# 80 
 
 
Guidance for Industry: Studies to 
Evaluate the Utility of Anti-Salmonella 
Chemical Food Additives in Feeds
 
 
FINAL GUIDANCE 
 
 
This final guidance is intended to provide advice on the standards upon studies to 
establish the utility of anti-Salmonella chemical food additives for achieving their 
intended technical effect of maintaining feeds Salmonella-negative should be based.  
 
Comments and suggestions regarding the document should be submitted to Division of 
Dockets Management (HFA-305), Food and Drug Administration, 5630 Fishers Lane, 
Rm. 1061, Rockville, MD 20852.   All comments should be identified with the Docket 
No. 94D-0147. Submit electronic comments to http://www.regulations.gov. 
 
For questions regarding this guidance document, contact Terry Proescholdt, Feed Safety 
Team, HFV-222, Center for Veterinary Medicine, Food and Drug Administration, 7500 
Standish Place, Rockville, MD 20855, 240-453-6838.  e-mail: 
 
terry.proescholdt@fda.hhs.gov.
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Veterinary Medicine (CVM) 
November 21, 2002 
 
   
Table of Contents 
 
 
 
I.  Introduction  ...........................................................................................  3 
 
II.  General Considerations  .............................................................  4 
 
III.  Laboratory Trials  ...............................................................................  5 
 
  General   ......................................................................................................  5 
 
  Dose titration (dose determination) trial   .....................................................  5 
 
  Study of ability to maintain feeds Salmonella-negative  ........................................  8 
 
IV.  Field Trials  ...........................................................................................  9 
 
V.  Collection and Analysis of Samples   .........................................................  10 
 
VI.  References  ............................................................................................  13 
 
 
  2  This final guidance represents our current thinking on this matter.  It does not create or 
confer any rights for or on any person and does not operate to bind the Food and Drug 
Administration (FDA) or public.  An alternative approach may be used if such approach 
satisfies the requirements of the applicable statutes and regulations. 
 
Introduction 
 
Food-borne illnesses can have severe negative impacts on human and animal health, and 
can be fatal.  The resultant economic losses can be quite significant.  Food-borne 
illnesses occur worldwide and can be caused by microbes like Salmonella.  In the United 
States, 76 million cases of food-borne illnesses are estimated to occur annually and to 
result in 325,000 hospitalizations and 5,000 deaths.  Annual economic losses due to 
medical expenses, lost production, and loss of life have been estimated to be as much as 
$35 billion (Ref. 3, 5, 10, 14, 16).    
 
Salmonella are enteric bacteria that cause a significant proportion of food-borne illnesses.  
Expressions of the illnesses caused by Salmonella in humans and other animals range 
from mild to severe diarrhea to anorexia, fever, nervous and respiratory signs, abortion, 
depression, shock, and death.  There are over 2,000 serotypes of Salmonella.  Food 
animals are significant sources of Salmonella infection for humans, and feeds are 
important sources of Salmonella for food animals (Ref. 5, 7, 9, 11, 16).   
 
Under section 402(a) of the Federal Food, Drug, and Cosmetic Act (21 U.S.C. 342(a)) 
animal feed, feed ingredients, and pet food containing Salmonella are adulterated.  
Additionally, 21 CFR 500.35 provides that animal byproducts intended for use in animal 
feed that are contaminated with Salmonella bacteria will be regarded as adulterated.  
FDA's policy is that feeds must be Salmonella-negative as shown by a sampling plan.  
See, for example, CVMs action on a food additive petition published in the Federal 
Register of September 28, 1995 (60 FR 50098).  It is also FDAs policy that the sampling 
plan should follow specifications in the 8th edition of FDAs Bacteriological Analytical 
Manual for fifteen 100 gram (g) samples, or more, to be randomly collected from foods 
for analysis for Salmonella (Ref. 2).  Thus, a feed is said to be Salmonella-negative if 
fifteen 100 gram (g) samples randomly collected from it test negative for Salmonella.   
 
