Abstract

We have analyzed antigenic variation of seven M. agalactiae wild strains using different sera from naturally infected sheep. Only 30 day sera recognized all surface proteins and inhibited the growth of mycoplasmas. Furthermore, we have observed that two strongly immunogenic proteins: 55 and 35 kDa were digested using 500 μg/ml of trypsin. These two bands are immunoprecipitated together with four other proteins but only the 35 kDa protein is recognized by eluted antibodies.

Introduction

Mycoplasma agalactiae is the agent of contagious agalactia (C.A.) involving primary mastitis and secondary arthritis and keratoconjunctivitis in sheep and goats [1, 2]. The impressive diffusion of this disease in the entire Mediterranean basin is due to primitive herding practices, to the inefficiency of antimicrobial therapies and to the adoption of very few prophylactic measures. Control and eradication of C.A. can be obtained through better diagnostic tests and through a more efficient vaccine. Some DNA probes [3, 4] and PCR assays [5–7] are already in use for its identification and diagnosis but very little is known on the pathogenetic mechanisms of M. agalactiae. In general, the detailed analysis of mycoplasmal components has led to a better understanding of the course of infection, disease development and the immunological responses in infected animals [8, 9]. Interest in M. agalactiae antigens, from the veterinary point of view, has been mainly directed toward developing better serological diagnostic tests. Furthermore, the isolation of specific antigens may also lead to the development of new vaccines, based on well defined protective antigens.

The Mycoplasma genus is characterized by the absence of a cell wall and the presence of a trilaminar membrane which mediates all interactions with the environment. In particular, membrane proteins are involved in adhesion to host cells surface receptors and induction of immunoresponse [10, 11].

In recent papers genomic and antigenic variability of French and Italian clones of M. agalactiae have been compared using pulsed field gel electrophoresis and immunoblotting analysis [12, 13]. On the other hand, Bergonier et al. [14], using species-specific monoclonal antibodies, has demonstrated that M. agalactiae possesses a capacity for phenotypic diversification of its surface antigens.

The aim of our research is to identify and characterize the principal antigens of M. agalactiae utilizing sera from infected sheep at different stages of infection.

Materials and methods

Mycoplasma isolates and growth conditions

M. agalactiae strains and their origin are listed in Table 1. All strains were cloned [15], identified by indirect immunofluorescence [16] and by PCR assay [6]. Mycoplasmas were grown at 37°C in modified Hayflick medium containing 10% equine serum. Cells were harvested from logarithmic-phase broth cultures by centrifugation at 20 000×g for 30 min and washed twice with phosphate buffered saline (PBS, 0.1 M phosphate, 0.33 M NaCl, pH 7.4). The final pellet was resuspended in one-tenth of the original culture volume and used immediately or stored at −80°C. Protein concentration of washed whole cell suspension was determined using the DC protein assay reagent (Bio-Rad, Richmond, CA) according to manufacturer's protocol.

1

Mycoplasma agalactiae isolates analyzed in this study

Isolate Geographic origin Clinical condition Year of isolation 
PG2, type strain (bga-Jena)    
32/M Sardinia (Sassari) mastitis, arthritis, keratitis 1991 
25/Or Sardinia (Oristano) mastitis, arthritis, keratitis 1991 
124/M Sardinia (Sassari) mastitis 1995 
248/Or Sardinia (Oristano) mastitis 1996 
2894/Nu Sardinia (Nuoro) mastitis 1996 
2697/Nu Sardinia (Nuoro) mastitis 1996 
233/M Sardinia (Sassari) mastitis 1996 
Isolate Geographic origin Clinical condition Year of isolation 
PG2, type strain (bga-Jena)    
32/M Sardinia (Sassari) mastitis, arthritis, keratitis 1991 
25/Or Sardinia (Oristano) mastitis, arthritis, keratitis 1991 
124/M Sardinia (Sassari) mastitis 1995 
248/Or Sardinia (Oristano) mastitis 1996 
2894/Nu Sardinia (Nuoro) mastitis 1996 
2697/Nu Sardinia (Nuoro) mastitis 1996 
233/M Sardinia (Sassari) mastitis 1996 

Antisera

We used 90 sera collected from infected sheep from different areas of Sardinia. Most sera came from sheep who had been sick for a while or had had a relapse. For five sheep, however, date of infection was known so, samples were collected on the first, fifteenth and thirtieth day after the appearance of the first clinical symptoms (i.e. mammary glands enlargement and altered milk production).

