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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).

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.