Abstract The first step to detect Salmonella in feed and other dry contaminated samples is a pre-enrichment broth that can assist in the recovery of small numbers of stressed Salmonella cells. A previous study demonstrated that incubation of feed and feed ingredients in commonly used pre-enrichment media resulted in a low pH that injured or killed the Salmonella. The objective of this study was to determine which environmental samples and pre-enrichment and selective broths could interfere with the accuracy of Salmonella detection by allowing pH to drop. Samples were collected from commercial poultry operations. Triplicate 10-g subunits were dispensed into sterile 18-oz Whirl Pak bags and 90 mL of each of the media [lactose broth (LB), buffered peptone water (BPW), Universal Pre-enrichment (UP), minimal salts (M-9), tetrathionate broth (TT) and Rappaport-Vassiliadis broth (RV)] were added to the bags and incubated 24 h at 37°C (or 42°C for RV and TT). The pH was measured after incubation. With turkey litter, fluff, and eggshells, regardless of media, the pH never went below 5.7. Regardless of sample type, the lowest pH was 6.1, 6.2, and 6.4 for UP, M-9, and BPW, respectively. For the pre-enrichment medium commonly used in the US, (LB) the pH dropped to 4.7 to 4.9 with broiler litter and 4.2 for boot covers used in turkey or broiler houses. Many researchers testing these sample types are unaware of this potential for pH change during pre-enrichment and may be underestimating the presence of Salmonella. DESCRIPTION OF PROBLEM For many years, it was believed that Salmonella in feed was not a common source for contamination of poultry. However, published surveys have reported that feed can account for more than 50% of the isolates present in breeders , broilers  and layers [3, 4]. When the level of a target organism (such as Salmonella) is low in number and possibly injured by a variety of stresses such as dryness, heat, or chemicals, the first step in the laboratory procedure is to use a nonselective pre-enrichment medium so that the injured organism can recover and proliferate to much higher numbers of healthy cells. However, when the sample being tested has a considerable amount of fermentable substrates, and a rather large array of background extraneous microbes, problems can occur. Many of these non-Salmonella microorganisms can convert these fermentable substrates to acid(s) and the pH of the pre-enrichment media can become extremely acidic which may injure and/or kill the target bacteria. Many researchers testing these types of samples are completely unaware of the hostile acidity of the pre-enrichment medium. Pre-enrichment media were developed and used to increase the chances of detecting injured bacterial cells, but may in fact be responsible for injury and death of the target organism. When this occurs, it may well produce a false negative result and that means that the incidence and level of Salmonella in these sample types may be vastly underestimated. When Salmonella goes undetected as a false negative then a false sense of security can result. A previous study demonstrated that incubation of feed or feed ingredients in pre-enrichment media can result in generation of an acidic environment (pH of 4 to 5) . Acid injury can prevent detection of Salmonella in feed and feed ingredients . Incubation in a medium at pH 4 was shown to be lethal to isolates of S. Enteritidis, S. Typhimurium, S. Heidelberg and S. Kentucky . The objective of this study was to determine if incubation of poultry house or hatchery environmental samples cause a change in pH of common Salmonella pre-enrichment or selective enrichment broths, and potentially interfere with Salmonella detection. MATERIALS AND METHODS Sample Collection and Processing Samples were collected from commercial poultry operations by the University of Georgia Poultry Disease Research Center in Athens, GA. Samples included used turkey litter, used broiler litter, and soiled plastic boot covers that had been used in turkey and broiler pens. Samples from hatching cabinets included fluff and eggshells. Litter and eggshells were finely ground in a sterile Waring blender to obtain a uniform particle size prior to subsampling. Samples were mixed and subsampled using a Humboldt sample splitter to obtained 3 replicate 10-g samples