Feeds can be maintained Salmonella-negative in several ways, including the addition of 
chemical substances to the feeds.  Such chemical substances are deemed to be food 
additives under section 201(s) (21 USC 321(s)) of the Federal Food, Drug, and Cosmetic 
Act (the Act) unless they are generally recognized as safe (GRAS) for use against 
Salmonella in feed (Ref. 6).  According to section 409(a) (21 USC 348(a)) of the Act, a 
food additive is deemed to be unsafe, unless it and its intended use conform to the terms 
of an exemption or existing regulation and/or (for food contact substances) an effective 
notification prescribing the conditions under which such additive may be safely used. 
 
Companies or individuals (sponsors) intending to market a food additive for use against 
Salmonella in feeds may file, under section 409(b) of the Act, a food additive petition to 
establish the safety and utility of the food additive for such use.  Details of the 
  3  regulations governing the filing of food additive petitions and the data they must include 
can be found in 21 CFR 571.1 (Ref. 4).  Briefly, a food additive petition must contain 
data establishing that the food additive is safe and will have the intended effect.  The 
amount of food additive necessary to accomplish this effect must also be identified.  21 
CFR 570.17 specifies the conditions under which a food additive or food containing a 
food additive intended for investigational use shall be deemed to be exempt from the 
requirements of section 409 of the Act.    
 
Our experience with food additive petitions indicates a need for consistency in evaluating 
the ability of anti-Salmonella food additives to achieve their intended effect of 
maintaining feeds Salmonella-negative.  Thus, the objective of this guidance is to help 
assure that appropriate studies are conducted by sponsors to evaluate the utility of anti-
Salmonella food additives, and that uniform review and decision-making are 
accomplished by CVM.  This should facilitate the approval process for such food 
additives. 
 
General Considerations 
 
This guidance pertains only to studies designed to establish the utility of chemical food 
additives in maintaining feeds and/or feed ingredients Salmonella-negative.  For the 
purpose of this guidance, the term feed will be used to mean complete feeds (those that 
can be fed as the sole ration) and/or feed ingredients that are recognized by the 
Association of American Feed Control Officials (AAFCO) for use in manufacturing 
complete feeds.  Sponsors of anti-Salmonella food additives may propose the food 
additive for use only in complete feeds, only in feed ingredients, or in both.  
 
Experiments designed for this purpose should be conducted in two main stages consisting 
of laboratory and field trials.  Protocols should be written for the experiments and contain 
a clearly expressed statement of purpose.  The protocols should also specify details of the 
materials, methods, and statistical tools that will be used in the trial.  The experiments 
should be designed to use feeds that are usually consumed by the proposed target 
animals.  The target animals should be specified.  For food additives containing two or 
more chemicals, the composition of the food additive and a list of all substances used 
must be specified (21 CFR 571.1(c)(paragraph A of the petition form)). 
 
Sponsors should submit protocols for review by CVM 90 days before the initiation of 
trials.  Submitting protocols to CVM for review and comment before conducting the 
trials generally facilitates the approval process for a food additive. 
 
Data should be collected and recorded in ways that enhance confidence in the integrity of 
data collected.  Sponsors are referred to CVM's guidance number 58 entitled "Guidance 
for Industry for Good Target Animal Study Practices: Clinical Investigators and 
Monitors" for detailed information about record keeping and record retention.  A copy of 
the guidance can be requested from CVMs Communication Staff (HFV-12) at 240-276-
9300.  Copies can also be downloaded from the Internet at 
  4  http://www.fda.gov/AnimalVeterinary/GuidanceComplianceEnforcement/GuidanceforIn
dustry/default.htm.  
  
To facilitate CVM's review of statistical procedures, raw data used by the sponsor in 
statistical analyses should be included in the petition for the food additive and should 
also be captured on a diskette, preferably in the form of an IBM compatible DOS ASCII 
file.  The diskette should also be included in the food additive petition. 
 
