Abstract
Monoclonal antibody (MAb) PK-2 inhibited the in vitro growth of nine Mycoplasma mycoides subsp. mycoides small-colony strains. In contrast to the results with polyclonal antisera, growth inhibition by MAb PK-2 was specific for M. mycoides subsp. mycoides small-colony strains and constituted a reliable means of distinguishing them from other mycoplasmas.
Mycoplasma mycoides subsp. mycoides small-colony (MmmSC) strains cause contagious bovine pleuropneumonia (CBPP) in cattle and buffalo (8, 17), which is one of the most serious diseases of cattle in some parts of the world. The disease, once widespread, has been eradicated from many countries and is now mainly confined to the African continent, affecting most of the countries south of the Sahara and north of South Africa (14). However, in recent years there have been outbreaks of the disease in Spain, Portugal, and Italy (14). It is thought to exist in the Middle East and Asia (14).
Differentiation of MmmSC strains by serological and biochemical means has been difficult. This difficulty is caused by immunological cross-reactions and biochemical similarities of MmmSC strains with M. mycoides subsp. mycoides large-colony (MmmLC) strains from cattle (4, 5, 16), and even other animal Mycoplasma strains, including M. mycoides subsp. capri. Even growth inhibition with polyclonal antibodies (6) and indirect immunofluorescence (7), assays which are considered species specific and which are used to classify Mycoplasma isolates (9), cannot differentiate MmmSC strains from MmmLC strains. The situation has been made even more complex by the isolation of MmmSC variants from sheep and goats (1). There is no disease currently associated with MmmLC strains from cattle; however, MmmLC strains cause pleuropneumonia and associated arthritis in goats. MmmLC strains have a worldwide distribution and have been isolated in the United States, Europe, Australia, India, and Africa (5). It is therefore important to distinguish MmmSC strains from MmmLC strains, particularly in regions currently free of CBPP. This communication describes a growth-inhibiting monoclonal antibody (MAb) which makes the necessary differentiation between MmmSC and MmmLC strains from cattle and between MmmSC strains and several other Mycoplasma species.
MAbs.
Production and testing of MAbs for growth-inhibiting activity to MmmSC strains and initial characterization of the epitope recognized by the MAbs have been described (12). All the MAbs were of the immunoglobulin M isotype and recognized a carbohydrate epitope. One MAb, PK-2, was selected for further evaluation of its growth-inhibiting effect on different Mycoplasma species and strains.
Strains of Mycoplasma and their source.
The strains were from three sources: the National Veterinary Research Center, Muguga (NVRC-M), Kenya; the National Veterinary Research Centre, Kabete (NVRC-K), Kenya; and R. H. Leach, National Collection of Type Cultures (NCTC), Corrindale, England. The MmmSC strains (SC group) included T419 (NVRC-M), T1M44 (T1 vaccine strain) (NVRC-M), B467/92 Kabete (NVRC-K), Gladysdale (NCTC), Poumarat 4813 (NCTC), B613/87 (NCTC), B101/93 (NVRC-M), Oremit (NVRC-M), and U716 (NVRC-M). The MmmLC strains (LC group) included VR1/3172 LB (NCTC), 78/441 LC (NCTC), and Y goat M207/86 (NCTC). The MAb was also tested against other members of the M. mycoides cluster, which included the following strains: M. mycoides subsp. capri (capri group) Pendik (NCTC) and BQT (NCTC); Mycoplasma capricolum subsp. capricolum (capricolum group) M4528/76 (NCTC), 74/3220 (NCTC), ZT 14 (NCTC), and 4528 (NCTC); bovine serogroup 7 (BSG7 group) strains Poumarat BSG7 (NCTC), L2917 BSG7 (NCTC), and PG 50 (NVRC-K); and M. capricolum subsp. capripneumoniae (capripneumoniae group) G22 (NVRC-K), G94/83 (NVRC-K), and G280/80 (NVRC-K).
In vitro growth inhibition.