of each. Replicate subsamples (10-g) of fluff were obtained using the sample splitter. Boot covers were cut into small pieces of 1 cm2 using sterile scissors and mixed well. Triplicate 10-g subunits were collected from each sample and placed into a sterile 18-oz WhirlPak bag . Broth Media The media tested were as follows: M-9 , lactose broth (LB) , Rappaport-Vassiliadis broth (RV), Universal pre-enrichment broth (UPB) , tetrathionate broth (TT), and buffered peptone water (BPW). Each medium (90 mL) was dispensed into WhirlPak bags, the initial pH was measured using a pH meter  and recorded; pre-weighed 10-g samples were added to each type of media. The pH of each medium was re-measured immediately after the samples were added (time 0). Samples were incubated at 37°C (or 42°C for RV and TT) and the pH was measured and recorded at 1, 18, 24, and 48 h of incubation. Statistical Analysis For broiler fluff, broiler eggshells, broiler boot covers, and turkey boot covers, one experiment with 3 replicate samples were collected (n = 3). For litter (both broiler and turkey) 2 experiments each with 3 replicate samples were collected (n = 6). Means and standard deviations were calculated. To test the effect of broth on pH, data was subjected to a generalized linear model; means were separated with Duncan's multiple range test. Significance is reported at P < 0.05. Statistical analyses were conducted using Statistica 12 . RESULTS AND DISCUSSION The initial pH of the various media tested were BPW (6.88–7.01), LB (6.88–6.90), M-9 (6.90–7.10), RV (5.03–5.16), UP (6.10–6.30) and TT (7.43–7.54). The pH after 24 h at 35°C with a variety of environmental samples and an assortment of various pre-enrichment and selective enrichment broths are shown in Table 1. For 3 of the pre-enrichment media, UP, M-9, and BPW, the lowest pH was reached at 24 h regardless of sample type and was 6.1, 6.2, and 6.4, respectively. For the other pre-enrichment medium LB, the pH dropped to 4.7 to 4.9 with broiler litter and to 4.2 for turkey and broiler boot covers. For one of the 2 selective enrichment broths, RV, the pH ranged from 4.8 to 6.2 and only dropped to 4.8 with turkey boot covers. For the other selective medium, TT, the pH ranged between 5.4 and 6.1. There was very little, if any, difference in the pH values at 18 and 48 h when compared to 24 h (data not shown). With turkey litter, fluff and eggshells, regardless of pre-enrichment or selective enrichment used, the pH never went below 5.7. Table 1. The pH (mean ± SD) of various bacteriological growth media at 24 h at 35°C or 42°C with an assortment of environmental poultry samples. pH in Various Media Sample BPW1 LB2 M-93 UP4 RV5 TT6 Turkey Litter (n = 6) 6.64 ± 0.09a 6.35 ± 0.03b 6.50 ± 0.01c 6.44 ± 0.01c 6.05 ± 0.04d 6.03 ± 0.02d Broiler Litter (n = 6) 6.42 ± 0.01a 4.81 ± 0.05d 6.27 ± 0.26a,b 6.14 ± 0.03b 5.44 ± 0.05c 5.62 ± 0.12c Turkey Boot Covers (n = 3) 6.52 ± 0.01a 4.17 ± 0.04f 6.40 ± 0.03b 6.23 ± 0.02c 4.80 ± 0.04e 5.41 ± 0.04d Broiler Boot Covers (n = 3) 6.31 ± 0.06a n/a 6.15 ± 0.08b 6.13 ± 0.01b 5.46 ± 0.01d 5.75 ± 0.04c Fluff (n = 3) 6.65 ± 0.01a 5.67 ± 0.22c 6.50 ± 0.03a,b 6.37 ± 0.02b 5.72 ± 0.08c 5.72 ± 0.02c Eggshell (n = 3) 7.00 ± 0.01a 5.96 ± 0.21c 6.75 ± 0.04b 6.64 ± 0.01b 6.02 ± 0.06c 5.71 ± 0.01d pH in Various Media Sample BPW1 LB2 M-93 UP4 RV5 TT6 Turkey Litter (n = 6) 6.64 ± 0.09a 6.35 ± 0.03b 6.50 ± 0.01c 6.44 ± 0.01c 6.05 ± 0.04d 6.03 ± 0.02d Broiler Litter (n = 6) 6.42 ± 0.01a 4.81 ± 0.05d 6.27 ± 0.26a,b 6.14 ± 0.03b 5.44 ± 0.05c 5.62 ± 0.12c Turkey Boot Covers (n = 3) 6.52 ± 0.01a 4.17 ± 0.04f 6.40 ± 0.03b 6.23 ± 0.02c 4.80 ± 0.04e 5.41 ± 0.04d Broiler Boot Covers (n = 3) 6.31 ± 0.06a n/a 6.15 ± 0.08b 6.13 ± 0.01b 5.46 ± 0.01d 5.75 ± 0.04c Fluff (n = 3) 6.65 ± 0.01a 5.67 ± 0.22c 6.50 ± 0.03a,b 6.37 ± 0.02b 5.72 ± 0.08c 5.72 ± 0.02c Eggshell (n = 3) 7.00 ± 0.01a 5.96 ± 0.21c 6.75 ± 0.04b 6.64 ± 0.01b 6.02 ± 0.06c 5.71 ± 0.01d 1BPW = buffered peptone water 35°C 2LB = lactose broth 35°C 3M-9 = minimal salts medium 35°C 4UP = Universal Pre-enrichment broth 35°C 5RV = Rappaport-Vassiliadis broth 42°C 6TT = tetrathionate broth 42°C n/a = not analyzed a–fvalues within rows with no-like superscripts are different by GLM and Duncan's multiple range test, P < 0.05. View Large The media that resisted the drop to a harmful pH, independent of