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1.  A survey of Mycoplasma agalactiae in dairy sheep farms in Spain 
Background
Contagious Agalactia (CA) is one of the major animal health problems in small ruminants because of its economic significance. Currently, four Mycoplasma spp. have been associated with this syndrome: M. agalactiae, M. mycoides subsp. capri, M. capricolum subsp. capricolum and M. putrefaciens. Their presence has been evaluated in several studies conducted in CA-endemic countries. However, previous Spanish studies have been focused on caprine CA, and there is a knowledge gap regarding which Mycoplasma species are present in sheep flocks from Spain, which has the second highest number of sheep amongst the 27 European Union member states. Consequently, we investigated the presence and geographic distribution of the four CA-causing mycoplasmas in Spanish dairy sheep farms. This is the first time such an investigation has been performed.
Results
Three hundred thirty nine out of 922 sheep flocks were positive for M. agalactiae by real time PCR (36.8%) and 85 by microbiological identification (9.2%). Interestingly, all 597 milk samples assessed for the presence of M. mycoides subsp. capri, M. capricolum subsp. capricolum and M. putrefaciens tested negative. To evaluate the intermittent excretion of the pathogen in milk, we sampled 391 additional farms from 2 to 5 times, resulting that in 26.3% of the cases a previously positive farm tested negative in a later sampling.
Conclusions
M. agalactiae was the only Mycoplasma species detected in the study area showing a high frequency of presence and wide distribution. Therefore, the establishment of a permanent surveillance network is advantageous, as well as the implementation of control and prevention measures to hinder the dissemination of M. agalactiae and to prevent the entrance of other Mycoplasma species.
doi:10.1186/1746-6148-8-171
PMCID: PMC3514350  PMID: 23006445
Mycoplasma agalactiae; Contagious agalactia; Real time PCR; Sheep; Dairy; Spain
2.  Comparative assessment of two commonly used commercial ELISA tests for the serological diagnosis of contagious agalactia of small ruminants caused by Mycoplasma agalactiae 
Background
Contagious agalactia (CA) of sheep and goats caused by Mycoplasma agalactiae is a widely occurring economically important disease that is difficult to control. The ELISA is commonly used for the serological detection of CA but it has some limitations and the performance of the available tests have not been properly evaluated.
Two commercial ELISA kits are widely used, one involving a fusion protein as target antigen and the other a total antigen. The objectives were to compare these tests by evaluating:
i. Their diagnostic sensitivity and specificity, the relevance of the recommended cut-off points, the correlation between the two tests, and, the correlation between serology data and the milk shedding of M. agalatiae;
ii. The influence of extrinsic factors such as the targeted animal species, geographical origin of the samples, intra-specific variability of M. agalactiae and concurrent mycoplasma infections.
A sample of 5900 animals from 211 farms with continuous CA monitoring for 20 years and no prior vaccination history was used. The infection status was known from prior bacteriological, epidemiological and serological monitoring with a complementary immunoblotting test.
Results
The average diagnostic sensitivity was 56% [51.8–59.8] for the fusion protein ELISA and 84% [81.3–87.2] for the total antigen ELISA, with noteworthy flock-related variations. The average diagnostic specificity for the fusion protein ELISA was 100% [99.9–100], and for the total antigen ELISA differed significantly between goats and sheep: 99.3% [97.4–99.9] and 95.7% [93.8–97.2] respectively.
Experimental inoculations with different M. agalactiae strains revealed that the ELISA kits poorly detected the antibody response to certain strains. Furthermore, test performances varied according to the host species or geographical origin of the samples.
Finally, the correlation between milk shedding of M. agalactiae and the presence of detectable antibodies in the blood was poor.
Conclusions
These serological tests are not interchangeable. The choice of a test will depend on the objectives (early detection of infection or disease control program), on the prevalence of infection and the control protocol used. Given the variety of factors that may influence performance, a preliminary assessment of the test in a given situation is recommended prior to widespread use.
doi:10.1186/1746-6148-8-109
PMCID: PMC3439703  PMID: 22776779
3.  Development of a Sensitive and Specific Enzyme-Linked Immunosorbent Assay Based on Recombinant Antigens for Rapid Detection of Antibodies against Mycoplasma agalactiae in Sheep▿ †  
Clinical and Vaccine Immunology  2007;14(4):420-425.