Growth inhibition test

The test was performed as described by Clyde [17] using 25 μl of each serum from all 90 infected sheep. Hayflick agar plates were incubated for 7 days at 37°C in a humidified chamber and observed daily with a Zeiss Axiovert 35 inverted-light microscope. The zones of growth inhibition were measured on the third and on the fifth day and confirmed on the seventh day.

Triton X-114 phase partitioning

Extraction of integral membrane proteins was carried out through TX-114 phase partitioning as described by Bordier [18] and adapted with some modifications [19]. Extracted proteins were processed for SDS-PAGE and immunoblotting.

Trypsin treatment of intact mycoplasmas

Intact mycoplasmas from fresh logarithmic-phase broth cultures were washed and resuspended in PBS pH 7.4 to a final concentration of 2 mg/ml. Samples were treated with 25, 50, 100, 250 and 500 μg/ml trypsin (GibcoBRL) and incubated at 37°C for 15, 30 and 45 min. Reactions were stopped adding 200 μl fetal bovine serum (GibcoBRL). After further washings with PBS, mycoplasmas were cultured on Hayflick agar and then processed for SDS-PAGE and immunoblotting. Control suspensions were similarly treated omitting the trypsin step.

Immunoprecipitation

Protein A preparation: 400 μg Affi-Gel Protein A agarose (Bio-Rad) was washed several times with PBS containing 1% (v/v) Triton X-100 (IBI, New Haven, CT). After decantation, protein A was incubated with PBS-1% Triton X-100 at 4°C with gentle rocking for at least 6 h. After centrifugation at 600×g for 1 min, Protein A was resuspended in 300 μl of PBS-1% Triton X-100 and used immediately as described below.

Immunocomplex: samples of whole cell proteins (300 μg) from fresh log-phase broth cultures were incubated with 1 ml of pooled 30 days infected sheep sera (diluted 1:1 in PBS pH 7.4) for 2 h at 4°C with gentle agitation. After centrifugation at 20 000×g for 15 min at 4°C, pellets were washed four times with PBS pH 7.4. The immunocomplex was precipitated by adding Affi-Gel Protein A agarose in PBS-1% Triton X-100 (as described above) and incubated overnight at 4°C. After several washings with PBS-1% Triton X-100, the precipitated antigens were resuspended in sample buffer (2% SDS, 5% 2-β-mercaptoethanol, 10% glycerol, 62.5 mM Tris (pH 6.8)), boiled for 5 min and subsequently processed for SDS-PAGE and immunoblotting as described below.

The same procedure was followed for mycoplasmas treated in advance with 500 μg/ml Trypsin for 15, 30 and 45 min. Negative controls were similarly treated omitting the serum exposure.

Elution of antibodies from intact mycoplasmas

Aliquots of whole cell proteins (300 μg) were incubated with pooled 30 day sera as described above. After four washings, pellets were resuspended in 200 mM ice-cold glycine pH 3.0 for 5 min, pipetting up and down to aid the separation from antibodies. After centrifugation at 20 000×g for 5 min at 4°C, the supernatant was neutralized with 1 M Imidazole (Sigma Chemical Co) pH 7.4 and used immediately. The same procedure was followed with mycoplasmas previously incubated for 15, 30 and 45 min with 500 μg/ml trypsin.

SDS-PAGE and immunoblotting

Gel electrophoresis of proteins was carried out on 8% or on 12% (w/v) polyacrylamide gel containing 0.1% SDS, according to Laemmli [20]. The apparent molecular mass of mycoplasma proteins was determined by the use of markers (Kaleidoscope prestained standards, Bio-Rad).

