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ANNEX U USDA Performance Standards for Salmonella

U.1 INTRODUCTION

The "Pathogen Reduction/HACCP Systems" Final Rule requires that certain types of meat products made in FSIS inspected establishments meet USDA "Performance Standards" for Salmonella published under sections 9CFR310.25(b) and 9CFR381.94(b) of the US Federal Register. The FSIS performs unannounced sampling and testing of products manufactured in US facilities in order to assess their compliance with the standards. If an establishment cannot produce meat which conforms with the prescribed Performance Standard, a process which can ultimately lead to the withdrawal of inspection is initiated. The FSIS will also use these test results to update baseline reference standards for Salmonella in US products and to update the Performance Standards for Salmonella.

Canadian establishments eligible to export meat or meat products to the US which are amenable to a USDA Performance Standard for Salmonella (see section 11.7.3(2)(a) on the USA) must manufacture these products in accordance to the applicable standard. In order to demonstrate this, the establishment shall conduct testing of products for Salmonella according to a written sampling program which meets the requirements set out in this Annex.

The Canadian Food Inspection Agency (CFIA) exercises government direction and oversight of establishments’ Salmonella testing programs. If an establishment’s testing shows that it is not manufacturing product which complies with the USDA Performance Standard for Salmonella, a process is initiated which may lead to the removal of the establishment from the list of eligible establishments. If the establishment fails to meet the requirements for Salmonella testing set out in this Annex, the CFIA will initiate the process set out in section Q.3 of Annex Q.

The CFIA intends to eventually use Canadian Performance Standard for Salmonella. Pending their publication and acceptance, the Performance Standards published by the USDA will be used in Canadian establishments.

Differences between USDA Performance Standard for Salmonella and Generic E. coli (biotype I) testing requirements:

The purpose of USDA Performance Standard for Salmonella is different than that of generic E. coli (Biotype I) testing requirements. As a result, the consequences of unsatisfactory test results differ.

USDA Performance Standards for Salmonella were designed to verify that establishments meet USDA’s pathogen reduction goals. FSIS selected Salmonella as a performance indicator for four reasons:

The requirement for generic E. coli (biotype I) testing is meant to achieve the implementation of microbiological-based Statistical Process Controls (SPC) in establishments preparing specific types of meat products. Generic E. coli (biotype I) as the organism to test because it is easy to test for and is common enough to provide a good indication of process control.

Because generic E. coli (biotype I) testing requirements are designed around Performance Verification Criteria (PVC) which are often establishment specific, the same test result may be lead to a different conclusion in two establishments which have different PVC. Generic E. coli (biotype I) is done on an ongoing basis using a moving window approach. Failure in meeting PVC is a deficiency only when the establishment is unable or unwilling to successfully bring their process back "in control" (refer to annex T.2.9 for details on handling this type of situation.)

Salmonella testing serves a different purpose. It is done to verify that the establishment is manufacturing product in accordance with the prescribed Performance Standard. It is not a method for SPC. A moving window approach is not used, instead a series of stand alone samples is taken. Unlike E. coli, individual tests are evaluated as "+" or "-" for the presence of Salmonella, no matter the actual number of Salmonella detected. If the number of "+" results during the series exceeds the maximum allowed during three consecutive series of tests, this is considered as an inspection finding of a serious deficiency, even if the establishment is making every reasonable effort to correct the problem and is dealt with as a more serious matter than failure to meet E. coli PVC.

U.1.1 Canadian establishments which must implement a Salmonella testing program in order to satisfy US requirements:

All Canadian registered establishments listed under section 11.7.3(2)(a) on the USA which manufacture a product amenable to USDA Performance Standards for Salmonella must produce product which meets the applicable Performance Standard(s).

With the exception of the following types of establishments, ALL establishments must implement a testing program.

ESTABLISHMENTS WHICH ARE NOT REQUIRED TO CONDUCT TESTING:

For example, an establishment which makes ground beef 20 times a year, ground chicken 20 times a year and ground turkey 30 times a year is only required to test the class of product which is made more than 26 times during the year (ground turkey).

Base this determination on how often the class of animal was slaughtered during the previous year as well as on predicted production information for the coming year (i.e., additions to the plant.)

U.1.2 Products amenable to USDA Performance Standard for Salmonella:

If a USDA Performance Standard for Salmonella has been published for the class of product,

all corresponding products made within the establishment must comply with the standard.

Table U.1.2 lists all USDA Performance Standards for Salmonella published to date.

USDA Performance Standards were based on microbial baseline studies conducted by the USDA. Further baseline studies will be performed and Performance Standards for new classes of product will be added to this list.

Table U.1.2 - USDA Performance Standard for Salmonella by class of product

Note 1 - Does not apply to meat which is ground as a processing step in the fabrication of a processed meat product (for example, beef ground to make Salami) or to Mechanically Separated Meats.

U.1.3 Requirement to manufacture product which complies with the applicable Performance Standard for Salmonella and to conduct annual testing

Establishments which manufacture a meat product amenable to a USDA Performance Standards for Salmonella are required to manufacture the product in conformance with the applicable standard at all times. In order to demonstrate this, the establishment shall conduct regular Salmonella testing at least once per year in accordance with the requirements set out in this annex.

If an establishment manufactures products which correspond to more than one class of USDA Performance Standards for Salmonella, all of the establishment’s products must meet the applicable Performance Standard. However, the establishment is not required to perform an annual set of tests on each class of product. A decision regarding which of the classes will be sampled during the year will be made by the CFIA V/IIC (see section U.1.6.1 for details.)

U.1.4 [Reserved]

U.1.5 Responsibilities under these testing requirements

U.1.5.1 Responsibility of the establishment operator:

Establishments must prepare a written sampling program for Salmonella which adheres to the sample collection, preparation and shipping procedures and technical criteria set out in this Annex (refer to Section U.2 for details.) After the program has been reviewed by the CFIA V/IIC using Form 4A of the BCC, the establishment is required to conduct their sampling activities in accordance with their written program and must :

(1) Manufacture products which complies with the applicable published USDA/FSIS Performance Standard for Salmonella;

(2) Present CFIA with a written annual Salmonella testing program which meets the requirements of this annex;

(3) Conduct the Salmonella testing program on the class(es) of product identified by the CFIA V/IIC in conformity with the accepted written program and the requirements of this annex;

(4) Arrange for laboratory results to be sent simultaneously and directly to the CFIA V/IIC;

(5) Keep records and test results in a manner consistent with the requirements outlined in Section U.4. Compile testing results to show the status of compliance with the Performance Standard for Salmonella;

(6) Initiate appropriate corrective action if the number of positive Salmonella test results exceeds the maximum number of positives per set (see U.2.2);

(7) Make available to CFIA inspection staff on request all information relevant to the establishment’s Salmonella sampling program (including written procedures, laboratory reports and analytical results) for purposes of compliance verification; and

(8) Assume the cost for sampling, testing and other activities related to the program.

Establishments must only use laboratories which meet the accreditation criteria set out in Section U.3 and which employ the methodology described in this annex for the detection and identification of Salmonella. Finally, the establishment must make arrangements with the laboratory so that samples of all Salmonella positive cultures are available to the CFIA upon request.

U.1.5.2 Responsibility of accredited laboratories:

(9) Maintain laboratory accreditation under the Standards Council of Canada (SCC) National Standards System "General Requirements for the Accreditation of Calibration and Testing Laboratories" (ref. CAN-P-4C, October 1991; ISO/IEC Guide 25-1990) and "Guidelines for the Accreditation of Agricultural and Food Products Testing Laboratories" (ref. CAN-P-1587, November 1998);

1. Demonstrate ongoing proficiency for Salmonella testing through the "Microbiology Proficiency Testing Program" jointly administered by the SCC and the CFIA;
2. Conduct testing for Salmonella according to the technical requirements outlined in this Annex; and
3. Send results for each individual test simultaneously and directly to the CFIA V/IIC for the establishment;
4. Make available to the CFIA V/IIC information on the condition of samples received.

U.1.5.3 Responsibility of the CFIA:

The CFIA:

• requires and verifies that the laboratories used for analysis of samples are accredited by the Standards Council of Canada under the National Standards System "General Requirements for the Accreditation of Calibration and Testing Laboratories" (CAN-P-4C OCTOBER 1991, ISO/IEC Guide 25-1990) and the "Guidelines for the Accreditation of Agricultural and Food Products Testing Laboratories (CAN-P-1587 November 1998) for the reporting of Salmonella. test results;
• on an annual basis, verifies that the establishment’s written Salmonella testing programs meets the requirements of this Annex and determine the scope of Salmonella testing at the establishment through the use of Form 4A of the Basic Compliance Checklist (BCC) and the "determination of class(es) to sample for Salmonella" worksheet;
• during each week of Salmonella testing, randomly performs on-site verification of the establishment Salmonella testing activities (e.g., sample taking, preparation and shipment, etc.) using part 4B of the BCC to verify adherence to the written program and to requirements;
• obtains copies for each individual test results simultaneously and directly from the laboratory;
• directly receives test results, evaluates and compares them to those of the establishment to verify that the establishment is properly compiling results and informing the CFIA whenever the number of Salmonella positive results exceeds the specified limit;
• V/IIC compiles data on Salmonella results and sends a report to H.Q. for the production of Canadian national Salmonella baselines and Performance Standards;
• reviews the establishment’s proposed actions when the limit is exceeded;
• V/IIC takes appropriate compliance action (see Q.3);
• V/IIC requires, as directed by the PND, that the operator conducts a supplementary of Salmonella tests if process is deemed out of control.

