9 C.F.R. Subpart A—Blood Testing Procedures
Title 9 - Animals and Animal Products
1 The procedure described is a modification of the method reported in the Proceedings of the U.S. Live Stock Sanitary Association, November 30 to December 2, 1932, pp. 487 to 491. (a) The blood samples should be collected and delivered as follows: (1) The blood samples should be taken by properly qualified and authorized persons only, and in containers provided by the laboratory. The containers should be stout-walled test tubes, preferably (2) Sufficient blood should be procured by making a small incision in the large median wing vein with a small sharp lancet and allowing the blood to run into the tube, or by the use of a small syringe (with 20 or 21 gage needle) which is properly cleansed between bleedings with physiological saline solution. To facilitate the separation of the serum, the tubes should be placed in a slanted position until the blood has solidified. After the blood has completely clotted, they should be packed and shipped by mail (special delivery), rapid express, or by messenger, to the laboratory. All labeling must be clear and permanent, and may be done with a suitable pencil on etched portions of the tube, or by means of fast-gum labels. (3) The blood samples must reach the laboratory in a fresh and unhemolyzed condition. Hemolyzed samples should be rejected. It is imperative, therefore, to cool the tubes immediately after slanting and clotting, and unless they reach the laboratory within a few hours, to pack them with ice in special containers, or use some other cooling system which will insure their preservation during transportation. In severe cold seasons, extreme precautions must be exercised to prevent freezing and consequent laking. The samples must be placed in cold (5 °to 10 °C.) storage, immediately upon arrival at the laboratory. (b) The antigen shall consist of representative strains of S. pullorum which are of known antigenic composition, high agglutinability, but are not sensitive to negative and nonspecific sera. The stock cultures may be maintained satisfactorily by transferring to new sloped agar at least once a month and keeping at 18 °to 25 °C. (average room temperature) in a dark closet or chest, following incubation for from 24 to 36 hours at 37 °C. The antigenic composition and purity of the stock cultures should be checked consistently. (c) A medium which has been used satisfactorily has the following composition: (d) Large 1–inch test tubes, Kolle flasks, or Blake bottles should be streaked liberally over the entire agar surface with inoculum from 48–hour slant agar cultures prepared from the stock cultures of the selected strains. The antigen-growing tubes or bottles should be incubated 48 hours at 37 °C., and the surface growth washed off with sufficient phenolized (0.5 percent) saline (0.85 percent) solution to make a heavy suspension. The suspension should be filtered free of clumps through a thin layer of absorbent cotton in a Buchner funnel with the aid of suction. The antigens of the separate strains should be combined in equal volume-density and stored in the refrigerator (5 °to 10 °C.) in tightly stoppered bottles. (e) Thiosulfate-Glycerin (TG) medium may be used as an alternate medium for the preparation of tube agglutination antigen. The TG medium, formerly used for the preparation of stained, whole-blood antigen, is described in more detail in the article by A. D. MacDonald, Recent Developments in Pullorum Antigen for the Rapid, Whole-Blood Test, Report of the Conference of the National Poultry Improvement Plan, pages 122–127, 1941. This medium provides a tube antigen of excellent specificity and greatly increases the yield of antigen from a given amount of medium. The TG medium has the following composition: Large 1–inch test tubes, Kolle flasks, Blake bottles, or Erlenmeyer flasks should be seeded over the entire agar surface with inoculum from 24–hour beef infusion broth cultures prepared from the stock cultures of the selected strains. The antigen-growing tubes or bottles should be incubated 96 hours at 37 °C., and the surface growth washed off with sufficient phenolized (0.5 percent) saline (0.85 percent) solution to make a heavy suspension. The suspension should be filtered free of clumps through a thin layer of absorbent cotton in a Buchner funnel with the aid of suction. The antigen then should be centrifuged. The mass of bacteria should be removed from the centrifuge tubes or bowl and resuspended in saline (0.85 percent) solution containing 0.5 percent phenol. After the bacterial mass has been uniformly suspended in the diluent, it should be again passed through a cotton pad in a Buchner funnel without the aid of suction. The antigens of the separate strains should be combined in equal volume-density and stored in the refrigerator (5 °to 10 °C.) in tightly stoppered bottles. (f) The diluted antigen to be used in the routine testing should be prepared from the stock antigen by dilution of the latter with physiological (0.85 percent) saline solution containing 0.25 percent of phenol to a turbidity corresponding to 0.75–1.00 on the McFarland nephelometer scale. The hydrogen-ion concentration of the diluted antigen should be corrected to pH 8.2 to 8.5 by the addition of dilute sodium hydroxide. New diluted antigen should be prepared each day and kept cold. The diluted antigen may be employed in 2 cc. quantities in 4 by (g) The maximum serum dilution employed must not exceed 1:50 for chickens, nor 1:25 for turkeys. The available data indicate that 1:25 dilution is the most efficient. In all official reports on the blood test, the serum dilutions shall be indicated. The sera should be introduced into the agglutination tubes in the desired amounts with well-cleaned serological pipettes or special serum-delivery devices which do not permit the mixing of different sera. The antigen and serum should be well mixed before incubation. The serum and antigen mixture must be incubated for at least 20 hours at 37 °C. (h) The results shall be recorded as:
N, or − (negative) when the serum-antigen mixture remains uniformly turbid. P, or + (positive) when there is a distinct clumping of the antigen, and the liquid between the agglutinated particles is clear. S, or ? (suspicious) when the agglutination is only partial or incomplete. M, or missing, when samples listed on the original record sheet are missing. H, or hemolyzed, when blood samples are hemolyzed and cannot be tested. B, or broken, when sample tubes are broken and no serum can be obtained. (Some allowance must always be made for the difference in sensitiveness of different antigens and different set-ups, and therefore, a certain amount of independent, intelligent judgment must be exercised at all times. Also, the histories of the flocks require consideration. In flocks where individuals show a suspicious agglutination, it is desirable to examine representative birds bacteriologically to determine the presence or absence of S. pullorum.) [36 FR 23121, Dec. 3, 1971. Redesignated at 44 FR 61586, Oct. 26, 1979, as amended at 59 FR 12799, Mar. 18, 1994] 2 The procedure described is a modification of the method reported by Runnels, Coon, Farley, and Thorpe, Amer. Vet. Med. Assoc. Jour. 