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2001 Poultry Science Association, Inc.

EFFECT OF DRY LITTER AND AIRFLOWIN REDUCING SALMONELLA AND

ESCHERICHIA COLI POPULATIONS IN THEBROILER PRODUCTION ENVIRONMENT

C. L. ERIKSSON DE REZENDE, E. T. MALLINSON,N. L. TABLANTE, R. MORALES,1 and A. PARK

Virginia-Maryland Regional College of Veterinary Medicine,

University of Maryland, College Park, MD 20742

L. E. CARR

Department of Biological Resources Engineering,University of Maryland, College Park, MD 20742

S. W. JOSEPH2

Department of Cell Biology and Molecular Genetics,

University of Maryland, College Park, MD 20742

Phone: 301-405-5452

FAX: 301-314-9489

email: [email protected]

Primary Audience: Flock Supervisors, Veterinarians, Extension Agents, Food Scientists,Quality Assurance Personnel, Epidemiologists.

SUMMARYBroiler farm management should include properly maintained watering devices and appropriate

house ventilation practices for an optimal preharvest production environment. An effective strategy

to prevent dripping water and spillage and to ensure a modest but continuous and uniform

flow of air directly over the litter surface appears to create an unfavorable environment for

enteric bacteria.

Qualitative and quantitative findings have revealed that Salmonella contamination loci are not

equally distributed in the broiler litter. Higher Salmonella and Escherichia coli counts were detected

in litter samples with water activity (aw) greater than 0.90 and percentage moisture content (MC)

greater than 35%. At reduced aw and MC levels, the numbers of viable Salmonella cells were low,

further reflecting the importance of preventing excessively damp areas (e.g., cake) in broiler

litter. Additionally, litter surface airflow measurements suggested that those areas exposed to

modest air velocities (100 to 150 ft/min or approximately 1.5 mph) had drier litter and reduced

E. coli populations.

Key words: Airflow, litter management, litter moisture, risk reduction, Salmonella, water activity

2001 J. Appl. Poult. Res. 10:245251

1 Presently at Research Triangle Park, NC 27709.2 To whom correspondence should be addressed.

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JAPR: Research Report246

DESCRIPTION OF PROBLEMHigh water parameters in the litter can create

favorable conditions for the multiplication of

enteric pathogens such as Salmonella and E.coli. Indeed, previous drag swab (DS) studies

[1, 2] have shown that broiler litter surfaces

with water activity (aw) greater than 0.90 were

associated with increased Salmonella recovery

rates. Furthermore, the risk of Salmonella con-

tamination on processed broiler carcasses is re-

duced when carcasses originate from farms with

low detectable levels of Salmonella [3]. Com-

bined, these observations suggest that lower awlevels at the litter surface likely contribute to

lower Salmonella loads on carcasses [3]. There-

fore, the transmission of Salmonella from the

farm to the processing plant and potentially to

marketed carcasses may be diminished and con-

trolled by implementing management strategies

that reduce bacterial loads in the production en-

vironment. Identification of Salmonella and E.

coli multiplication hot spots in the litter should

enhance poultry health and quality control.

This study, based on the quantitative Salmo-

nella and E. coli data obtained from litter sam-ples with various levels of aw and moisture con-

tent (MC), identified regions in litter that consti-

tute multiplication hot spots (i.e., litter with

aw from 0.90 to 0.95 or MC from 35 to 50%).

The measurement of airflow velocities over litter

surfaces further revealed that moderately in-

creased, rates of airflow directly over the litter

surface generally contribute to drier litter and,

consequently, to decreased viable Salmonella

and E. coli populations.

MATERIALS AND METHODSSAMPLING

Brood chambers of 13 commercial broiler

houses from four different farms located on the

Delmarva Peninsula were visited on a single or

on multiple occasions during the growout pe-

riod, except for one farm, which was visited

during a subsequent growout period. On all

farms, except for one, sampling was restrictedto the brood chambers, because preliminary data

(not included) indicated that they had the highest

prevalence of Salmonella-positive DS. At each

visit the brood chamber and, in some cases the

entire house, was sampled using four to six DS

per chamber to obtain an overall perspective of

the Salmonella status of the house. Swabs were

placed in 1,000-mL Whirl-Pak bags and then

placed inside an insulated, refrigerated box. The

swabs were processed, and broth suspensions

were cultured qualitatively as described by

Opara et al. [1]. Next, 96 litter samples were

collected as described by Mallinson et al. [4].

