Heterogeneity of Mycoplasma iowae determined by restriction enzyme analysis

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1 J Vet Diagn Invest 1: (1989) Heterogeneity of Mycoplasma iowae determined by restriction enzyme analysis Shaohua Zhao, Richard Yamamoto Abstract. Strains of Mycoplasma iowae were homogeneous in some characteristics and heterogeneous in others. Thus, the biochemical tests, immunofluorescence, and protein profiling by sodium dodecyl sulfatepolyacrylamide gel electrophoresis were group-specific tests. However, some minor differences in protein patterns were seen among strains. The growth inhibition test tended to be strain-specific. Hemagglutination titers were very low and unstable for the majority of strains. One strain (RY-65) with a stable high-titer hemagglutinin failed to react in the hemagglutination-inhibition test against immune sera to the reference strains. Restriction endonuclease DNA analyses was the most useful method to differentiate 1 strain from another. Although early studies suggested that Mycoplasma iowae was pathogenic for turkeys, 5,21 only in recent years has there been renewed interest in this agent as a potential pathogen of turkeys. Field and laboratory observations suggest that M. iowae can cause mortality of turkey embryos 6,14,15 and airsacculitis, 5,16,22 stunting and leg deformities, 2 and exudative tenosynovitis in turkeys. 22 Most of the studies have suggested that some isolates of M. iowae are more pathogenic than others 2,5,15,22 In early studies avian mycoplasmas were classified by serotypes (serovars) using capital letters. Serovars from A to S were recognized. 5 As more detailed biochemical and antigenic analyses were conducted, some of the serovars were found to be related and were combined. Serovars I, J, K, N, Q, and R were combined into a single serovar l and later named M. iowae. 9 Therefore, serovars I, J, K, N, Q, and R may now be considered as strains of M. iowae. The purpose of this study was to confirm and expand on the biochemical, serological, and other characteristics of strains of M. iowae. The study emphasized the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and restriction endonuclease analyses to more clearly differentiate strains of this species. Materials and methods Strains of M. iowae. The M. iowae standard reference strains, field isolates, and other avian mycoplasmas used in the study are listed in Table 1. They were grown using standard PPLO base medium + 10% fresh yeast extract + 12% horse serum and standard culture conditions. 8,23 Each culture was purified by a filtration cloning technique. * From the Department of Epidemiology and Preventive Medicine, School of Veterinary Medicine, University of California, Davis, CA Received for publication September 25, Biochemical tests. Each mycoplasma was examined for glucose fermentation, arginine activity, growth in bile salt, and phosphatase activity using standard procedures. 1,8,l7 Serological tests. The serological tests used to identify the isolates were the growth-inhibition (GI), 3,4 indirect fluorescent antibody (FA), 7 hemagglutination (HA), 20,21 and hemagglutination-inhibition (HI) 2o,2l tests. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE was performed as described, l2 with modifications. l0 Restriction endonuclease analyses (REA). The REA was performed as described, l3 with modifications as follows. Mycoplasma cells were washed 3 times in saline, and 500 µg of cell protein was resuspended in 3 ml of 10 mm Tris + 1 mm EDTA, ph 7.4 (TE), and lysed by adding 10% SDS to a final concentration of 1%. RNase A c was added to a final concentration of 25 µg/ml and the mixture was incubated for 30 min at room temperature. Proteinase K c was then added (25 µg/ml) and the mixture was incubated at 37 C for 30 min. Following incubation, the DNA was extracted 3 times with an equal volume of neutralized phenol and once 165 Reference strains* Table 1. I (695) J (693) K (1805) N (PHND-13) Q (L3-10) R (D2497) M. iowae Strains of avian mycoplasmas studied. Field strains RY-65 RY-66 NL-12 Other Mycoplasma species M. gallisepticum (Strain S6) M. synoviae (Strain 1853) * The reference strains were kindly supplied by Dr. K. R. Rhoades. a In an earlier study it was demonstrated that the strain designated K (1805) consisted of a mixture of M. iowae and M. gallinaceum. 23 The M. iowae component of this culture was used in the present study. Isolated from semen of adult turkeys.

