During catching 11/20 3/20 3/20 2.17B 2.49 Trial 3  Preharvest 1 wk 5/20 0/20 5/20 2.08 3.76  Preharvest 1 d 7/20 0/20 7/20 1.43 3.48  During catching 8/20 1/20 8/20 2.74 4.00 Overall  Preharvest 1 wk 36/60 13/60 19/60 1.47 3.10  Preharvest 1 d 32/60 10/60 17/60 0.98 3.03  During catching 30/60 8/60 16/60 1.77 3.19 1 EB = Enterobacteriaceae. 2 APC = Aerobic plate count. 3 Counts are reported as Log10 CFU/mL. A,B Values within a Trial and within a column with differing superscripts are significantly different (P ≤ 0.05). View Large Table 1. Broiler Respiratory Tract and Ceca Microbiology Prior to and During Broiler Catching. Respiratory tracts Sampling time Ceca Salmonella Prevalence Salmonella Prevalence EB1 Prevalence EB Counts3 APC2 Counts Trial 1  Preharvest 1 wk 17/20 11/20A 8/20 1.80 2.77  Preharvest 1 d 12/20 10/20A,B 8/20 0.68 2.83  During catching 11/20 4/20B 5/20 <0.01 3.11 Trial 2  Preharvest 1 wk 14/20 2/20 6/20 0.52A 2.77  Preharvest 1 d 13/20 0/20 2/20 0.58A,B 2.78  During catching 11/20 3/20 3/20 2.17B 2.49 Trial 3  Preharvest 1 wk 5/20 0/20 5/20 2.08 3.76  Preharvest 1 d 7/20 0/20 7/20 1.43 3.48  During catching 8/20 1/20 8/20 2.74 4.00 Overall  Preharvest 1 wk 36/60 13/60 19/60 1.47 3.10  Preharvest 1 d 32/60 10/60 17/60 0.98 3.03  During catching 30/60 8/60 16/60 1.77 3.19 Respiratory tracts Sampling time Ceca Salmonella Prevalence Salmonella Prevalence EB1 Prevalence EB Counts3 APC2 Counts Trial 1  Preharvest 1 wk 17/20 11/20A 8/20 1.80 2.77  Preharvest 1 d 12/20 10/20A,B 8/20 0.68 2.83  During catching 11/20 4/20B 5/20 <0.01 3.11 Trial 2  Preharvest 1 wk 14/20 2/20 6/20 0.52A 2.77  Preharvest 1 d 13/20 0/20 2/20 0.58A,B 2.78  During catching 11/20 3/20 3/20 2.17B 2.49 Trial 3  Preharvest 1 wk 5/20 0/20 5/20 2.08 3.76  Preharvest 1 d 7/20 0/20 7/20 1.43 3.48  During catching 8/20 1/20 8/20 2.74 4.00 Overall  Preharvest 1 wk 36/60 13/60 19/60 1.47 3.10  Preharvest 1 d 32/60 10/60 17/60 0.98 3.03  During catching 30/60 8/60 16/60 1.77 3.19 1 EB = Enterobacteriaceae. 2 APC = Aerobic plate count. 3 Counts are reported as Log10 CFU/mL. A,B Values within a Trial and within a column with differing superscripts are significantly different (P ≤ 0.05). View Large Broiler litter Salmonella recovery has been shown to peak around 3 to 4 wk and diminish thereafter [15–17]. The lack of significant difference between ceca Salmonella prevalence was not unexpected because of the short time between sampling time points (6 or 7 d). However, the presence of Salmonella in 25 to 85% of the broiler ceca sampled indicated that Salmonella was likely continuously being shed into the litter. The respiratory tract results from Trial 1 in this study suggest that respiratory tract Salmonella prevalence may decrease with catching, but this result was not consistent in the later 2 trials. One possible explanation for the inconsistency between trials could be the lower combined prevalence of respiratory tract Salmonella detected in Trials 2 and 3 (8%, 5/60 and 2%, 1/60, respectively) compared to the higher combined prevalence in Trial 1 (42%, 25/60). Potential differing levels of Salmonella in the house litter, differing times of year (April, August, May), age (5 to 6 wk vs 6 to 7 wk), and differing housing construction and ventilation may all have contributed to the inconsistency among trials. There are also inconsistent results in previous studies evaluating dust concentrations in broiler houses ranging from 0.03 to 6.5 mg/m3 [18,19]. These differences were attributed to sampling time, bird activity, and house ventilation differences due to ambient temperature [19]. In caged turkeys exposed to Salmonella contaminated fecal dust for 2 or 4 h (105 cfu/g), Harbaugh [20] reported very low recovery (≥1/16 turkeys) from respiratory tract samples (nasal passages, infraorbital sinuses, trachea, or air sacs); and when the dust exposure level was elevated to 107 or 109 cfu/g then 0 to 6/8 turkeys had positive samples. In contrast, lungs (31 to 94% positive) and