Identification of conserved Mycoplasma agalactiae surface antigens by immunoproteomics.

Contagious agalactia represents one of the most relevant infectious diseases of dairy sheep, with Mycoplasma agalactiae being the primary etiological agent. The early, sensitive, and specific identification of infected animals, as well as the development of efficient prophylactic tools, remain challenging. Here, we present a comprehensive characterization of M. agalactiae antigens focusing on those shared among different isolates. Leveraging on previous proteomic data obtained on individual strains, we adopted a strategy entailing sample pooling to optimize the identification of conserved proteins that induce an immune response. The liposoluble proteins from previously characterized field isolates and the type strain PG2T were enriched by Triton X-114 fractionation, pooled, analysed by one-dimensional (1D) and two-dimensional (2D) electrophoresis, and subjected to western immunoblotting against sheep sera collected during natural infection with M. agalactiae. Immunodominant antigens were identified by Matrix-Assisted Laser Desorption-Time-Of-Flight-Mass Spectrometry (MALDI-TOF-MS). This combined immunoproteomic approach confirmed the role of several known immunogens, including P80, P48, and P40, and most variable surface proteins (Vpmas), and unveiled novel immunodominant, conserved antigens, including MAG_1000, MAG_2220, MAG_1980, phnD, MAG_4740, and MAG_2430. Genomic context, functional prediction, subcellular localization, and invariable expression of these proteins in all isolates suggest their possible involvement in bacterial pathogenicity and metabolism. Moreover, most of the identified antigens elicit a host humoral response since the early stages of infection, persisting for at least 270 days. The immunodominant, conserved antigen panel identified in this work supports the development of effective vaccines and diagnostic tools with higher sensitivity and specificity in all the natural infection stages.

Novel antigenic proteins of Mycoplasma agalactiae as potential vaccine and serodiagnostic candidates.

Contagious agalactia (CA) is a serious disease notifiable to the World Organisation for Animal Health (OIE) causing severe economic losses to sheep and goat producers worldwide. Mycoplasma agalactiae, considered as its main etiological agent, inflicts a variety of symptoms in infected animals, including keratoconjunctivitis, mastitis, arthritis, ankylosis, abortions, stillbirths and granular vulvovaginitis. Despite its significance, developing a successful vaccine remains elusive, mostly due to the lack of knowledge about M. agalactiae's pathogenicity factors and pathogenic mechanisms, including its "core" antigens. The aim of this study was to identify, characterize and express antigenic proteins of M. agalactiae as potential vaccine candidates. Predicted proteins of type strain PG2 were analyzed using bioinformatic algorithms to assess their cellular localization and to identify their linear and conformational epitopes for B cells. Out of a total of 156 predicted membrane proteins, three were shortlisted as potential antigenic surface proteins, namely [MAG_1560 (WP_011949336.1), MAG_6130 (WP_011949770.1) and P40 (WP_011949418.1)]. These proteins were expressed in recombinant Escherichia coli strains. Purified proteins were evaluated for their antigenicity using Western blot and ELISA using sera of M. agalactiae-naturally infected and non-infected sheep and goats. All 3 proteins were specifically recognized by the tested sera of M. agalactiae-infected animals. Also, specific rabbit antisera raised against each of these 3 proteins confirm their membrane localization using TritonX-114 phase partioning, Western and colony immunoblotting. In conclusion, our study successfully identified P40 (as proof of concept and validation) and two novel antigenic M. agalactiae proteins as potential candidates for developing effective CA vaccines.

Xer1-independent mechanisms of Vpma phase variation in Mycoplasma agalactiae are triggered by Vpma-specific antibodies.

