Stratification of bacterial concentrations, from upper to lower, in broiler litter

DESCRIPTION OF PROBLEM

Broiler litter contains high levels of bacteria, from 108 to 1011 cfu/g of litter [1–4]. Common potentially pathogenic bacteria found in broiler litter are Escherichia coli, Staphylococcus spp., and Clostridium spp., less common are Salmo-nella [2, 3, 5–7]. Staphylococcus spp. have been found to occur in the highest concentrations and represent the highest percentage of total bacteri-al population, which can be as high as 29.1% of

the total bacteria present within the litter [2, 3, 8, 9]. Escherichia coli is commonly used as an in-dicator of fecal contamination, and is commonly found in broiler litter in significant populations, between 105 and 1010 cfu/g litter [2].

Salmonella, E. coli, Clostridium perfringens, and Staphylococcus spp. are all known to cause significant numbers of foodborne illnesses in people [10]. Salmonella enterica strains are the leading cause of bacterial foodborne illness in the United States, causing more than 1 million

© 2013 Poultry Science Association, Inc.

Stratification of bacterial concentrations, from upper to lower, in broiler litter

Z. T. Williams and K. S. Macklin 1

Department of Poultry Science, Auburn University, Auburn, AL 36849

Primary Audience: Flock Supervisors, Researchers

SUMMARY

There have been numerous research studies concerning the different types of bacteria in broiler litter. However, very little is known about the spatial distribution of bacteria from the top to the bottom of the litter bed. Two experiments were conducted, one of a single flock in a pen trial, and the second on a commercial broiler farm for 8 consecutive flocks. Core litter samples were taken and 3 fractions of litter were removed from each core for bacterial analysis: the up-per 5.08 cm of litter, the exact middle of the litter, and the lower section of litter directly on top of the dirt pad, being sure to include some dirt pad. Core litter samples were taken specifically at 7 d before placement then again at 1, 7, 14, 21, 35, and 49 d of age in experiment 1, and 14 and 35 d of age in experiment 2. Core samples were removed from 6 pens in the first experi-ment. In experiment 2, core litter samples were taken near the evaporative cooling cells, the environmental control room or middle of the house, and near the exhaust fans. Litter fractions were analyzed for Staphylococcus spp., Clostridium perfringens, Escherichia coli, Salmonellaspp., total aerobes, and total anaerobes. A decrease was observed from the top to middle and from middle to bottom fractions of litter for all bacteria. From upper to lower, all bacteria con-centrations were reduced by at least 99%. Therefore, the concentration of bacterial populations within broiler litter could shift dramatically within only a few inches of litter and the lower fraction of litter holds relatively less bacteria than the upper or middle fractions.

Key words: litter , bacteria stratification , management

2013 J. Appl. Poult. Res. 22 :492–498 http://dx.doi.org/ 10.3382/japr.2012-00705

1 Corresponding author: [email protected]

493WILLIAMS AND MACKLIN: LITTER BACTERIA DISTRIBUTION

cases per year [10]. Of these cases, approxi-mately 221,000 are of poultry-type meat origin [11]. Escherichia coli strains account for ap-proximately 200,000 cases of human food-borne illnesses per year, an estimated 7% are of poul-try meat origin [10, 11]. Clostridium perfringens causes 965,000 causes of human foodborne ill-nesses a year, and Staphylococcus spp. account for approximately 240,000 cases [10]. Staphy-lococcus spp. are ubiquitous in the environment and are found in high numbers in poultry litter [2, 8]. Of these bacteria, 3 can cause illness in poultry: E. coli, C. perfringens, and Staphylo-coccus spp. Clostridium perfringens can cause necrotic enteritis and gangrenous dermatitis in broilers, both of which can result in morbidity or mortality and lead to a significant performance loss [12, 13]. Escherichia coli can cause coli-bacillosis; though this disease is not typically as severe as diseases caused by C. perfringens, it does lead to live performance reduction. Staphy-lococcus spp. infections are known to cause cellulitis and swollen hocks in poultry, both of which can lead to either impaired growth or a downgrade in the final product [14]. Staphylo-coccus spp. are noted as being sources of numer-ous antibiotic-resistant genes that are capable of being passed to other competent bacteria [15, 16].