Electrophoresed proteins were transferred to nitrocellulose membranes (0.45 μm pore size, Sigma) in a Trans-Blot-semidry-apparatus (Bio-Rad) as described by the manufacturer. Blots were incubated 2 h at r.t. in PBS-2% skim milk (Difco, Detroit, MI, USA) and then incubated 1 h at 37°C in M. agalactiae positive antiserum. After several washings with PBS-2% skim milk, blots were incubated for at least 1 h at 37°C in peroxidase-conjugated anti-sheep (heavy and light chain specific, Kirkegaard and Perry, Gaithersburg, MD). After five more washes, blots were developed with 4-chloro-1-naphthol/H2O2. Color reaction was stopped by washing blots in water.

Results and discussion

Antigenic analysis during infection

In order to study antigenic variation of M. agalactiae during natural infection, we used sera isolated from just infected sheep (collected in the first, fifteenth and thirtieth day of appearance of the first clinical symptoms) and from relapsed sheep. Western immunoblot analysis was performed using these sera and pooled M. agalactiae strains as shown in Fig. 1. The first day serum (Fig. 1, lane B) has antibodies against 80 and 55 kDa proteins. As the infection proceeds, sera react with more and more protein bands. Thirtieth day serum reacts with the greatest number of immunogenic proteins, even more than serum from relapsed sheep (Fig. 1, lane D).

1

Immunoblots showing antigenic profiles of M. agalactiae as natural infection progresses. Lane A: negative control. Lane B: one day sera after the appearance of clinical symptoms. Lane C: fifteen day sera. Lane D: thirty day sera. lane E: sera from relapsed sheep. Proteins’ relative molecular sizes (kilodaltons) appear at left.

1

Immunoblots showing antigenic profiles of M. agalactiae as natural infection progresses. Lane A: negative control. Lane B: one day sera after the appearance of clinical symptoms. Lane C: fifteen day sera. Lane D: thirty day sera. lane E: sera from relapsed sheep. Proteins’ relative molecular sizes (kilodaltons) appear at left.

First, fifteenth and thirtieth day sera and those from relapsed sheep have been used for the growth inhibition test on Hayflick agar (Fig. 2). First day serum does not inhibit mycoplasmas growth. A slight inhibition is obtained with the fifteenth day serum while thirtieth day serum inhibits growth even more than sera from relapsed sheep. Moreover thirtieth day and relapsed sheep sera inhibit mycoplasmas growth at all dilutions tested (107, 106, 105 and 104 CCU/ml). The fact that first and fifteenth day sera do not inhibit mycoplasmas growth could be caused by low serum titer given the test relative insensitivity [15], or to the fact that these membrane antigens cannot produce neutralizing antibodies during early infection.

2

Growth inhibition test using M. agalactiae culture and one day sera (A), fifteen day sera (B), thirty day sera (C) and relapsed sheep sera (D).

2

Growth inhibition test using M. agalactiae culture and one day sera (A), fifteen day sera (B), thirty day sera (C) and relapsed sheep sera (D).

Analysis of TX-114 phase fractioned mycoplasmas by SDS-PAGE followed by immunoblotting with pooled antisera from 30 day naturally infected sheep was compared with immunoblots of whole cell proteins (Fig. 3). The lack of any difference in bands number and size between lane A and lane D, suggests that the most common M. agalactiae antigens are integral membrane proteins.

3

Immunoblots of whole cell proteins (A), TX-114 phase proteins (D), aqueous phase proteins (B) and TX-114 insoluble material (C) from M. agalactiae conjugated with thirty day sera. Proteins’ relative molecular sizes appear at left.

3

Immunoblots of whole cell proteins (A), TX-114 phase proteins (D), aqueous phase proteins (B) and TX-114 insoluble material (C) from M. agalactiae conjugated with thirty day sera. Proteins’ relative molecular sizes appear at left.