Despite their small genomes mycoplasmas maintain large multigene families devoted to surface antigenic variation. Although implicated as important factors for mycoplasma pathogenicity and persistence, the role of these antigenic switches in host immune evasion has never been unequivocally proven in these minimalist microbes. Mycoplasma agalactiae exhibits antigenic variation due to Xer1-mediated site-specific DNA inversions of vpma genes encoding abundant multiple surface lipoproteins. To evaluate the biological significance of Vpma oscillations the xer1 recombinase gene has been disrupted in earlier studies to abolish Vpma switching and to generate stable phase-locked mutants (PLMs) steadily expressing a single Vpma product. However, in previous animal infection studies, surprisingly these PLMs switched to new different Vpma phenotypes. The aim of the current study was to demonstrate the influence of anti-Vpma antibodies on change of Vpma expression in PLMs as well as on the wildtype strain. In in vitro assays it is shown that wild type M. agalactiae escapes the negative effects of Vpma-specific antibodies by high-frequency Xer1-mediated switching to alternative Vpma phenoytpes. Even for Xer1-disrupted PLMs that stably expressed the same Vpma for several in vitro generations, the presence of the corresponding Vpma-specific antibody caused repression of the target Vpma and induction of new Vpma phenotypes by novel complex vpma rearrangements like intragenic deletions and gene chimeras. These Xer1-independent vpma recombinations correlated very well with similar PLM switches observed in vivo in an earlier independent study, clearly demonstrating that Vpma phase variation is necessary to express 'Vpma immune evasion proteins' in order to escape the immune response and to survive in the immunocompetent host. The data clearly demonstrate that although the Xer1 recombinase is the sole factor responsible for Vpma switching of wild type M. agalactiae in vitro, other alternative molecular switches operate in its absence under the selective pressure of the immune response. Furthermore, this evasion from the immune attack of the host involves complex vpma rearrangements, a causal relationship that was so far never demonstrated for M. agalactiae, thereby illustrating novel features of its regulation under immune pressure. The results are anticipated to have a direct impact on understanding the in vivo role of surface antigenic variation systems and the immune evasion tactics of other pathogenic mycoplasma species.

Vpma phase variation is important for survival and persistence of Mycoplasma agalactiae in the immunocompetent host.

Despite very small genomes, mycoplasmas retain large multigene families encoding variable antigens whose exact role in pathogenesis needs to be proven. To understand their in vivo significance, we used Mycoplasma agalactiae as a model exhibiting high-frequency variations of a family of immunodominant Vpma lipoproteins via Xer1-mediated site-specific recombinations. Phase-Locked Mutants (PLMs) expressing single stable Vpma products served as first breakthrough tools in mycoplasmology to study the role of such sophisticated antigenic variation systems. Comparing the general clinical features of sheep infected with a mixture of phase-invariable PLMs (PLMU and PLMY) and the wild type strain, it was earlier concluded that Vpma phase variation is not necessary for infection. Conversely, the current study demonstrates the in vivo indispensability of Vpma switching as inferred from the Vpma phenotypic and genotypic analyses of reisolates obtained during sheep infection and necropsy. PLMY and PLMU stably expressing VpmaY and VpmaU, respectively, for numerous in vitro generations, switched to new Vpma phenotypes inside the sheep. Molecular genetic analysis of selected 'switchover' clones confirmed xer1 disruption and revealed complex new rearrangements like chimeras, deletions and duplications in the vpma loci that were previously unknown in type strain PG2. Another novel finding is the differential infection potential of Vpma variants, as local infection sites demonstrated an almost complete dominance of PLMY over PLMU especially during early stages of both conjunctival and intramammary co-challenge infections, indicating a comparatively better in vivo fitness of VpmaY expressors. The data suggest that Vpma antigenic variation is imperative for survival and persistence inside the immunocompetent host, and although Xer1 is necessary for causing Vpma variation in vitro, it is not a virulence factor because alternative Xer1-independent mechanisms operate in vivo, likely under the selection pressure of the host-induced immune response. This singular study highlights exciting new aspects of mycoplasma antigenic variation systems, including the regulation of expression by host factors.

Cathelicidin production and release by mammary epithelial cells during infectious mastitis.

Cathelicidins are well-characterized antimicrobial peptides (AMPs) that are present in significant amounts in mastitic milk. Neutrophils are believed to be the main producers of these AMPs, while the role of mammary epithelial cells (MECs) in their production and release is still unclear. In this work, cathelicidin production patterns were investigated in mammary tissues of ewes infected by Staphylococcus aureus, Streptococcus uberis, or Mycoplasma agalactiae, with a combined approach including immunohistochemistry, immune-colocalization, and fluorescent in situ hybridization. Our results confirm that MECs produce and release cathelicidins in response to different mastitis pathogens. As opposed to neutrophils, however, MECs do not seem to store the preformed protein precursor in their cytoplasm, but appear to synthesize and release it only upon exposure to the microorganisms. Cathelicidin production by MECs appears to occur before leukocyte influx in the milk, suggesting a role for these cells in the initial response of the mammary epithelium to microbial infection. Once in the milk, infiltrating neutrophils release massive amounts of cathelicidin by degranulation and production of neutrophil extracellular traps, acting as the main contributor for cathelicidin abundance in mastitic milk. Taken together, our results support the active contribution of MECs to cathelicidin production and release, and reinforce the value of cathelicidins as sensitive and pathogen-independent mastitis markers.