70 (N.S. 23): 660–662 (1927). (a) The procedure for the collection and delivery of blood samples in the rapid serum test is the same as that described in §147.1(a). (b) The selection and maintenance of suitable strains of S. pullorum and the composition of a satisfactory medium are described in §147.1 (b) and (c). (c) Large 1–inch test tubes, Kolle flasks, or Blake bottles are streaked liberally from 48–hour slant-agar cultures prepared from stock cultures of the selected strains. (d) The antigen-growing tubes or bottles should be incubated 48 hours at 37 °C., and the surface growth washed off with a very slight amount of 12 percent solution of sodium chloride containing 0.25 to 0.5 percent phenol, filtered through lightly packed sterile absorbent cotton placed in the apex of a sterile funnel. (e) The washings should be adjusted (using 12 percent sodium chloride containing 0.25 to 0.5 percent phenol) so that the turbidity is 50 times greater than tube 0.75 of McFarland's nephelometer, or to a reading of 7 mm. by the Gates nephelometer. (f) The individual strain antigens should be tested with negative sera for their insensitivity and with positive sera for high agglutinability in comparison with known satisfactory antigen. The antigens of the separate strains should be combined in equal volume-density and stored in the refrigerator (5 °to 10 °C.) in tightly stoppered bottles. (g) The tests should be conducted on a suitable, smooth plate. The serum-antigen dilution should be made so that the dilution will not exceed 1:50 when compared to the standard tube agglutination test. When testing turkey blood samples, it is desirable to use a serum-antigen dilution equivalent to the 1:25 in the tube method. The serum should be added to the antigen and mixed thoroughly by use of the tip of the serum pipette. Most strong positive reactions will be plainly evident within 15 to 20 seconds. The final reading should be made at the end of 2 or 3 minutes. Heating the plate at approximately 37 °C. will hasten agglutination. Before reading, the plate should be rotated several times. (h) The results shall be recorded as described in §147.1(h). [36 FR 23121, Dec. 3, 1971. Redesignated at 44 FR 61586, Oct. 26, 1979, as amended at 59 FR 12799, Mar. 18, 1994] 3 The procedure described is a modification of the method reported by Schaffer, MacDonald, Hall, and Bunyea, Jour. Amer. Vet. Med. Assoc. 79 (N. S. 32): 236–240 (1931). (a) The description of the preparation of antigen is not herein included because the antigen is a proprietary product produced only under license from the Secretary of Agriculture. (b) A loop for measuring the correct quantity of blood can usually be obtained from the manufacturer of the antigen. A satisfactory loop may be made from a piece of No. 20 gage nichrome wire, 2 (c) A drop of antigen should be placed on the testing plate. A loopful of blood should be taken up from the wing vein. When submerged in the blood and then carefully withdrawn, the loop becomes properly filled. On looking down edgewise at the filled loop, one observes that the blood appears to bulge. The loopful of blood then should be stirred into the drop of antigen, and the mixture spread to a diameter of about 1 inch. The loop then should be rinsed in clean water and dried by touching it to a piece of clean blotting paper, if necessary. The test plate should be rocked from side to side a few times to mix the antigen and blood thoroughly, and to facilitate agglutination. The antigen should be used according to the directions of the producer. (d) Various degrees of reaction are observed in this as in other agglutination tests. The greater the agglutinating ability of the blood, the more rapid the clumping and the larger the clumps. A positive reaction consists of a definite clumping of the antigen surrounded by clear spaces. Such reaction is easily distinguished against a white background. A somewhat weaker reaction consists of small but still clearly visible clumps of antigen surrounded by spaces only partially clear. Between this point and a negative or homogeneous smear, there sometimes occurs a very fine granulation barely visible to the naked eye; this should be disregarded in making a diagnosis. The very fine marginal clumping which may occur just before drying up is also regarded as negative. In a nonreactor, the smear remains homogeneous. (Allowance should be made for differences in the sensitiveness of different antigens and different set-ups, and therefore, a certain amount of independent, intelligent judgment must be exercised at all times. Also, the histories of the flocks require consideration. In flocks where individuals show a suspicious agglutination, it is desirable to examine representative birds bacteriologically to determine the presence or absence of S. pullorum.) [36 FR 23121, Dec. 3, 1971. Redesignated at 44 FR 61586, Oct. 26, 1979, as amended at 59 FR 12799, Mar. 18, 1994] Routinely, the microagglutination test is applied as a single-dilution test and only a single 18–24 hour reading is made. (a) The procedure for the collection and delivery of blood samples in the microagglutination test is the same as that described in §147.1(a). A method that has proven advantageous is to transfer the serum samples from the blood clot to a microplate as described in “Applied Microbiology,” volume 24, No. 4, October 1972, pages 671–672. The dilutions are then performed according to paragraphs (d) or (e) of this section. (b) Stained microtest antigen for pullorum-typhoid is supplied as concentrated stock suspension and must be approved by the Department.4 4 Information as to criteria and procedures for approval of concentrated stock suspension of stained microtest antigens may be obtained from the National Poultry Improvement Plan, Veterinary Services, APHIS, USDA, 1498 Klondike Road, Suite 200, Conyers, GA 30094. (c) Available data indicate that a 1:40 dilution for the microagglutination test is most efficient for the detection of pullorum-typhoid agglutinins in both chickens and turkeys. In all official reports on the blood test, the serum dilutions shall be indicated. (d) The recommended procedure for the 1:40 dilution in the microagglutination test is as follows: (1) Add 100 microliters (0.10 cc.) of 0.85 percent physiological saline to each well of the microplate. (2) Using a microdiluter or a multimicrodiluter handle fitted with twelve 10 microliter microdiluters, transfer 5 microliters (0.005 cc.) of the serum sample from the collected specimen to the corresponding well of the microplate. This is accomplished by touching the surface of the serum sample with the microdiluter and then transferring and mixing with the diluent in the microplate well. The microdiluter is removed, blotted, touched to the surface of the distilled water wash, and again blotted. Other acceptable methods of serum delivery are described in “Applied Microbiology,” volume 21, No. 3, March 1971, pages 394–399. (3) Dilute the microtest antigens with 0.50 percent phenolized saline and add 100 microliters (0.1 cc.) to each microplate well. (4) Seal each plate with a plastic sealer or place unsealed in a tight incubation box as described in “Applied Microbiology,” volume 23, No. 5, May 1972, pages 931–937. Incubate at 