small intestines (44 to 88% positive) had very high recovery of Salmonella at all 3 dust exposure levels. Cross contamination from Salmonella-inoculated turkeys to non-inoculated turkeys by fans was less successful with the highest recovery of 14% for the respiratory tract samples but no recovery for the trachea samples. In the current study, dust levels prior to and during catching visually appeared to increase but were not directly measured. The levels of dust present during catching may depend on the outside ambient temperature. During hot weather, ventilation rates are at maximum levels to prevent bird heat stress; however, during cold weather ventilation rates are at lower levels to prevent bird chilling. This difference in ventilation could significantly impact levels of dust present during broiler catching. Ambient temperatures for Trials 1, 2, and 3 were 26°C, 31°C, and 29°C (79°F, 88°F, 84°F), respectively. This would indicate that ventilation rates may have been at higher levels than they would have been during the winter months. Although it would seem that increased ventilation would decrease dust [21], some authors have suggested that increased ventilation rates may cause resuspension of dust particles [22]. This is further demonstrated by Chinivasagam et al. when greater levels of Escherichia coli were detected in air from tunnel ventilated broiler houses during the summer in comparison to winter [23]. Broiler activity can also significantly impact the levels of dust present in the growout house. Calvet et al. [24] determined a direct cause-effect relationship between animal activity and dust concentration (r2 = 0.89) where an increase in activity led to an increase in dust concentrations. Although dust concentrations were not measured in the current study, it is anticipated that there were greater levels of dust during catching than the day prior to catching. It is interesting to note that broilers with Salmonella-positive respiratory tracts did not always have Salmonella-positive ceca. There were 26% (8/31) of broilers that had positive respiratory tracts that were ceca Salmonella-negative. Of these 8 carcasses that were respiratory tract Salmonella-positive/ceca Salmonella-negative, 5 were detected from broilers collected during catching. In comparison overall, 23% (23/98) of the broilers were Salmonella-positive for both the respiratory tracts and the ceca. Kallapura [6] also reported dissimilar recovery from trachea and ceca for the same broiler; from 100 commercial broilers there were 28 Salmonella-positive trachea and only 10 Salmonella-positive ceca (Trial 3), and in contrast from 150 broilers only 3 Salmonella-positive trachea and 27 Salmonella-positive ceca (Trial 5). This indicates that the presence of Salmonella within the respiratory tract but not the ceca may have been due to means other than systemic intestinal colonization, such as dust in the air. However, the route by which Salmonella became present in the respiratory tract or the duration of persistence was not examined within the scope of this work. It was expected that the increased dust in the air would lead to increased levels of EB and APC within the broiler respiratory tracts between 1 d prior to catching and during catching. However, this did not occur. Overall counts of EB at 1.41 log10 CFU/mL and APC at 3.11 log10 CFU/mL were similar to a previous study where respiratory tracts were sampled for E. coli, coliforms, and aerobes following slaughter and prior to scalding [11]. A lack of significant difference between bacterial levels prior to and following catching may have been due to the length of time (1 to 2 h) between collecting the broilers at the farm and respiratory tract sampling. The mucus and cilia lining the trachea are continuously removing dust particles, bacteria, and toxins from the air within the trachea into the pharynx where they are swallowed and enter the esophagus. This mucus coating may facilitate the passage through