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Detection of Salmonella in Environmental Samples from Poultry Houses

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New Microbiological Methods
(Proposed for inclusion in the Bacteriological Analytical Manual [BAM])

Proposed Rule: Prevention of Salmonella Enteritidis in Shell Eggs During Production Federal Register, September 22, 2004

 

INTRODUCTION

This method, for the detection of all Salmonella serotypes in environmental samples, has been used primarily for finding Salmonella Enteritidis (SE) in the hen houses. Salmonella Enteritidis (SE) foodborne outbreaks in human beings have been associated predominantly with eggs, when a vehicle has been identified. Due to the characteristics of SE organisms shed into the egg from the laying hen, environmental analytical methods for detecting SE were developed for the houses in which the laying hens are maintained. Environmental testing is used to verify that an on-farm egg safety program is functioning in the reduction of SE in laying hen houses, thereby reducing the number of SE contaminated eggs. Environmental testing is also used in hen houses that have been implicated in tracebacks from foodborne SE outbreaks. For further information on SE refer to Salmonella enterica Serovar Enteritidis in Humans and Animals: The Epidemiology, Pathogenesis, and Control. Editors: A. M. Sadeed, Richard K. Gast, Morris Potter, and Patrick G. Wall; Iowa State University Press, Ames, Iowa, 1998, 516pp.

  1.  Sampling

    (The sampling method described here is an interim method and, therefore, may not be identical to the sampling method included in any FDA final rule on shell egg safety during production.)

    1. Sampling for verification of an on-farm egg program

      Manure is the preferred sample type. Use a 10 cm x 10 cm (4 in x 4 in) 12 ply sterile gauze pad which is aseptically attached to a pole by clips or to a string. The clips should be disinfected before each use with 70% ethanol. The gauze pad and string, or gauze pad alone, should be sterilized by autoclaving. Moisten pad with canned evaporated milk, canned skim (fat free) evaporated milk or canned lowfat evaporated milk. Disinfect the top of the canned milk with 70% ethanol before opening. The can opener should also be disinfected with 70% ethanol. Place a gauze pad over the opened can of milk to deter flies from contaminating the milk. Use sterile gloves when handling and moistening the pad.

      Drag the moistened gauze pad over the manure the entire length of one side of the row/bank. Take another gauze pad and drag the other side of the row/bank. Repeat this procedure on all rows/banks of the house.

      Place each pad in a separate whirlpak-type bag with sufficient milk to keep the pad wet (no more than a tablespoon or approximately 15 ml). If the collection time is greater than 6 h, the use of a cooler with frozen gel packs is recommended for storage of the samples prior to shipment to the laboratory.

    2. Sampling for outbreak traceback

      An analytical unit is a 10 cm x 10 cm (4 in x 4 in) 12 ply sterile gauze pad that has been moistened with canned evaporated milk, canned skim (fat free) evaporated milk or canned lowfat evaporated milk. Disinfect can as directed above. This pad is used to sample a designated area of the laying hen house environment. Designated sampling areas are manure piles/ pits/scrapers, eggbelts and de-escalators, and fans or walkways.

      For manure sampling, follow the procedure for verification of egg program (see A.1 above).

      For eggbelt and de-escalator sampling, use aseptic technique to moisten a gauze pad with canned evaporated milk, canned skim (fat free) evaporated milk or canned lowfat evaporated milk. Use a 70% ethanol solution to disinfect the top of the canned milk before opening. The can opener should also be disinfected using 70% ethanol. Place a gauze pad over the opened can of milk to prevent flies from contaminating the milk. Use sterile gloves when handling and when moistening the pad. With gauze pad in hand, which has been surgically gloved using sterile gloves, swab multiple areas of approximately 10 to 15 cm (4 to 6 in) in length on all eggbelts from all reachable tiers/levels on the side of a bank/row of cages for at least 27.4 meters (30 feet). With the same gauze pad, swab multiple areas of all de- escalators (fingers, belt or baskets). Place one moistened gauze pad in each bag. Repeat the procedure for all sides of all rows/banks.

      For fans, using aseptic technique moisten a gauze pad with canned evaporated milk, canned skim (fat free) evaporated milk or canned lowfat evaporated milk. Use the same procedures as with eggbelts to disinfect the top of the canned milk and the can opener. Use sterile gloves when handling and when moistening the pad. With gauze pad in hand, which has been surgically gloved using sterile gloves, swab areas on the left, right, top and bottom of the frame/housing of the fan. If the fan is off, swab the blades of the fan. For walkways, use aseptic technique to moisten gauze pad on a string or pole and drag the pad along the length of a walkway. Use the same procedures for handling of the gauze pad, for moistening, and for handling the milk, as for gauze pads used for manure sampling. Place each pad in a whirlpak-type bag. For the fan or walkway, take two representative samples.

      Place each pad in a separate whirlpak-type bag with sufficient milk to keep the pad wet (no more than tablespoon or approximately 15 ml). If the collection time is greater than 6 h, the use of a cooler with frozen gel packs is recommended for sample storage prior to shipment.

    The laboratory will analyze each sample for the presence of Salmonella Enteritidis according to methods described for Salmonella spp. from laying hen houses (see D-E, below). The analytical unit is used in its entirety so that there is no remainder.

    1. Shipment and receipt

      All samples should be labeled to indicate 1) farm, 2) house and 3) type (eggbelt, manure or fan/walkway).

      The samples should be placed in an insulated transport container with frozen gel packs to keep the samples cool, but not frozen. Shipment must contain appropriate collection reports for the laboratory.

      Upon receipt at the laboratory, the samples should be checked for completeness of information and condition of sample. If the samples can not be processed at the time of receipt, store samples in a refrigerator kept at 7 ± 3° C (45° F).