U.1.6 CFIA verification of testing and related BCC tasks

U.1.6.1 Review of the establishment’s written program by the CFIA (BCC part 4A)

Once a year, prior to the commencement of the upcoming calendar year, the CFIA V/IIC completes part 4A of the Basic Compliance Checklist (see annex Q, section Q.4) and the "determination of class(es) to sample for Salmonella" worksheet (copy provided in section U.6) in order to verify that the establishments written testing program for Salmonella is satisfactory and to determine which class(es) of product will be sampled for Salmonella.

A copy of the completed checklist and worksheet will be kept by the V/IIC. In cases where the V/IIC finds that a condition is not being met, they are to document the situation and advise the Program Network Director (PND). No testing shall begin until all of the items specified in BCC part 4A have been assessed as satisfactory by the V/IIC.

The "determination of class(es) to sample for Salmonella" worksheet is designed to allow for a uniform determination of which class(es) to sample at establishments and takes the following factors into consideration :

• results of previous Salmonella testing;
• results of generic E. coli (biotype I) testing;
• production volumes and types of products made at the establishment;
• modifications to manufacturing practices;
• products made at the establishment which have not been tested previously;
• other relevant information; and
• any specific testing instructions received from the CFIA Program Network.

The worksheet is used in the following manner :

• the establishment makes available to the V/IIC production information for each (Performance Standard for Salmonella) class of product made at the establishment;
• this information is used by the V/IIC to complete the worksheet;
• the worksheet will assign a points value to each class of product made at the establishment;
• testing for Salmonella shall be done on:
• the class of product with the highest point score (in case of tie, testing for Salmonella will be done on the product which is produced in greatest quantity by weight); as well as
• all classes of products which have a total point score of 5 or more.
• the V/IIC and the establishments will set the date when the testing of the class(es) will commence and this date will be recorded on the form.

Testing may begin at any point during the year, however the selection of the starting date needs to be based on valid reasons and these reasons are to be stated in the written program.

U.1.6.2 Verification of the establishment’s implemented program by the CFIA (BCC part 4B)

A special inspection task exists for verifying that the establishment is conducting Salmonella testing in accordance with its written program. Part 4B of the Basic Compliance Checklist (see annex Q, section Q.4) is used for this. The V/IIC will conduct random on-site verification at least once during each week that Salmonella testing takes place at the establishment. In cases where the V/IIC finds that a condition is not being met, they are to document the situation and advise the Program Network Director (PND).

U.2 TECHNICAL REQUIREMENTS

U.2.1 Written Protocol And Procedures; Written Records

The technical requirements outlined in this section were developed using information published by the USDA in the US Federal Register (Volume 61, No. 144/ July 25, 1996, page 38917) under the title "FSIS Sample Collection Guidelines and Procedure for Isolation and Identification of Salmonella from Raw Meat and Poultry Products" and further information which was provided directly to the CFIA by the USDA.

The operator shall have a written program for Salmonella sampling done at the establishment and make it available to the CFIA V/IIC upon request. The written program shall contain procedures covering all the establishment’s areas of responsibility. The procedures shall be detailed to the extent necessary to enable on-site verification. The following information must be included in the written program:

• The list of products manufactured at the establishment which are covered by a Performance Standard for Salmonella (classified by Salmonella sampling class of product);
• Information regarding the sampling plan used and the selection of samples, e.g., how many samples to be taken, who carries out the sampling, when and where sampling will be done, how random sampling of carcasses will be achieved;
• Information regarding pre-sample preparation: e.g., check list of tasks to be performed prior to sample collection, materials needed for sample collection, verification of the suitability of materials before testing (ex: sponges checked to ensure they will support growth);
• The sampling procedure used;
• The shipping procedures used: e.g., who packages, where is packaging done, where are samples kept pending shipment, who ships samples; where are samples shipped (laboratory)/how are samples shipped (shipping agent);
• The means to tamper-proof and protect the integrity of samples: e.g., how samples are handled/packaged and shipped to ensure temperature maintenance and tamper proofing;
• The identity of the laboratory used to provide results and proof of the laboratory’s current accreditation for Salmonella testing;
• The procedure for providing the CFIA V/IIC directly and simultaneously with Salmonella test results;
• The procedures for compiling test results and record keeping: e.g., who receives test results, who reviews and compiles data, where are lab reports/worksheets kept, how long are records kept;
• The operator’s internal monitoring and/or verification activities to verify that the program is delivered as intended;
• The corrective action procedures to be taken for items such as: inconclusive test results, non-compliances with the program that the operator identifies during their internal verification activities, for Salmonella positive results; for a cumulative number of Salmonella positive test results which exceed the "fail" threshold for a set of tests;
• The CFIA notification procedures: e.g., who in the company will notify the V/IIC when the tests are completed/if the number of positive tests within a set exceeds the "fail" threshold.

Reference may be made to an establishment’s general sample collection procedures provided that the references are to the procedure specific sections and that details specific to Salmonella sampling procedures are provided. Procedures may also be written into the establishment's HACCP plans.

U.2.2 USDA Performance Standards for Salmonella by class of product

Number of tests per set ("n" value); Maximum number of positives per set ("c" value)

The USDA Performance Standards for Salmonella were developed by the USDA Food Safety and Inspection Service (FSIS) based on a statistical analysis of the data collected by USDA through Salmonella baseline studies performed in the United States. Because the percentage of Salmonella positive results was different between the different classes of product tested during the baseline study, USDA developed Performance Standard and sampling plans for each class of product.

Table U.2.2

USDA Performance Standards for Salmonella by class of product

(source: Federal Register, Vol 61, No. 144, page 38865 + 38867)

U.2.2.1 Sampling Rates and Frequency of Sampling

Table U.2.2 indicates the number of samples to be collected and tested in order to complete one set of tests. Sampling plans are designed to ensure similar accuracy of results between product classes with different prevalence levels for Salmonella. The number of tests to be done varies between classes of product because of statistical requirements.

Tests must be taken on consecutive days until the set of tests is completed: Once a set of tests has been started by the operator, samples shall be taken by the operator at the rate of one sample taken at random on each consecutive production day until the number of conclusive laboratory test results (i.e., either negative or positive) for Salmonella is equal to the "n" value published in Table U.2.2 for the class of product.

On the day that the first sample is to be taken, the operator shall advise the CFIA V/IIC that a set of Salmonella tests has begun. The operator must conclude the set of tests once it is started. It is not acceptable to stop testing once a set has begun or to re-start testing.

Procedure for dealing with laboratories which are unable to begin the analysis of certain samples within 24 hours of sample taking because of week-end and holiday closures: if the laboratory used is closed on a given day and this makes it impossible to initiate the analysis of samples within 24 hours of the sample being taken, the following additional procedures shall be used:

For poultry carcasses: the carcasses shall continue to be selected at random during each production day. They shall be held under refrigeration and shall be kept under strict control at the establishment until the earliest time at which the samples could be taken and shipped to the laboratory for analysis within 24 hours of shipment.

For example: if the laboratory is closed on Sunday, but the establishment operates 7 days a week, daily random carcass selection shall still be performed on Saturday and on Sunday. The carcasses shall be kept under refrigeration and under control of the operator at the establishment. The samples are to be taken and shipped by the operator to the laboratory the next business day when the laboratory can conduct the tests within 24 hours of shipment.

For red meat carcasses: A carcass shall still be selected for sampling at random from each day of production, but when the laboratory cannot begin the analysis of the sample within 24 hours, the operator shall:

• randomly select the carcass after 12 hours or more of chilling and,
• place the selected carcass in a designated area and delay the sample collection on the carcass until 24 hours before the laboratory can receive and initiate analysis of samples.

For example: The establishment operates on Friday and Saturday (it is closed on Sunday) and the laboratory is closed on Saturday and Sunday. As usual, a carcass is selected at random among the carcasses produced on Friday and another carcass among Saturday’s production, and both of the selected carcasses are placed and kept in a designated area. The selected carcasses are sampled at the earliest time at which and the samples can be sent to the laboratory for analysis within 24 hours of shipment.

For ground meat samples: the sample shall continue to be selected at random during each production day. They shall be held under refrigeration and will be prepared for shipment, but shall be kept under strict control at the establishment until the earliest time at which it can be shipped to the laboratory for analysis within 24 hours of shipment.