Laboratory Trials 
 
General 
 
The utility of a food additive against Salmonella can be influenced by several factors.  
The factors include the type and other characteristics of the medium in which the additive 
and Salmonella are made to interact, the mode of contamination of medium by 
Salmonella, type and concentration of Salmonella, concentration of the additive, and the 
duration of contact between Salmonella and the food additive.  Pertinent medium 
characteristics include water activity, pH, temperature, and oxygen content (Ref. 18).  It 
is possible that manipulation of these factors could, in the absence of uniform testing 
methods, result in over-estimation or under-estimation of the anti-Salmonella capabilities 
of food additives.  To ensure that the data relied on by the FDA to approve anti-
Salmonella food additives are relevant, the FDA believes that the additives should be 
subjected to a uniform testing method. 
 
The goal of the uniform testing method is to determine, in a consistent manner, whether 
or not an anti-Salmonella chemical food additive is able to maintain feeds Salmonella-
negative for a specific period of time, even if the feeds are repeatedly exposed to 
Salmonella during the period.  Sponsors should conduct two laboratory experiments.  The 
first should be a "dose titration" trial to determine the range of effective doses of the food 
additive.  The second should consist of a "prevention of recontamination" trial to assess 
the ability of the minimum effective dose to maintain feeds Salmonella-negative despite 
repeated exposure of the food additive-treated feeds to the microorganism.  Specific 
issues that should be addressed for each of the two trials follow: 
 
Dose titration (dose determination) trial 
 
CVM considers the "dose titration" trial to be pivotal.  The main purpose of the trial is to 
determine the range of doses over which the food additive is effective against Salmonella 
in feeds.  As much as it is possible, the range determined should include the minimum 
and maximum effective doses.  Various concentrations of the food additive should be 
tested in feed against an inoculum containing a known concentration of a mixture of 
Salmonella serotypes.   
 
The concentrations of food additive used should be multiples of the proposed effective 
dose.  A minimum of six concentrations of the food additive in feed is recommended.  
One should be at zero, another at the proposed effective dose, and two each below and 
  5  above the proposed effective dose (e.g., 0, 0.25X, 0.50X, X, 2.00X, and 4.00X; where X 
is the proposed effective dose).  When using data from the trials to plot the dose response 
curve, the dependent variable would typically be the concentration of Salmonella in the 
sample of treated feed while the independent variable would typically be the 
concentration of food additive in the feed. 
 
The feed used should be Salmonella-negative.  A feed is considered to be Salmonella-
negative if every one of 15 or more representative 100 g samples collected from it is 
shown by laboratory analysis to have no detectable levels of Salmonella.  Samples of the 
feed, or 25 g subsamples randomly collected from each sample, may be analyzed for 
Salmonella individually or after combination into three or more pools consisting of a 
maximum of 3 samples  (300 g) or 15 subsamples (375 g) per pool.  Samples should be 
analyzed for Salmonella either by the methods described in the 8th edition of FDAs 
Bacteriological Analytical Manual (Ref. 1) or by other methods whose sensitivity and 
specificity are equal to or higher than that in the manual.   
 
Because the anti-Salmonella effects of chemical food additives can be influenced by the 
presence of other microbes in the food additive-treated feeds, it is recommended that the 
general microbial load of the experimental feeds should be ascertained by conducting the 
following additional analyses on the samples: 
 
  a)  Aerobic plate count (Ref. 13) 
  b)  Escherichia coli count (Ref. 8) 
  c)  Fungal spore count (Ref. 17) 
 
The Salmonella-negative feed should be divided into three or more replicates.  Each 
replicate should be divided into seven experimental lots of feed.  All seven lots should be 
separately treated with appropriate amounts of the food additive, assayed to determine 
the concentration of food additive that is actually present, and then inoculated with 
Salmonella.  One lot should serve as a negative control (containing neither Salmonella 
nor the food additive), and another as a positive control (containing Salmonella but not 
the food additive).  Each lot should then be subdivided into twelve or more units.  The 
units should be randomly assigned to one of four groups containing equal number of 
units.  There should be three or more units per group.  Throughout the trial, the positive 
and negative controls should be handled exactly like the other experimental units of feed.  
 
