Electrophoretic Analysis of Indian Isolates of Mycoplasma agalactiae and Mycoplasma bovis by SDS-PAGE and Immunoblotting

Mycoplasma agalactiae and Mycoplasma bovis both are responsible for respiratory conditions in sheep and goats. M. agalactiae is a major pathogen of sheep and goats and accounts for almost 90% of outbreaks of contagious agalactia syndrome in goats and almost 100% in sheep. On the basis of clinical signs and cultural, morphological, and biochemical characterization it is almost impossible to differentiate between both the species. Moreover, due to presence of genomic and proteomic similarity most of the time routine diagnostic tests fail to differentiate between them. Hence the present study was conducted to find out the protein profile of isolates of both the species by SDS-PAGE and to find out the cross-reacting as well as differentiating immunogenic proteins by Immunoblotting, which can be of immunoprophylactic as well as diagnostic values. The study revealed 6-7 major immunogenic cross-reactive proteins with the presence of two important non-cross-reacting species specific polypeptides particularly 25.50 and 24.54 kDa in M. agalactiae and M. bovis, respectively, that might be of diagnostic values.


Introduction
Mycoplasmas are the smallest, simplest, and self-replicating organisms with the minimum set of organelles required for the growth and replication. Till now more than one hundred species of mycoplasmas [1,2], widespread in nature as pathogens of human, animals, birds, fishes, reptiles, arthropods, and plants, have been recognized [1][2][3]. Out of these species Mycoplasma agalactiae and Mycoplasma bovis both are important pathogens of small ruminants [3][4][5]. M. agalactiae accounts for almost 90% of outbreaks of contagious agalactia syndrome in goats [6] and almost 100% in sheep [4,7]. Contagious agalactia causes high economic losses due to loss in milk yield and kids/lambs because of abortions, neonatal deaths, and loss of animals [4,8,9]. In contrast to M. agalactiae, M. bovis is supposed to be a major pathogen in calf pneumonia complex and the isolation rates of it have been reported in the range of 23 to 35% [2,10,11]; however recently it has been observed in the respiratory infection of sheep with significant mortality [3]. M. agalactiae and M. bovis are two closely related species [11,12] and were earlier regarded As subspecies of the same species [11]. Moreover, it is difficult to differentiate M. agalactiae and M. bovis on the basis of morphological, biochemical, and traditionally used serological tests due to many common antigens and receptors on cell surface for antibodies [5,[11][12][13]. Hence, the present study was designed to identify the non-cross-reactive as well as cross-reacting protein antigens of both the species as a prospective antigen for their detection through serological tests.  [10]. For the colony characteristics solid media were prepared by the addition of 1.2% Bacto Agar (Difco) in respective liquid medium [10]. (WCA). WCA were prepared as per earlier prescribed method [14] with slight modifications. Actively growing 2 to 5 mL of M. agalactiae and M. bovis culture was inoculated in 10 mL liquid medium and incubated at 37 ∘ C for 48 hours. The growth was confirmed by the change in pH (change of color red to yellowish orange). This growth was subsequently transferred to larger volume of media and incubated for 4 to 5 days to obtain sufficient growth. Simultaneously the growth was checked for purity on Robertson Cooked Meat (RCM), Sabouraud's Dextrose Agar (SDA), and Blood Agar media. The growth was centrifuged at 10,000 rpm for 25 minutes using a refrigerated centrifuge (Sorvell, RC-5C). The pellets were washed thrice with PBS (pH 7.2) and finally suspended in 10 mL of PBS (pH 7.2). The protein concentrations of WCA were estimated by the standard method [15]. (SSA). Sonicated antigens of all the isolates were prepared from the whole cell antigen by the method described earlier [16] with slight modification. For the preparation of SSA from WCA, the whole cell antigens were diluted in PBS (pH 7.2) and sonicated with MSE-Soniprep 150 at 10 microns by applying 10 jerks, each of 90 seconds with the interval of 30 seconds, on ice. Thus prepared sonicated antigens were further centrifuged at 13,000 rpm for 30 minutes at 4 ∘ C (Biofuge, Fresco). The supernatant was separated carefully and was used as SSA. The protein concentration of SSA was estimated by the previously described procedure [15].

Production of Hyperimmune Sera.
To assess the immunogenicity of WCA and SSA polypeptides through Immunoblotting, the polyclonal hyperimmune serum was raised against M. agalactiae (RPNS 216) and M. bovis (NC 317) in white New Zealand rabbits (obtained from LAR, IVRI, Izatnagar), according to the standard protocol [17]. These antisera were tested by slide agglutination test [18] and titer was estimated by indirect haemagglutination (IHA) [19] with slight modification [20]. Sera were finally filtered through 0.20 m filter (Sartorius) and stored at −20 ∘ C for further use.  bovis (NC 317), under denaturating, reducing conditions, was performed by the method of Lammli [21] with recommended modifications [14]. Electrophoresis was carried out using 12.5% separating and 4% stacking gel at 50 volts for 1 h and at 100-110 volts afterwards till the end of run. The determination of molecular weights was based on the distance migrated by the polypeptides in the gels in comparison to the distance migrated by polypeptide markers of known molecular weights (Biored) [22].