For example: if the laboratory is closed on Sunday, but the establishment operates 7 days a week, a daily sample shall still be taken at random on Saturday and on Sunday. The samples shall be packaged and stored kept under refrigeration and under control of the operator at the establishment. The samples are to be shipped by the operator to the laboratory the next business day when the laboratory can conduct the tests within 24 hours of shipment.

Inconclusive test results and "make-up" samples: Inconclusive laboratory results will not be counted. For each inconclusive result (for example, a sample received after 24 hours or in unsatisfactory conditions), a "make-up" sample must be added to the set. These samples will be taken in the same manner as regular samples on the consecutive production day(s) following the initial number of samples.

U.2.2.2 Assessment of an individual set of tests

A set of tests is concluded when the total number of conclusive (i.e. negative or positive) laboratory results is equal to the "n" value specified in table U.2.2 for the class of product. The evaluation of the establishment’s performance is based the total number of Salmonella positive results found within the set of results. In order to have successfully completed the set of tests, the establishment must:

• have a number of valid and conclusive (i.e., negative or positive) laboratory results equal to the number of samples to be sampled within the set ("n" value); and
• have a total number of Salmonella positive test results which does not exceed the maximum number of Salmonella positive results permitted for a single set of samples ("c" value).

If the number of Salmonella positive test results exceeds the "c" value, the establishment has not successfully completed the set of tests and further actions are to take place.

U.2.2.3 Conclusion of tests for the year

The establishment shall notify the CFIA V/IIC when the set of tests is completed. The V/IIC is to review the establishment’s data. If the data is satisfactory and the total number of Salmonella positive results for the set of tests is equal or less than the published "c" value for the class of product, no further testing of this class of product is required for this year.

If the establishment has committed in its annual Salmonella testing program to also test other classes of product, testing for each of the other classes must still be completed.

If an establishment has not completed the number of sample required to complete the set by the end of the calendar year (for example, establishments which produce low volumes of product), they shall continue to collect samples during the following production days until they have the number of results required to complete the set. If the results for this set are negative, the establishment will begin any testing specified for the new calendar year (as provided for under section U.1.6). If the set is positive, the provisions of the following sections apply.

U.2.2.4 Actions when the number of positive test results during the first set of tests exceeds the maximum number of positives per set ("c" value)

There are two possible reasons for this occurring during the first set of tests:

1. the establishment is producing product which has a Salmonella incidence greater than the USA national prevalence used for setting the Performance Standard; OR
2. the establishment is producing product which has a Salmonella incidence lower than the USA national prevalence used for setting the Performance Standard, but "fails" because of statistical reasons related to how the Performance Standards are designed.

When the number of positive test results exceeds the specified "c" value, the establishment

shall investigate the possible causes for the results (ex: lack of adherence to the HACCP plan, procedures or CCP’s which are not sufficiently effective, etc.) and draw up an action plan to correct the situation. The establishment shall communicate its findings and action plan to the V/IIC in writing within 5 working days after the "C" value has been exceeded.

If the set of tests has not yet been completed, the establishment shall continue to take samples at the normal rate until the required number of test results (number of tests equal to the "n" value) have been obtained. This is important as it will allow for a complete analysis of the situation. If further positives are identified, the establishment shall review its action plan, amend it as required and inform the V/IIC of the changes made.

Within 5 working days of receiving the final results for the set of tests, a report shall be prepared by the establishment and given to the V/IIC. All action plans as well as a summary of testing results shall be attached to the report. The V/IIC is to forward the report to the Program Network Director (PND) and attach their comments.

The PND reviews this information to decide on when the operator shall begin the second set of tests. Depending on the total number of positive results, the time lines contained within the establishment’s action plan and the corrective actions which have been initiated by the operator, the PND may provide the operator with time to complete the implementation of the corrective actions before starting the second set of tests. However, should the establishment’s action plan be unsatisfactory or be not adhered to, the PND will instruct that the establishment begin the second set of tests immediately.

The second set of tests shall be performed in the same way as the first and test results assessed in the same manner. If the total number of Salmonella positive test results for the second set of tests does not exceed the maximum number of positives per set ("c" value), the establishment is said to have successfully completed the set of tests and no further testing for the class of product will be required for the year. If not, see U.2.2.5.

U.2.2.5 Actions when the number of positive test results during a second set of tests exceeds the maximum number of positives ("c" value)

If during the second set of tests, the number of positive test results for Salmonella exceeds the maximum number of positives allowed during the set ("c" value), the establishment shall immediately notify the V/IIC.

This type of situation is believed to indicate a strong likelihood of serious shortcomings in the establishment's HACCP plan(s) for the class of product sampled. The establishment shall immediately conduct a systematic review of all HACCP plans for the products within the class sampled and shall immediately initiate any corrective actions that are indicated as a result of this review.

The establishment shall communicate the results of this investigation along with an action plan to the V/IIC in writing within 5 working days of having notified the V/IIC that the number of Salmonella results exceeding the maximum number per set ("c" value). As per FSEP requirements, all changes to the establishment HACCP System must be recorded in the HACCP System logbook and must be reviewed by CFIA at the time of the next HACCP System audit.

The V/IIC is to provide the PND a copy of the establishment’s report and attach their comments. Inspection activity frequencies for the affected product(s) and process(es) should be reviewed. The PND will also initiate any further appropriate actions.

The establishment shall continue to take samples for Salmonella until it has completed the set of samples (i.e. has a number of conclusive test results equal to the "n" value). Any further Salmonella positive test results shall be immediately communicated to the V/IIC. If further positives are identified, the establishment shall review its action plan, amend it as required and inform the V/IIC of the changes made. The V/IIC will review the results and the establishment operator's written response and report their findings to the PND.

When there have been two consecutive sets of Salmonella tests where the number of Salmonella positive test results for each set of tests has exceeded the maximum number of positives (i.e. greater than "c" value), a third and final set of tests will be required. The date when this third set of tests must begin will be communicated to the establishment by the PND. Provided that the establishment has presented a reasonable time within its action plan for the implementation of changes to its HACCP system and adheres to this action plan, the PND may set the beginning date for the third set of testing after the implementation and validation of the HACCP System changes and the verification and recognition of these changes by the CFIA.

The third set of tests shall be performed in the same way as the first two and the results shall be assessed in the same manner. If the number of positive results for the third set of tests does not exceed the maximum number of positives per set ("c" value), no further testing for the class of product will be required for the year. If not, see U.2.2.6.

U.2.2.6 Actions required when the number of Salmonella positive test results for the third set of tests exceeds the maximum number of positives per set ("c" value)

If the number of positive test results for Salmonella exceeds the maximum number of positives per set ("c" value), the establishment shall immediately notify the V/IIC. This situation is considered as a very strong indication of a likely failure of HACCP plans for the products sampled and that there may also be serious shortcomings in overall establishment operations. The V/IIC immediately notifies the PND and the PND notifies the Director, Food of Animal Origin Division (FAOD). The Director, FAOD removes the establishment from the list of establishments eligible to export to the USA (see Q.3.3)

A full FSEP-HACCP System audit is to take place at the establishment along with a simultaneous in-depth investigation conducted by a team including Program Network Center staff acting on behalf of headquarters. These activities need to take place in an expeditious manner after the number of positive results for the third set of tests exceeds the maximum number of positives per set ("c" value) and may take place before the entire set of samples has been completed.

The in-depth investigation will be aimed at determining the causes of the repeated test results and assessing if the establishment is meeting USDA export requirements as well as Canadian domestic requirements. The investigation team will report its findings to the Director, FAOD.

U.2.3 Pre-sampling Preparation

Sample collection will be carried out by the individual designated in the establishment's written protocol. Sampling supplies, such as sterile gloves, sterile sampling solutions, hand soap, sanitizing solution, etc., as well as specific materials needed for sampling different carcass types (i.e., specimen sponges in bags and template if sampling cattle or swine carcasses), will need to be assembled prior to beginning sample collection.

When sponges are used, a verification of the sponges suitability must have been done. Sponges must not possess antimicrobial properties which might reduce bacterial counts. Supplier certification (e.g., letters of guarantee) shall be obtained for each new lot or in-house verification of each new lot of sponges shall be performed.

Such verification typically should consist of immersing a sponge for a number of hours within a seeded bath of transport medium (p. ex.: Buffered Peptone Water) to simulate shipping conditions. The bath containing indicator organisms in a known quantity is cultured at the end of the period to verify if the concentration of bacteria has decreased significantly, which would indicate that substances within the sponge are killing bacteria.

For cattle and swine carcass sampling, a template will be needed to mark off the area to sample. The template can be made of metal or aluminum foil, brown paper, flexible plastic, etc. Some disposable templates may come sterilized and individually prepackaged. To make a reusable template, cut out a 10 cm x 10 cm from a sheet larger than the area to be sampled. (Refer to Annex T, Section T.2.12, Illustration 1). If a reusable template is used, it will need to be sanitized with an approved sanitizing solution [e.g., hypochlorite (bleach) solution or alcohol]. However, the template needs to be completely dry before placing it on the carcass. Aluminum foil or paper templates can be used once and discarded. The foil for the template should be stored in a manner to prevent contamination.