Before use in growth inhibition assays, MAb PK-2 was isolated from hybridoma culture supernatants by gel filtration (19). The isolated PK-2 (2.0 mg/ml) was tested for growth inhibition of 24 mycoplasmas belonging to the M. mycoides cluster as described earlier (19). Briefly, 1 ml of log phase broth culture of test Mycoplasma organisms was spread evenly on Newing's tryptose agar plates and allowed to dry for 10 min. Wells were punched into the agar, and 50 μl of MAb PK-2 was added and allowed to seep into the agar. Plates were incubated at 37°C for 3 to 5 days until the colonies were visible. The plates were examined with a low-power stereo microscope for zones of inhibition. MAb PK-2 caused growth inhibition of nine MmmSC strains (Table 1), and these strains were not inhibited by an unrelated immunoglobulin M control MAb (WM-25) which reacts with a carbohydrate epitope and inhibits the growth of M. capricolum subsp. capripneumoniae (19, 20). In contrast, MAb PK-2 did not inhibit growth of three MmmLC strains from cattle. Neither were 15 other Mycoplasma strains inhibited with MAb PK-2 even at Mycoplasma concentrations 102- to 103-fold lower than the concentration used for MmmSC isolates (Table 1). That MAb PK-2 did not inhibit the growth of non-MmmSC strains suggests that these Mycoplasma strains do not possess the epitope recognized by PK-2 or that, if they do, the epitope either is not exposed on the live mycoplasma or has changed to the extent that PK-2 binding does not inhibit growth. The three MmmLC strains and the two M. mycoides subsp. capri strains included in Table 1 have been notoriously difficult to differentiate from MmmSC strains by other assays (10, 18). All of the Mycoplasma strains that were not inhibited by MAb PK-2 in Table 1 were inhibited by either polyclonal antiserum to each of the five Mycoplasma groups (SC, LC, capri, capricolum, and BSG7) or a MAb to the M. capricolum subsp. capripneumoniae group (19).
TABLE 1.
Growth inhibition of MmmSC strains and other mycoplasmas with monoclonal and polyclonal antibodies to MmmSC strains
| Mycoplasma strain | Growth range (CFU/ml) | No. of isolates with growth inhibition/no. tested
|
|||
|---|---|---|---|---|---|
| MAb PK-2 | Control MAb | Polyclonal antiserum to:
|
|||
| MmmLC strains | MmmSC strains | ||||
| MmmSC | 4.5 × 107–2.5 × 109 | 9/9 | 0/9 | 9/9 | 9/9 |
| MmmLC | 1.5 × 102–2.5 × 103 | 0/3 | 0/3 | 3/3 | 1/3 |
| Othersa | 3.6 × 103–1.7 × 104 | 0/12 | 2/12 | 0/12 | 0/12 |
The non-MmmSC and non-MmmLC strains were as follows: M. mycoides subsp. capri Pendik (NCTC) and BQT (NCTC); M. capricolum subsp. capricolum M4528/76 (NCTC), 74/3220 (NCTC), ZT 14 (NCTC), and 4528 (NCTC); bovine serogroup 7 strains Poumarat BSG7 (NCTC), L2917 BSG7 (NCTC), and PG 50 (NVRC-K); and M. capricolum subsp. capripneumoniae G22 (NVRC-K), G94/83 (NVRC-K), and G280/80 (NVRC-K). The growth of these mycoplasmas was inhibited by the homologous antiserum.
Polyclonal antiserum to the MmmSC strain group caused growth inhibition of MmmSC strains and the Y goat strain of the MmmLC strain group while polyclonal antiserum to MmmLC strains caused growth inhibition of MmmLC strains and of MmmSC strains to a similar level. This confirms the problem of differentiating MmmSC strains from MmmLC strains by growth inhibition using polyclonal antiserum.