  2.  Equipment and materials

     

    1. Sterile 12 ply gauze pads, 10 cm x 10 cm (4 in x 4 in)
    2. Sterile gloves
    3. Incubator, 35 ± 2° C
    4. Water bath, circulating, thermostatically controlled, 42.0 ± 0.2° C
    5. Canned evaporated milk, canned skim (fat free) evaporated milk or canned lowfat evaporated milk
    6. Can opener
    7. Whirlpak-type bags, 15 cm x 22.5 cm (6 in x 9 in)
    8. Sterile 12 ply gauze pad 10 cm x 10 cm (4 in x 4 in) on a string
    9. Poles for manure dragging
    10. Ethanol, 70%
    11. Transport container with gel packs
    12. Appropriate tyvek suits, bouffant caps, boots for biosecurity measures for entry into laying hen houses
  3.  Media and reagents

    Buffered peptone water (BPW) Tetrathionate (TT) broth
    Rappaport-Vassiliadis (RV) medium
    Brilliant green novobiocin (BGN) agar
    Xylose lysine Tergitol 4 (XLT-4) agar
    Bismuth sulfite (BS) agar

    The media and reagents listed are those that are specific for environmental sampling of hen houses. Refer to BAM Chapter 5 for all other media and reagents mentioned in the procedure.

  4.  Preparation of test portions

    Aseptically add 100 ml of buffered peptone water to the whirlpak-type bag containing the environmental gauze pad. Shake the bag vigorously in an up-and-down motion at least 10 times in a 30 cm (1 ft) arc in approximately 30 sec. Incubate 24 ± 2 h at 35° C.

  5.  Isolation After incubation, shake the bag containing the gauze pad and BPW.

    Transfer 1 ml of the incubated pre-enrichment into 10 ml of tetrathionate (TT) broth.

    Transfer 0.1 ml of the incubated pre-enrichment to 10 ml of Rappaport-Vassilliadis (RV) medium.

    Incubate the TT broth and the RV medium 24 ± 2 h at 42.0 ± 0.2° C (water bath).

    After incubation, streak a loopful of TT broth onto brilliant green with novobiocin (BGN) agar, xylose lysine Tergitol 4 (XLT4) agar and bismuth sulfite (BS) agar plates.

    Repeat this procedure with the RV medium.

    Incubate all plates for 24 ± 2 h at 35° C.

    Examine plates for presence of colonies that may be Salmonella.

    Pick at least 5 typical colonies from each plate and incubate on TSI and LIA slants at 24 ± 2 h at 35° C. Cap tubes loosely to maintain aerobic conditions while incubating slants to prevent excessive H2S production. Salmonella in culture typically produces alkaline (red) slant and acid (yellow) butt, with or without production of H2S (blackening of agar) in TSI. In LIA, Salmonella typically produces alkaline (purple) reaction in butt of tube. Consider only distinct yellow in butt of tubes as acidic (negative) reaction. Do not eliminate cultures that produce discoloration in butt of tube solely on this basis. Most Salmonella cultures produce H2S in LIA. Some non-Salmonella cultures produce a brick-red reaction in LIA slants

    If typical colonies are present on the BS agar after the 24 ± 2 h incubation, then pick at least 5 colonies. Irrespective of whether or not BS agar plates are picked at 24 ± 2 h, reincubate BS agar plates an additional 24 ± 2 h. After 48 ± 2 h incubation, pick at least 5 typical colonies, if present, from the BS agar plates, only if colonies picked from the BS agar plates incubated for 24 ± 2 h give atypical reactions in TSI and LIA slants that result in culture being discarded as not being Salmonella.

     

At this point continue with BAM Chapter 5, section D10 (Food and Drug Administration Bacteriological Analytical Manual, 8th Ed., rev. A. 1998. AOAC International, Gaithersburg, MD).

 


 

Buffered Peptone Water
Peptone10 g
Sodium chloride5 g
Sodium phosphate, dibasic3.5 g
Potassium phosphate, monobasic1.5 g
Distilled water1 liter

Autoclave 15 minutes at 121 C
Final pH 7.2 ± 0.2
Store at room temperature


 

Brillant Green Agar with Novobiocin
Proteose peptone10 g
Yeast extract3 g
Lactose10 g
Saccharose10 g
Sodium chloride5 g
Agar20 g
Brillant green0.0125 g
Phenol red0.08 g
Distilled water1 liter

Suspend ingredients in 1 liter distilled water.
Autoclave 121° C for 15 minutes
Add l ml of 20 mg/ml novobiocin to 1 l basal medium and swirl thoroughly, dispensing 20 ml portions into sterile 15 x 100 mm petri plates.
Final pH 6.9 ± 0.2
Store plates at 2-8° C


 

Xylose Lysine Tergitol 4 Agar
Proteose peptone1.6 g
Yeast extract3 g
L-lysine5 g
Xylose3.75 g
Lactose7.5 g
Saccharose7.5 g
Ferric ammonium citrate0.8 g
Sodium thiosulfate6.8 g
Sodium chloride5 g
Agar18 g
Phenol red0.08 g
Distilled water1 liter

Suspend ingredients in 1 l distilled water. Add 4.6 ml XLT agar supplement (27% solution of Tergitol 4). Heat to boiling to dissolve completely. Do not autoclave. Cool to 45-50° C in a water bath and dispense 20 ml portions into sterile 15 x 100 mm petri plates.
Final pH 7.4 ± 0.2


For methods information contact Wallace H Andrews
For general information contact Marilyn F Balmer
At FDA, 5100 Paint Branch Parkway, College Park, MD 20740


Prevention of Salmonella Enteritidis in Shell Eggs During Production; Proposed Rule Federal Register, September 22, 2004