37°C. for 18–24 hours. (5) Read the test results as described in paragraph (f) of this section. (e) The recommended procedure for a microagglutination test titration is as follows: (1) Add 50 microliters (0.05cc.) of 0.85 percent physiological saline to each well of the microplate. (2) To the wells representative of the lowest dilution in the titration, add an additional 50 microliters (0.05 cc.) of 0.85 percent physiological saline making a total of 100 microliters in these wells. (3) Transfer each serum sample as described in §147.5(d)(2) of this section to the first well containing 100 microliters (0.10cc.) in the titration, which represents the lowest dilution. (4) Make twofold serial dilutions of each serum by transferring 50 microliters (0.05cc.) of diluted serum from one well to the next using twelve 50 microliter microdiluters fitted in a multimicrodiluter handle. When transfers have been made to all of the wells of the desired series, the 50 microliters remaining in the microdiluters are removed by blotting, touching the microdiluters to the surface of the distilled water wash, and blotting again. (5) Dilute the desired microtest antigen with 0.50 percent phenolized saline and add 50 microliters (0.05 cc.) to each microplate well. (6) Seal each plate with a plastic sealer or place the unsealed microplates in a tight incubation box and incubate at 37 °C. for 18–24 hours. (7) Read the test results as described in paragraph (f) of this section. (f) Read the test results with the aid of a reading mirror. Results are interpreted as follows: (1) N, or − (negative) when the microplate well has a large, distinct button of stained cells; or (2) P, or + (positive) when the microplate well reveals no antigen button; or (3) S, or ? (suspicious) when the microplate well has a small button. Suspicious reactions may tend to be more positive than negative [±] or vice versa [±] and can be so noted if desired. [41 FR 48726, Nov. 5, 1976. Redesignated at 44 FR 61586, Oct. 26, 1979, and amended at 57 FR 57342, Dec. 4, 1992; 59 FR 12799, Mar. 18, 1994; 59 FR 67617, Dec. 30, 1994; 61 FR 11521, Mar. 21, 1996; 63 FR 3, Jan. 2, 1998; 67 FR 8469, Feb. 25, 2002] The macroagglutination tests for Mycoplasma antibodies, as described in “Standard Methods for Testing Avian Sera for the Presence of Mycoplasma Gallisepticum Antibodies” published by the Agricultural Research Service, USDA, March 1966, and the microagglutination tests, as reported in the Proceedings, Sixteenth Annual Meeting of the American Association of Veterinary Laboratory Diagnosticians, 1973, shall be the official tests. Procedures for isolation and identification of Mycoplasma may be found in Isolation and Identification of Avian Pathogens, published by the American Association of Avian Pathologists and §§147.15 and 147.16. (a) The status of a flock for Mycoplasma shall be determined according to the following criteria: (1) If the tube agglutination or the serum plate test is negative, the flock qualifies. (2) If the tube agglutination or the serum plate test is positive, the hemaglutination inhibition (HI) test and/or the Serum Plate Dilution (SPD) test shall be conducted. Provided, that for egg-type and meat-type chicken and waterfowl, exhibition poultry, and game bird flocks, if more than 50 percent of the samples are positive for either Mycoplasma gallisepticum, M. synoviae, or both, the HI and/or the SPD test shall be conducted on 10 percent of the positive samples or 25 positive samples, whichever is greater. The results of the HI and/or SPD tests must be followed by the action prescribed in paragraphs (a)(3), (a)(4), and (a)(5) of this section. (3) If the tube agglutination or serum plate tests are positive and HI and/or the SPD tests are negative, the flock shall be retested in accordance with paragraph (a)(6) of this section. (4) If HI titers of 1:40 or SPD titers of 1:5 are found, the flock shall be considered suspicious and shall be retested in accordance with paragraph (a)(6) of this section. (5) If HI titers of 1:80, positive enzyme-labeled immunosorbent assay (ELISA) titers, or SPD titers of 1:10 or higher are found, the Official State Agency shall presume the flock to be infected. If the indicated titers are found, tracheal swabs from 30 randomly selected birds shall be taken promptly and cultured individually or a PCR-based procedure conducted on these specimens for Mycoplasma, and additional tests conducted in accordance with paragraph (a)(6) of this section before final determination of the flock status is made. (6) Fourteen days after the previous bleeding date, all birds or a random sample comprised of 75 birds shall be tested by the serum plate or tube agglutination test. Tested birds shall be identified by numbered bands. (7) If the tube agglutination test or serum plate test is negative for the Mycoplasma for which the flock was tested, the flock qualifies. (8) If the tube agglutination or serum plate test is positive on the retest, the HI and/or SPD test shall be conducted on the reacting samples. (9) On the retest, if the tube agglutination or serum plate tests are positive at the same or higher rate and the HI or SPD tests are negative, the flock shall be considered suspicious and shall be retested in accordance with paragraph (a)(6) of this section. (10) On the retest if HI titers of 1:80 and/or SPD titers of 1:10 or higher are found, the flock shall be considered infected: Provided, That, at the discretion of the Official State Agency, additional tests may be conducted in accordance with paragraph (a)(6) of this section before final determination of the flock status is made. (11) If HI titers of 1:80 and/or SPD titers of 1:10 or higher are found on the second retest, the flock shall be considered infected for the Mycoplasma for which it was tested. (12) If the tube agglutination or serum plate tests are found on the second retest to be positive at the same or higher rate and the HI and/or SPD tests are negative, the flock should be considered infected: Provided, That if the status of the flock is considered to be equivocal, the Official State Agency may examine reactors by the in vivo bio-assay, PCR-based procedures, and/or culture procedures before final determination of the flock status is made. (13) If the in vivo bio-assay, PCR-based procedures, and culture procedures are negative, the Official State Agency may qualify the flock for the classification for which it was tested. (14) If the in vivo bio-assay, PCR-based procedures, or culture procedures are positive, the flock will be considered infected. However, the following considerations may apply: (i) In PCR-positive flocks for which there are other negative mycoplasma test results, the flock's mycoplasma status should be confirmed through either seroconversion or culture isolation of the organism, or through both methods, before final determination of the flock's status is made. (ii) In flocks for which only the bio-assay is positive, additional in vivo bio-assay, PCR-based procedures, or cultural examinations may be conducted by the Official State Agency before final determination of the flock's status is made. (15) If the in vivo bio-assay, PCR-based procedures, or cultures are positive on retest, the flock shall be considered