The method used in inoculating experimental feeds with Salmonella can influence the 
outcome of the dose titration study.  Three methods are currently available.  Each 
involves inoculation with one of the following: 
 
  a)  Materials naturally contaminated with Salmonella 
 
  b)  Materials contaminated with Salmonella in a way that simulates natural  
    contamination (simulated naturally-contaminated materials) 
 
  c)  Broth cultures of Salmonella 
  6   
Contamination or recontamination of feeds with Salmonella can occur directly through 
several avenues including infected or contaminated wild birds, rats, mice, cockroaches, 
and indirectly through contaminated inanimate objects like dust (Ref. 18).  Thus, it 
appears logical to use naturally-contaminated materials (e.g., feces) from these sources as 
inoculi for experimental feeds.  However, CVM believes that the use of this natural 
method of contamination would be incompatible with the goal of uniform testing because 
the concentration of Salmonella in such materials varies widely.   
 
Broth cultures can be manipulated to provide inoculi containing fairly uniform amounts 
of Salmonella, and their use in inoculation will significantly reduce the variability in 
Salmonella concentration associated with the natural method.  However, the direct 
addition of such inoculi to experimental feeds may result in an over-estimation of the 
ability of the food additive to achieve its intended effect, because Salmonella present in 
broth culture-inoculated feeds has been shown to be less resistant to insults than that 
present in naturally-contaminated feeds (Ref. 15).   
 
The use of a method involving inoculation with simulated naturally-contaminated 
materials will overcome, to a large extent, the variability in Salmonella concentration and 
the reduction in Salmonella resistance associated with the natural and broth culture 
methods.  Therefore, this method involving the inoculation of feeds with simulated 
naturally-contaminated materials is recommended.  
 
A technique for producing simulated naturally-contaminated meat and bone meal 
containing stable populations of Salmonella has been described by Liu and his co-
workers (Ref. 12).  They also showed that the Salmonella present in feeds inoculated 
with the meal was more resistant to insults than that present in feeds directly inoculated 
with broth cultures of Salmonella.  Briefly, in the Liu et al technique, a sterile suspension 
of meat and bone meal inoculated with Salmonella was incubated for 48 hr at 37 C.  
After incubation, the suspension was centrifuged and the sediment dried, ground to a fine 
powder and stored at 4 C pending its use as the inoculum for experimental feeds.  The 
population of Salmonella in the meal was reported to remain relatively stable over an 11-
month period.  To produce simulated naturally-contaminated materials for use as inoculi 
for experimental feeds in the dose titration study, sponsors should use the Liu et al 
technique or any other similar one that is shown to equally or more effectively ensure the 
stability of Salmonella even during cold storage. 
 
The level of Salmonella in the inoculum should be high enough to ensure a final 
concentration of 104 colony-forming units (CFUs) or greater of Salmonella per gram of 
experimental feed.  The inoculum should contain equal amounts of American Type 
Culture Collection (ATCC) cultures of Salmonella typhimurium, Salmonella senftenberg, 
Salmonella montevideo, and Salmonella enteritidis.  The identity of the serotype in each 
ATCC culture should be independently confirmed prior to its use in the trial.  S. 
typhimurium is being recommended because of its universal nature, S. senftenberg 
because it has been reported to be one of the more resistant serotypes (Ref. 12), S. 
montivideo because it is reported to be the serotype most frequently isolated from feeds 
  7  and is said to have a relatively high resistance (Ref. 10), and S. enteritidis because it is a 
major public health concern.  Other Salmonella serotypes that are of animal or public 
health significance could be included by the sponsor, if desired.  
 
Because the antimicrobial activity of organic acids and other chemicals appears to be 
time-dependent, the dose titration trial should be designed to demonstrate the nature of 
that time-dependency when the chemicals are used as antimicrobial food additives.  The 
design should also allow a determination of shortest interval of time it takes for the 
additive to exert its effect against Salmonella.  The preferred method for achieving this is 
the generation of a series of dose titration curves using data obtained from the analyses of 
groups of units of feed at 1, 4, 8, 24 and 48 hours after inoculation of the feeds with 
Salmonella.  The units of feed used are obtained, as described earlier, by subdividing 
each lot of experimental feed into 12 or more equal portions.   
 
