01998 Applied Poultry Science, l n c
MICROBIOLOGICAL
SURVEY
OF GEORGIA
POULTRY
LITTER
SCOTT A. MARTIN' and MARK A. M C C A "
Department of Animal and Dairy Science, 308 Livestock-Poultry Bldg.,
The University of Georgia,Athens, GA 30602-2771
Phone: (706) 542-1065
FAX: (706) 542-0399
W. DOUGLAS WALTMAN I1
Georgia Poultry Laboratoy, Oakwoocl: GA 30566
Primary Audience: Nutritionists, Researchers, Veterinarians, Feed
Manufacturers
of the poultry industry is beneficial because
DESCRIPTION
OF PROBLEM
it is readily available, economical, and of
Lack of regular rainfall often leads to a
shortage of high quality forages in the southeastern United States. Consequently, during
dry years many beef producers rely on alternative feedstuffs to provide the necessary
nutrients for their animals. One of these alternatives includes poultry litter. This by-product
1
To whom correspondence should be addressed
some nutritionalvalue to the ruminant animal,
particularly as a source of nitrogen.
Some concern has been raised by Georgia
beef producers regarding the safety of feeding
large quantities of poultry litter to beef cattle.
It is well known that poultry raised in confiiement, especially chicks, is susceptible to in-
Field Report
91
MARTIN et al.
fection by pathogenic microorganisms, especially Salmonella typhimurium [l, 21. Therefore, it is possible that these pathogenic
microbes could be prevalent in poultry litter
fed to ruminants and eventually passed on to
humans via consumption of beef products.
Because it is assumed that harmful microorganisms are killed during the composting of
poultry litter prior to feeding (although in
some cases, the litter is not composted prior to
feeding), limited research has been conducted
to evaluate whether there is a significant
pathogen problem in litter fed to cattle. The
objective of this study was to collect samples
of poultry litter from different locations in
Georgia and to analyze each sample for the
presence of pathogenic bacteria using selective microbiological media. In addition, analyses were conducted to determine the nutrient
content of each sample.
(MAC) = yields counts of Gram-negative
bacteria and lactose fermenting (coliform)
bacteria; Baird-Parker agar (BP) = yields
counts of Staphylococcus aureus; MAC
sorbitol (MACS) = yields presumptive
Escherichia coli 0157/H7 bacteria. The composition of each selective medium is detailed
in the Difco Manual [3].
Total aerobic bacteria, S. aureus, Gramnegative bacteria, and coliforms were quantitated by inoculating the tryptose, BP, MAC,
and MACS plates with 0.5 mL of diluted
sample using a Spiral Plating instrument
(Spiral Systems, Cincinnati, OH). These
plates were incubated at 35 to 37°C for 40 to
48 hr. To screen for the presence of E. coli
0157/H7, 0.5 mL of each dilution (1:lO and
1:lOOO) was inoculated onto two MACS plates.
The plates were incubated at 35°C for 48 hr.
Ten clear colonies were picked from each
dilution and transferred to a MAC plate. Lactose fermenting colonies were biochemically
identified as E. coli and tested for agglutination with 0157/H7 antisera.
The salmonellae isolation procedures
used in this study included three separate
techniques, two selective enrichment media,
and two plating media. The combination of
these media and methods have been shown
to be extremely effective [4]. Presence or
absence of Salmonella was determined by
adding 25 g of litter to 225 mL of tetrathionate
(TT)broth in a sterile whirlpak bag. Litter
samples plus TT broth were incubated at 35
t o 3 7 T for 24 hr and then inoculated onto
brilliant green agar with 20,uuglmL novobiocin
(BGN) and xylose lysine tergitol 4 (XLT4)
agar plates. In addition to this 24 hr treatment,
the TT broth mixture was left at 25°C for 5
to 7 days. Furthermore, the 1 : l O dilution of
titter sample in buffered peptone water was
incubated at 35 to 37°C for 24 hr (preenrichment). One mL of this solution was
then transferred to 10 mL of TT broth and
0.1 mL was inoculated into RappaportVassiliadis (RV) broth [5]. The TT and RV
enrichments were incubated at 41°C for 24 hr
and then inoculated onto BGN and XLT4
plates. All BGN and XLT4 plates were incubated at 35 to 37°C for 24 hr. Salmonella
suspect colonies were transferred to triple
sugar iron agar [3] and biochemically identified and serologically typed for somatic antigens. If Salmonella were not detected after
MATERIALS
AND METHODS
Eighty-six poultry litter samples were
obtained from locations throughout the state
of Georgia (Figure 1). Samples 1 to 41 were
collected during spring and summer 1994,
while samples 42 to 86 were collected during
winter 1995. Out of the 86 litter samples, 64
were composted, 18were not composted, and
no determination could be made about 4 samples. The sources providing the titter samples
included home produced samples (53%),
locally (within a county or adjoining county)
produced samples (31%), or samples purchased from a commercial broker (16%). For
samples that were composted, the time periods used for composting were Il month
(26% of samples), 2 months (28% of samples),
3 months (18% of samples), or 1 4 months
(28% of samples).