Previous research into litter bacterial levels was conducted by the grab-sampling method, which typically examines only the top 2 inches of litter. The grab sampling method is conducted by scraping the heel of a gloved hand across the top of the litter; this litter is then placed in a bag and can possibly be mixed with litter from other areas [2, 3, 7]. To date, little research has been performed that examines the remaining portion of litter. Research that has been conducted on the middle and bottom fractions of litter has of-fered some interesting findings. Litter samples from the bottom 2 inches of litter have yielded different E. coli isolates than the top portion of litter [17]. An examination of total aerobes, an-aerobes, and coliforms yielded a direct relation-ship between litter depth and bacterial concen-tration, with the researchers finding significantly decreased bacterial numbers in the middle and lower fractions of broiler litter [18].

The objective of this study was to find the spatial differences in bacterial concentrations of

total bacteria and a few select pathogenic bac-teria of importance to the poultry industry in 3 fractions of litter: upper, middle, and lower. We examined 2 hypotheses: (1) that total bacterial concentrations decrease in the middle and lower litter fractions, and (2) that the lower fraction of litter provides a more anaerobic condition and has more or an equal concentration of anaerobic bacteria. Initially these differences were mea-sured at the Auburn University Poultry Science Research Unit. A larger, year-long study was also performed in which these parameters were measured in 2 commercial broiler houses that had clean bedding added at the initiation of the trial.

MATERIALS AND METHODS

Auburn University Poultry Science Research Unit

Core litter samples were taken during a sin-gle, 49-d broiler flock on the Auburn University Poultry Science Research Unit. Core litter sam-ples were taken from each of 6 individual floor pens. Samples were taken 1 wk before chick placement, on the day of placement, and then at 7, 14, 21, 35, and 49 d of age. Each pen had litter that was previously used for at least 1 grow-out, a nipple watering system, and a single gravity-type feeder.

Commercial Broiler Farm

Litter samples were taken from a single broil-er farm over the course of 1 yr, for a total of 8 consecutive flocks, starting July 10, 2010 and ending July 27, 2011. This farm was located in northeast Alabama, and consisted of four 50 × 500 ft and two 60 × 600 ft solid sidewall houses. The 50 × 500 ft houses were 7-yr-old and the 60 × 600 ft houses were new, with the first flock being placed in July 2010. New pine shaving lit-ter was added to the houses to be sampled at the initiation of the study.

Litter samples were taken twice per grow-out flock at 14 and 35 d of age. Core litter samples were taken from one 50 × 500 ft house and one 60 × 600 ft house. Cores were taken in the open area in the middle of the house near the evaporative cooling cells, at the center of house (near control room), and near the exhaust fans (Figure 1).

494 JAPR: Research Report

Litter Sampling and Microbiological Methods

Core litter samples were taken using 1 of 6 sterile 5.08-cm (inside diameter), 60.96-cm long PVC pipes. Each PVC pipe had a 2.54 × 25.4-cm window cut into the side to allow for litter removal, these windows were sealed with duct tape during sample harvesting. Measurement markings were made on the side of the window so that litter depth could be determined, this would serve later as a guide when removing litter. Pipes were driven down through the lit-ter and into the hard dirt pad underneath, which would serve to plug the bottom of the pipe so litter would not fall out. After each sample had been taken, the PVC pipe was sealed on top and bottom with duct tape for transport to the labo-ratory.

From each core sample, 10 g of litter was taken from upper, middle, and lower and placed into Whirl-Pak bags [19]. Top litter was con-sidered that which was directly on top of the core, middle was litter halfway down the core, and bottom was litter directly above and includ-ing the hard dirt pad. To each sample, 90 mL of PBS [20] was added and then homogenized in a stomacher for 60 s [21]. Dilutions (1:10) were performed in tubes containing 9 mL of PBS until a sufficient dilution was achieved.

From the dilution tubes, 100 µL was spread-plated onto duplicate plates of differential me-dia. Three dilutions per sample were plated onto aerobes, plate count agar [20]; anaerobes, anaer-

obic agar [20]; C. perfringens, Oxoid tryptose sulfite cycloserine agar [22]; E. coli, MacConk-ey agar [20]; Staphyloccocus, Baird Parker agar with tellurite [23]; and Salmonella enriched in tetrathionate broth, Hajna [20] before streaking for isolation on Xylose-Lysine-Tergitol 4 agar [20]. Total anaerobes and C. perfringens plates were incubated in an anaerobic chamber at 37°C in 5% H2, 5% CO2, and 90% N2 for 24 h. All oth-er plates were incubated at 37°C in an aerobic atmosphere for 24 h, except for Staphyloccocus plates, which were incubated for 48 h to allow colonies to grow larger. After incubation, plates were removed from the incubator and bacterial colonies enumerated.

