JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1988, p. 672-674 Vol. 26, No. 4 0095-1137/88/040672-03$02.00/0 Copyright © 1988, American Society for Microbiology Evaluation of the Rapid Test for Identification of and agalactiae Isolated from Bovine Mammary Glandst JEFFREY L. WATTS* AND W. E. OWENS Mastitis Research Laboratory, Hill Farm Research Station, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Route 1, Box 10, Homer, Louisiana 71040 Received 29 September 1987/Accepted 5 January 1988 A latex agglutination test system (Rapid Mastitis Test [RMT]; Immucell, Portland, Maine) containing reagents for the identification of Staphylococcus aureus and from bovine intramam- mary was evaluated with 527 staphylococcal and 267 streptococcal isolates. The RMT Staphylococcus aureus reagent detected 94.2% of 242 Staphylococcus aureus isolates, 80% of 25 Staphylococcus intermedius isolates, and 42.8% of 21 tube -positive Staphylococcus hyicus isolates. All Streptococcus agalactiae isolates were correctly identified by the RMT Streptococcus agalactiae reagent. Cross-reactions were observed with one Streptococcus dysgalactiae and three Streptococcus uberis strains. The RMT was found to be an acceptable method for the detection of Staphylococcus aureus and Streptococcus agalactiae isolated from bovine mammary glands. The occurrence of coagulase-positive staphylococci other than Staphylococcus aureus requires biochemical testing for species level identification.

Bovine mastitis is the most important economic disease in lococcus warneri ATCC 27836, Staphylococcus haemoly- the dairy industry, with losses in the United States placed at ticus ATCC 29970, Staphylococcus sciuri ATCC 29062, 2 billion dollars per year (8). Staphylococcus aureus and Streptococcus agalactiae ATCC 27956, Streptococcus aga- Streptococcus agalactiae are the most frequently isolated lactiae Cornell 48, Streptococcus dysgalactiae ATCC 27957, from intramammary infections (IMI) in many dairy Streptococcus equinus ATCC 27960, (Strep- herds (reviewed in reference 3). Accurate, rapid identifica- tococcus) faecalis ATCC 27959, Streptococcus uberis tion of these organisms permits implementation of specific ATCC 27958, Streptococcus uberis ATCC 19436, Strepto- control measures. uberis NCFB 2018, and Streptococcus uberis NCFB Recommended methods (4, 12) for the identification of 2038. Since only four bovine Staphylococcus intermedius Staphylococcus aureus and Streptococcus agalactiae iso- strains were available, an additional 20 canine strains ob- lated from bovine IMI emphasize the tube coagulase test tained from H. U. Cox, Louisiana State University School of (TC) and CAMP test. These procedures, while technically Veterinary Medicine, Baton Rouge, were included in the nondemanding, require 24 h for results. Latex agglutination study. All other strains were isolated from bovine IMI. tests for the identification of Staphylococcus aureus and All isolates were stored in Trypticase soy broth (BBL group B streptococci (Streptococcus agalactiae) have been Microbiology Systems, Cockeysville, Md.) containing 20% widely accepted in human clinical laboratories (1, 2, 13, 18). glycerin at -20°C. Prior to testing, all isolates were subcul- Recently, the Rapid Mastitis Test (RMT; Immucell, Port- tured twice on Trypticase soy agar (BBL) containing 5% land, Maine) containing latex agglutination reagents for the bovine blood and 0.1% esculin Chemical St. identification of Staphylococcus aureus and Streptococcus (Sigma Co., agalactiae from bovine IMI has become available. The Louis, Mo.). All staphylococcal isolates were identified purpose of this study was to evaluate the accuracy of the previously by using the STAPH-Trac system (Analytab RMT for the identification of Staphylococcus aureus and Products, Plainview, N.Y.) or a previously described con- Streptococcus agalactiae isolated from bovine mammary ventional method (17). All streptococcal isolates were glands. grouped by using Phadebact streptococcus coagglutination reagents for Lancefield groups A, B, C, D, and G (Pharmacia Diagnostics, Piscataway, N.J.). The accuracy ofthis product MATERIALS AND METHODS was established previously (15). Streptococcal isolates were Bacteria. A total of 794 bacterial isolates (527 staphylo- identified by previously described methods (12). cocci and 267 streptococci) were used in the study. The Conventional testing. All Staphylococcus aureus, Staphy- following reference strains were also included: Staphylococ- lococcus intermedius, and