Since the area enclosed by the template will be sampled, take care not to touch this area with anything other than the sampling sponge. Using dirty or contaminated material may lead to erroneous results. If an autoclave is available, paper or aluminum foil templates can be wrapped in autoclavable paper and sterilized.

Sterile sampling solutions, Buffered Peptone Water (BPW), can be stored at room temperature. However, at least on the day prior to sample collection, check solutions for cloudiness. DO NOT use solutions that are cloudy, turbid or contain particulate matter. Place the number of containers of sampling solution (BPW) that will be needed for the next day's sampling in the refrigerator. Ensure that shipping containers, coolant packs and shipping documents are prepared as required.

U.2.4 Random Selection of Samples:

The operator’s written procedure must clearly explain how the random selection of samples is achieved. Use of random numbers tables or similar random number generating systems (p. ex.: computer-generated, calculator generate, drawing cards, etc.) is required. To ensure that all carcasses (or ground product) have an equal chance of being selected, the procedure must ensure:

1. that the TIME when sample is to be selected is determined in a random manner (alternatively, random selection of the carcass sequence number achieves this) ;
2. that, where there is more than one possible site for taking the sample, the SITE is determined in a random manner. For example:
   • Selection of beef and/or hog carcasses when selection is done within coolers (12 or more hours after slaughter ) or on transfer chains, grading chains or other rails: the selection of the cooler and site within the cooler where the carcass is to be selected for the sampling period (i.e. day’s production) is determined in a random manner; AND the selection of the half-carcass or side of carcass to sample is randomly determined;
   • Selection of poultry carcass when the selection is done after chilling, at the end of the drip line or last readily accessible point prior to packaging ^{2 &} : that the selection of the chiller/drip line/packaging line where the sample is to be selected for the sampling period (i.e. day’s production) is determined in a random manner;
   • Selection of raw ground product where there is more than one grinding machine: that the selection of the grinder where the sample is to be selected for the sampling period (i.e. day’s production) is determined in a random manner. Raw ground product samples shall be randomly selected and collected after the grinding process and, if possible, before any addition of spices or seasonings, but prior to final packaging.
3. where the sample selection procedure involves selecting a half-carcass or carcass at a randomly determined sampling time/sampling site, the following procedure is used to eliminate bias and ensure random selection of the beef half-carcass, hog carcass or poultry carcass.

After having identified the half-carcass or carcass selected by the random procedure from the predetermined point, count back five (5) half-carcasses/carcass and select the next half-carcass/carcass for sampling. Each half-carcass/carcass must have an equal chance of being selected. The reason for counting back five half-carcasses is to avoid any possible bias during selection.

Additionally, for poultry carcasses ONLY: because only WHOLE (untrimmed) carcasses are to be sampled, if the fifth carcass is not a WHOLE (untrimmed) carcass, count back an additional five carcasses for sample selection. Repeat if necessary.

If more than one shift is operating at the plant, the sample can be taken on any shift, provided the above requirements have been met.

U.2.5 Aseptic Techniques/Sampling:

Extraneous organisms from the environment, hands, clothing, sample containers, sampling devices, etc., may lead to erroneous analytical results. Stringent requirements for microbiological analysis are necessary, therefore, use of aseptic sampling techniques and clean, sanitized equipment and supplies are of utmost importance.

There should be an area designated for preparing sampling supplies, etc. A stainless steel, wheeled cart or table would be useful during sampling. A small tote or caddy could be moved to the location of sampling and could be used for carrying supplies, supporting sample bags when adding sterile solutions to sample bags, etc.

Sterile gloves should be used for collecting samples. The only items which may contact the external surface of the glove are the exposed sample being collected and/or the sterile sample utensil (specimen sponge). Keep in mind that the outside surfaces of the sample container are not sterile. Do not handle the inside surface of the sterile sample containers. Do not touch anything else. The following procedure for putting on sterile gloves can be followed when collecting samples:

(a) Peel open the package of sterile gloves from the top without contaminating (touching, breathing on, contacting, etc.) the exterior of the gloves.

(b) Remove the first glove by holding it from the wrist-side opening inner surface. Avoid any contact with the outer surface of the glove. Insert the washed and sanitized hand into the glove, taking care not to puncture the glove.

(c) Remove the second glove by holding it by the cuff (outer surface). Avoid contaminating the outside of the first glove when pulling on the second glove. Take care not to touch the face, skin, clothes and other non-sterile surfaces.

(d) If at any time you are concerned that a glove may be contaminated, discard it and begin again with Step (a) above.

U.2.5.1 Preparation for Sample Collection:

Prior to collecting samples, review appropriate sampling steps, random selection procedures, and other information that will aid in sample collection.

On the day prior to sample collection, after checking for cloudiness/ turbidity, place the BPW containers that will be needed for the next day's sampling in the refrigerator/cooler. If samples are to be shipped to an off-site facility, pre-chill shipping container and refrigerator packs.

On the day of sampling, gather sample collection bags, sterile gloves, sanitizer, hand soap, sterile solutions for sampling, and specific materials listed under the (i) Materials section of the sample collection section for the type of carcass or ground product to be sampled. Ensure that all sampling supplies are on hand and readily available before beginning sample collection.

Label the sample bags before starting the sampling procedure. Use permanent ink. If you are using paper labels, it is important that the label be applied to the bag at normal room temperature; it will not stick if applied in the cooler.

Outer clothing (frocks, gloves, head gear, etc.) worn in other areas of the plant should be removed before entering the sampling area or preparing to collect samples. Replace outer clothing removed earlier with clean garments (i.e., laboratory coat) that have not been directly exposed to areas of the plant outside of the sampling area.

Sanitize the sample work area surfaces by wiping with a clean disposable paper towel dipped in a freshly prepared 500 ppm (parts per million) sodium hypochlorite solution (0.05% sodium hypochlorite) or other approved sanitizer which provides an equivalent available chlorine concentration. The sample work area surfaces must be free of standing liquid before sample supplies and/or product containers are placed on them.

Before sampling, thoroughly wash and scrub hands to the mid-forearm. Use antibacterial hand soap. If available, this should include a sanitizer at 50 ppm equivalence available chlorine. Dry the hands using disposable paper towels.

U.2.5.2 Specific Sample Collection Procedures:

The following procedures shall be used in the collection of samples for Salmonella testing. They are derived from the same ones that FSIS uses for its sampling activities in the USA. To prevent contamination of samples and to ensure national uniformity of results they must be strictly adhered to.

U.2.5.2.1. Sample Collection Procedures - Raw Ground Product

This method applies to raw ground beef, ground chicken and ground turkey. It will also be the one used for collecting fresh pork sausage samples.

(i) Materials

1. 2 sterile self-sealing-type bags, stomacher bags or equivalent.
2. Sterile gloves.
3. Plastic cable-tie-wrap or thick rubber band for securing bag.

(ii) Collection

Ensure that all supplies are on hand and readily available. Use the predetermined random selection procedure to select sample. Samples of raw ground product will be collected after the grinding process, and, if possible, before the addition of any spices or seasonings, but prior to final packaging.

1. Put on sterile gloves.
2. Aseptically collect approximately 250 grams of ground product, if possible, before the addition of any spices or seasonings, but just prior to final packaging. (Sample will be about the size of an orange). Use the sterile sampling bag, taking care not to contaminate the inside of the bag with your gloved hand.
3. Close the bag tightly by twisting the top and securing it with the plastic cable-tie-wrap or rubber band or securely closing the bag seal.
4. Place bagged sample inside a second bag and close the outer bag tightly.
5. Follow the procedure in section U.2.6, "Sample Shipment".

U.2.5.2.2 Sample Collection Procedure - Cattle half-carcasses

(i) Materials

1. Sterile specimen sponge in sterile bag wire top sealable sample bag or equivalent
2. 10 ml sterile Buffered Peptone Water (BPW)
3. Sterile self-sealing (ziplock-type) bag or stomacher bag
4. Template for 100 cm² sampling area
5. Sterile gloves
6. Wheeled ladder, sampling platform, or step ladder
7. Sanitizing solution
8. Small tote or caddy for carrying supplies

(ii) Collection

Read sections U.2.3 "Pre-sampling Preparation" and U.2.5.1 "Preparation for Sample Collection" before beginning the sampling procedure. Use pre-determined random selection procedures for selecting the half-carcass to be sampled (randomly selected sequence number or sampling site).

A sampling sponge (which usually comes dehydrated and prepackaged in a sterile bag) will be used to sample all three sites on the carcass (flank, brisket, and rump - refer to Annex T, Section T.2.12, Illustration 2). It is important to swab the areas in the order of least to most contamination in order to avoid spreading any contamination.