Data were analyzed in SAS 9.2, using the GLM function at P ≤ 0.05, for effects due to bird age, flock number, house, location within house, litter depth, and interactions [24]. Significant differences were separated by either t-test (bird age) or Tukey’s honestly significant difference test. Bacterial counts were log10-transformed be-fore analysis. Results given are composite data from all litter samples taken.

RESULTS AND DISCUSSION

Auburn University Poultry Research Unit

As shown in Table 1, all bacteria decreased in concentration from the upper to middle and bot-tom fractions (P < 0.05). For all bacteria tested, except C. perfringens, there was an approximate

Figure 1. Graphical representation of the layout of commercial broiler houses sampled during the second study. Evaporative cooling cells are on the left, the control room is in the middle, and exhaust fans are on the right. Solid lines represent water lines and dashed lines represent feed lines. Each X marks approximate sampling location where core litter samples were taken.


2 log or 99% decrease between the upper and lower litter fractions (P < 0.05). Total aerobic bacteria and Staphylococcus concentrations de-creased by approximately 1 log from upper to middle and 1 log from middle to lower. Anaero-bic bacteria and E. coli decreased by 1.4 and 1.7 logs, respectively, from the upper to middle frac-tions. Anaerobic bacteria concentration was fur-ther reduced from the middle to lower fraction by 1 log. Escherichia coli concentration only decreased by 0.4 log from the middle to lower fraction. Clostridium perfringens was found in low concentrations, less than 0.5 log10 cfu/g of litter, in any fraction.

As birds aged, bacteria increased in number, as much as 5 log in the case of E. coli (Table 2). At 35 and 49 d of age, a numerical decrease

was observed in all bacteria concentrations from 21 d, and a significant decrease in E. coli (P < 0.001).

Commercial Broiler Farm

In agreement with the results from the short-term study described in the previous section, a significant decrease was observed for all bac-teria sampled between the upper, middle, and lower litter fractions (P < 0.0001; Table 3). Total aerobic bacteria and Staphylococcus concen-trations had a 1 log decrease from the upper to middle fraction and a 1.5 log decrease from the middle to lower fraction (P < 0.0001). Anaero-bic bacteria had a 1.3 log decrease between the upper and middle fraction, but a 2.5 log decrease

Table 1. Composite litter fraction results for preliminary trial

Litter fraction

Bacteria1,2,3

Aerobic Anaerobic Staphylococcus spp.Escherichia

coliClostridium perfringens

Upper 8.6x 7.1x 8.7x 4.4x 0.4y

Middle 7.6y 5.7y 7.6y 2.7y 0.1yz

Lower 6.8z 4.5z 6.6z 2.3z 0.0z

SEM 0.3 0.3 0.1 0.1 0.1P-value 0.010 <0.001 0.008 0.001 0.044x–zMeans within a column with different superscripts differ (P < 0.05).1Log10 cfu/g of litter.2Means are composited from all sampling times.3Values are means with n = 42.

Table 2. Bacteria concentrations during a 49-d broiler grow out

Bird age (d)

Bacteria1,2,3



−74 7.7 3.5z 5.7yz 1.2y 0.01 7.2 5.0xyz 5.1z 0.4z 0.07 7.4 6.7xy 7.2xy 3.7w 0.214 6.9 6.4xy 7.6x 5.4v 0.121 8.4 7.1x 8.5x 5.5v 0.635 8.2 5.9xyz 8.5x 3.6w 0.049 7.8 4.7xyz 8.3x 1.9x 0.3SEM 0.5 0.5 0.1 0.1 0.2P-value 0.514 <0.001 0.015 <0.001 0.156v–z Means within a column with different superscripts differ (P < 0.05).1Log10 cfu/g of litter.2Values are means with n = 36.3Means are composited from all litter fractions.4Seven days before placement.


from the middle to lower fraction (P < 0.0001). Escherichia coli decreased by 2.6 logs from the upper to middle fraction and 1.3 logs from the middle to lower fraction (P < 0.0001); C. per-fringens decreased by 0.6 log from the upper to middle fractions and 0.9 log from the middle to lower fraction (P < 0.0001).

In Table 4, differences in mean concentra-tions for each sampling location are shown. The only statistical difference is the higher concen-tration of total aerobes found at the middle of the house than at the evaporative cooling cells (P < 0.001). No Salmonella were recovered in either study (data not shown).