infected for the mycoplasma for which it was tested. (b) [Reserved] [40 FR 1504, Jan. 8, 1975, as amended at 41 FR 48726, Nov. 5, 1976. Redesignated at 44 FR 61586, Oct. 26, 1979, and amended at 47 FR 21993, May 20, 1982; 50 FR 19900, May 13, 1985; 54 FR 23957, June 5, 1989; 59 FR 12799, Mar. 18, 1994; 61 FR 11521, Mar. 21, 1996; 62 FR 44070, Aug. 19, 1997; 63 FR 3, Jan. 2, 1998; 65 FR 8019, Feb. 17, 2000] The serum plate agglutination test, the tube agglutination test, and the enzyme-linked immunosorbent assay (ELISA) test should be considered basic screening tests for mycoplasma antibodies. The test selected will depend on preference, laboratory facilities, and availability of antigen. These three tests, though quite accurate, determine flock status rather than individual bird status, since occasional reactions are nonspecific. Under normal circumstances, the rate of such nonspecific reactions is low. Nonspecific reactions may occasionally be high, 5 For additional information on mycoplasma test procedures, refer to the following references: Proc. 77th Annual Meeting, U.S. Animal Health Association, 1973; Isolation and Identification of Avian Pathogens, 3rd Edition; Methods for Examining Poultry Biologics and for Identifying and Quantifying Avian Pathogens, 1991. (a) Serum plate agglutination test. (1) The serum plate agglutination test for mycoplasma is conducted by contacting and mixing 0.02 ml of test serum with 0.03 ml of serum plate antigen on a glass at room temperature. The standard procedure is: (i) Allow antigen and test serums to warm up to room temperature before use. (ii) Dispense test serums in 0.02 ml amounts with a pipette or standardized loop (rinsed between samples) to 1 (iii) Dispense 0.03 ml of antigen beside the test serum on each square. Hold antigen dispensing bottle vertically. (iv) Mix the serum and antigen, using a multimixing device if large numbers are to be run at one time. (v) Rotate the plate for 5 seconds. At the end of the first minute, rotate the plate again for 5 seconds and read 55 seconds later. (2) A positive reaction is characterized by the formation of definite clumps, usually starting at the periphery of the mixture. Most samples that are highly positive will react well within the 2-minute test period. Reactions thereafter should be considered negative, although partial agglutination at 3 and 5 minutes may warrant further retesting. High-quality antigen contacted with negative serum will usually dry up on the plate without visible clumping. Whenever samples are run, the antigen should be tested against known positive and negative control serums. Standard reference antigens and negative and positive titered sera are available from the National Veterinary Services Laboratories (NVSL), P.O. Box 884, Ames, Iowa 50010. (3) Since it is difficult to measure uniform amounts of serum with a calibrated loop, this technique should not be used in conducting an official test. (b) Serum plate dilution test. (1) The serum plate dilution (SPD) test may be used to evaluate possible nonspecific reactions, gain additional information to evaluate positive plate tests occurring in an unexpected manner, and/or to evaluate the level of mycoplasma antibodies present in the serum sample. If sufficient serum is available, the following method would provide the dilutions required to conduct the test. (i) Rack three tubes and put 0.8 ml of phosphate-buffered saline (PBS) in tube 1 and 0.5 ml of PBS in tubes 2 and 3. (ii) Pipette 0.2 ml of the test serum into tube 1 and discard the pipette. (iii) With a pipette, mix the serum and PBS in tube 1 and withdraw 0.5 ml and add to tube 2. (iv) Repeat the process in step (iii), mixing the contents of tube 2 and transferring 0.5 ml to tube 3. (v) Conduct the test, as described for the serum plate test in paragraph (a), on the undiluted sample and on samples in tubes 1, 2, and 3 after proper mixing of each dilution. (vi) To assist in the evaluation of the test, conduct concurrent SPD tests using both positive 1:80 and positive 1:160 HI sera for the mycoplasma being tested. The antigen should be pretested for reactivity with standard serum at the 1:5 and 1:10 dilution. (vii) Interpretation of the SPD test results should be based on the criteria in §147.6. (c) Tube agglutination test. (1) The mycoplasma tube agglutination test is conducted by mixing 0.08 ml of test serum with 1.0 ml of diluted (1:20) antigen in a tube and allowing the mixture to react for 18–24 hours at 37° C. The diluent will be the standard phosphate-buffered saline with phenol. This solution is made up as follows: The pH of the buffered phenolized saline will be 7.1–7.2 if all reagents are accurately measured. The stock tube antigen is diluted 1:20 with buffered phenolized saline. The procedures for the tube test are as follows: (i) Rack 12×75 mm clean tubes and identify the tubes according to the sample to be tested. (ii) Add 0.08 ml of the individual test serum to each tube. This will create approximately a 1:12.5 screening dilution of test serum when 1.0 ml of diluted antigen is added. The use of a pipetting device will insure proper mixing of serum and antigen. (iii) To interpret positive reactions to the 1:12.5 dilution, two additional dilutions may be made by adding 0.04 ml of serum for 1:25 dilution and 0.02 ml of serum for 1:50 dilution, with the addition of 1.0 ml of diluted antigen as indicated in paragraph (c)(1)(ii) of this section. (iv) Shake racks and incubate test systems for 18–24 hours at 37° C. (2) Tests are read against a dark background under indirect fluorescent light. Regarded as a positive reaction is a clearing of the supernatant fluid, with visible sediment in the bottom of the tube. Incomplete reactions are suspect. Positive and negative control serums should be incorporated into each day's run of tests. Reactions at 1:25 or greater are considered positive. They should be confirmed by the HI test. Incubation for periods greater than 24 hours may be helpful in evaluating suspicious reactions and need for possible retesting or other diagnostic tests. (d) Hemagglutination Inhibition (HI) test. The mycoplasma HI test is conducted by the constant-antigen, decreasing-serum method. This method requires using a 4-hemagglutination (HA) unit of diluted antigen. Differences in the number of HA units used will change the titers of positive sera markedly. Standard HA antigens for Mycoplasma gallisepticum, M. synoviae, and M. meleagridis are available from NVSL. The antigen has been titrated and diluted to approximately 1:640. The HA titration of each sample should be checked as described in paragraph (d)(2) on initial use or after long storage. To maintain HA activity, the undiluted HA antigen should be stored at −60° to −70° C. The test procedures are illustrated in Tables 2 and 3 of this paragraph. (1) Preparation of materials. (i) Prepare phosphate-buffered saline (PBS) as follows: The pH of the PBS will be 7.1–7.2 if all reagents are accurately measured. (ii) Collect the turkey or chicken red blood cells (RBC's) in Alsever's solution which has been prepared as follows: The sodium citrate and sodium chloride are dissolved in 800 ml distilled water and sterilized at 15 lbs. pressure for 15 minutes. Dissolve the dextrose in 200 ml