MICROBIAL ANALYSIS
Wenty-five grams of each sample was
transferred into a sterile whirlpak bag and
225 mL of sterile 1% buffered peptone water
was added. The sample was allowed to sit
for 30 to 60 min at room temperature with
frequent shaking. One mL of this sample
(1:lO dilution) was transferred to 99 mL of
sterile saline to make a 1:lOOO dilution. The
following media were inoculated with the
diluted sample preparations (1:lO and
1:lOOO): tryptose agar = yields total aerobic
bacteria plate counts; MacConkey agar
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MICROBIOLOGY OF POULTRY LITTER
92
FIGURE 1. Poultry litter samples obtained from Georgia counties marked in black. These included: Baker,
Carroll, Chattahoochee, Coffee, Colquitt, Columbia, Coweta, Dougherty, Elbert, Emanuel, floyd, Franklin,
Gilmer, Habersham, Haralson, Heard, Jackson, Jefferson, Lamar, Lanier, Lincoln, Long, Lumpkin, Macon,
Madison, Marion, Monroe, Polk, Tattnall, Walker, Ware, Wilcox, and Wilkinson counties.
the initial 24 hr incubation, 0.5 to 1.0 mL of
the TT broth that had been stored at 25°C for
5 to 7 days was transferred to a fresh tube
of TT broth and incubated for 24 hr at 35 to
37°C and processed as described above.
Mold and filamentous fungi were quantitated by inoculating Sabouraud dextrose agar
plates supplemented with 50pg/mL chloramphenicol and incubated at 37°C for 4 days [3].
All enumerations were performed in duplicate
and are expressed as number of CFU/g of
litter.
1
NUTRIENT ANALYSIS
The nutrient content of all Litter samples
was determined using standard procedures
by The University of Georgia Soil Testing
Laboratory (Athens, GA). The following
nutrients were measured: moisture, crude
protein, bound protein, phosphorus, potas-
Field Report
MARTIN ef al.
sium, calcium, manganese, iron, aluminum,
copper, zinc, sodium, and ash.
93
Almost all of the samples had extremely low
or no Gram-negative bacteria with the exception of Sample 19. Only three samples had
quantifiable coliformsand in two of these samples non-pathogenic E. coli was the predominant bacterium. Even though very sensitive
isolation procedures were used, neither E. coli
0157/H7 or Salmonella was detected in any
litter sample. Mold was detected in 23 samples, but in most of the samples mold contamination was very low.
Compared to the first 41 samples collected during spring and summer 1994
(Table l), the 45 samples collected during
winter 1995had more mold contaminationand
higher total bacterial counts (Table 2). This
finding most likely results from a higher moisture environment in the winter than in the
spring and summer. However, there was no
consistent effect of composting vs. noncomposting, length of composting time, or
source (home, local, broker) of litter on microbial numbers.