Sensitivity of surface proteins to trypsin treatment

In order to understand if antibodies reactive proteins contain trypsin sensitive sites, we treated mycoplasmas with increasing concentrations of trypsin for different lengths of time. After 15 min treatments with 25, 50 or 100 μg/ml trypsin we did not detect any alteration in the protein electrophoresis pattern. Only using 250 and 500 μg/ml trypsin for 15 min we noted some modifications. Therefore, we digested all seven wild strains and type strain with 500 μg/ml of trypsin for 15, 30 and 45 min (Fig. 4). After 15 min of trypsin digestion, the band corresponding to the 55 kDa protein decreased in intensity or completely disappeared. On the contrary, the 50 kDa protein band, remained constant and in some strains it actually increased in intensity. All the proteins in the range 38–45 kDa, disappeared altogether as do those in the range 18–32 kDa. The 32 kDa band, very strong in undigested strains, decreased in intensity after 15 min trypsinizing treatment and a 35 kDa band appeared (Fig. 4, panel B). After 30–45 min of trypsin treatment proteins of apparent molecular mass 120, 80, 70, 50, 32, 30 and 18 Kda remained constant. The 32 KDa protein was completely trypsinized after 45 min in strain 32/M (Fig. 4, panel D, lane 1) and partially in strain 233/M (Fig. 4, panel D, lane 7). Reference strain PG2 not trypsinized has a pattern different from wild strains (Fig. 4, panel A, lane 8) showing a very immunogenic specific 40 kDa protein. On the contrary with 15 min trypsin treatment even this strain pattern becomes very similar to that of the seven wild strains even if the 35, 32, 30 and 18 kDa bands were missing.

4

Immunoblots of whole cell proteins (panel A) from M. agalactiae strain 32/M (lane 1), M. agalactiae strain 248/Or (lane 2), M. agalactiae strain 25/Or (lane 3), M. agalactiae strain 124/M (lane 4), M. agalactiae strain 2894/Nu (lane 5), M. agalactiae strain 2697/Nu (lane 6), M. agalactiae strain 233/M (lane 7), and PG2 type strain (lane 8) with 30 day infected sheep sera. Panel B shows the same strains after 15 min treatment with 500 μg/ml trypsin. Panel C: after 30 min treatment with 500 μg/ml trypsin. Panel D: after 45 min treatment with 500 μg/ml trypsin. Proteins’ relative molecular sizes appear at left next to Kaleidoscope prestained standard (Bio-Rad).

4

Immunoblots of whole cell proteins (panel A) from M. agalactiae strain 32/M (lane 1), M. agalactiae strain 248/Or (lane 2), M. agalactiae strain 25/Or (lane 3), M. agalactiae strain 124/M (lane 4), M. agalactiae strain 2894/Nu (lane 5), M. agalactiae strain 2697/Nu (lane 6), M. agalactiae strain 233/M (lane 7), and PG2 type strain (lane 8) with 30 day infected sheep sera. Panel B shows the same strains after 15 min treatment with 500 μg/ml trypsin. Panel C: after 30 min treatment with 500 μg/ml trypsin. Panel D: after 45 min treatment with 500 μg/ml trypsin. Proteins’ relative molecular sizes appear at left next to Kaleidoscope prestained standard (Bio-Rad).

Membrane integrity though does not seem to be compromised by trypsin action since mycoplasmas treated with 500 μg/ml trypsin for 15, 30 and 45 min retain their ability to grow on Hayflick agar.

Trypsin digestion suggests that many M. agalactiae proteins are accessible to this enzyme because they are surface-exposed membrane components and are arginine-lysine rich.

Analysis of major antigens by immunoprecipitation

Immunoprecipitation technique, using intact bacteria, allowed us to exclusively obtain antigens represent exocytoplasmatic domains [21]. We carried out immunoprecipitations using pooled sera from thirty day infected sheep with trypsinized mycoplasmas (500 μg/ml trypsin for 15, 30 and 45 min) and whole mycoplasmas. The pattern obtained is the same for all the strains analyzed (Fig. 5). The main immunoprecipitated antigens seem to have the following molecular masses: 80, 70, 55, 35, 32 and 30 kDa.