the sample type were BPW, M-9, and UP. BPW was slightly better than the other 2. With LB, the pH dropped as low as 4.2 and consistently below pH 5.0 for broiler litter and boot covers (both turkey and broiler boot covers). In this study, BPW seems to be the best media for pre-enrichment of dry contaminated samples. The results from this study demonstrate that the pH of some pre-enrichment and selective enrichment media with an assortment of sample types can become very acidic, to the point of being lethal or resulting in significant injury to many cells of the target organism . Obviously, the death of a cell due to acid injury would reduce the ability to detect Salmonella in a sample and significant acid injury could also result in a false negative result. Many researchers testing this type of sample may be completely unaware of the hostile acidic conditions that can occur in the pre-enrichment or selective enrichment media. As a result, the incidence and level of Salmonella in these sample types may be underestimated. This can result in a false sense of security when the presence of Salmonella in many of these samples is not detected. CONCLUSIONS AND APPLICATIONS When analyzing dry poultry environmental samples, a drop in pH in some pre-enrichment media creating an acidic condition, can result in death or injury of the target organism (Salmonella). This situation reduces the ability to detect Salmonella and could produce a false negative result. Many researchers analyzing these types of samples are unaware of the hostile acidic nature of the pre-enrichment media and do not realize that the laboratory procedure is resulting in an underestimation of Salmonella in samples. The procedures used for detecting Salmonella should be revisited to include the international standards currently in use. Footnotes Primary Audience: Researchers, Regulators, Feed Manufacturers, Veterinarians REFERENCES AND NOTES 1. Jensen E. L., Rosales G.. 2002. Salmonella control in primary breeders. Poult. E-Digest 2: 1– 8. 2. Corry J. E. L., Allen V. M., Hudson W. R., Breslin M. F., Davies R. H.. 2002. Sources of Salmonella on broiler carcasses during transportation and processing: modes of contamination and methods of control. J. Appl. Microbiol. 92: 424– 432. Google Scholar CrossRef Search ADS PubMed 3. Shirota K., Katoh H., Murase T., Ito T., Otsuki K.. 2000. Salmonella contamination in commercial layer feed in Japan. J. Vet. Med. Sci. 62: 789– 791. Google Scholar CrossRef Search ADS PubMed 4. Shirota K., Katoh H., Musase T., Ito T., Otsuki K.. 2001. Monitoring of layer feed and eggs for Salmonella in eastern Japan between 1993 and 1998. J. Food Prot. 64: 734– 737. Google Scholar CrossRef Search ADS PubMed 5. Cox N. A., Cason J. A., Buhr R. J., Richardson K. E., Richardson L. J., Rigsby L. L., Fedorka-Cray P. J.. 2013. Variations in preenrichment pH of poultry feed and feed ingredients after incubation periods up to 48 hours. J. Appl. Poult. Res. 22: 190– 195. Google Scholar CrossRef Search ADS 6. Cox N. A., Richardson K. E., Cosby D. E., Berrang M. E., Cason J. A., Rigsby L. L., Holcombe N., DeRome L.. 2016. Injury and death of various Salmonella serotypes due to acidic conditions. J. Appl. Poult. Res. 25: 62– 66. Google Scholar CrossRef Search ADS 7. Whirl Pak 18-oz/532-mL Write-On, puncture roof tabs sample bags . Nasco Inc., Fort Atkinson, WI. 8. Gomez R. F., Sinskey A. J., Davies R., Labuza T. P.. 1973. Minimal medium recovery of heated Salmonella typohimurium LT2. J. Gen. Microbiol. 74: 267– 274. Google Scholar CrossRef Search ADS PubMed 9. North W. R. Jr. 1961. Lactose preenrichment method for isolation of Salmonella from dried egg albumin. Its use in a survey of commercially produced albumin. Appl. Microbiol. 9: 188– 195. Google Scholar PubMed 10. Bailey J. S., Cox N. A.. 1992. Universal preenrichment broth for the simultaneous detection of Salmonella and Listeria in foods. J. Food Prot. 55: 256– 259. Google Scholar CrossRef Search ADS 11. VWR Model SB70P, VWR Scientific, Westbury, NY. 12. Statistica 12. Statsoft, Tulsa, OK. Published by Oxford University Press on behalf of Poultry Science Association 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.
Journal of Applied Poultry Research – Oxford University Press
Published: Mar 1, 2018
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