Comprehensive RNA-Seq Profiling to Evaluate the Sheep Mammary Gland Transcriptome in Response to Experimental Mycoplasma agalactiae Infection.

Mycoplasma agalactiae is a worldwide serious pathogen of small ruminants that usually spreads through the mammary route causing acute to subacute mastitis progressing to chronic persistent disease that is hard to eradicate. Knowledge of mechanisms of its pathogenesis and persistence in the mammary gland are still insufficient, especially the host-pathogen interplay that enables it to reside in a chronic subclinical state. This study reports transcriptome profiling of mammary tissue from udders of sheep experimentally infected with M. agalactiae type strain PG2 in comparison with uninfected control animals using Illumina RNA-sequencing (RNA-Seq). Several differentially expressed genes (DEGs) were observed in the infected udders and RT-qPCR analyses of selected DEGs showed their expression profiles to be in agreement with results from RNA-Seq. Gene Ontology (GO) analysis revealed majority of the DEGs to be associated with mycoplasma defense responses that are directly or indirectly involved in host innate and adaptive immune responses. Similar RNA-Seq analyses were also performed with spleen cells of the same sheep to know the specific systemic transcriptome responses. Spleen cells exhibited a comparatively lower number of DEGs suggesting a less prominent host response in this organ. To our knowledge this is the first study that describes host transcriptomics of M. agalactiae infection and the related immune-inflammatory responses. The data provides useful information to further dissect the molecular genetic mechanisms underlying mycoplasma mastitis, which is a prerequisite for designing effective intervention strategies.

Synthesis of Recombinant P48 of Mycoplasma agalactiae by Site Directed Mutagenesis and its Immunological Characterization.

Contagious agalactia caused by Mycoplasma agalactiae is an economically important disease of sheep and goats and has been prevalent worldwide including India. The present study was undertaken to evaluate the membrane protein P48 of M. agalactiae for specific diagnosis of disease. For this, p48 gene of the organism was amplified by PCR and subjected to site directed mutagenesis to convert three TGA codons to TGG's and, subsequently, cloned into prokaryotic expression vector pPRO EX HTb. Purified recombinant P48 protein reacted to anti-P48 serum in western blotting, which confirmed its immunogenic nature. Furthermore, the immune-blotting of the cell lysates from various Indian isolates of M. agalactiae against anti-P48 serum resulted in a single band at âˆ¼ 48 kDa among all isolates, indicating the conserved nature of P48 antigen in M. agalactiae. Also, the cross reactivity of P48 antigen among various Mycoplasma spp. was checked by western blotting which revealed reactivity only with M. agalactiae and M. bovis. Hence, this antigen could be exploited to differentiate M. agalactiae from other pathogenic Mycoplasma species except M. bovis. However, the inability of P48 to distinguish M. agalactiae from M. bovis does not downgrade the significance of P48 as the two species are usually host specific.

Detecting asymptomatic rams infected with Mycoplasma agalactiae in ovine artificial insemination centers.

Mycoplasma agalactiae (Ma) is the main causative agent of ovine contagious agalactia, which is a serious disease of small ruminants. In endemic areas, its most common clinical situation consists of chronically infected herds, and asymptomatic infected individuals represent an epidemiological risk regarding the transmission of this disease. The aim of this work was to detect the presence of asymptomatic rams infected with Ma in different artificial insemination centers, and to determine the most effective way to identify these individuals so as to implement adequate surveillance protocols. For this purpose, 215 rams and 14 teaser sheep were sampled taking auricular, nasal, and vaginal swabs and serum samples. In addition, ejaculates from 147 rams were analyzed. These samples were subjected to specific culture and molecular techniques to isolate and identify mycoplasmas, and to a serological test to detect antibodies against Ma. Mycoplasma agalactiae was detected in 47 (4.4%) of the 1077 samples analyzed, and also one individual resulted seropositive. Thus, 37 (17.2%) of the 215 studied rams were infected with Ma. The specimens which proportionally yielded the greatest number of positive results for this pathogen were semen samples (13.6%), followed by nasal swabs (5.8%). In contrast, the sampling of the external auricular canal and the serological analyses resulted insufficient to effectively detect infected individuals. Asymptomatic rams infected with Ma were detected in all the analyzed artificial insemination centers, highlighting the need to implement adequate surveillance protocols to prevent the presence of these individuals in these centers, reducing the risk of transmitting contagious agalactia.