Non-destructive surface sampling will be conducted as follows:

1. Ensure that all bags have been pre-labeled and all supplies are on hand, including the sampling template. (An assistant may be helpful during the sampling process.)
2. Position the wheeled ladder, sampling platform, or step ladder near the carcass so the rump sample area (Annex T, Section T.2.12, Illustration 2) is within easy reach from the ladder.
3. IF a reusable template is used, immerse the sampling template in an approved sanitizing solution for at least 1-2 minutes. Just prior to swabbing the first sample site on the carcass (step 13), retrieve the sampling template from the sanitizing solution. Shake ALL excess solution from the utensil, then protect the portion of the template that will contact the carcass from contamination.
4. Locate the flank, brisket, and rump sampling sites using illustrations and directions in Annex T, Section T.2.12 (Illustration 2) (cattle carcass sampling locations).
5. While holding the sponge bag at the top corner by the wire closure, tear off the clear, perforated strip at the top of the bag.
6. Remove the cap from sterile BPW bottle, being careful not to touch the bottle opening.
7. Carefully pour the contents of the sterile BPW bottle (approximately 10 ml) into the sponge bag to moisten the sponge.
8. Close the top of the bag by pressing the wire closures together. Use hand pressure from the outside of the bag and carefully massage the sponge until it is FULLY HYDRATED (moistened).
9. With the bag still closed, carefully push the moistened sponge to the upper portion of the bag orienting one narrow end of the sponge up toward the opening of the bag. Do NOT open the bag or touch the sponge with your fingers. While holding the bag, gently squeeze any excess fluid from the sponge using hand pressure from the outside. The whole sponge should still be in the bag.
10. Open the bag containing the sponge, being careful not to touch the inner surface of the bag with your fingers. The wire closure at the top of the bag should keep the bag open. Set bag aside.
11. Put on a pair of sterile gloves.
12. Carefully remove the moistened sponge from the bag with the thumb and fingers (index and middle) of your sampling hand.
13. With the other hand, retrieve the template by the outer edge, taking care not to contaminate the inner edges of the sampling area of the template.
14. Locate the flank sampling area (Annex T, Section T.2.12, Illustration 2). Place the template over this location.
15. Hold the template in place with one gloved hand (Remember, only the sponge should touch the sampling area. Take care not to contaminate this area with your hands)
16. With the other hand, wipe the sponge over the enclosed sampling area (10 cm x 10 cm) for a total of approximately 10 times in the vertical and 10 times in the horizontal directions. Avoid touching the template, especially if it has been treated with an anti-microbial agent. The pressure for swabbing would be as if you were removing dried blood from the carcass. However, the pressure should not be too hard as to crumble or destroy the sponge. (Note: The template may need to be "rolled" from side to side during swabbing since the surface of the carcass is not flat. This ensures that the 100 cm² area is enclosed while swabbing.)
17. Repeat steps 14-16 for the brisket area, using the SAME side or surface of the sponge used to swab the flank area.
18. After swabbing the brisket area, transfer the template to the same hand holding the sponge. Do not contaminate the sponge or inner edges of the sampling area of the template.
19. Climb the ladder or platform, holding onto the handrail with the hand used to hold the template. Once at a convenient and safe height for sampling the rump, transfer template back to "climbing" hand (hand used to hold onto the rail while climbing the ladder), taking care not to contaminate the inner edges of the template.
20. Repeat steps 14-16 for the rump area, using the "clean" surface or side (the side that was NOT previously used to swab the flank/brisket areas) of the sponge.
21. After swabbing the rump area, carefully place the sponge back in the sponge sample bag, taking care not to touch the sponge to the outside of the sample bag.
22. While holding the handrail, climb down from the ladder.
23. Expel excess air from the bag containing the sponge and fold down the top edge of the bag 3 or 4 times to close. Secure the bag by folding the attached wire tie back against the bag. Place closed sponge bag into second bag and close the second bag securely.
24. Follow the procedure in section U.2.6, "Sample Shipment".

U.2.5.2.3 Sample Collection Procedure - Swine carcasses

(i) Materials

1. Sterile specimen sponge in sterile sealable wire-top bag or equivalent
2. 10 ml sterile Buffered Peptone Water (BPW)
3. Sterile self sealing (ziplock-type) bag or stomacher bag
4. Template for 100 cm² sampling area
5. Sterile gloves
6. Wheeled ladder, sampling platform, or step ladder
7. Sanitizing solution
8. Small tote or caddy for carrying supplies

(ii) Collection

Read sections U.2.3 "Pre-sampling Preparation" and U.2.5.1 "Preparation for Sample Collection" before beginning the sampling procedure. Use pre-determined random selection procedures for selecting the carcass/side of carcass to be sampled (randomly selected sequence number or sampling site).

A sampling sponge (which usually comes dehydrated and prepackaged in a sterile bag) will be used to sample all three sites on the carcass (ham, belly and jowls - refer to Annex T, Section T.2.12, Illustration 3). It is important to swab the areas in the order of least to most contamination in order to avoid spreading any contamination.

Nondestructive surface sampling will be conducted as follows:

1. Ensure that all bags have been pre-labeled and all supplies are on hand, including the sampling template. (An assistant may be helpful during the sampling process.)
2. Position the wheeled ladder, sampling platform, or step ladder near the carcass so the ham sample area (Annex T, Section T.2.12, Illustration 3) is within easy reach from the ladder.
3. IF a reusable template is used, immerse the sampling template in an approved sanitizing solution for at least 1-2 minutes. Just prior to swabbing the first sample site on the carcass (step 13), retrieve the sampling template from the sanitizing solution. Shake ALL excess solution from the utensil, then protect the portion of the template that will contact the carcass from contamination.
4. Locate the ham, belly and jowl sampling sites using illustrations and directions in (Annex T, Section T.2.12, Illustration 3)(swine carcass sampling locations).
5. While holding the sponge bag at the top corner by the wire closure, tear off the clear, perforated strip at the top of the bag.
6. Remove the cap from sterile BPW bottle, being careful not to touch the bottle opening.
7. Carefully pour the contents of the sterile BPW bottle (approximately 10 ml) into the sponge bag to moisten the sponge.
8. Close the top of the bag by pressing the wire closures together. Use hand pressure from the outside of the bag and carefully massage the sponge until it is FULLY HYDRATED (moistened).
9. With the bag still closed, carefully push the moistened sponge to the upper portion of the bag orienting one narrow end of the sponge up toward the opening of the bag. Do NOT open the bag or touch the sponge with your fingers. While holding the bag, gently squeeze any excess fluid from the sponge using hand pressure from the outside. The whole sponge should still be in the bag.
10. Open the bag containing the sponge, being careful not to touch the inner surface of the bag with your fingers. The wire closure at the top of the bag should keep the bag open. Set bag aside.
11. Put on a pair of sterile gloves.
12. Carefully remove the moistened sponge from the bag with the thumb and fingers (index and middle) of your sampling hand.
13. With the other hand, retrieve the template by the outer edge, taking care not to contaminate the inner edges of the sampling area of the template.
14. Locate the belly sampling area (Annex T, Section T.2.12, Illustration 3). Place the template over this location.
15. Hold the template in place with one gloved hand (Remember, only the sponge should touch the sampling area. Take care not to contaminate this area with your hands).
16. With the other hand, wipe the sponge over the enclosed sampling area (10 cm x 10 cm) for a total of approximately 10 times in the vertical and 10 times in the horizontal directions. Avoid coming into contact with the template, especially if it has been treated with an anti-microbial agent. The pressure for swabbing would be as if you were removing dried blood from the carcass. However, the pressure should not be too hard as to crumble or destroy the sponge. (Note: The template may need to be "rolled" from side to side during swabbing since the surface of the carcass is not flat. This ensures that the 100 cm² area is enclosed while swabbing.)
17. After swabbing the belly area, transfer the template to the same hand holding the sponge. Do not contaminate the sponge or inner edges of the sampling area of the template.
18. Climb the ladder or platform, holding onto the handrail with the hand used to hold the template. Once at a convenient and safe height for sampling the ham, transfer template back to "climbing" hand (hand used to hold onto the rail while climbing the ladder), taking care not to contaminate the inner edges of the template.
19. Repeat steps 14-16 for the ham area, using the SAME surface of the sponge used to swab the belly area.
20. After swabbing the ham area, carefully place the template back to the same hand that is holding the sponge. Do not contaminate the inner edges of the sampling area of the template.
21. While holding the handrail, with the hand not used for sampling, climb down from the ladder.
22. Transfer the template back to the "climbing" hand (hand used to hold onto the rail while descending the ladder), taking care not to contaminate the inner edges of the template.
23. Repeat steps 14-16 for the jowl area, using the "clean" surface or side (the side that was NOT previously used to swab the belly and ham areas).
24. After swabbing the jowl area, carefully place the sponge back into the sponge bag. Do not touch the surface of the sponge to the outside of the sponge bag.
25. Press wire closures on the sponge bag together, expel the excess air, then fold over the top of the bag 3 or 4 times. Close the bag with attached wire by bending the wire tie back against the bag to secure it.
26. Follow the procedure in section U.2.6, "Sample Shipment".