Each sample comprised 5 to 10 pooled collec-

tions from wet and dry areas (five samples from

areas under nipple drinkers and five from areas

5 ft away). These areas were selected because

they had comparable moisture levels between

houses on the same farm and between farms.

Furthermore, pooled samples were used to in-crease the probability of detecting Salmonella

from the litter. These pooled litter samples were

collected and placed into 36-oz Zip-Lock bags,

using separate disposable surgical gloves for

each pool. Samples were collected from identical

sites for physical (aw and MC) and bacterial

(Salmonella and E. coli) analyses.

AIRFLOW MEASUREMENT

Airflow patterns over each sampled areawere measured using a Digital Hygro thermome-

ter, anemometer, data-logging instrument ac-

cording to the manufacturers instructions [5].

The instruments anemometer, located at the end

of a one-meter handle bar, was held approxi-

mately 1 in. from the litter base at the center of

the area from which a particular litter pool was

collected. Each air velocity reading represented

the average flow of air at a particular location

during a 15-s interval.

Aw AND MC MEASUREMENTS

AND LITTER PROCESSING

The aw and MC were measured as previously

described [6]. Litter samples were processed ac-

cording to Mallinson et al. [4]. Briefly, litter

samples and DS were immediately transported

back to the laboratory in an insulated container

for processing. Thirty grams from each litter

sample was suspended in 300-mL of buffered

peptone water (BPW) contained in 500-mLpolypropylene wide-mouth bottles [7]. Samples

were vigorously shaken for 2 min and lightly

filtered. Three 80-mL aliquots were divided

among three 100-mL Snap-Cap jars [8], and two

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ERIKSSON ET AL.: SALMONELLA-ESCHERICHIA COLI REDUCTION 247

10-mL aliquots of the filtrate were poured into

two 15-mL centrifuge tubes. The jars and tubes

were immediately flash-frozen at 70C.

SALMONELLA QUANTIFICATION

Litter samples were prescreened for further

Salmonella quantification as previously de-

scribed [4]. Briefly, Salmonella screening was

performed by qualitatively testing for this organ-

ism using two of the three 80-mL litter suspen-

sions for each sample. The purpose of prescreen-

ing was to preserve materials and to avoid per-

forming the laborious five-tube most probable

number (MPN) procedure on samples that were

essentially Salmonella-negative. The last 80-mL

suspension of those samples positive for Salmo-

nella at prescreening were quantified for Salmo-

nella by using the five-tube MPN procedure [9,

10]. After primary and selective enrichment in

BPW and tetrathionate-Hajna [11] broth, respec-

tively, all MPN tubes were plated on Miller-

Mallinson (MM) [12] agar and incubated at

35C. Plates were read after 24 and 48 h of incu-

bation.

E. COLIQUANTIFICATION

E. coli quantification was performed by

thawing one 10-mL aliquot suspension of litter,

which was serially diluted and spread-plated (0.1

mL) on eosin-methylene-blue agar (EMB) [11].

The plates were incubated at 35C for 24 hr

and typical E. coli colonies were counted. The

smaller (10-ml) litter suspension aliquot was

used for E. coli quantification because of the

higher load of this organism in the litter.

STATISTICAL ANALYSIS

We determined the significant difference of

the means of aw, MC levels, and E. coli popula-

tions for litter samples collected from areas ex-

posed to varying airflow levels by using the

directional Students t test [13].

RESULTS AND DISCUSSIONAs reported by Hayes et al. [14], the DS

method was more reliable for qualitatively de-termining the Salmonella status of a house than

culturing litter samples. When compared to the

overall high percentage of negative litter culture

results, these observations suggested that Salmo-

nella spp. were not uniformly distributed

throughout the litter surface. In this study, Sal-

monella spp. were detected in only 17.7% of

all collected litter samples (Table 1). The DS

technique proved to be more sensitive than cul-

ture of litter samples for establishing the Salmo-

nella status of a broiler house with 2.4 greater

detection (42.5%) in commercial houses (Table

1). Because the DS method involves sampling

from a large surface area in the house, it is more

likely to be in contact with the contaminated

areas.