Mycoplasma lipoproteins are major determinants of neutrophil extracellular trap formation.

Neutrophil granulocytes are paramount to innate responses as major effectors of acute inflammation. Among the various strategies enacted by neutrophils to eliminate microbes NETosis is a novel distinct antimicrobial activity in which an interlacement of chromatin fibres rich in granule-derived antimicrobial peptides and enzymes is extruded (NETs, neutrophils extracellular traps ). NETs contribute to the pathogenesis of acute and chronic inflammatory disorders. The interactions of mycoplasmas and innate immune cells, in particular neutrophil granulocytes, are poorly defined. Here, we describe NET formation in vivo in the mammary gland and milk of sheep naturally infected by Mycoplasma agalactiae. Also, we assess the contribution of liposoluble proteins, the most abundant component of the Mycoplasma membrane, in inducing NETosis. We demonstrate that Mycoplasma liposoluble proteins induce NET release at levels comparable to what observed with other stimuli, such as lipopolysaccharides and phorbol 12-myristate 13-acetate. Stimulation of neutrophils with synthetic diacylated lipopeptides based on the M. agalactiae P48, P80, and MAG_1000 proteins, combined in a mix or used individually, suggests that NETosis might not be dependent on a specific lipopeptide sequence. Also, NETosis is partially abolished when TLR2 is blocked with specific antibodies. The results presented in this work provide evidences for the mechanisms underlying NET activation in mycoplasma infections, and on their contribution to pathogenesis of mycoplasmosis.

Differential cytokine expression in natural and experimental mastitis induced by Mycoplasma agalactiae in dairy goats.

Cytokines, primarily produced by macrophages and lymphocytes, mobilize the immune system in response to infection, particularly at mucosal surfaces. Knowledge of the pathogenesis and persistence of Mycoplasma agalactiae (Ma) in the mammary gland is still insufficient. The aim of this study was to elucidate the role of cytokines in the pathogenesis of caprine mastitis caused by Ma. Cytokine expression was evaluated by immunohistochemical methods in the inflammatory lesions of 10 (5 naturally and 5 experimentally infected) goats with Ma-induced mastitis. Immunolabelling for IL-10, IFN-γ, IL-4 and TNF-α was observed in inflammatory cells within the lumen of acini and ducts and in the interstitial spaces and was usually associated with the presence of Ma antigen. The results suggest that cytokines play a role in the pathophysiological processes during Ma infection as differential expression of these cytokines was detected in relation to the course of the infection.

Development of a specific immunomagnetic capture-PCR for rapid detection of viable Mycoplasma agalactiae in sheep milk samples.

AIMS: To develop an immunomagnetic capture (IMC) to detect viable Mycoplasma agalactiae in routine ovine milk samples. METHODS AND RESULTS: Polyclonal antibodies against two M. agalactiae membrane surface proteins (P80 and P55) were covalently conjugated to magnetic beads (MBs) to form MB-Ab80 and MB-Ab55. Mycoplasma agalactiae cells were captured by a specific antigen-antibody reaction and magnetic separation. Immunomagnetic capture (IMC) was used to isolate and concentrate M. agalactiae in serial decimal dilutions and in artificially contaminated milk to facilitate subsequent detection by PCR. A 375-bp fragment of M. agalactiae was amplified using a pair of M. agalactiae-specific primers in PCR. The limit of detection of IMC-PCR method ranged from 10 to 10(2)  CCU ml(-1) when mycoplasmas were resuspended in PBS and from 10(2) to 10(3)  CCU ml(-1) when mycoplasmas were resuspended in uncontaminated ovine milk. This study also describes the application of IMC-PCR method to test for M. agalactiae in 516 milk samples collected from sheep with suspected contagious agalactia. Its performance was evaluated relative to culture. CONCLUSIONS: This report has demonstrated for the first time, the effective use of rapid and reliable IMC combined with PCR assay for the detection of viable M. agalactiae. SIGNIFICANCE AND IMPACT OF THE STUDY: The method IMC-PCR provides an alternative to conventional microbiological detection, method and it could be applied to quick detection of M. agalactiae in routine sheep milk samples.