5

Immunoblots of whole cell proteins from M. agalactiae strain 248/M (lane A); lane B: immunoprecipitated proteins; lane C: immunoprecipitated proteins after 15 min treatment with 500 μg/ml trypsin. All proteins were electrophoresed on 8% polyacrylamide gel.

5

Immunoblots of whole cell proteins from M. agalactiae strain 248/M (lane A); lane B: immunoprecipitated proteins; lane C: immunoprecipitated proteins after 15 min treatment with 500 μg/ml trypsin. All proteins were electrophoresed on 8% polyacrylamide gel.

Trypsin treatment decreased protein bands signal intensity. After 45 min of treatment there is almost no signal. With the PG2 strain 80, 70 and 55 kDa protein bands are immunoprecipitated while 35, 32 and 30 kDa protein bands disappeared (data not shown).

Antibody elution

Antibodies were separated from immunocomplexes formed by whole or trypsinized mycoplasmas (500 μg/ml trypsin for 15, 30 and 45 min) and 30 day sera. Using whole mycoplasmas, antibodies reacted mainly with 35 and 32 kDa protein bands. This holds true for all the wild strains with the exception of 32/M strain. In this case eluted antibodies recognize only the 35 kDa protein band. After 15 min trypsin treatment eluted antibodies react only weakly with the 35 kDa band. On the contrary the 32 kDa band remains unchanged while new bands appeared with 80 and 50 kDa apparent molecular mass. This pattern doesn't change after prolonged trypsin treatment (Fig. 6).

6

Immunoblots of whole cell proteins from M. agalactiae strain 248/Or (lane A) with eluted antibodies from whole mycoplasmas (lane B), from mycoplasmas digested with 500 μg/ml trypsin for 15 min (lane C), 30 min (lane D) and 45 min (lane E).

6

Immunoblots of whole cell proteins from M. agalactiae strain 248/Or (lane A) with eluted antibodies from whole mycoplasmas (lane B), from mycoplasmas digested with 500 μg/ml trypsin for 15 min (lane C), 30 min (lane D) and 45 min (lane E).

In the type strain PG2 antibodies react mainly with the 40 kDa protein band and weakly with the 80, 35 and 32. After 15 min trypsin exposure 35, 32 and 40 kDa bands disappear completely while the 80 kDa band remains unchanged (data not shown).

In conclusion:

(1) The 80 kDa protein: it is not trypsinized, it is immunoprecipitated but it is recognized by eluted antibodies only after trypsinization of 35 kDa band. It is present in all the strains analyzed [13] and it appears at the first signs of infection. The 80 kDa protein could be a good candidate for serological diagnostic Kits.

(2) The 70 kDa protein: it is present in all strains, it is immunoprecipitated and it is particularly evident after trypsin treatment.

(3) The 55 kDa protein: it is strongly immunogenic, it is present since the beginning of infection even if it is lacking in 20% of analyzed [13], it immunoprecipitates even while it is not recognized by eluted antibodies. We are conducting further studies to clarify if this trypsin-sensitive protein plays an important role in M. agalactiae adhesion to epithelial ovine mammary cells.

(4) The 35 kDa protein: it is strongly immunogenic, it is trypsin digested, it immunoprecipites and it is recognized by eluted antibodies. Also for this protein we are conducting studies on its role in pathogenesis of M. agalactiae.

(5) The 32 and 30 kDa proteins: generally they are not digested by trypsin, they immunoprecipitated but only the 32 kDa protein is recognized by eluted antibodies.

Acknowledgements

This work was supported by grants of Ministero della Sanità (Health Ministry), Ricerca Finalizzata (Aim-specific research) 1993. We thank Dr. Iana Fiori and Dr. Paola Naitana who have signalled new agalactia infections and have provided us with the sera. Furthermore, we thank P. Melis for support and helpful discussion and F. Masia, P. Cassano and L. Saccu for technical assistance.

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