The decrease in concentration of all bacteria types measured between the upper, middle, and lower fractions of litter show that, in just a few inches of litter, the overall bacterial load can de-crease by 99% or more. These findings are in agreement with Barker et al. [18], who found a significant decrease in total aerobes, anaerobes, and coliform bacteria between the upper and

lower fractions of litter. This observation was expected, as the upper fraction of litter receives a constant supply of new bacteria from excreta; in addition, it receives ample water, nutrients, and oxygen. The most significant decrease was observed in the lower fraction of litter, which contained a portion of the dirt pad. In previous studies it was shown that litter bacterial loads up to 1011 cfu/g of litter by the grab sampling method at various locations within the house in-cluding, but not limited to, feed and water lines, side walls, evaporative cooling cells and exhaust fans. The researchers would then extrapolate those results to the entire quantity of litter in the house [1–4].

Based on the data in this paper, grab sample findings may be inadequate to describe the litter bacteria community simply because the bacte-ria are not evenly distributed, and that beyond the upper 2 inches of litter bacterial numbers are significantly reduced by several orders of mag-nitude. Bacterial concentrations being lower in

Table 3. Bacteria concentrations in each litter fraction (upper, middle, lower) on a commercial broiler grow-out farm

Fraction

Bacteria1,2,3



Upper 8.5x 6.1x 8.5x 4.4x 1.6x

Middle 7.7y 4.8y 7.3y 1.8y 1.0y

Lower 6.1z 2.3z 5.7z 0.5z 0.1z

SEM 0.1 0.2 0.1 0.2 0.1P-value <0.0001 <0.0001 <0.0001 <0.0001 <0.0001x–zMeans within a column with different superscripts differ (P < 0.05).1Log10 cfu/g of litter.2Means are composited from all sampling times, house, and house location.3Values are means with n = 96.

Table 4. Differences in broiler litter bacteria concentration at 3 different locations within a commercial broiler grow-out house at the exhaust fans (EF), middle of house (M), and evaporative cooling cells (ECC)

Location

Bacteria1,2

Aerobic Anaerobic Staphylococcus spp. E. coli C. perfringens

EF 7.5yz 4.5 7.1 2.5 0.9M 7.6z 4.4 7.2 2.2 1.0ECC 7.3z 4.3 7.2 2.0 0.7SEM 0.1 0.2 0.1 0.1 0.1P-value 0.0536 0.7649 0.6921 0.0944 0.4802y,zMeans within a column with different superscripts differ (P < 0.05).1Log10 cfu/g of litter.2Values are means with n = 96.


this paper than reported in previous research could be attributed to the small sampling area. A larger sampling area in the case of grab sam-pling would allow for areas of higher bacterial populations to be added to the samples (i.e., water lines, feed lines, or any other areas with higher bird density). As core samples were taken in the center of the house, this area would have less bird density and, therefore, fewer bacteria. It was hypothesized that the lower depth of lit-ter would have reduced oxygen content and, therefore, yield a more anaerobic environment. However, based on data shown here, we dispute that hypothesis, as less than 3 log10 cfu/g of litter were found in the lower fraction of litter. This could also be attributed to sampling technique, as strict anaerobic conditions were not kept dur-ing sampling and true anaerobes would be killed, leaving only spore-forming (e.g., Clostridium) and facultative anaerobes. The major contribut-ing factor to the higher bacterial concentrations in upper fraction of litter is simply the constant shedding of feces onto the surface of the litter. Bacteria either do not survive or do not progress into the middle and lower fractions of litter.

The increase observed in bacteria concen-trations over the flock age is in agreement with Macklin et al. [4]. Before chick placement, the researchers observed total aerobic bacteria lev-els of 105 cfu/g of litter, which then increased to 108 immediately after bird placement, and reached their highest concentration during wk 4 at 1010 cfu/g, followed by a subsequent decline. Based on this observation, once birds are placed and begin shedding bacteria through fecal mate-rial onto the litter, the number of bacteria rapidly increases but will level off around 3 to 4 wk of age. Once the upper limit for bacteria has been reached, the decline may mean that the litter can only support a certain number of bacteria de-spite the constant addition of new bacteria.

CONCLUSIONS AND APPLICATIONS

1. An overall decrease in the concentration of bacteria takes place from the upper fraction to lower fraction of broiler lit-ter, regardless of the location sampled (evaporative cooling cells, middle of house, and exhaust fans).

2. Based on the results of this study, and assuming that the bacterial numbers are consistent throughout the house, remov-al of the top few inches of litter would reduce litter bacteria concentrations by several logs, potentially reducing the number of pathogenic bacteria present; this would also allow farmers to add considerably less new litter in a house, leading to a significant cost reduction.

3. With the decrease in total anaerobic bacteria, from upper to lower, it can be concluded that the bottom fraction of the litter bed is not more anaerobic than the top, as has been reported.

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Stratification of bacterial concentrations, from upper to lower, in broiler litter