Mycoplasma agalactiae MAG_5040 is a Mg2+-dependent, sugar-nonspecific SNase recognised by the host humoral response during natural infection.

In this study the enzymatic activity of Mycoplasma agalactiae MAG_5040, a magnesium-dependent nuclease homologue to the staphylococcal SNase was characterized and its antigenicity during natural infections was established. A UGA corrected version of MAG_5040, lacking the region encoding the signal peptide, was expressed in Escherichia coli as a GST fusion protein. Recombinant GST-MAG_5040 exhibits nuclease activity similar to typical sugar-nonspecific endo- and exonucleases, with DNA as the preferred substrate and optimal activity in the presence of 20 mM MgCl2 at temperatures ranging from 37 to 45°C. According to in silico analyses, the position of the gene encoding MAG_5040 is consistently located upstream an ABC transporter, in most sequenced mycoplasmas belonging to the Mycoplasma hominis group. In M. agalactiae, MAG_5040 is transcribed in a polycistronic RNA together with the ABC transporter components and with MAG_5030, which is predicted to be a sugar solute binding protein by 3D modeling and homology search. In a natural model of sheep and goats infection, anti-MAG_5040 antibodies were detected up to 9 months post infection. Taking into account its enzymatic activity, MAG_5040 could play a key role in Mycoplasma agalactiae survival into the host, contributing to host pathogenicity. The identification of MAG_5040 opens new perspectives for the development of suitable tools for the control of contagious agalactia in small ruminants.

Timing of activation of CD4+ memory cells as a possible marker to establish the efficacy of vaccines against contagious agalactia in sheep.

Mycoplasma agalactiae is a major pathogen of sheep and goats in many areas of the world and particularly in Mediterranean countries. It causes contagious agalactia, an infectious disease primarily affecting mammary glands. Many vaccines against the pathogen are currently under development. The aim of the study was to investigate the involvement of T cell-mediated immunity during vaccination and challenge experiments against Mycoplasma agalactiae. A comparison of the antigen-specific expansion of interferon gamma positive T cell memory and naïve subsets was performed between vaccinated and non-vaccinated sheep to identify cellular subsets whose activation was different between protected and non-protected sheep. Data reported in this manuscript demonstrated that two out of the three vaccines used in this study protected sheep from the disease. In the protected groups CD4(+) memory interferon-γ(+) T cells underwent an early expansion (p<0.05 when compared to unprotected groups), whilst memory CD8(+) Interferon-γ(+) T cells increased in non-protected animals 7 days after infection (p<0.05). γδ(+) Interferon-γ(+) T cells reached peaks of expansion in infected and in two vaccinated groups thus indicating that these cells are not preferentially involved in protection or pathogenesis (p<0.05). Hereby we propose that the early activation of CD4(+) memory Interferon-γ(+) T cells could be considered as a marker of protection from the disease as well as a tool to establish vaccine efficacy.

A relevant long-term impact of the circulation of a potentially contaminated vaccine on the distribution of scrapie in Italy. Results from a retrospective cohort study.

A sudden increase in the incidence of scrapie in Italy in 1997 was subsequently linked to the use of a potentially infected vaccine against contagious agalactia. The relative risk for the exposed farms ranged between 6 and 40. The aim of this study was to assess the long-term impact of exposure to the potentially scrapie-contaminated vaccine on the Italian classical scrapie epidemic. We carried out a retrospective cohort study, fitting mixed-effects Poisson regression models, dividing national geographic areas into exposure categories on the basis of the vaccine circulation levels. We took into account the sensitivity of the surveillance system applied in the different areas. The population attributable fraction (PAF) was used to assess the impact on the total population of farms associated with the effect of circulation of the vaccine. The provinces where the vaccine was more often sold were noted to have a higher level of disease when compared to those provinces where the vaccine was sold less often (incidence rate ratio [IRR]: 2.7; 95% confidence interval [CI]: 1.1-6.5). The population attributable fraction was high (68.4%). Standardization techniques allowed to account for the potential of geographical variability in the sensitivity of the Italian surveillance system. Although the number of the directly exposed farms was limited, an important long-term impact of the vaccine circulation could be quantified in terms of secondary outbreaks likely due to the exchange of animals from directly exposed flocks.

Role of Vpma phase variation in Mycoplasma agalactiae pathogenesis.