Staphylococcus hyicus isolates cus aureus ATCC 29740, Staphylococcus aureus ATCC were tested for the presence of clumping factor (CF). A 25923, Staphylococcus hyicus ATCC 11249, Staphylococ- single, well-isolated colony was mixed with 1 drop of EDTA- cus chromogenes NADC-A3, Staphylococcus epidermidis treated rabbit plasma (BBL) on a clean glass slide. The slide ATCC 14990, Staphylococcus warneri ATCC e155, Staphy- was rocked for approximately 10 s and examined for clump- ing. Strains which could not be easily emulsified were * Corresponding author. considered autoagglutinators and were classified as CF neg- t Approved for publication by the Director of the Louisiana ative. Agricultural Experiment Station as manuscript number 87-80-1467. CF-negative isolates were tested by using the TC. A 672 VOL. 26, 1988 RAPID MASTITIS TEST FOR S. AUREUS AND S. AGALACTIAE 673

TABLE 1. Comparison of the RMT Staphylococcus aureus reagent and conventional coagulase tests for identification of staphylococci isolated from bovine mammary glands Organism (no. of No. (%) of strains No. (%) of strains positive by the following conventional method(s): strains tested) RMT positive CF TC4 TC24 All S. aureus (242) 228 (94.2) 207 (85.5) 26 (10.7) 7 (2.9) 240 (99.2) S. intermedius (25) 20 (80.0) 13 (52.0) 0 (0.0) 12 (48.0) 25 (100.0) S. hyicus (100) 9 (9.0) o (0.0) 4 (4.0) 17 (17.0) 21 (21.0) a None of the following were positive (no. of strains): S. chromogenes (50), S. epidermidis (62), S. warneri (18), S. haemolyticus (4), S. hominis (13), S. sciuri (3), and S. xylosus (10). single, well-isolated colony was used to inoculate 0.5 ml of uberis strains. Cross-reactions were not observed with EDTA-treated rabbit plasma in a sterile test tube. The tube Streptococcus equinus or strains. was incubated at 35°C and examined at 4 h (TC4) and 24 h (TC24) for clot formation. All other staphylococcal species were tested for coagulase production by the TC. DISCUSSION RMT. The RMT was conducted in accordance with the instructions of the manufacturer. Staphylococci were tested The control of specific pathogens within individual dairy by placing 1 drop of Staphylococcus aureus latex reagent in herds requires accurate identification of infectious agents. the center of an etched circle in a black glass slide. One to Confirmatory testing by the TC and by the CAMP test for the two staphylococcal colonies were removed from a bovine identification of Staphylococcus aureus and Streptococcus blood with a wooden applicator stick and mixed agalactiae, respectively, may be logistically difficult for with the reagent. The glass slide was rocked for 1 min. No many laboratories. Latex agglutination tests are technically agglutination or slight agglutination (1+) was considered nondemanding, rapid, and accurate (1, 2, 10, 13, 15, 18). negative. Visible, distinct agglutination ('2+) was consid- The RMT correctly identified 94.2% of 242 Staphylococ- ered positive. Streptococcal isolates were tested in a similar cus aureus isolates, a value which was lower than that fashion, except that three to four streptococcal colonies and obtained in previous evaluations of similar reagents with Streptococcus agalactiae latex reagent were used. A nega- human isolates (1, 2, 13) or veterinary isolates (5, 10). The tive control provided by the manufacturer was used to RMT was not as sensitive as TC24 but was comparable in ensure reagent performance. sensitivity to CF and TC4 combined. Thus, the RMT is useful for screening large numbers of Staphylococcus aureus RESULTS isolates. However, small numbers of isolates require the TC for the detection of coagulase production. The results obtained with the RMT Staphylococcus au- The TC is considered confirmatory for the identification of reus reagent and conventional coagulase tests for 527 human Staphylococcus aureus (1, 2). However, the presence staphylococcal isolates are summarized in Table 1. The of the coagulase-positive species Staphylococcus interme- RMT Staphylococcus aureus reagent yielded positive results dius and the coagulase-variable species Staphylococcus with 228 of 242 (94.2%) Staphylococcus aureus isolates, hyicus lowers the specificity of the TC in veterinary bacte- whereas conventional coagulase tests detected 240 (99.2%) riology. Additionally, the sensitivity of latex agglutination isolates. A total of 207 (85.5%) Staphylococcus aureus tests, such as the RMT, which detect CF and protein A is isolates were identified by CF; an additional 33 isolates reduced, as these