2.7.
Immunoblotting. The WCA and SSA of all the isolates separated on 12.5% (w/v) SDS-PAGE slabs [21] were transferred electrophoretically on nitrocellulose membrane papers (NCP) (Sartorius). Then these membranes were crossblotted with the hyperimmune serum raised against M. agalactiae (RPNS 216) and M. bovis (NC 317) separately by the previously described method [23] to find out cross-reactive polypeptides.

Results
The results of SDS-PAGE profiles of the two isolates of M. agalactiae and one of M. bovis have been presented in  (Figures 1 and 2). Moreover, the isolate of M. bovis was having exclusive polypeptide of 85.10, 51.29, 40.74, and 24.54 kDa along with 79.43 kDa in SSA only (Figures 1 and 2).
When Immunoblotting was performed with the hyperimmune serum against M. agalactiae RPNS 216 of M. bovis NC 317 the immunoblots revealed almost similar patterns of immunogenic polypeptides with the difference of some immunogenic polypeptides. As usual isolates revealed more immunogenic polypeptides with homologous hyperimmune serum, namely, 13 each in M. agalactiae isolates ( Figures  3 and 5) and 13 and 14 in WCA and SSA of M. bovis (Figures 5 and 6). The numbers of cross-reactive antigens were comparatively lesser with 5 numbers of polypeptides in M. agalactiae isolates (Figures 5 and 6) and 7 in M. bovis isolate (Figures 3 and 4). The immunoblot profiles of both the isolates of M. agalactiae were identical with both homologous and heterologous hyperimmune serum (Figures  4 and 6). The immunogenic polypeptides of molecular  (Figures 3, 4, 5, and 6).

Discussion
The immunologic cross-reactivity among members of the mycoplasmatales is well documented and tests, namely, immunodiffusion, agglutination, two-dimensional immunoelectrophoresis, counter current immunoelectrophoresis, ELISA, immunobinding assay, polyacrylamide gel electrophoresis [1,5,11,12,24], and variety of other methods with the use of polyclonal serum, failed due to many common antigens and receptors on cell surface for antibodies [1,5,13]. The results of the present study are also in the concurrence of previous findings revealing many cross-reactive polypeptides [5,11,12,24]. Moreover 52 and 60 common polypeptides were observed in almost similar range and pattern in M. agalactiae [25] and M. bovis [26,27] with the presence of 5 to 7 major proteins in the range of 90 to 20 kDa. Similarly almost identical patterns with minor differences were recorded in M. agalactiae and M. bovis with one-third polypeptides of identical molecular masses [12]. However, in an earlier report, no common protein band but areas of homology did exist among different mycoplasmal species including M. agalactiae and M. bovis [13].
Based on previous information identification of species specific immunogenic proteins is always of diagnostic as well as prophylactic values. For that Immunoblotting, a sensitive and specific confirmatory test for the identification and isolation of immunogenic proteins was performed with homo-and heterologous hyperimmune serum [23]. The presence of 5-7 major immunogenic proteins (Figures 3, 4  5, and 6) obtained during the Immunoblotting might be of diagnostic as well as protective values. These findings are in the concurrence of many earlier reports in various species of mycoplasmas M. ovipneumoniae [26], M. mycoides cluster [28], M. hominis [29], M. hyopneumoniae and M. flocculare [22], M. agalactiae [12,14,30], and M. bovis [12,31,32]. As protein patterns obtained by SDS-PAGE provide an indirect measure of the coding capacity of mycoplasmal genome and comparison sought to provide an approximate reflection of probable genomic capacity. However, each band may consist of many proteins of similar molecular weight as separation using SDS is dependent on molecular weight only [33]. Hence the presence of six to seven major immunogenic polypeptides in all the three isolates of both the species particularly the polypeptides of 60.25 and 28.84 kDa can be of immunoprophylactic use against both the species. Similarly the presence of species specific polypeptides particularly 25.50 and 24.54 kDa in M. agalactiae and M. bovis, respectively, might be of diagnostic values. However to authenticate the findings studies with more number of isolates from different geographical region are required.

Conclusions
It can be concluded from the present study that both the isolates of M. agalactiae have almost similar protein profile and presence of identical immunogenic polypeptides. Moreover, protein profile of M. agalactiae and M. bovis are almost similar with the presence of many cross-reactive major polypeptides. These can be differentiated by the presence of certain species specific immunogenic proteins or by using the monoclonal antibodies raised against those proteins in the diagnostics. However, a large number of isolates are required to be examined before these conclusions are put to practice.
Veterinary Medicine International 5

Conflict of Interests
There is no conflict of interests among the authors.