Compared with other bacterial pathogens, the molecular mechanisms of mycoplasma pathogenicity are largely unknown. Several studies in the past have shown that pathogenic mycoplasmas are equipped with sophisticated genetic systems that allow them to undergo high-frequency surface antigenic variations. Although never clearly proven, these variable mycoplasma surface components are often implicated in host immune evasion and adaptation. Vpma surface lipoproteins of the ruminant pathogen Mycoplasma agalactiae are encoded on a genomic pathogenicity island-like locus and are considered as one of the well-characterized model systems of mycoplasma surface antigenic variation. The present study assesses the role of these phase-variable Vpmas in the molecular pathogenesis of M. agalactiae by testing the wild-type strain PG2 in comparison with the xer1-disrupted Vpma 'phase-locked' mutants in sheep infection models. The data clearly illustrate that although Xer1 recombinase is not a virulence factor of M. agalactiae and Vpma phase variation is not necessary for establishing an infection, it might critically influence the survival and persistence of the pathogen under natural field conditions, mainly due to a better capacity for dissemination and evoking systemic responses. This is the first study where mycoplasma 'phase-locked' mutants are tested in vivo to elucidate the role of phase variation during infection.

Chronological and immunohistochemical characterization of the mammary immunoinflammatory response in experimental caprine contagious agalactia.

To explore the pathogenesis of caprine contagious agalactia (CA), we assessed the ability of Mycoplasma agalactiae (Ma) to modulate the immune system in host tissues by immunohistochemically and chronologically characterizing the main cell subsets present during the mammary immunoinflammatory response. Fifteen lactating goats were inoculated with 10(10) colony-forming units (cfu) of Ma and killed 5, 15 or 45 days post-inoculation (dpi). Blood was taken before necropsy to determine antibodies and milk to determine mycoplasma number. Cells in mammary tissue expressing lysozyme, myeloid-histiocyte antigen (Mac387), major histocompatibility complex class II antigen, immunoglobulin G (IgG), IgA, and CD3, CD4 and CD8 lymphocytes were determined by immunohistochemistry. Results indicate an innate immune response in animals sacrificed at 5dpi, characterized by an abundance of Mac387+ and lysozyme+ cells, that was unable to block or control Ma infection. Elevated numbers of all the cell subsets of the specific immune response (MHC-II+, IgG+, IgA+, CD3+, CD4+ and CD8+ cells) were observed during the subacute stage of the inflammatory process, represented by the 15dpi group. However, these findings could not be correlated with an intense antibody response in blood. The chronic stage of the inflammatory process observed in the goats killed at 45dpi was mainly characterized by expansion of the CD8 compartment at the expense of the CD4 subset leading to a reduced CD4/CD8 ratio. These results contribute to establishing the basic morphological and immunohistochemical characterization of the local immune response against Ma in the goat's mammary gland.

Long-term immunogenicity and protection against Mycoplasma agalactiae induced by an oil adjuvant vaccine in sheep.

The long-term protective immunity of an inactivated mineral-oil adjuvanted Mycoplasma agalactiae vaccine was evaluated in sheep. The antigen suspension was emulsified with a mixture of three mineral oils (Montanide ISA-563, Marcol-52, Montane-80 at the ratio of 30%, 63%, and 7%, respectively). Twenty-two animals were divided in 2 groups (A and B) and immunised with two doses of the vaccine (group A, n=14) or used as unvaccinated control (group B, n=8). Five months after the second vaccination, seven animals of group A and four animals of group B were challenged by nasal route with M. agalactiae. The remaining seven vaccinated and four control animals were challenged intranasally eight months after vaccination. The vaccine was able to induce a full-protective immunity preventing the clinical signs of contagious agalactia and the infection by M. agalactiae in all groups of animals irrespective of the time of challenge after booster administration.

Genetic immunization with the immunodominant antigen P48 of Mycoplasma agalactiae stimulates a mixed adaptive immune response in BALBc mice.

A DNA vaccine against contagious agalactia was developed for the first time, encoding the P48 of Mycoplasma agalactiae. Specific immune responses elicited in BALB/c mice were evaluated. Both total IgG and IgG1 were detected in mice vaccinated with pVAX1/P48. Proliferation of mononuclear cells of the spleen, levels of gamma interferon, interleukin-12, and interleukin-2 mRNAs were enhanced in immunized animals. Results indicate that pVAX1/P48 vaccination induced both T(h)1 and T(h)2 immune responses. Nucleic acid immunization could be a new strategy against M. agalactiae infections and may be potentially used to develop vaccines for other Mycoplasma diseases.