distilled water, sterilize by Seitz or other type of filtration and then add aseptically to the sterile sodium citrate and sodium chloride solution. (iii) From a turkey(s) or chicken(s) known to be free of the mycoplasma being tested, withdraw sufficient blood with a syringe containing Alsever's solution to give a ratio of 1 part blood to 5 parts Alsever's solution (e.g., 8 ml blood in 40 ml of Alsever's solution). Centrifuge the blood suspension at 1,000 rpm for 10 minutes and remove the Alsever's solution or supernatant with a pipette. (iv) Wash the RBC's two times in 10 or more parts of Alsever's solution, centrifuging after each washing. Centrifugation is at 1,000 rpm for 10 minutes. The supernatant fluid is removed and the RBC deposit resuspended to give a 25 percent suspension of packed RBC's in Alsever's solution. (In testing either chicken or turkey sera, the homologous RBC system must be used; i.e., use chicken cells when testing chicken serum and turkey cells when testing turkey serum.) If this suspension is kept refrigerated, it should keep for 7 or 8 days after the blood has been collected. (v) For the test, 1 ml of the 25 percent RBC's is added to 99 ml of buffered saline to make a 0.25 percent RBC suspension. (2) Hemagglutination (HA) antigen titration. The HA stock antigen is stored at −70 ° C in PBS buffer containing 25 percent glycerin (vol/vol) in a concentrated suspension (i.e., 320–640 HA units/ml) in screwtype vials. Under such conditions, potency will be retained for years. There will be a rapid loss of titer if improperly stored. The titer of HA antigen is determined as illustrated in Table 1 and described in paragraphs (d)(2)(i) through (x) of this section. (i) Rack a series of 11 chemically clean 12×75 mm test tubes. Label the tubes 1–11 left to right. (ii) Put 0.8 ml of PBS in tube 1 and 0.5 ml of PBS in each of tubes 2–11. (iii) Add 0.2 ml of antigen to tube 1. This will make a 1:5 dilution of antigen. Discard pipette. (iv) Mix contents of tube 1 thoroughly with a clean pipette, and transfer 0.5 ml to tube 2. This will make a 1:10 dilution of antigen in tube 2. Discard pipette. (v) Continue making serial twofold dilutions of antigen, changing pipettes after each transfer, through tube 10. This will result in a series of twofold dilutions ranging from 1:5 to 1:2560. Discard 0.5 ml of antigen dilution from tube 10. (vi) Add 0.5 ,ml of 0.25 percent RBC's to tubes 1–11. Tube 11 will serve as PBS/RBC control. (vii) Shake the rack and incubate at room temperature until the cells in the PBS/RBC control tube have settled into a compact button at the bottom of the tube. (viii) If turkey sera is also to be tested for HI titer, repeat steps outlined in paragraphs (d)(2)(i) through (vii) of this section, using 0.25 percent turkey RBC's. (ix) The end point of the titration is the highest dilution of antigen that produces complete agglutination of the RBC's, as evidenced by the formation of a thin sheet of cells covering the concave bottom of the tube. For example, if complete agglutination is produced through tube 8 (a dilution of 1:640 of antigen), the antigen would be said to titer 640, the reciprocal of the dilution. (x) Specificity of HA antigen should be determined by conducting HI tests with specific chicken sera of variable HI titers. Specific turkey sera of varying HI titers should be used if turkey sera is also to be tested. (3) Reagents for mycoplasma HI test. (i) Eight-unit antigen (Dilution factor for stock antigen is established by dividing titer by 8; i.e., 640 antigen is diluted 1:80 in PBS to make 8-unit antigen.) (ii) Four-unit antigen (made by diluting surplus 8-unit antigen 1:2 with PBS). (iii) PBS at pH 7.0. (iv) Unknown test serums. (v) Positive control serum of known titer (should be from the same species as the unknown). (vi) Negative control serum (should be from the same species as the unknown). (vii) Solution of 0.25 percent washed RBC's. (4) Test outline. (i) Rack 10 chemically clean 12×75 mm tubes for each serum, including controls, to be tested. Identify each row of tubes, and label tubes in each row 1–10, left to right. In row 1, add tube 11 for a PBS/RBC control. (ii) Put 0.8 ml of PBS in tube 1 of each test row; put 0.5 ml of 8-unit antigen in tube 2 of each test row; put 0.5 ml of 4-unit antigen in each of tubes 3–10 in each test row; and put 0.5 ml of PBS in tube 11. (iii) Add 0.2 ml of test serum to tube 1. This tube will be the serum control in the test system. (iv) Mix and make 0.5 ml transfers from tube 1 through tube 10. This will result in serial twofold dilutions of serum starting with 1:5 and ending with 1:2560. Discard 0.5 ml from tube 10. (v) Rack five tubes in which to set up an antigen control. (vi) In tube 1, put 1.0 ml of 4-unit antigen; put 0.5 ml of PBS in tubes 2–5. (vii) Make 0.5 ml serial transfers from tube 1 through tube 5, changing pipettes after each transfer. Discard 0.5 ml from tube 5. This will result in a series of tubes respectively containing 4, 2, 1, (viii) After 20–30 minutes at room temperature to permit antibody-antigen reaction, add 0.5 ml of 0.25 percent washed RBC's to each tube. Shake racks and incubate as for HA titration. (ix) In this test system, positive serum should inhibit the HA activity of the antigen, while negative serum should have no effect. Inhibition will be evidenced by the formation of a free-flowing bottom of cells in the botton of the tube. The titer of the serum can be calculated as the reciprocal of the highest dilution of serum that produces complete HI. Controls should read as follows: (A) Serum control (tube 1). Cells should settle out. (B) PBS/RBC control (tube 11). Cells should settle out. (C) Antigen control. HA in tubes 1–3. Cells should settle out in tubes 4–5. (D) Positive and negative serum control. Positive control should inhibit to its known titer; negative control should have no inhibitory effect. (x) With this test system and 4 units of antigen, HI titers of 80 or above are considered positive and titers of 40 are strongly suspicious. However, titers of 10 or 20 are usually negative. Sample test results are illustrated in Table 4 in this paragraph. (xi) If serological results from agglutination tests complemented by the HI test are inconclusive, cultural examination, bio-assay, or retesting of samples after an interval of at least 21 days may be indicated. (e) Procedure for mycolplasma hemagglutination inhibition tests using microtiter technique—(1) Procedure No. 1. The microtiter mycoplasma HI test was developed from the tube HI test described in §147.7(d). Refer to these procedures for preparation of materials not listed below. (i) Materials needed. (A) Microtiter equipment (minimal); i.e., microplates, microdiluters, micropipettes, go-no-go diluter delivery tester, (0.05 ml). (B) Phosphate-buffered saline (PBS). (C) Reagents from NVSL; i.e., HA antigen and negative and positive titered sera for the mycoplasma to be tested. (D) Homologous red blood cells (RBC's) suspension 0.5 percent (2 ml of 25 percent RBC's to 98 ml of PBS) obtained from birds free of the mycoplasma to be tested. (See paragraphs (d)(1)(ii) through (v) of this section for preparation of RBC's.) (ii) Microtiter hemagglutination (HA) antigen titration. (A) Mark off two rows of 