We developed a new recombinant enzyme-linked immunosorbent assay (rELISA) for serodiagnosis of contagious agalactia (CA), a disease caused by Mycoplasma agalactiae in sheep and goats. The assay is based on two M. agalactiae surface proteins, namely, P80 and P55. Identification of these immunodominant and common antigens was accomplished by examining the antibody response elicited in sheep during experimental infection and comparing it to the protein expression profiles of 75 M. agalactiae field strains. Our rELISA was tested with 343 sera, collected from sheep with a laboratory-confirmed diagnosis of CA (n = 223) and from healthy animals (n = 120). All sera had previously been tested by Western blotting (WB) for reactivity against M. agalactiae. In addition, our rELISA was compared with a commercial routine ELISA based on inactivated antigens (CHEKiT). Among the 223 samples that were WB positive for M. agalactiae, 209 (93.7%) tested positive for rP80-P55 with our ELISA, whereas only 164 (73.8%) tested positive with the CHEKiT ELISA. Among the 120 samples tested that were WB negative for M. agalactiae, 96.7% were confirmed as negative with our rELISA, while only 75.8% were confirmed as negative with the CHEKiT ELISA. A comparison of the results with receiver operating characteristic curves indicated that the differences observed between our rELISA and the CHEKiT ELISA are statistically significant. The use of recombinant peptides instead of inactivated antigens could significantly improve the discrimination of positive and negative animals, bringing significant advantages in controlling the import/export of live animals and helping in eradication of this economically detrimental disease.
doi:10.1128/CVI.00439-06
PMCID: PMC1865618  PMID: 17287317
4.  Characterization and Analysis of a Stable Serotype-Associated Membrane Protein (P30) of Mycoplasma agalactiae 
Journal of Clinical Microbiology  2001;39(8):2814-2822.
The gene for a 30-kDa immunodominant antigen, P30, of Mycoplasma agalactiae was cloned from type strain PG2 and expressed in Escherichia coli. P30 is encoded on a monocistronic operon determined by two −10 boxes and a possible −35 region constituting the potential promoter, and a transcription termination site. The gene for the 266-amino-acid protein is preceded by a polypurine-rich region designed as the consensus sequence for a ribosome-binding site. Analysis of the amino acid sequence of P30 revealed the presence of a recognition site for a prokaryotic signal peptidase II at amino acid (aa) 24, indicating that P30 is a transmembrane protein. Moreover, Triton X-114 phase partitioning of M. agalactiae PG2 total antigen revealed that P30 is strongly hydrophobic and hence a possible membrane component. Immunoblot analysis using the monospecific polyclonal anti-P30-His serum indicated that P30 is specific to M. agalactiae. Furthermore, PCR amplification with specific primers for p30 and Southern blot analysis revealed the presence of the gene in all M. agalactiae strains tested and its absence in the other mycoplasma species. Among 27 strains of M. agalactiae studied, 20 strains belonging to the common serotypes A to D, including PG2, expressed P30 or part of it as detected by the monospecific polyclonal anti-P30 antibodies. The other seven strains belonging to the rarely isolated serotypes E to H were negative for P30. The p30 gene was sequenced in 15 strains of M. agalactiae, 10 of which expressed P30 or at least part of it and 5 of which did not express P30. The negative strains carried mutations in both −10 boxes of the promoters. These mutations seem to be responsible for the lack of P30 expression in these strains. Analysis of sera from sheep that were experimentally infected with M. agalactiae revealed that P30 induced a strong and persistent immune response which was still very high two months after infection. In contrast, currently used enzyme-linked immunosorbent assay serology gave only low titers.
doi:10.1128/JCM.39.8.2814-2822.2001
PMCID: PMC88244  PMID: 11473997
5.  Mapping Antigenic Sites of an Immunodominant Surface Lipoprotein of Mycoplasma agalactiae, AvgC, with the Use of Synthetic Peptides  
Infection and Immunity  2002;70(1):171-176.
As a first step toward the design of an epitope vaccine to prevent contagious agalactia, the strongly immunogenic 55-kDa protein of Mycoplasma agalactiae was studied and found to correspond to the AvgC protein encoded by the avgC gene. The avg genes of M. agalactiae, which encode four variable surface lipoproteins, display a significant homology to the vsp (variable membrane surface lipoproteins) genes of the bovine pathogen Mycoplasma bovis at their promoter region as well as their N-terminus-encoding regions. Some members of the Vsp family are known to be involved in cytoadhesion to host cells. In order to localize immunogenic peptides in the AvgC antigen, the protein sequence was submitted to epitope prediction analysis, and five sets of overlapping peptides, corresponding to five selected regions, were synthesized by Spot synthesis. Reactive peptides were selected by immunobinding assay with sera from infected sheep. The three most immunogenic epitopes were shown to be surface exposed by immunoprecipitation assays, and one of these was specifically recognized by all tested sera. Our study indicates that selected epitopes of the AvgC lipoprotein may be used to develop a peptide-based vaccine which is effective against M. agalactiae infection.
doi:10.1128/IAI.70.1.171-176.2002
PMCID: PMC127643  PMID: 11748179
6.  Mycoplasma agalactiae MAG_5040 is a Mg2+-Dependent, Sugar-Nonspecific SNase Recognised by the Host Humoral Response during Natural Infection 
PLoS ONE  2013;8(2):e57775.