2 166 Zhao, Yamamoto Figure 1. SDS-PAGE of proteins from various strains of M. iowae in 10% gel. Lane 1 = molecular mass standards (Kd = kilodalton); 2 = I-695; 3 = J-693; 4 = K-1805; 5 = N-PHND-13; 6 = Q-L3-10; 7 = R-D-2493; 8 = RY-66; 9 = RY-65; 10 = NL-12. with an equal volume of chloroform : isoamyl alcohol (24: 1). The DNA solution was precipitated with 2/3 volume of 5 M ammonium acetate and 2 volumes of absolute ethanol in a dry ice-alcohol bath for 30 min (or at -20 C overnight), pelleted by centrifugation (8,000 rpm for 15 min), resuspended in TE, and again precipitated with 1/10 volume of 3 M sodium acetate and 2 volumes of absolute ethanol. After overnight incubation at -20 C, DNA was collected by centrifugation, resuspended in 1 ml TE, and then centrifuged to equilibrium in a cesium chloride gradient containing 450 µg/ ml ethidium bromide at 110,000 x g for 42 hr at 14 C. The DNA bands were removed from the gradient and ethidium bromide was removed from the DNA by extraction with water-saturated n-butanol. Cesium chloride was removed by dialysis in TE buffer. The DNA concentration was determined by measuring the absorbance of the preparation at 260 nm. Purified DNA were stored at -20 C until used. Restriction enzymes EcoRI d and Hind III d were used to digest the mycoplasma DNA. The concentration of agarose gel was 0.7%. One microgram of DNA from each strain was digested with 20 units of the enzyme (2 µ1) at 37 C for 2 hr. Electrophoresis was performed in Tris-acetate-EDTA (TAE) Table 2. Results of growth inhibition and fluorescent antibody tests. Serologic test* I M. iowae Other Mycoplasma species Reference strains Field strains MG MS J K N Q R RY-65 RY-66 NL-12 (S6) (1853) GI-anti-I GI-anti-Q FA-anti-I * GI-anti-I = growth inhibition using anti-1 immune serum, GI-anti-Q = growth inhibition using anti-q immune serum, FA-anti-I = fluorescent antibody using anti-1 immune serum. The zone of growth inhibition is given in millimeters. MG = M. gallisepticum, MS = M. synoviae.

3 Heterogeneity of Mycoplasma iowae 167 Figure 2. EcoRI (lanes 2-7) and Hind III (lanes 8-13) restriction enzyme cleavage patterns of strains of M. iowae in 0.7% gel. Lane 1 = lambda Hind III-digested DNA; 2 = I-695; 3 = J-693; 4 = K-l 805; 5 = N-PHND-13; 6 = Q-L3-10; 7 = R-D-2497; 8 = I-695; 9 = J-693; 10 = K- 1805; 11 = N-PHND-13; 12 = Q-L-l 3; 13 = R-D-2469; 14 = lambda Hind III-digested DNA. buffer at 50 V and about ma for 3 hr. Gels were stained with ethidium bromide for 20 min, destained 15 min with distilled water, visualized under ultraviolet light, and then photographed. Results Biochemical tests. The 6 standard reference (I, J, K, N, Q, and R) and 3 field (RY-65, RY-66, and NL- 12) strains of M. iowae utilized glucose and arginine, grew in 1% bile salt, and were negative for phosphatase activity. The Mycoplasma gallisepticum and Mycoplasma synoviae cultures that served as controls were positive only for glucose. Serological tests. The results of the GI and FA tests are shown in Table 2. Rabbit anti-m. iowae strains I-695 and Q-L3-10 immune sera were used as antibodies in the GI test. Based on a zone of growth inhibition of less than 2.0 mm as negative, the anti-i- 695 serum inhibited reference strains I, K, N, Q, and R and field strain NL-12. Strain J and field strains RY- 66 and RY-65 were not inhibited. When rabbit anti- Q strain antibody was used, the Q, K, and field strains RY-66 and NL-12 were inhibited. The anti-i-695 immune serum reacted with all reference and field strains in the FA test. The HA activity of the M. iowae strains is shown in Table 3. Hemagglutination titers were observed in reference strains I, K, N, Q, and R and field strains RY- 65, RY-66, and NL-12. However, except for RY-65, the HA activity was unstable and no longer detectable 24 hr after the antigens were prepared and stored at 4 C, -20 C, or -70 C. Although not shown in Table 3, an HI test was performed with antigen prepared from strain RY-65 because the HA activity of this strain was stable for at least 45 days. Turkey immune sera a to each of the M. iowae strains (I, J, K, N, Q, and R) did not react in the HI test using the HA antigen prepared from strain RY-65. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results of SDS-PAGE showed that the protein patterns of the reference strains I, J, K, N, Q, Table 3. Hemagglutination activity of M. iowae strains. Reference strains HA titer Field strains HA titer I 1:8 J Neg K 1:256 N 1:32 Q 1:2 R 1:16 RY-65 1:512 RY-66 1:2 NL-12 1:2