10 wells each for antigen titer (HA is done in duplicate). (B) Mark last well in each row for cell controls. (C) Prepare in small test tube (12×75 mm) a starting dilution of antigen by combining 0.1 ml antigen with 0.9 ml PBS. This is a 1:10 dilution. (D) Add 0.05 ml PBS to all wells, including cell controls. (E) Add 0.05 ml antigen (1:10 dilution) with diluters to the first well in both rows, mix thoroughly, transfer diluter to second well of each row and mix, continuing through the 10th well of each row. With mixture in diluter from last well, check diluter on go-no-go card, then place diluter in distilled water. If diluter checks out, antigen dilution will be 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560, 1:5120. (F) Add 0.05 ml of 0.5 percent RBC suspension to all wells using a 0.05 dropper. (G) Seal plate (if plate is to be held over 2 hours); shake and allow to stand at room temperature until cells in cell control gather in compact button. The titer is the highest dilution in which agglutination is complete. The dilution contains 1 HA unit in 0.05 ml. (H) Prepare a dilution of antigen which contains 8 HA units in 0.05 ml. Example: if the antigen titer is 1:640, then that dilution contains 1 HA unit per 0.05 ml. Then 640÷8=80, or a dilution of 1:80 containing 8 HA units. Or 640÷4=160, a dilution of 1:160 containing 4 HA units per 0.05 ml. (iii) Microtiter HI test. (A) Prepare two dilutions of antigen, one containing 8 HA units per 0.05 ml and one containing 4 HA units per 0.05 ml. The 4-unit antigen can be prepared from the 8-unit antigen by mixing with equal parts of PBS. (B) Mark off one row of 8 wells for each test. (C) Prepare a 1:5 dilution of each sera to be tested in a small test tube (12×75 mm): 0.1 ml serum plus 0.4 ml PBS or 0.05 ml serum plus 0.20 ml PBS. (D) Add 0.05 ml PBS with the 0.05 ml dropper to the first well in each row. (E) Add 0.05 ml of 8-unit antigen to well 2 in each row. (F) Add 0.05 ml of 4-unit antigen to well 3 through 8 for each row. (G) For each serum to be tested, load 0.05 ml diluter with 1:5 dilution as prepared in paragraph (iii) above and place in first well of row. (H) Mix well and transfer loaded diluter to well 2. Continue serial twofold dilutions through well number 8. (I) Well 1 (serum dilution of 1:10) is serum control. Well 2=1:20 dilution; well 3=1:40 dilution; well 4=1:80 dilution; well 5=1:160 dilution; well 6=1:320 dilution; well 7=1:640 dilution; and well 8=1:1280 dilution. (J) Antigen control. (1) Mark off 6 wells for antigen controls. (2) Add 0.05 ml PBS to wells 2, 3, 4, 5, and 6. (3) Add 0.05 ml 8-unit antigen to wells 1 and 2. (4) With empty diluter, mix contents of well 2. Continue serial twofold dilutions through well 6. (5) Well 1 contains 8 units; well 2 contains 4 units; well 3 contains 2 units; well 4 contains 1 unit; well 5 contains (6) Mark off two wells for cell controls and add 0.05 ml PBS to each. (7) After 20–30 minutes at average room temperature (20°–23°C) to permit antibody-antigen reaction, add 0.05 ml of a 0.5 percent suspension of RBC's to all wells. (8) Seal all wells (if wells are to be held over 2 hours). Shake the plate thoroughly. (9) Incubate at room temperature for 30–45 minutes. (K) Interpretation: The HI titer is the highest serum dilution exhibiting complete inhibition of hemagglutination as indicated by flowing of cells when the plate is tilted. Serum having a titer of 1:80 or greater is considered positive. A titer of 1:40 or 1:20 is suspicious. (2) Procedure No. 2. Purpose: To test for antibodies to avian mycoplasma by hemagglutination inhibition (HI). The test uses the constant antigen, titered-sera method for measuring antibodies to M. gallisepticum, M. synoviae, or M. meleagridis. (i) Materials needed. (A) M. gallisepticum, M. synoviae, and/or M. meleagridis HI antigens. (B) Positive and negative control sera. (C) Phosphate buffered saline (PBS). (D) Microtiter plates, 96-well, U-bottom. (E) 12-channel pipettor (Titerek). (F) 50 μL pipettor (Pipetman P200). (G) Pipette tips. (H) 0.5 percent homologous red blood cells (RBC's) in PBS (use RBC's from the same species being tested). (I) Plate-sealing tape. (J) Mirrored plate reader. (ii) Microtiter hemagglutination antigen (HA) titration. (A) Perform standard hemagglutination test (HA) on mycoplasma antigen to determine titer of antigen. (1) Dispense 50 μL of PBS into each well of 3 rows of a 96-well microtiter plate. (2) Dispense 50 μL of stock antigen into the wells of 2 rows. (3) Perform serial two-fold dilutions (50 μL) using a 12-channel pipettor. The dilution series will be from 1:2 to 1:4096. (4) Add 50 μL of 0.5 percent homologous RBC's to each well of all 3 rows. The row with no antigen serves as an RBC control. (B) Incubate at room temperature (approximately 30 minutes) until the control RBC's give tight buttons. The HA titer is read as the last well to give a complete lawn (hemagglutination). (C) Dilute stock antigen to 4 HA units for the HI test. The dilution required to give 4 HA units is calculated by dividing the stock antigen HA titer by 8. (Example: 1:320 HA units ÷ 8 = 40, dilute stock antigen 1:40.) (iii) Hemagglutination inhibition assay. (A) Label one column (A to H) of a 96-well, U-bottom microtiter plate for each sample, each positive and negative control sera, antigen backtitration, and RBC control. (B) Add 40 μL of PBS to the top row of wells (row A) of the plate. (C) Add 25 μL of PBS to all remaining wells of the plate. (D) Add 10 μL of each test sera to well A of each column (making a 1:5 sera dilution). (E) Serially dilute 25 μL from well A through H using a 12-channel pipettor. Discard the final 25 μL. Row A = 1:5...row H = 1:640. (F) With an Oxford doser, add 25 μL of 4 HA unit antigen to wells B through H. Well A serves as sera control. (G) Prepare an antigen backtitration by adding 25 μL of PBS to each well of one column. Add 25 μL of diluted antigen to well A and serially dilute 25 μL from wells A to D. This prepares 1:2, 1:4, 1:8, and 1:16 dilutions. (It is recommended that the antigen control backtitration be performed before the diluted antigen is used in the assay. Dilution problems could be detected and corrected before the inappropriately diluted antigen is used in the assay.) (H) Leave a column of wells blank for an RBC control. (I) Agitate gently and incubate for 30 minutes at room temperature. (J) Add 50 μL of 0.5 percent RBC's to all wells. Note: Do not agitate after RBC's have been added (agitation may result in false positive reactions by causing the RBC's to fall, resulting in “false” buttons). (K) Cover the plate with sealing tape. Incubate at room temperature for 30 minutes or until control RBC's give a tight button. (L) Read the reaction on a mirrored plate reader. (iv) Results. (A) The titer is reported as the reciprocal of the last dilution to give a tight button of RBC's. The final dilution scheme includes the antigen in the dilution calculation and is as follows: B=1:20, C=1:40, D=1:80, E=1:160, F=1:320, G=1:640, H=1:1,280. (B) For the assay to be valid: (1) The positive control sera must give a result within one dilution of the previously determined titer. (2) The negative control sera must be negative. (3) The backtitration of the antigen must be 1:4 or 1:8. (4) The