The average as well as range of nutrient
(moisture, protein, minerals, ash) content for
all 86 litter samples appears in Table 3. Crude
protein was over 6-fold higher than bound
protein and the average moisture content was
21.9%. All samples contained similar amounts
(2 to 3%) of phosphorus, potassium, and calcium, while manganese concentrations were
much lower. Sodium, aluminum, and iron concentrations were much higher than concentrations of zinc and copper; and ash averaged
RESULTS
AND DISCUSSION
The enumeration and identification of
microorganisms in the first 41 poultry Litter
samples appear in Table 1. The number of
total bacteria in these samples ranged between 1,200 and 84,OOO,OOO CFu/g, but only
11 of the litter samples had total bacterial
counts of more than l,OOO,OOO CFU/g. Fifteen
of the samples had counts less than
100,OOO CFU/g. Under the conditions used
in our study, the predominant bacteria in these
litter samples was Sfaphylococcus xylosus.
Almost all of the samples had extremely
low or no Gram-negative bacteria (except
Sample 2), and only two samples had quantifiable coliforms with non-pathogenic E. coli
being the predominant bacterium. Even after
pre-enrichment only 10 samples had coliforms
(data not shown). E. coli 0157/H7 was not
found in any litter sample and no Salmonella
were isolated in spite of extremely sensitivt:
isolation procedures. Mold in most of the samples was extremely low with only 13 samples
showing the presence of mold.
In the second survey, 45 poultry litter
samples were analyzed (Table 2). Fifteen of
the litter samples had total bacterial counts
of greater than 10 million CFU/g. Another 6
Litter samples had total bacterial counts between l and 10 million CFU/g. Ten litter samples had total bacterial counts of less than
100,OOO CFU/g. The predominant bacteria in
these samples was Staphylococcus xylosus.
30.4%.
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MICROBIOLOGY OF POULTRY LITTER
94
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MARTIN et al.
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MICROBIOLOGY OF POULTRY LITTER
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MARTIN et al.
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MICROBIOLOGY OF POULTRY LITTER
98
NUTRIENTS (Dry Basis)
I
I
AVERAGE
RANGE
Moisture, %
21.9
10.143.4
Crude Protein, %
27.9
15.041.5
Bound Protein, %
4.1
1.4-13.2
Phosphorus, %
2.1
1.0-5.3
Potassium, %
3.0
1.0-4.7
Calcium, %
3.0
1.1-8.1
Manganese, %
0.63
0.27-1.75
Iron, ppm
3859
1025-9869
Aluminum, pprn
3957
684-9919
Copper, PPm
Zinc, pprn
557
484
52-1306
160-1422
Sodium, ppm
8200
3278-14,344
Ash, %
30.4
14.4-69.2
CONCLUSIONS
AND APPLICATIONS
1. Based on a survey of 86 Georgia poultry litter sources, no pathogenic bacteria were detected
in any of the samples.
2. It appears that poultry litter is not asource of harmful pathogenic bacteria when fed to beef
cattle, but is a good source of crude protein and some minerals.
3. The results from this study provide important information that can be used to educate the
public should the practice of feeding poultry litter to domestic ruminants come under
scrutiny.
~~
REFERENCES
AND NOTES
1. Hinlon, A, Jr., D.E C o d e r , G.E Spates, J.O.
Norman, RL Ziprin, RC. Beier, and J.R Dthach,
1990. Biological control of Salmonella
. in
young chickens. Avian Dis. M626-633.
2. Stavric, S. and J.-Y. JYAoust, 1993. Undefined and
defined bacterial preparations for the com titive exclusion of Salmonella in pouItIy: A review. !?Food Prot.
56173-180.
3. Difco Manual. 10th Edition. Difco Laboratories,
Detroit, MI 48232.
4. Waitman, W.D., A.M. Home, and C.Plrkle, 1995.
Comparative analysis of media and methods for isolating
Salmonella from poultry and environmental samples.
Pages 1-14 in: Proc. Symp. Diagnosis of
Infections. United States Anim. Health Assn. and Amer.
Assn. of Laboratory Veterinary Diagnosticians.
5. Vassiliadis, P., 1983. Rappaport-Vassiliadis (RV)
enrichment medium for the isolation of salmonellas: An
overview. J. Appl. Bacteriol. 54:69-76.
ACKNOWLEDGEMENTS
Financial support for this study was provided by
the Georgia Beef Board and The University of Georgia
Agricultural Experiment Station. The authors express
their ap reciation to the countyagents that provided the
poultry Etter samples.