two species lack one or both of these required the TC. Of these, 26 (10.7%) were TC4 positive and components. Staphylococcus intermedius does not produce 7 (2.9%) were TC24 positive. Two encapsulated Staphylo- protein A, and only 14% of strains produce CF (9). The RMT coccus aureus strains were negative by the RMT and con- detected 20 of 25 (80%) Staphylococcus intermedius strains ventional methods. included in this study, a value which is much higher than the Of 25 Staphylococcus intermedius strains tested, 20 (80%) 43.2% previously reported by Cox et al. (5) with a latex were positive by the RMT. All isolates were identified by agglutination reagent developed with human clinical isolates. conventional tests, with 13 (52%) being CF positive and 12 Overall, 13 (52%) Staphylococcus intermedius strains were (48%) being TC24 positive. The stock strain and four bovine CF positive, a value which is much higher than the 14% strains of Staphylococcus intermedius were CF positive, reported in the species description (9) and which concurs whereas only 10 of 20 canine strains were CF positive. The with the 52.3 and 51.4% for CF-positive strains reported by remaining canine Staphylococcus intermedius strains were Cox et al. (5, 6). However, the stock strain and all 4 bovine TC24 positive. A total of 100 Staphylococcus hyicus isolates, including 21 TC-positive strains, were used in the study. Of the 21 TABLE 2. Performance of the RMT Sîreptococcus agalactiae TC-positive strains, 9 were RMT positive. No Staphylococ- reagent in identification of streptococci isolated from bovine cus hyicus isolates were CF positive. Isolates representing mammary glands coagulase-negative staphylococcal species, including the Organism (no. of strains RMT TC-negative Staphylococcus hyicus strains, were negative strains tested) psiti ve by the RMT and conventional methods. positive The results obtained with the RMT Streptococcus agalac- S. agalactiae (84) ...... 84 (100.0) tiae reagent for 267 streptococcal isolates are presented in S. dysgalactiae (42) ...... 1 (2.4) Table 2. All 84 Streptococcus agalactiae isolates were RMT S. uberis (103) ...... 3 (2.9) S...... 0 positive. Strong agglutination (3 +) was observed with 1 of 42 equinus (20) (0.0) Streptococcus dysgalactiae and 3 of 103 Streptococcus E.faecalis (18) . . 0 (0.0) 674 WATTS AND OWENS J. CLIN. MICROBIOL. strains were CF positive and RMT positive, whereas only 10 LITERATURE CITED canine strains were CF positive, with 4 yielding negative 1. Baker, J. S., M. A. Bormann, and D. H. Bourdreau. 1985. RMT results. Evaluation of various rapid agglutination methods for the iden- Staphylococcus hyicus does not produce CF (7), but tification of Staphylococcus aureus. J. Clin. Microbiol. 21:726- reports vary on protein A production by this species (11, as 729. cited in reference 14). None of the Staphylococcus hyicus 2. Berke, A., and R. C. Tilton. 1986. Evaluation of rapid coagulase strains in the present study were CF positive, although 21 methods for identification of Staphylococcus aureus. J. Clin. were TC positive. Of these, nine (42.8%) were detected by Microbiol. 23:916-919. 3. Bramley, A. J., and F. H. Dodd. 1984. Reviews of the progress the RMT. Hogan et al. (10) determined that 19 of 63 (30.2%) of dairy science: mastitis control-progress and prospects. J. Staphylococcus hyicus strains were TC positive. However, Dairy Res. 51:481-521. only 1.6% were detected by a latex agglutination reagent. 4. Brown, R. W., D. A. Barnum, D. E. Jasper, J. S. McDonald, and Protein A production by Staphylococcus hyicus strains was W. D. Schultze. 1981. Microbiological procedures for use in the not determined in the present study or in the study of Hogan diagnosis of bovine mastitis, p. 16-20. National Mastitis Coun- et al. (10). cil, Arlington, Va. Hogan et al. (10) suggested that latex agglutination tests 5. Cox, H. U., S. S. Newman, A. F. Roy, and J. D. Hoskins. 1984. were an acceptable method for the differentiation of Staph- Species of Staphylococcus isolated from animal infections. ylococcus aureus from Staphylococcus hyicus. As Staphy- Cornell Vet. 74:124-135. 6. Cox, H. U., S. S. Newman, A. F. Roy, J. D. Hoskins, and C. S. lococcus intermedius is infrequently isolated from bovine Foil. 1985. Comparison of coagulase test methods for identifi- IMI, isolates positive in the RMT are probably Staphylococ- cation of Staphylococcus intermedius from dogs. Am. J. Vet. cus aureus. However, additional character determinations, Res. 46:1522-1525. such as testing for and pigment production or 7. Devriese, L. A., V. Hajek, P. Oeding, S. A. Meyer, and K. H. biochemical testing, are necessary to differentiate Staphylo- Schleifer. 