RBC control must give tight, non-hemolyzed buttons. (5) Sera controls (well A of each test sera) must not have non-specific agglutination or hemolysis. If negative, report as “negative with non-specific agglutination or non-specific hemolysis” or “unable to evaluate due to non-specific agglutination or hemolysis” or treat the serum to remove the non-specific agglutination and repeat the test. (See paragraph (e)(2)(v) of this section.) (v) Treatment to remove non-specific agglutination—(A) Purpose. Treatment of serum to remove non-specific agglutination that is interfering with HI assays. (B) Specimen. Serum. (C) Materials. Homologous RBC's (chicken or turkey), 50 percent solution PBS, centrifuge, incubator, 4C (refrigerator). (D) Procedure. (1) Prepare a 1:5 dilution of test serum by adding 50 μL of serum to 200 μL of PBS. (2) Prepare a 50 percent solution of RBC's by adding equal volumes of packed RBC's to PBS. Mix well. (3) Add 25 μL of 50 percent RBC solution to the serum dilutions. (4) Vortex gently to mix. (5) Incubate at 4 °C for 1 hour. (6) Centrifuge to pellet the RBC's. (7) Use the supernatant to perform the HI assay. Modify the dilution scheme in the assay to consider the initial 1:5 dilution prepared in the treatment. For the 1:5 dilution scheme, do not add PBS to row A. Add 50 μL of the 1:5 treated supernatant to row A. Serially dilute 25 μL from rows A through H. This prepares a serum dilution of 1:10 through 1:640 in rows B through H. [49 FR 19803, May 10, 1984, as amended at 57 FR 57342, Dec. 4, 1992; 59 FR 12799, Mar. 18, 1994; 63 FR 3, Jan. 2, 1998; 67 FR 8469, Feb. 25, 2002] The following testing provisions may be used for retaining the classification U.S. M. Gallisepticum Clean under §145.23(c)(1)(ii)(C) and §145.33(c)(1)(ii)(C), and for retaining the classification U.S. M. Synoviae Clean under §145.23(e)(1)(ii)(b) and §145.33(e)(1)(ii)(b) of this chapter. (a) Under the supervision of an Authorized Agent or State Inspector, the eggs which are used in egg yolk testing must be selected from the premises where the breeding flock is located, must include a representative sample of 30 eggs collected from a single day's production from the flock, must be identified as to flock of origin and pen, and must be delivered to an authorized laboratory for preparation for diagnostic testing. (b) The authorized laboratory must identify each egg as to the breeding flock and pen from which it originated, and maintain this identity through each of the following: (1) Crack the egg on the round end with a blunt instrument. (2) Place the contents of the egg in an open dish (or a receptacle to expose the yolk) and prick the yolk with a needle. (3) Using a 1 ml syringe without a needle, aspirate 0.5 ml of egg yolk from the opening in the yolk. (4) Dispense the yolk material in a tube. Aspirate and dispense 0.5 ml of PBS (phosphate-buffered saline) into the same tube, and place in a rack. (5) After all the eggs are sampled, place the rack of tubes on a vortex shaker for 30 seconds. (6) Centrifuge the samples at 2500 RPM (1000×g) for 30 minutes. (7) Test the resultant supernatant for M. gallisepticum and M. synoviae by using test procedures specified for detecting IgG antibodies set forth for testing serum in §147.7 (for these tests the resultant supernatant would be substituted for serum); except that a single 1:20 dilution hemagglutination inhibition (HI) test may be used as a screening test in accordance with the procedures set forth in §147.7. Note: For evaluating the test results of any egg yolk test, it should be remembered that a 1:2 dilution of the yolk in saline was made of the original specimen. [50 FR 19900, May 13, 1985; 63 FR 3, Jan. 2, 1998] (a) The agar gel immunodiffusion (AGID) test should be considered the basic screening test for antibodies to Type A influenza viruses. The AGID test is used to detect circulating antibodies to Type A influenza group-specific antigens, namely the ribonucleoprotein (RNP) and matrix (M) proteins. Therefore, this test will detect antibodies to all influenza A viruses, regardless of subtype. The AGID test can also be used as a group-specific test to identify isolates as Type A influenza viruses. The method used is similar to that described by Beard.6 6 Beard, C.W. Demonstration of type-specific influenza antibody in mammalian and avian sera by immunodiffusion. Bull. Wld. Hlth. Org. 42:779–785. 1970. (1) Materials needed. (i) Refrigerator (4 °C). (ii) Freezer (−20 °C). (iii) Incubator or airtight container for room temperature (approximately 25 °C) incubations. (iv) Autoclave. (v) Hot plate/stirrer and magnetic stir bar (optional). (vi) Vacuum pump. (vii) Microscope illuminator or other appropriate light source for viewing results. (viii) Immunodiffusion template cutter, seven-well pattern (a center well surrounded by six evenly spaced wells). Wells are 5.3 mm in diameter and 2.4 mm apart. (ix) Top loading balance (capable of measuring 0.1 gm differences). (x) Pipetting device capable of delivering 50μl portions. (xi) Common laboratory supplies and glassware—Erlenmeyer flasks, graduated cylinders, pipettes, 100 × 15 mm or 60 × 15 mm petri dishes, flexible vacuum tubing, side-arm flask (500 mL or larger), and a 12- or 14-gauge blunt-ended cannula. (2) Reagents needed. (i) Phosphate buffered saline (PBS), 0.01M, pH 7.2 (NVSL media #30054 or equivalent). (ii) Agarose (Type II Medium grade, Sigma Chemical Co. Cat.# A–6877 or equivalent). (iii) Avian influenza AGID antigen and positive control antiserum approved by the Department and the Official State Agency. (iv) Strong positive, weak positive, and negative control antisera approved by the Department and the Official State Agency (negative control antisera optional). (3) Preparing the avian influenza AGID agar. (i) Weigh 9 gm of agarose and 80 gm of NaCl and add to 1 liter of PBS (0.01 M, pH 7.2) in a 2 liter Erlenmeyer flask. (ii) To mix the agar, either: (A) Autoclave the mixture for 10 minutes and mix the contents by swirling after removing from the autoclave to ensure a homogeneous mixture of ingredients; or (B) Dissolve the mixture by bringing to a boil on a hot plate using a magnetic stir bar to mix the contents in the flask while heating. After boiling, allow the agar to cool at room temperature (approximately 25 °C) for 10 to 15 minutes before dispensing into petri plates. (iii) Agar can be dispensed into small quantities (daily working volumes) and stored in airtight containers at 4 °C for several weeks, and melted and dispensed into plates as needed. Note: Do not use agar if microbial contamination or precipitate is observed. (4) Performing the AGID—(i) Detection of serum antibodies. (A) Dispense 15 to 17 mL of melted agar into a 100 × 15 mm petri plate or 5 to 6 mL agar into a 60 × 15 mm petri plate using a 25 mL pipette. The agar thickness should be approximately 2.8 mm. (B) Allow plates to cool in a relatively dust-free environment with the lids off to permit the escape of water vapor. The lids should be left off for at least 15 minutes, but not longer than 30 minutes, as electrolyte concentration of the agar may change due to evaporation and adversely affect formation of precipitin lines. Note: Plates should be used within 24 hours after they are poured. (C) Record