4 Figure 3. EcoRI restriction enzyme cleavage pattern of strains of M. iowae (lanes 2-10) and of M. gallisepticum (lane 11) and M. synoviae (lane 12) in 0.7% gel. Lane 1 = lambda Hind III-digested DNA, 2 = I-695; 3 = J-693; 4 = K-l 805; 5 = N-PHND-13; 6 = Q-L3-10; 7 = R-D-2497; 8 = RY-66; 9 = RY-65; 10 = NL-12; 11 = M. gallisepticum (strain S6); 12 = M. synoviae (strain 1853). and R were very similar, with only minor variations between 42 and 66 kd (Fig. 1, lanes 2-7). Three field strains also showed protein patterns similar to the reference strains, but field strains RY-66 and NL-12 showed 1 heavy band at about the 116-kd level and an additional band between 42 and 66 kd that were absent in the reference strains (Fig. 1, lanes 8-10). Restriction endonuclease analyses. The REA results showed that DNA patterns of the 6 reference strains (I, J, K, N, Q, and R) digested by EcoRI were quite different from each other above the 6.6-kb level, but they shared many common bands, particularly below the 6.6-kb level, where the DNA patterns of all reference strains were almost identical (Fig. 2, lanes 2-7). Similar to the EcoRI results, Hind III-digested DNA s of all reference strains were different from each other (Fig. 2, lanes 8-13). Two field strains (RY-66 and NL-12) showed identical patterns, and were similar but not identical to reference strain N (Fig. 3, lanes 5, 8, and 10). Strain RY-65 was identical to reference strain N (Fig. 3, lanes 5 and 9). Discussion The results reported here confirm previous work that indicated that strains of M. iowae were homogeneous in some characteristics and quite heterogeneous in others. l7 The biochemical tests, particularly growth in medium containing 1% bile, were group-specific. The FA and SDS-PAGE were group-specific, with minor exceptions in the latter. The GI test tended to be strainspecific; therefore, the use of an antiserum prepared from a single strain to identify an unknown culture may yield false negative results. The HA of members of this species generally were of low titer and unstable. One strain, RY-65, did maintain a high HA titer but failed to react in the HI test against sera of turkeys infected with reference strains I, J, K, N, Q, and R. Because the REA pattern of strain RY-65 was similar to reference strain N and the HI is highly strain-specific for M. gallisepticum, 11 positive results were expected with at least the sera from turkeys infected with reference strain N. Similar inconsistent results were ob-

5 Heterogeneity of Mycoplasma iowae 169 served when strains of M. gallisepticum were studied 4. DaMassa AJ: 1983, Identification of avian mycoplasma isolates by REA and HI (Kleven, personal communication). by growth-inhibition. Proc 32nd West Poult Conf, Coop Ext, Earlier, studies 17 University of California, Davis, CA, pp showed that the agglutination test was 5. Dierks RE, Newman JA, Pomeroy BS: 1967, Characterization highly strain-specific for M. iowae. This finding was of avian mycoplasma. NY Acad Sci 143:170-l89. unique in that the agglutination test is considered to 6. Edson RK: 1980, Mycoplasma meleagridis infection of turkeys: be a group-specific test with regard to other avian my- motivation, methods, and predictive tools for eradication. PhD coplasmas. l9 These findings with regard to the HI and Thesis, University of California, Davis, CA. 7. Jones GL, Herbert GA, Cherry WB: 1978, Fluorescent antitions do exist among members of M. iowae. Communicable Disease Center, Atlanta, GA (HEW Pub1 78- agglutination tests indicate that large antigenic variabody techniques and bacteria applications. US Dep HEW, PHS, Restriction endonuclease DNA profiling was the 8364). method of choice for showing variations among strains 8. Jordan FTW: 1983, Recovery and identification of avian mycoplasmas. Diagnostic mycoplasmology. In: Methods in my- of M. iowae. The 6 reference strains (I, J, K, N, Q, and coplasmology, ed. Tully JG, Razin S, vol. 2, pp Academic Press, New York, NY. R) showed unique restriction patterns with either EcoRI or Hind III digestion. In addition, 2 field isolates (RY- 9. Jordan FTW, Erno H, Cottew GS, et al.: 1982, Characterization 66 and NL-12) differed in their patterns from the 6 and taxonomic description of five mycoplasma serovars (serotypes) of avian origin and their elevation to species rank and reference strains and thus could be considered as a new strain of M. iowae. Because some strains of M. iowae further evaluation of the taxonomic status of Mycoplasma synare more pathogenic than others, 2,5,15,22 it may be possible to correlate their restriction patterns with their relative pathogenicity. However, further studies are needed to determine the validity of this hypothesis. Acknowledgements This research was supported in part by funds provided by US Department of Agriculture Special Grant 87-CRSR ; the US Department of Agriculture Animal Health Act of 1977, Public Law (Formula Funds); and the California Turkey Industry Federation. This paper was prepared by the senior author in partial fulfillment of the Master of Preventive Veterinary Medicine (MPVM) degree. We are indebted to Dr. K. R. Rhoades, Ms. Herrad B. Ortmayer, and Drs. Mazhar I. Kahn, A. J. DaMassa, and Patrick J. Blackall for providing valuable advice and/or technical support. oviae. 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