U.2.5.2.4 Sample Collection Procedure - Chicken Carcasses

(i) Materials

1. 2 Sterile 3500 millilitre (ml) stomacher-type bags, self-sealing (ziplock-type) bags or equivalent. (The bags must be sterile and should be large enough to hold the carcass while rinsing.)
2. 400 ml sterile, Buffered Peptone Water (BPW)
3. Plastic tie wraps or equivalent (if needed to secure the bag).
4. Sterile gloves.

(ii) Collection

Read sections U.2.3. "Pre-sampling Preparation" and U.2.5.1 "Preparation for Sample Collection" before beginning the sampling procedure. Use the predetermined random selection procedure to select the carcass to sample. The randomly selected bird will be collected after the chiller, at the end of the drip line as follows:

1. Ensure all sampling supplies are present and have been properly labeled. An assistant may be helpful during sampling.
2. Open a large stomacher-type bag without touching the sterile interior of the bag. (Rubbing the top edges of the bag between the thumb and forefinger will cause the opening to gap for easy opening.)
3. Put on sterile gloves.
4. With one hand, push up through the bottom of the sampling bag to form a "glove" over one hand with which to grab the bird, while using your other hand to pull the bag back over the hand that will grab the bird. This should be done aseptically without touching the exposed interior of the bag.
5. Using the hand with the bag reversed over it, pick up the bird by the legs (hocks) through the stomacher bag. (The bag functions as a 'glove' for grabbing the bird's legs.) Take care not to contaminate the exposed interior surface of the bag. Allow any excess fluid to drain before reversing the bag back over the bird. (Alternately, have an assistant hold open the bag. Using your gloved hand, pick up the bird by the legs, allow any fluid to drain, and place the bird in the sampling bag.)
6. Rest the bottom of the bag on a flat surface. While still holding the top of the bag slightly open, add the sterile BPW (400 ml) to the bag containing the carcass, pouring the solution over the carcass. (Alternately, with the aid of an assistant holding the bag open, add the sterile BPW (400 ml) to the bag containing the carcass, pouring the solution over the carcass.)
7. Expel most of the air from the bag, then close the top of the bag. While securely holding the bag, rinse the bird inside and out using a rocking motion for 30 shakes (approximately one minute). This is done by holding the bird through the bottom of the bag with one hand and the closed top of the bag with the other hand. Hold the bird securely and rock it in an arcing motion, alternating the weight of the bird from one hand to the other (motion like drawing an invisible rainbow or arch), assuring that all surfaces (interior and exterior of the carcass) are rinsed.
8. Rest the bag with the bird on a flat surface and, while still supporting the bird, open the bag.
9. With a gloved hand, remove the carcass from the bag. Since the carcass was rinsed with a sterile solution, it can be returned to the chill tank. Be sure not to touch the interior of the bag with your gloved hand.
10. Secure the top of the bag so that the rinse fluid will not spill out or become contaminated. (Alternately, at least 30 millilitres of rinse fluid can be poured into a sterile leak-proof container to be sent to the lab for analysis.)
11. Place the sample bag (or leak-proof container) into another bag and secure the opening of the outer bag.
12. Shipment of sample to the Accredited Laboratory: see section U.2.6, "Sample Shipment".

U.2.5.2.5 Sample Collection Procedure - Turkey Carcasses

NOTE: At this time the Performance Standard for Turkey carcasses has not been published. The following procedure is for information purposes only. It is likely that this procedure will be modified to allow for swabbing of turkey carcasses instead of the rinse technique described here.

(i) Materials

1. 1 Sterile 3500 ml stomacher-type bag, self-sealing (ziplock-type) bag or equivalent. (The bag must be sterile and should be large enough to hold the carcass while rinsing. The bags used by FSIS for their Salmonella sampling program measure approximately 18" x 24". Large turkeys should be placed in a plain, clear polypropylene autoclave bag , about 24" x 30" to 36").
2. 600 ml sterile, Buffered Peptone Water (BPW)
3. Plastic tie wraps or thick rubber bands or equivalent, if needed to secure sample bag
4. Sterile gloves

(ii) Collection

Read sections U.2.3. "Pre-sampling Preparation" and U.2.5.1 "Preparation for Sample Collection" before beginning the sampling procedure. Ensure that all supplies are on hand and readily available. An assistant will be needed to hold the bag for collecting the bird. An assistant will be needed to hold the bag for collecting the bird. Use the predetermined random selection procedure to select the carcass to be sampled. The randomly selected bird will be collected after the chiller, at the end of the drip line as follows:

1. Ensure that all supplies are on hand and readily available. An assistant will be needed to hold the bag for collecting the bird.
2. Have an assistant open the large sterile stomacher-type bag (designated for rinsing the carcass) and be ready to receive the turkey carcass. (Rubbing the top edges of the bag between the thumb and index finger will cause the opening to gap open). Alternately: If no assistant is available, place the closed large sampling bag into a bucket or pail (e.g., use the bag to "line" a bucket like a trash-can liner), then open the bag. The bucket will be used as a holder or stand to support the bag. Do not contaminate the inner surfaces of the sampling bag.
3. Put on sterile gloves.
4. Remove the selected turkey from the drip line by grasping it by the legs and allowing any fluid to drain from the cavity.
5. Place the turkey carcass, vent side up, into a sterile sampling bag. Only the carcass should come in contact with the inside of the bag.
6. Manipulate the loose neck skin on the carcass through the bag and position it over the neck bone area to act as a cushion and prevent puncturing of the bag. The assistant will need to support the carcass with one hand on the bottom of the bag.
7. While still supporting the bottom of the bag, have the assistant open the bag with the other hand. Alternately, rest the bottom of the bag on a pre-sanitized surface (i.e., a table), and while still supporting the carcass in the bag, open the bag with the other hand.
8. Add the sterile BPW (600 ml) to the bag containing the carcass, pouring the diluent over the carcass.
9. Take the bag from the assistant and expel excess air from the bag and close the top. While securely holding the bag, rinse the bird inside and out using a rocking motion for 30 shakes (approximately one minute). This is done by holding the carcass through the bag with one hand and the closed top of the bag with the other hand. Holding the bird securely with both hands, rock in an arcing motion alternating the weight of the bird from one hand to the other (motion like drawing an invisible rainbow or arch), assuring that all surfaces (interior and exterior of the carcass) are rinsed.
10. Hand the bag back to the assistant.
11. With a gloved hand, remove the carcass from the bag letting excess fluid drain back into the bag. Since the carcass was rinsed with a sterile solution, it can be returned to the chill tank. Be sure not to touch the interior of the bag with your gloved hand.
12. Expel excess air, taking care not to expel any rinse fluid. Secure the top of the bag so that the rinse fluid will not spill out or become contaminated. (Alternately, at least 30 millilitres of rinse fluid can be poured into a sterile, leak-proof container and sent to the lab for analysis.)
13. Place the sample bag (or container) into another bag and secure the opening of the outer bag.
14. Shipment of samples to the Accredited Laboratory: follow the procedure in section U.2.6, "Sample Shipment".

U.2.6. Sample shipment

Samples must be sent to a laboratory authorized to provide results. See Section U.3. Samples must be collected and shipped for analysis as outlined in section U.2.2.1.

The establishment operator must have procedures detailing how protection from temperature abuse of the sample and possible tampering will be ensured.

A- Protection from temperature abuse:

The establishment’s written program must describe the procedures used to ensure that the sample temperature arrives at the laboratory as low as possible but without freezing. Laboratories should not be performing analysis on samples which are reaching the laboratory above 10°C. To help ensure that samples arrive at the laboratory at the proper temperature, a procedure similar to the one used by FSIS and described here should be used.

1. Prechill shipping container by placing the open shipping container in the refrigerator at least the day before sampling.
2. Place the appropriately-labeled double-bagged sample in the prechilled shipper in an upright position to prevent spillage. Newspaper may be used for cushioning the sample and holding it in the upright position. Ensure that the sample is maintained at refrigeration temperature to prevent multiplication of any microorganisms present and to provide the most accurate results.
3. Place a corrugated cardboard pad on top of the sample. Next, place the frozen gel pack(s) on top of the corrugated pad to prevent direct contact of frozen gel packs with the sample. Use sufficient frozen coolant to keep the sample refrigerated during shipment to the designated laboratory. Insert a foam plug and press it down to minimize shipper head space.
4. Ship sample (via overnight delivery or courier) to the accredited laboratory.

B- Protection from tampering:

The establishment’s written Salmonella sampling program must describe procedures for ensuring tamper-evidency of samples submitted to the independent laboratory. This would include the following: handling and packaging/shipping procedures for samples, personnel involved, location of sample prior to shipment, tamper-proof shipping devices used (e.g., seals), etc.

The V/IIC will review these written procedures before sampling begins to verify that samples cannot be tampered with between the time of sampling and analysis. The V/IIC also monitors these activities on an ongoing basis during testing to verify that written procedures are adhered to and are effective.