the acid environments of the proventriculus and gizzard (pH 2) of young chicks and result in intestinal colonization [4,5,16]. An in vitro tracheal clearance assay reported a clearance velocity of 2.39 mm/min for 19- to 20-d chicken embryos [25]. Using this clearance rate, the entire broiler trachea (15 cm glottis to syrinx) would clear every 1.05 min. In humans for particles ≤6 μm, a certain fraction was retained in the trachea for more than 24 h [26]. When anesthetized White Leghorn roosters were exposed to airborne particles for 30 min and immediately euthanized, the largest particles (3.7 to 7 μm) were captured in the head and anterior trachea [27]. Tracheal clearance of dust inhaled during catching may contribute to downstream contamination during transport and processing. In future work, direct comparisons of dust levels, respiratory tract bacteria, and processed carcasses should be examined to determine if there is any significant relationship. CONCLUSION AND APPLICATIONS The aerosolized dust that occurred during catching broiler chickens did not significantly impact Salmonella prevalence, EB prevalence and counts, or aerobic bacterial counts when compared to broilers collected 1 d prior to harvest. Twenty-six percent of broilers with Salmonella-positive respiratory tracts did not have cecal Salmonella colonization. There is the potential for downstream poultry product contamination from the presence of Salmonella within broiler respiratory tracts; however, this relationship needs to be demonstrated in future research. Note Primary Audience: Researchers, Veterinarians, Poultry Farm Managers, Quality Assurance Personnel REFERENCES AND NOTES 1. Marin C. , Balasch S. , Vega S. , Lainez M. . 2011 . Sources of Salmonella contamination during broiler production in Eastern Spain . Prev. Vet. Med. 98 : 39 – 45 . Google Scholar CrossRef Search ADS PubMed 2. Mueller-Doblies D. , Sayers A. R. , Carrique-Mas J. J. , Davies R. H. . 2009 . Comparison of sampling methods to detect Salmonella infection of turkey flocks . J. Appl. Microbiol. 107 : 635 – 645 . Google Scholar CrossRef Search ADS PubMed 3. O’Brien K. M. , Chimenti M. S. , Farnell M. , Tabler T. , Bair T. , Bray J. L. , Nonnenmann M. 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Following ISODS sampling, 120 mL of 1% BPW (Difco, Becton, Dickinson and Co., Sparks, MD) was added to each bag and manually mixed . Then samples were incubated 24 h at 37°C. Following enrichment, 0.5 ml of each rinsate was transferred into 9.5 ml of tetrathionate broth Hajna (TT; BD), and an additional 0.1 mL was transferred into 9.9 mL of Rappaport-Vassiliadis R10 broth (RV; Acumedia, Lansing, MI). These broths were then incubated at 42°C for 24 h. Two 10-μL loops from each of the two broths were streaked for isolation onto brilliant green sulfur (BGS; Acumedia) and xylose lysine Tergitol 4 agar (XLT4; BD) plates, and the plates were incubated for 24 h at 37°C. Presumptive colonies were selected, stabbed individually into triple sugar iron and lysine iron agar slants (Acumedia), and then incubated at 37°C for 24 h. After incubation, the presumptive-positive colonies were subjected to Salmonella O antiserum Poly A-I & Vi agglutination assay . 10. Berrang M. E. , Meinersmann R. 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Scheuch G. , Stahlhofen W. . 1987 . Particle deposition of inhaled aerosol boluses in the upper human airways . J. Aerosol. Sci. 18 : 725 – 727 . Google Scholar CrossRef Search ADS 27. Hayter R. B. , Besch E. L. . 1974 . Airborne-particle deposition in the respiratory tract of chickens . Poult. Sci. 53 : 1507 – 1511 . Google Scholar CrossRef Search ADS PubMed © 2018 Poultry Science Association Inc. This article is published and distributed under the term of oxford University Press, standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Journal of Applied Poultry ResearchOxford University Press

Published: Jun 6, 2018

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