1978. Staphylococcus hyicus (Sompolinsky 1953) coccus aureus from Staphylococcus hyicus. comb. nov. and Staphylococcus hyicus subsp. chromogenes Hogan et al. (10) reported that cross-reactions occurred subsp. nov. Int. J. Syst. Bacteriol. 28:482-490. with Staphylococcus xylosus strains and a latex agglutina- 8. Eberhart, R. J., R. J. Harmon, D. E. Jasper, R. P. Natzke, S. C. tion reagent. No cross-reactions were observed in the pres- Nickerson, J. K. Reneau, E. H. Row, K. L. Smith, and S. B. Spencer. 1987. Current concepts of bovine mastitis. National ent study with coagulase-negative staphylococcal strains, Mastitis Council, Arlington, Va. including 10 Staphylococcus xylosus strains. However, non- 9. Hajek, V. 1976. Staphylococcus intermedjus, a new species specific agglutination or autoagglutination occurred with the isolated from animals. Int. J. Syst. Bacteriol. 26:401-408. Staphylococcus aureus reagent when large amounts of 10. Hogan, J. S., A. Cornetta, and J. W. Pankey. 1986. Comparison staphylococcal colony material were used. This problem was of four test procedures to identify Staphylococcus aureus iso- alleviated by holding the applicator stick perpendicular to lated from bovine intramammary infections. Am. J. Vet. Res. the plate and touching the end to the colony until no 47:2017-2020. additional colony material adhered. This procedure limited 11. Lachica, R. V. F., C. A. Genigeorgis, and P. D. Hoeprich. 1979. the amount of colony material used and minimized the Occurrence of protein A in Staphylococcus aureus and closely related Staphylococcus species. J. Clin. Microbiol. 10:752-753. autoagglutination phenomenon, providing easier interpreta- 12. Pedersen, P. S., J. A. Madsen, W. Heeschen, F. K. Neave, tion of results. F. H. S. Newbould, and W. D. Schultze. 1981. and The Streptococcus agalactiae reagent produced clear-cut identification of mastitis bacteria, p. 21-22. In F. H. Dodd (ed.), reactions with all Streptococcus agalactiae isolates. One Laboratory methods for use in mastitis work. International Streptococcus dysgalactiae strain (group C) cross-reacted Dairy Federation, Brussels. with the RMT Streptococcus agalactiae reagent. Poutrel 13. Pennell, D. R., J. A. Rott-Petri, and T. A. Kurzynski. 1984. (15) reported cross-reactions between group C streptococci Evaluation of three commercial agglutination tests for the and group B latex agglutination reagents. Three Streptococ- identification of Staphylococcus aureus. J. Clin. Microbiol. 10: cus uberis strains cross-reacted with the RMT Streptococcus 614-617. 14. Phillips, W. E., and W. E. Kloos. 1981. Identification of coagu- agalactiae reagent. Again, cross-reactions between Strepto- lase-positive Staphylococcus intermedius and Staphylococcus coccus uberis and group B antisera have been reported (16). hyicus subsp. hyicus isolates from veterinary clinical speci- Streptococcus dysgalactiae and Streptococcus uberis are mens. J. Clin. Microbiol. 14:671-673. easily differentiated from Streptococcus agalactiae on the 15. Poutrel, B. 1983. Comparative evaluation of commercial latex basis of hippurate and esculin hydrolysis and the CAMP agglutination and coagglutination reagents for groups B, C, and reaction (4, 12). D mastitis streptococci. Am. J. Vet. Res. 44:490-492. The RMT appears to be an accurate, rapid system for the 16. Shuman, R. D., N. Nord, R. W. Brown, and G. E. Wessman. detection of coagulase-positive staphylococci and Strepto- 1971. Biochemical and serological characteristics of Lancefield coccus agalactiae isolated from bovine IMI. However, the groups E, P, and U streptococci. Cornell Vet. 62:540-568. 17. Watts, J. L., and S. C. Nickerson. 1986. A comparison of the occurrence of coagulase-positive staphylococci other than STAPH-Ident and STAPH-Trac systems to conventional meth- Staphylococcus aureus in veterinary sources requires bio- ods in the identification of staphylococci isolated from bovine chemical testing for species level identification. Addition- udders. Vet. Microbiol. 12:179-187. ally, the RMT is specifically designed and marketed for use 18. Webb, B. J., and C. J. Baker. 1980. Commercial latex aggluti- in mastitis bacteriology; this fact should enhance acceptance nation test for rapid diagnosis of group B streptococcal of the product in veterinary laboratories. in infants. J. Clin. Microbiol. 12:442-444.