U.3 LABORATORY ACCREDITATION REQUIREMENTS AND ANALYTICAL METHODS

Laboratories eligible to provide results:

To be eligible to provide results, laboratories must be accredited by the Standard Council of Canada (SCC) and have on their scope of approved methods, the method(s) of analysis prescribed by the USDA-FSIS. USDA-FSIS methods include a rapid screening procedure (MLG 4C.01- true negative are obtained in 24 hours) and a cultural confirmation method that must be used when potentially positive samples are identified. The cultural confirmation method is described below but laboratories should refer to the USDA-FSIS website at the following address to ensure that the most current version is used:

http://www.fsis.usda.gov/Science/Microbiological_Lab_Guidebook/index.asp

The methods should be referred to in accredited laboratories’ scope as: FSIS Procedure for the Use of the BAX System PCR Assay for screening Salmonella in Raw Meat, Carcass Sponge Samples, Whole Bird Rinses, Ready-to-Eat Meat and Poultry Products, and Pasteurized Egg Products (MLG 4C.01, as last amended) and Isolation And Identification of Salmonella From Meat, Poultry And Egg Products (MLG 4. 03, as last amended). Laboratory testing reports must indicate the specific USDA-FSIS method(s) used.

A list of accredited laboratories by SCC is available on SCC website at: http://palcan.scc.ca.

U.3.1 Equipment, Reagents, and Media Equipment

U.3.1.1 Equipment

1. Sterile scalpels, scissors, forceps, knives, spatulas, spoons, ruler or template, pipettes, petri dishes, test tubes
2. Sterile Stomacher 3500 bags (or equivalent) or plain, clear polypropylene autoclave bags (ca. 24'' x 30-36'')
3. Incubator, 36 ± 1°C
4. Incubator/Water bath, 42 ± 0.5 °C
5. A mechanical homogenization device. A Stomacher, used with sterile plastic bags, is acceptable. Some laboratories prefer to use a sterile Osterizer-type blender with sterilized cutting assemblies and adapters for use with sterile Mason jars.
6. Water bath, 48-50 °C
7. Glass slides, glass plate marked off in one-inch squares or agglutination ring slides
8. Balance, 2000 gram capacity, sensitivity of 0.1 gram
9. Inoculating needles and loops
10. Vortex mixer
11. Sterile sampling sponge and sponge bag

U.3.1.2 Reagents

1. Iodine solution for TT broth (Hajna)
2. Buffered Peptone Water (BPW) diluent
3. Methyl red reagent
4. O'Meara's V-P reagent, modified
5. Kovac's reagent
6. Ferric chloride, 10% aqueous solution
7. Sterile mineral oil
8. Saline, 0.85%
9. Saline, 0.85% with 0.6% formalin
10. Salmonella polyvalent O antiserum
11. Salmonella polyvalent H antiserum
12. Salmonella individual O grouping sera for groups A-I

U.3.1.3 Media

1. Buffered peptone water (BPW)
2. Tetrathionate broth (TT-Hajna)
3. Rappaport-Vassiliadis (RV) broth (4)--Merck Chemical Co., Cat. #7700 or equivalent
4. Brilliant green sulfa agar (BGS; contains 0.1% sodium sulfapyridine)
5. Double modified lysine iron agar (DMLIA; 2)
6. Triple sugar iron agar (TSI)
7. Lysine iron agar (LIA)
8. MR-VP Medium
9. Tryptone broth
10. Simmons citrate agar
11. Phenol red tartrate agar
12. Motility Medium
13. Christensen's urea agar
14. Carbohydrate fermentation media with Andrade's indicator
15. Decarboxylase test media (Moeller)
16. Malonate broth
17. KCN broth
18. Phenylalanine agar
19. Nutrient gelatin
20. Trypticase soy broth
21. Tryptose broth

U.3.2 Analytical Procedures

U.3.2.1 Sample Preparation for Analysis

The diverse nature of the samples which may require analysis (e.g., ground product versus a poultry carcass rinse sample) requires separate preparation procedures for each sample type. To ensure uniformity of results from different laboratories, these procedures shall be adhered to rigorously.

U.3.2.1.1 Raw Ground Product Sample Preparation

a. Use a sterile spoon or spatula to take portions of product from several areas of the sample to prepare a 25 g composite sample in a sterile plastic stomacher-type bag or blender jar. Use of a stomacher filter bag may facilitate pipetting after pre-enrichment.

b. Add 225 ml BPW. Homogenize for two minutes in a Stomacher or blender.

U.3.2.1.2 Beef or Pork Carcass Sponge Sample Preparation

Add 50 ml of BPW to the sample bag containing the sponge to bring the total amount of fluid to 60 ml and to ensure that the sponge is completely immersed. It is critical that the entire sponge be submerged before proceeding with the analytical process. Mix well.

U.3.2.1.3 Whole Chicken Carcass Rinse-Fluid Sample Preparation

a. Remove 30 ml of carcass-rinse fluid and place it in a sterile plastic bag or other sterile container.

b. Add 30 ml of BPW to the sample. Mix well.

U.3.2.1.4 Turkey Carcass Rinse-Fluid Sample Preparation

a. Remove 30 ml of carcass-rinse fluid and place it in a sterile plastic bag or other sterile container.

b. Add 30 ml of BPW to the sample. Mix well.

U.3.2.3 Detection Procedure

Sample/BPW suspensions prepared as directed in Sample preparation for analysis section (above) are the starting point for this step in the protocol. From this point on, sample suspensions of various types (e.g., whole bird rinse sample vs. raw ground product) can all be treated in the manner described below.

Note: If using a screening test, follow manufacturer's instruction for enrichment procedures. If an alternate enrichment scheme is to be used, verification of the effectiveness of this alternate enrichment protocol with the screening test should be received from the manufacturer of the screening test or by in-laboratory testing; records of such verification activities shall be maintained.

1. Incubate sample / BPW suspension at 36 ± 1°C for 20-24 hours.
2. a. Transfer 0.5 ml of the BPW sample pre-enrichment culture into 10 ml TT broth.

   b. Transfer 0.1 ml of the BPW sample pre-enrichment culture into 10 ml RV broth.

3. a. Incubate the TT enrichment culture at 42 ± 0.5°C for 22-24 hours.

   b. Incubate the RV enrichment culture at 42 ± 0.5°C for 22-24 hours.

4. Streak each enrichment culture onto both DMLIA and BGS agar plates. Do not subdivide plates for streaking multiple samples; streak the entire agar plate with a single sample enrichment.
5. Incubate plates at 36 ± 1°C
6. Examine plates after 22-24 hours of incubation. Re-incubate negative plates and re-examine them the following day.
7. Select and confirm suspect colonies as described in the sections for Isolation procedure through Biochemical testing procedures (below).

U.3.2.4 Isolation Procedure

1. 1. Pick typical well-isolated colonies.
2. a. BGS. Select colonies that are pink and opaque with a smooth appearance and an entire edge surrounded by a red color in the medium. On very crowded plates, look for colonies that appear tan against a green background.

   b. DMLIA. Select purple colonies with or without black centers. Since Salmonellae typically decarboxylate lysine and ferment neither lactose nor sucrose, the color of the medium reverts to purple.

3. Select three suspect colonies from each plate. Pick only from the surface and center of the colony. Avoid touching the agar because these selective media may suppress growth of organisms which are viable but not visible; such "sleeper" organisms can be picked up from the agar surface and carried forward onto media used for confirmation tests. If a plate is crowded and there are no well-isolated colonies available, re-streak from this plate directly onto fresh selective agar plates.

U.3.2.5 Initial Isolate Screening Procedure

1. Inoculate TSI and LIA slants consecutively with a single pick from a colony by stabbing the butts and streaking the slants in one operation. If screw-cap tubes are used, the caps must be loosened before incubation. Incubate at 36 ± 1°C for 24 ± 2 hours.
2. Examine TSI and LIA slants as sets. Note the colors of butts and slants, blackening of the media and presence of gas as indicated by gas pockets or cracking of the agar. Note also the appearance of the growth on the slants along the line of streak. Discard sets that show "swarming" from the original site of inoculation. Discard sets that show a reddish slant in LIA. Isolates giving typical Salmonella reactions should be confirmed by serological tests. Examine isolates which are suggestive, but not typical of Salmonella by a combination of biochemical and serological procedures. Confirm by biochemical tests ONLY those isolates that appear typical of Salmonellae, but do not react serologically. Refer to the following chart for assistance in making these determinations.

U.3.2.5 Serological Tests

All isolates giving TSI and LIA reactions which could be considered suggestive of Salmonella should be tested serologically. If the TSI and LIA reactions, together with the serological reactions, are indicative of Salmonella, confirmation may cease at this point. If, however, atypical TSI or LIA results and/or negative serological tests are encountered, biochemical testing is mandatory (see Biochemical testing procedure, below).

U.3.2.5.1 O Agglutination Tests

At a minimum, isolates should be tested with polyvalent O antiserum reactive with serogroups A through I. Following a positive reaction with polyvalent O antiserum, it is necessary to type the isolate using individual Salmonella antisera for O groups A through I. Testing for O groups A through I should encompass the majority of the Salmonella serotypes commonly recovered from meat and poultry products. Occasionally, however, an isolate which is typical of Salmonella (biochemically and Poly H serologically) but non-reactive with antisera to groups A through I will be recovered; such an isolate should be reported as "Salmonella non A-I" or "Salmonella O group beyond I".