Each unit is assigned to one of four groups containing three or more units.  The amount 
of inoculated feed constituting each individual unit should be enough to ensure its 
adequacy for quantitative and qualitative analyses for Salmonella.  The number of 
samples to be collected from each unit for Salmonella analysis should be determined by a 
statistical model that instills 99% confidence that Salmonella will be detected if present.  
The analysis of all of the inoculated feed in each unit, or the collection and analysis of 60 
or more representative 100 g samples from each, should be compatible with the model.  
In the latter case, the samples, or 25 g subsamples randomly collected from each sample, 
may be analyzed for Salmonella individually or composited for analysis into three or 
more pools consisting of a maximum of 3 samples (300 g) or 15 subsamples (375 g) per 
pool. 
 
Temperature, pH, water activity and other pertinent experimental conditions should be 
specified.  Because of the pivotal nature of the dose titration studies, sponsors are 
strongly encouraged to submit the results of the study to CVM for review and comment 
before initiating either the prevention of recontamination study or the field trial. 
 
Study of ability to maintain feeds Salmonella-negative  
 
The purpose of the second laboratory trial is to demonstrate the ability of the minimum 
effective level of the anti-Salmonella chemical food additive to maintain feeds 
Salmonella-negative for a specific period of time, even if the feeds are repeatedly 
exposed to Salmonella during the period.  For complete feeds or feed ingredients that, 
shortly after manufacture, are made directly available to animals or used to produce 
complete feeds, that time should be a minimum of 14 days.  For complete feeds or feed 
ingredients that are intended to be bagged, or stored in some other manner for sometime 
prior to use, a period such as 90 days is suggested. 
 
In this trial, the level of food additive determined in the dose titration trial to be the 
minimum effective dose should be added to Salmonella-negative feed, and challenged 
with Salmonella contained in a simulated naturally-contaminated material as described 
earlier under the dose titration study.  The challenge with Salmonella should be 
  8  conducted during day 1 of the experiment and again at one or more other days 
afterwards, including the last day of the time period for which the sponsor intends to 
claim the food additive as being effective.  Sixty or more representative 100 g samples 
each should be collected from the treated feed and its controls immediately before and 
again at two time intervals after each challenge with Salmonella and analyzed 
qualitatively for Salmonella.  The samples, or 25 g subsamples randomly collected from 
each sample, may be analyzed individually or composited into three or more pools 
consisting of a maximum of 3 samples (300 g) or 15 subsamples (375 g) per pool.  The 
first time interval for the collection of samples should be that at which the minimum dose 
of the food additive was shown to be effective in the dose titration trial, and the second 
one 24 hours afterwards.   
 
The quantity of feed used in the study should be enough to ensure its adequacy for the 
analysis for Salmonella.  As with the dose titration trial, temperature, pH, water activity, 
and other pertinent experimental conditions should be specified.  The trial should also 
include a control and a negative control, and contain an adequate number of replicates.  
The duration of the experiment and the number of challenges with Salmonella should be 
consistent with claims the sponsor intends to make for the food additive. 
 
Feeds used in both laboratory trials should consist of specified quantities of complete 
feeds (e.g., broiler starter) and/or feed ingredients (e.g., meat and bone meal).  They 
should either be obtained commercially or self-produced.  Details of the composition of 
complete feeds (including all added drugs and additives) and the techniques used for their 
manufacturing or processing should be specified.  The amount of feed assigned to each 
experimental group should be enough to permit the collection of all required samples. 
 
Field Trials 
 
The dose of food additive determined during the dose titration trial to be the dose that 
will achieve the intended effect should be further evaluated in field trials.  The purpose of 
field trials is to determine if the food additive could be safely used to achieve its intended 
technical effect of maintaining feeds Salmonella-negative under actual conditions of use.  
Such conditions of use would include those associated with the preparation, storage and 
consumption of food additive-treated feeds.   
 
The experiments should be conducted at feed-mills or farms with histories of recurrent 
contamination by Salmonella.  Experimental and control groups should be located on the 
same premises or farms and subjected to identical system of handling or management 
except for the addition of the food additive to the experimental group.  The feeds used 
should be Salmonella-negative.  Their detailed composition should be specified to 
include all added drugs and additives.  The quantities of feeds used, and the conditions 
under which they are manufactured or produced, stored, transported and consumed, 
should be the same as those expected if the food additive is approved for use.   
 