The mechanisms by which antibody inhibits the growth of Mycoplasma organisms are not clearly understood. It has been proposed that growth-inhibiting antibodies are directed against exposed surface membrane proteins (13). MmmSC cells are enclosed in a capsule of carbohydrate (11) composed of galactan (2, 3). It is not surprising, therefore, that attempts to make protein-reactive growth-inhibiting MAbs to MmmSC strains have not been successful. The first growth-inhibiting MAbs to MmmSC strains that were reported reacted with carbohydrate epitopes, as demonstrated by periodate sensitivity and proteinase K insensitivity of the recognized epitopes in Western blots (12). Recently, other growth inhibition of MmmSC strains by a pool of MAbs was reported, but the epitopes recognized were not described (18). The suggested involvement of galactan produced by MmmSC strains in the pathogenesis of CBPP (15) and the data demonstrating that growth of both MmmSC strains and M. capricolum subsp. capripneumoniae can be inhibited by MAb-recognizing carbohydrate epitopes (12, 19, 20) suggest that immune responses to carbohydrate epitopes will protect against these pleuropneumonia-causing Mycoplasma organisms. In addition, growth-inhibiting antibody PK-2 provides a reagent to evaluate the relationship between in vitro growth inhibition and protective immune responses.
Conclusions.
MAb PK-2 caused growth inhibition of nine MmmSC strains and did not inhibit either three MmmLC strains from cattle or 15 other strains belonging to the M. mycoides cluster. Use of MAb PK-2 in growth inhibition resolved the cross-reactions observed in this study, which were growth inhibition of MmmSC strains with polyclonal antibodies to MmmLC strains. These data suggest that growth inhibition by MAb PK-2 could be used to identify MmmSC isolates and differentiate them from MmmLC isolates from cattle. Although molecular techniques can be used to make this differentiation (10), growth inhibition with MAb PK-2 is a more suitable test for most laboratories where CBPP is a concern.
Acknowledgments
This research was conducted as part of the United States Agency for International Development Title XII Small Ruminant Collaborative Research Support Program under grant AID/DSAN/XII-G-0049 in a collaboration between the Kenya Agricultural Research Institute and Washington State University.
REFERENCES
- 1.Brando E. Isolation and identification of Mycoplasma mycoides subsp. mycoides SC strains in sheep and goats. Vet Rec. 1995;136:98–99. doi: 10.1136/vr.136.4.98. [DOI] [PubMed] [Google Scholar]
- 2.Butterry S H. Hapten inhibition of the reaction between Mycoplasma mycoides polysaccharide and bovine antisera. Immunochemistry. 1970;7:305–310. doi: 10.1016/0019-2791(70)90168-0. [DOI] [PubMed] [Google Scholar]
- 3.Butterry S H, Placket P. A specific polysaccharide from M. mycoides. J Gen Microbiol. 1960;23:357–368. doi: 10.1099/00221287-23-2-357. [DOI] [PubMed] [Google Scholar]
- 4.Cottew G S, Breard A, Da Massa A J, Erno H, Leach R H, Lefevre P C, Rodwell A W, Smith G R. Taxonomy of the Mycoplasma mycoides cluster from cattle. Isr J Med Sci. 1987;23:632–635. [PubMed] [Google Scholar]
- 5.Cottew G S, Yeats F R. Subdivision of Mycoplasma mycoides subsp. mycoides from cattle and goats into two types. Aust Vet J. 1978;54:293–296. doi: 10.1111/j.1751-0813.1978.tb02462.x. [DOI] [PubMed] [Google Scholar]