Follow the manufacturer's instructions enclosed with the antisera. Use growth from either the TSI or LIA slant. Test the isolate first using polyvalent O antiserum. Do not read agglutination tests with a hand lens. If there is agglutination with the saline control alone (autoagglutination), identify such an isolate by biochemical reactions. If the saline control does not agglutinate and the polyvalent serum does, identify the individual O group using the individual Salmonella O grouping antisera for groups A through I. Record positive results and proceed to H agglutination tests.

U.3.2.5.2 H Agglutination Tests

Inoculate Trypticase soy broth or Tryptose broth. Incubate at 36 ± 1 °C overnight or until growth has an approximate density of three on McFarland's scale. Add an equal amount of saline containing 0.6% formalin and let set one hour. Remove one ml to each of two 13 x 100 mm test tubes. To one of the tubes, add Salmonella polyvalent H serum in an amount indicated by the serum titer or according to the manufacturer's instructions. The other tube serves as an autoagglutination control. Incubate both tubes at 48-50°C in a water bath for up to one hour. Record presence or absence of agglutination. Alternatively, any other poly H agglutination test may be used as long as it gives results equivalent to the conventional tube agglutination procedure described above.

U.3.2.6 Biochemical Testing Procedures

Biochemical confirmation is only necessary with those isolates giving atypical TSI or LIA results and/or negative serological tests. Do the minimum number of tests needed to establish that an isolate can be discarded or that it is a member of the genus Salmonella. Exhaustive testing of any isolate from a sample that has already yielded a typical, easily identifiable Salmonella is unnecessary.

If further testing is necessary, inoculate the following media first: Tryptone broth, MR-VP medium, Simmons citrate agar, Christensen's urea agar, motility test medium, phenol red tartrate agar, and glucose, lactose, sucrose, salicin and dulcitol fermentation broths. Incubate at 36 ± 1°C and record reactions the following day. Test Tryptone broth with Kovac's reagent for indole production in 24 hours and, if negative, again in 48 hours. Do not perform the MR-VP test until 48 hours have elapsed. If results are ambiguous, repeat MR test after five days of incubation. Hold negative carbohydrate fermentation tests for 14 days.

Refer to "Edwards and Ewing's Identification of Enterobacteriaceae", 4th Edition (3), for biochemical reactions of Enterobacteriaceae and for fermentation media and test procedures.

Discard all isolates that give positive urea or VP reactions. Discard any isolate that has the following combination of characteristics: produces gas in glucose, produces indole but not H₂S, is MR positive, VP negative and citrate negative; such organisms are E. coli regardless of ability to ferment lactose in 48 hours.

Inoculate additional biochemical tests as necessary to eliminate other Enterobacteriaceae. Refer to Edwards and Ewing for details. Eliminate Providencia spp. by a positive phenylalanine reaction. Eliminate Hafnia alvei on the basis of the following biochemical pattern: indole negative; MR negative, and VP and citrate positive based on four days of incubation at 25 °C; fermentation of arabinose and rhamnose; failure to ferment adonitol, inositol, sorbitol, and raffinose.

Alternatively, any other biochemical test system may be used as long as it gives results equivalent to the conventional tests.

U.3.3 Quality Control Procedures

A minimum of three method controls be analyzed whenever meat or poultry products are being examined for the presence of Salmonellae. These controls should include a S. typhimurium (H₂S positive), S. senftenberg (H₂S negative), and an uninoculated media control. The inoculum level for the positive controls should approximate 30-300 CFU per container of enrichment medium. Inoculate positive controls at the end of each day's run. Incubate the three controls along with the samples, and analyze them in the same manner as the samples. Confirm at least one isolate recovered from each positive control sample.

U.3.3.1 Storage of Isolates

Do not store isolates on TSI agar because this tends to cause roughness of O antigens. For short-term (2-3 months) storage, inoculate a nutrient agar slant, incubate at 36 ± 1 °C overnight, and then store at 4-8& °C.

For long-term storage of isolates, subculture Salmonella isolates by stabbing nutrient agar (0.75% agar). Incubate at 36 ± 1 °C overnight, and then seal with hot paraffin-soaked corks. Household wax is better than embedding paraffin because it stays relatively soft at room temperature making the corks easy to remove. Store isolates in the dark at room temperature. Such isolates will remain viable for several years. Store "working" Salmonella stock cultures on nutrient agar slants. Transfer stocks monthly, incubate overnight at 36 ± 1 °C overnight, and then store them at 4-8& °C.

U.3.4 References

1. AOAC International. 1995. Official Methods of Analysis of AOAC International. P.A. Cunniff, ed. 16th Edition. Gaithersburg, MD.
2. Bailey, J. S., J. Y. Chiu, N.A. Cox, and R.W. Johnston. 1988. Improved selective procedure for detection of Salmonellae from poultry and sausage products. J. Food Protect. 51(5):391-396.
3. Ewing, W. H. 1986. "Edwards and Ewing's Identification of Enterobacteriaceae", 4th Edition. Elsevier Science Publishing Co., Inc., New York, NY.
4. Vassiliadis, P. 1983. The Rappaport-Vassiliadis (RV) enrichment medium for the isolation of Salmonellae: An overview. J. Appl. Bacteriol. 54:69-76.

U.4 REPORTING OF TEST RESULTS; RECORD KEEPING

A - Accredited laboratories:

Accredited laboratories performing Salmonella testing shall directly report test results in an expeditious and simultaneous manner both to the establishment of origin and directly to the CFIA V/IIC of the establishment. Laboratories shall also maintain records of tests performed as per usual procedures.

B - Registered Establishments:

The registered establishment shall have written procedures outlining how laboratory results are received, collated and filed at the establishment. There must be an organized system allowing for ready analysis of the set of tests: a suitably organized log book can suffice for this purpose. Proof of test results (e.g.,

reports of analysis) shall be kept at the establishment for a period of not less than 24 months. A notification procedure is required for identifying all positive results to the CFIA V/IIC.

C - CFIA V/IIC at the Registered Establishment:

The CFIA V/IIC shall keep the laboratory reports sent directly to themselves from the laboratory for a period of at least 2 years. In addition tracking all positives, the V/IIC will compare their results with the establishment’s to verify that the establishment is compiling the correct data in the appropriate manner. Laboratory results are compiled on the Salmonella Testing Results report of part 4 of The Basic Compliance Checklist (BCC). This report shall be sent to H.Q. as soon as each set of samples has been completed.

U.5 Flow Chart for analysing test results

FLOWHCART DIAGRAM 

U.6 Determination of product to be sampled for Salmonella - WORKSHEET

Instructions to the IIC/VIC: Use this form when completing Form 4A of the BCC. Have the establishment provide you with a listing of products made at the establishments which are amenable to a Salmonella Performance Standard. The products need to be grouped by Performance Standard class and ranked in descending volume of production (i.e., class most produced to class least produced) Complete 1 copy of this form for each class of product made at the establishment which is amenable to a Salmonella Performance Standard. Staple a copy of the completed forms to form 4A and keep in the IIC/VIC’s office.

IS the class of product exempt from testing? (Refer to section U.1.1)	YES NO
Question one - Has the class been tested previously?	Award :
- Yes	0 points
- No	4 points
If yes, what were the results of the testing for the previous year?
- passed first round of testing successfully	0 points
- failed first round but passed second round successfully	1 point
- failed first two rounds but passed third round successfully	3 points
- failed first, second and third round but successfully returned to compliance	5 points
Question two - Has the establishment modified its manufacturing practice for the class of product?
- Yes	2 points
- No	0 points
Question three - What were the results of generic E. coli (biotype I) testing for the previous year?
- consistently met or occasionally exceeded the Performance Verification Criteria (PVC)	0 points
- regularly exceeded the PVC (twice or more per month on average)	2 points
Question four - Have written instructions been received from the CFIA Program Network Center which indicate that testing for this class is to be performed during the upcoming year?
- Yes	5 points
- No	0 points
Testing for Salmonella shall be done on: the class of product with the highest point score (in case of tie, testing for Salmonella will be done on the product which is produced in greater quantity); and any class of products which have a total point score of 5 or more.

USA - ANNEX U - Section U.6 - Determination of product to be sampled for Salmonella - worksheet page 2

Determination of product to be sampled for Salmonella - WORKSHEET Est. No.: _____

Date :

CFIA Vet.-in-Charge /Insp.-in-Charge (Signature):

Annex C | Annex D | Annex D-1 | Annex E | Annex J (PDF) | Annex K (PDF) | Annex L | Annex M | Annex Q |
Annex R | Annex S | Annex T | Annex U | Annex W | Annex W-1 | Annex W-1 | Annex X | Annex Y |
Annex Z | Annex Z-1 | Annex Z-2 |