Samples should be collected for Salmonella analysis from the experimental and control 
groups of feeds before and after the addition of food additive, and sufficiently often 
  9  enough to substantiate the claim for the food additive.  The times of sample collection 
should be specified.  For trials involving feed ingredients, like meat and bone meal, 
samples should be collected from treated and untreated ingredients after their 
manufacture, during their storage, and immediately prior to their use in manufacturing 
complete feeds.  For trials involving complete feeds, like broiler or hog starter rations, 
samples should be collected from treated and untreated feeds after their manufacture at 
the feed-mill, during their storage, and during the period the feeds are made directly 
available for consumption by the target animals.   
 
The number of samples to be collected at each location or sampling interval should be 
determined by a statistical model that instills 95% confidence that Salmonella will be 
captured among samples collected even if the concentration of the microorganism in the 
feed was as low as 1 CFU/250 g.  The collection and analysis of 30 or more 
representative 100 g samples should be compatible with this model.  The samples, or 25 g 
subsamples from each, may be analyzed for Salmonella individually or after being 
composited into three or more pools consisting of a maximum of 3 samples (300 g) or 15 
subsamples (375 g) per pool.   
 
CVM has additional recommendations for field investigations involving anti-Salmonella 
chemical food additives intended for use in the feeds of aquatic animals.  Sponsors of 
such food additives are referred to CVM guidance number 53 entitled "Guidance for the 
Evaluation of the Utility of Food Additives in Diets Fed to Aquatic Animals."  A copy of 
the guidance can be requested from CVMs Communication Staff at 301-594-1755.  
Copies can also be downloaded from CVMs site on the Internet at 
http://www.fda.gov/cvm. 
   
Because food additives are approved for use on a nationwide basis, trials should be 
conducted in geographically different locations and during two or more different seasons 
of the year, one of which should be the summer. 
 
Collection and Analysis of Samples 
 
The sampling techniques employed in both the laboratory and field trials under this type 
of study are critical to the validity of results obtained.  Samples should be aseptically 
collected without unduly disturbing the experimental feeds or target animals consuming 
the feeds.  FDA's Bacteriological Analytical Manual contains pertinent information about 
sample handling (Ref. 2). 
 
The time of sample collection should be recorded and scheduled so that storage of 
samples prior to analysis is avoided as much as possible.  If it is necessary to store the 
samples, the conditions under which the samples are stored (e.g., temperature) should be 
specified and shown not to be detrimental to either the survival of Salmonella or the 
maintenance of the integrity of nutrients.  The duration of storage should also be 
specified. 
 
  10  Microbiological analysis for Salmonella should be initiated within 1 hour after sample 
collection and steps taken to ensure that pH and other critical parameters of the growth 
media that can influence growth of the microorganism are not modified by presence of 
the food additive.  In situations like field trials where it may not be feasible to initiate 
analysis within one hour of sample collection, the activity of food additive in samples 
used for Salmonella analysis should be neutralized or arrested immediately after sample 
collection.  The compound or technique used to neutralize or arrest activity should be one 
that has been shown not to have any negative impact on the survival of Salmonella.  If 
this is not achievable, sample analysis should be initiated at the earliest possible time 
after sample collection and the length of time between the collection of samples and their 
analysis for Salmonella should be specified. 
 
Laboratory analyses should be conducted "blind."  It is recommended that the 
experimental feeds should be prepared and coded by one person or team, and samples 
collected from the feeds analyzed by a different person or team unaware of the 
concentration of the food additive in the samples or the coding technique used to identify 
samples.  If it is necessary to transport samples, they should be packaged to ensure the 
maintenance of their microbiological integrity during transportation, and sent by the 
fastest means possible. 
 