the sample identification, reagent lot numbers, test date, and identification of personnel performing and reading the test. (D) Using the template, cut the agar after it has hardened. Up to seven template patterns can be cut in a 100×15 mm plate and two patterns can be cut in a 60×15 mm plate. (E) Remove the agar plugs by aspiration with a 12- to 14-gauge cannula connected to a side arm flask with a piece of silicone or rubber tubing that is connected to a vacuum pump with tubing. Adjust the vacuum so that the agar surrounding the wells is not disturbed when removing the plugs. (F) To prepare the wells, either: (1) Place 50 μl of avian influenza AGID antigen in the center well using a micropipette with an attached pipette tip. Place 50 μl AI AGID positive control antiserum in each of two opposite wells, and add 50 μl per well of test sera in the four remaining wells. This arrangement provides a positive control line on one side of the test serum, thus providing for the development of lines of identity (see figure 1); or (2) Place 50 μl AI AGID positive control antiserum in each of three alternate peripheral wells, and add 50 μl per well of test sera in the three remaining wells. This arrangement provides a positive control line on each side of the test serum, thus providing for the development of lines of identity on both sides of each test serum (see figure 2). Note: A pattern can be included with positive, weak positive, and negative reference serum in the test sera wells to aid in the interpretation of results (see figure 3). (G) Cover each plate after filling all wells and allow the plates to incubate for 24 hours at room temperature (approximately 25 °C) in a closed chamber to prevent evaporation. Humidity should be provided by placing a damp paper towel in the incubation chamber. Note: Temperature changes during migration may lead to artifacts. (ii) Interpretation of test results. (A) Remove the lid and examine reactions from above by placing the plate(s) over a black background, and illuminate the plate with a light source directed at an angle from below. A microscope illuminator works well and allows for varying intensities of light and positions. (B) The type of reaction will vary with the concentration of antibody in the sample being tested. The positive control serum line is the basis for reading the test. If the line is not distinct, the test is not valid and must be repeated. The following types of reactions are observed (see figure 3): (1) Negative reaction. The control lines continue into the test sample well without bending or with a slight bend away from the antigen well and toward the positive control serum well. (2) Positive reaction. The control lines join with, and form a continuous line (line of identity) with, the line between the test serum and antigen. The location of the line will depend on the concentration of antibodies in the test serum. Weakly positive samples may not produce a complete line between the antigen and test serum but may only cause the tip or end of the control line to bend inward toward the test well. (3) Non-specific lines. These lines occasionally are observed between the antigen and test serum well. The control lines will pass through the non-specific line and continue on into the test serum well. The non-specific line does not form a continuous line with positive control lines. (b) The enzyme-linked immunosorbent assay (ELISA) may be used as a screening test for avian influenza. Use only federally licensed ELISA kits and follow the manufacturer's instructions. All ELISA-positive serum samples must be confirmed with the AGID test conducted in accordance with paragraph (a) of this section. [65 FR 8019, Feb. 17, 2000]
Title 9: Animals and Animal Products
PART 147—AUXILIARY PROVISIONS ON NATIONAL POULTRY IMPROVEMENT PLAN
Subpart A—Blood Testing Procedures
§ 147.1 The standard tube agglutination test.1
Water 1,000 cc.Difco beef 4 gm. (0.4 percent). extractDifco Bacto- 10 gm. (1.0 percent). peptoneDifco dry- 20 gm. (2.0 percent). granular agar.Reaction_pH 6.8 to 7.2.
Beef infusion 1,000 cc.Difco Bacto- 20 gm. (2.0 percent). peptone Sodium 5 gm. (0.5 percent). thiosulfateAmmonium 5 gm. (0.5 percent). chlorideGlycerin, 20 cc. (2.0 percent). U.S.P. (95 percent)Difco dry- 30 gm. (3.0 percent). granular agar.Reaction_pH 6.8 to 7.2.
§ 147.2 The rapid serum test.2
§ 147.3 The stained-antigen, rapid, whole-blood test.3
§ 147.4 [Reserved]
§ 147.5 The microagglutination test for pullorum-typhoid.
§ 147.6 Procedure for determining the status of flocks reacting to tests for Mycoplasma gallisepticum, Mycoplasma synoviae, and Mycoplasma meleagridis.
§ 147.7 Standard test procedures for mycoplasma.5
------------------------------------------------------------------------ Grams------------------------------------------------------------------------Sodium hydroxide (C.P.)...................................... 0.15Sodium chloride (C.P.)....................................... 8.5Potassium dihydrogen phosphate (KH2 PO4) (C.P.).............. 0.68Phenol (Crystal) (C.P.)...................................... 2.5 Distilled water to make 1,000 ml------------------------------------------------------------------------
------------------------------------------------------------------------ Grams------------------------------------------------------------------------Sodium hydroxide (C.P.)...................................... 0.15Sodium chloride (C.P.)....................................... 8.5Potassium dihydrogen phosphate (KH2 PO4) (C.P.).............. 0.68 Distilled water to make 1,000 ml------------------------------------------------------------------------
------------------------------------------------------------------------ Grams------------------------------------------------------------------------Sodium citrate................................................ 8.0Sodium chloride............................................... 4.2Dextrose...................................................... 20.5Distilled water to make 1,000 ml..............................------------------------------------------------------------------------
Table 4_Sample Results of HI Tests [Tube and Serum Dilution]-------------------------------------------------------------------------------------------------------------------------------------------------------- 1 2 3 4 5 6 7 8 9 10 -------------------------------------------------------------------------------------------------------- 1:5 1:10 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 1:2560--------------------------------------------------------------------------------------------------------------------------------------------------------Serum A (HI neg.).............................. - + + + + + + + + +Serum B (HI 1:40).............................. - - - - + + + + + +Serum C (HI 1:160)............................. - - - - - - + + + +Serum D (HI 1:20).............................. - - - + + + + + + +--------------------------------------------------------------------------------------------------------------------------------------------------------+, HA.-, no HA or HI.
§ 147.8 Procedures for preparing egg yolk samples for diagnostic tests.
§ 147.9 Standard test procedures for avian influenza.