Although the DS method helps establish,

semi-quantitatively, the general prevalence of

Salmonella contamination in the house litter, it

does not adequately examine the direct contribu-tion of physico-chemical litter parameters on

Salmonella and E. coli populations. By collect-

ing data on specific conditions and correspond-

ing bacterial populations of litter samples, the

individual or cumulative effects of certain pa-

rameters (i.e., aw and airflow) on bacterial

growth can be identified. This approach can help

determine the optimal environmental conditions

for the production of healthier and safer poultry.

Previous studies [1, 6] have established thata relationship exists between litter regions with

high aw levels (>0.90) and the number ofSalmo-

nella-positive DS. However, the data presented

here further suggested that high litter MC also

contributed favorably to the presence of Salmo-

nella. Therefore, understanding the relationship

between these two water parameters becomes

important because both may serve as strong indi-

cators of high-risk conditions that support Sal-

monella contamination. We observed a curvilin-

ear relationship between aw and MC levels in

broiler litter (Figure 1). As MC levels decreased

from approximately 60 to 35%, aw decreased

TABLE 1. Comparisons between drag swab and litter

sampling for the detection of Salmonella on seven

broiler farmsA

POSITIVE FOR

DETECTION METHOD SALMONELLA (%)

Drag swab (n = 40) 42.5

Litter samplesB (n = 96) 17.7

AThe only houses that were included in the table were thosewhere Salmonella was detected by either or both methods.BThere was only one instance when a house was positiveby litter sampling but negative by the drag swab method.

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FIGURE 1. Relationship between water activity (aw)and percentage moisture content (%MC) in broiler littersamples (n = 96).

gradually almost linearly. Further, as MC levels

decreased to

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FIGURE 3. Quantification of Escherichia coli from littersamples grouped according to A) water activity (aw) andB) percentage moisture content (%MC) in broiler litter.

Salmonella could possibly be surviving and per-

sisting in the litter bed in a viable but non-culturable phase [18, 19]. Regardless, growth

conditions in the litter, such as elevated water

parameters, might promote greater survival and

notable multiplication of the organism.

Interestingly, litter samples with water pa-

rameters in the highest aw range (0.95 to 1.00)

were not associated with elevated Salmonella

populations. Possibly such conditions are less

than ideal for the growth ofSalmonella, perhaps

due to the creation of a hypotonic environmentor to the dilution of essential nutrients.

TABLE 2. Airflow, water activity (aw), percentage moisture content (%MC), and Escherichia coli populations for

litter surfaces of six commercial broiler flocks

LITTER SURFACESAVERAGE AIRFLOW

AIRFLOW RANGES AT LITTER SURFACE E. COLI CFU/

FPMA (n = 92B) (FPM) %MCC awD 10.0 g LITTERE

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significantly associated with lower MC, aw, and

E. coli population [13]. Because there were a

limited number of Salmonella-positive litter

samples, the direct impact of ventilation on the

reduction ofSalmonella populations was not de-

finitively established. Due to a similarity in re-

sponse to aw and MC changes, the results ob-

tained for E. coli can be extrapolated to Salmo-

nella (Figure 2). Although ventilation practices

varied considerably between farms (number,

placement, static pressure, running time of fans,

and curtain setting), a correlation seemed to exist

between the rate of air movement over a specific

litter location and its water parameters (Table

2). Locations where air movement was approxi-

mately zero tended to have higher water parame-ters, which might have increased the likelihood

of pronounced Salmonella multiplication in such

areas, should the organism have been in-

troduced.

It was also noted that, in contrast to houses

that were ventilated by wind and propeller fans,

tunnel ventilation houses (two observations) had

a higher (131 to 198 ft/min) and unvarying flow

of air over the entire litter surface in the brooder

chamber. This elevated and continuous flow ofair might have contributed to the low detection

of Salmonella from litter samples (1 positive of

26 collected). To achieve airflow of this magni-

tude (150 to 200 ft/min) the grower had four

tunnel fans running concurrently, generating an

in-house static pressure between 0.04 to 0.05.

Static pressure appears to be directly related to

CONCLUSIONS AND

APPLICATIONS

1. Careful attention must be given to aw and MC levels in broiler litter. According to recent data,

aw

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ACKNOWLEDGMENTS

We thank Russell Miller for his valuable technical assistance and

Evandro Missagia for hardware and software support. This research

was supported by a grant from the U.S. Poultry and Egg Association.

This research contributed to Regional Poultry Research Project

NE-127.

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