The concentration of Salmonella present in samples collected from feeds used in the dose 
titration trial should be determined either by the standard dilution and pour-plate 
technique, or the most probable number method.  Because the sensitivity of the standard 
dilution and pour-plate technique is low when used in isolating Salmonella from feeds 
containing viability-impaired and/or very small numbers of Salmonella, the samples 
analyzed by this technique should also be analyzed qualitatively for Salmonella using a 
procedure that includes pre-enrichment and enrichment.  
 
Samples of feed collected for Salmonella analysis during both the "prevention of 
recontamination" and field trials should be analyzed qualitatively to determine the 
presence or absence of the microorganism.  
 
CVM recommends the use of methods described in the FDA's Bacteriological Analytical 
Manual (Ref. 1).  Sponsors may use other analytical techniques, but those techniques 
should be demonstrated to be at least as sensitive and specific for Salmonella. 
  11  References 
 
 1. Andrews, W. H., G. A. June, P. S. Sherrod, T. S. Hammack, and R. M. Amaguana. 
Salmonella. FDA Bacteriological Analytical Manual, 8th ed., p. 5.01  5.20, 1998. U.S. 
Food and Drug Administration, Washington, D. C. 
 
 2. Andrews, W. H., and G. A. June. Food sampling and preparation of sample 
homogenate. FDA Bacteriological Analytical Manual, 8th ed., p. 1.01  1.10, 1998. U.S. 
Food and Drug Administration, Washington, D. C. 
 
 3. Busby, J.,and Roberts, T. Estimates of costs of illness caused by 7 food-borne 
pathogens. Food Review, April 1997. United States Department of Agriculture, 
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 6. Federal Food, Drug, and Cosmetic Act, as amended, February 1998. Title 21 of the 
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consequences. 1994. CAST Task Force Findings. Washington, D. C. 
 
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Escherichia coli and the coliform bacteria. FDA Bacteriological Analytical Manual, 8th 
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 9. Hui, Y.H., J. R. Gorham, K. D. Murrel, and D. O. Cliver (eds.). Food-borne diseases 
handbook. 1994. Marcell-Dekker, New York. 
 
10. Juven, B. J., N. A. Cox, J. S. Bailey, J. E. Thomson. O. W. Charles, and J. V. Shutze. 
Survival of Salmonella in dry food and feed. Journal of Food Protection 47: 445 - 448, 
1984. 
 
11. Lacey, R. W. Food poisoning: the rise continues. Br. Food J. 95: 25  31, 1993.   
 
12. Liu, T. S., G. H. Snoeyenbos, and V. L. Carlson. Thermal resistance of Salmonella 
senftenberg 775W in dry animal feeds. Avian Diseases 13: 611 - 613, 1969. 
 
  12  13. Maturin, L. J., and J. T. Peeler. Aerobic plate count. FDA Bacteriological Analytical 
Manual, 8th ed., p. 3.01  3.10, 1998. U. S. Food and Drug Administration, Washington, 
D. C. 
 
14. Mead, P. S., L. Slutsker, V. Dietz, L. F. McCaig, J. S. Bresee, C. Shapiro, P. M. 
Griffin, and R. V. Tauxe. Food-related illness and death in the United States. Emerging 
Infectious Diseases 5: 607  625, 1999. 
 
15. Rasmussen, O. G., R. Hansen, N. J. Jacobs and O. H. M. Wilder. Dry heat resistance 
of Salmonellae in rendered animal by-products. Poultry Science 43: 1151 - 1157, 1964. 
 
16. Tauxe, R. V. Emerging food-borne diseases: An evolving public health challenge. 
Emerging Infectious Diseases 3(4): 425  434, 1997. 
 
17. Tournas, V., M. E. Stack, P. B. Mislivec, H. A. Koch, and R. Bandler. Yeasts, molds, 
and mycotoxins. FDA Bacteriological Analytical Manual, 8th ed., p. 18.01  18.11, 1998. 
U. S. Food and Drug Administration, Washington, D. C. 
 
18. Williams, A. E. Paratyphoid infections. In: Diseases of Poultry, 8th ed. Hofstad, M. 
S., H. J. Barnes, B. W. Calnek, W. M. Reid, and H. W. Yoder, Jr., eds., p. 91 - 129, 1984. 
Iowa State University Press, Ames, Iowa, USA. 
  
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