- 6.Davis G, Read W C S. A modification of the growth-inhibition test and its use for detecting Mycoplasma mycoides var. mycoides. J Hyg. 1968;66:319–324. doi: 10.1017/s002217240004119x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Del Giudice R A, Robillard N F, Carski T R. Immunofluorescence identification of Mycoplasma on agar by use of incident illumination. J Bacteriol. 1967;93:1205–1209. doi: 10.1128/jb.93.4.1205-1209.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Edward D G, Freundt E A. The classification and nomenclature of organisms of the pleuropneumonia group. J Gen Microbiol. 1956;14:197–207. doi: 10.1099/00221287-14-1-197. [DOI] [PubMed] [Google Scholar]
- 9.Freundt E A, Edward D G H. Classification and taxonomy. In: Barile M F, Razin S, editors. The mycoplasmas. Vol. 1. New York, N.Y: Academic Press, Inc.; 1979. pp. 1–41. [Google Scholar]
- 10.Hotzel H, Sachse K, Pfutzner H. A PCR scheme for differentiation of organisms belonging to the Mycoplasma mycoides cluster. Vet Microbiol. 1996;49:31–43. doi: 10.1016/0378-1135(95)00176-x. [DOI] [PubMed] [Google Scholar]
- 11.Howard C J, Gourlay R N. An electron microscopic examination of certain bovine mycoplasmas stained with ruthenium red and demonstration of a capsule on Mycoplasma dispar. J Gen Microbiol. 1974;83:393–398. doi: 10.1099/00221287-83-2-393. [DOI] [PubMed] [Google Scholar]
- 12.Kiarie M K, Rurangirwa F R, Perryman L E, Jasmer D P, McGuire T C. Monoclonal antibodies to surface-exposed proteins of Mycoplasma mycoides subsp. mycoides (small-colony strain), which causes contagious bovine pleuropneumonia. Clin Diagn Lab Immunol. 1996;6:746–752. doi: 10.1128/cdli.3.6.746-752.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Levisohn S, Razin S. Isolation, ultrastructure and antigenicity of Mycoplasma gallisepticum membranes. J Hyg Camb. 1932;71:725–737. doi: 10.1017/s0022172400022981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Nicholas R A, Bashiruddin J B. Mycoplasma mycoides subsp. mycoides (small colony variant): the agent of contagious bovine pleuropneumonia and a member of the “Mycoplasma mycoides cluster.”. J Comp Pathol. 1995;113:1–27. doi: 10.1016/s0021-9975(05)80065-9. [DOI] [PubMed] [Google Scholar]
- 15.Nunes-Pwtisca J L, Costa Durao J F, Lage M, Goncalves J M, Azevedo Ramos M M, Baptista R, Galo A, Monteiro M, Caiado J, Silva E R, Mota J F, Afonso A. Pathogenesis and pathological features of contagious bovine pleuropneumonia. In: Regella J, editor. Contagious bovine pleuropneumonia. Luxembourg, Luxembourg: Commission of the European Communities; 1990. pp. 2–6. [Google Scholar]
- 16.Perreau P, Bind J L. Infection naturelle du veau par Mycoplasma mycoides subsp. mycoides (biotype chevre) Bull Acad Vet Fr. 1981;52:575–581. [Google Scholar]
- 17.Provost A, Perreau P, Breard A, Le Golf C, Martel J L, Cottew G S. Contagious bovine pleuropneumonia. Rev Sci Tech Off Int Epizoot. 1987;6:625–680. doi: 10.20506/rst.6.3.306. [DOI] [PubMed] [Google Scholar]
- 18.Rodriguez F, Fernandez A, Ball H J. Detection of Mycoplasma mycoides subspecies mycoides by growth-inhibition using monoclonal antibodies. Res Vet Sci. 1997;63:91–92. doi: 10.1016/s0034-5288(97)90165-x. [DOI] [PubMed] [Google Scholar]
- 19.Rurangirwa F R, McGuire T C, Musoke A J, Kibor A. Differentiation of F38 mycoplasmas causing contagious caprine pleuropneumonia with a growth-inhibiting monoclonal antibody. Infect Immun. 1987;55:3219–3220. doi: 10.1128/iai.55.12.3219-3220.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Rurangirwa F R, Wambugu A N, Kihara S M, McGuire T C. A Mycoplasma strain F38 growth-inhibiting monoclonal antibody (WM-25) identifies an epitope on a surface-exposed polysaccharide antigen. Infect Immun. 1995;63:1415–1420. doi: 10.1128/iai.63.4.1415-1420.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]