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.
doi:10.1371/journal.pone.0057775
PMCID: PMC3585158  PMID: 23469065
7.  Prevalence of contagious mastitis pathogens in bulk tank milk in Québec 
The Canadian Veterinary Journal  2012;53(10):1071-1078.
The objective of this study was to estimate the prevalence of mycoplasma, Staphylococcus aureus, and Streptococcus agalactiae in bulk tank milk (BTM) in Québec dairy herds. BTM was sampled 3 times a month in 117 randomly selected dairy herds. Samples were submitted for S. aureus, S. agalactiae, and mycoplasma and for direct mycoplasma detection by polymerase chain reaction (PCR). Mycoplasma spp. was identified at least once in 3 herds (2.6%) by primary culture and/or PCR and in 4 herds (3.4%) by enrichment culture and/or PCR. Staphylococcus aureus was isolated at least once in 99 (84.6%) and 112 (95.7%) herds in primary culture and after enrichment, respectively. Streptococcus agalactiae was isolated at least once in 9 (7.7%) and 10 (8.6%) herds in primary culture and after enrichment, respectively. Herd prevalence of mycoplasma was similar to that previously reported in Canada. Staphylococcus aureus is still by far the most important contagious mastitis pathogen.
PMCID: PMC3447309  PMID: 23543925
8.  Specific detection by PCR of Streptococcus agalactiae in milk. 
The aim of this study was to develop a simple and specific method for direct detection of Streptococcus agalactiae from cow's milk. The method was based on polymerase chain reaction (PCR) using species-specific and universal primers derived from the 16S rRNA gene. The amplification product was verified by restriction endonuclease digest and sequencing. Specific identification was proven on a collection of 147 S. agalactiae isolates of bovine and human origin. In addition, 17 strains belonging to different bacterial species that potentially can be found in milk samples also tested negative. The PCR developed was used for direct detection of S. agalactiae in milk, using for the first time with gram-positive bacteria the nucleic acid-binding properties of diatomaceous earth. The test, which has high specificity, high sensitivity (100 cfu/mL), and can be carried out in less than 24 h, represents an innovative diagnostic tool for the detection of S. agalactiae in milk.
Images
PMCID: PMC1189646  PMID: 11227199
9.  The indirect hemagglutination test for the detection of antibodies in cattle naturally infected mycoplasmas. 
Stable mycoplasma antigens for the indirect hemagglutination test (IHA) were prepared employing glutaraldehyde treated sheep erythrocytes sensitized with Mycoplasma agalactiae subsp. bovis and Mycoplasma bovigenitalium antigens. Employing these antigens mycoplasma antibodies were detected in sera from cattle which had mastitic symptoms due to natural infection with either M. agalactiae subsp. bovis or M. bovigenitalium. A total of 200 cows from four herds were examined at varying intervals for the presence of M. agalactiae subsp. bovis and for the detection of antibody using growth inhibition and IHA tests. Mycoplasmas were isolated from 37 animals. Growth inhibiting antibody was detected from 56 of the 200 animals. In the IHA tests, antibody titer greater than or equal to 1:80 were detected in 148 animals, 76 of these having antibody titers greater than or equal to 1:160, while sera of 116 normal control animals had no growth inhibiting antibody and none had IHA antibody titers greater than 1:40. M. bovigenitalium was isolated from the milk of three of 26 animals in a fifth herd during an outbreak of mastitis. Growth inhibiting antibodies were demonstrated in the sera of ten of the 26 animals. However, the IHA test detected antibody titers of greater than or equal to 1:160 in 13 animals and of 1:80 in one of the 26 animals. To determine the specificity of the IHA tests, M. agalactiae subsp. bovis and M. bovigenitalium antigens were reacted with rabbit hyperimmune typing sera produced against 12 species of bovine mycoplasmatales. Homologous antisera showed IHA antibody titers of 1:1280 and 1:2560 against M. agalactiae subsp. bovis and M. bovigenitalium respectively, whereas heterologous antisera showed IHA antibody titers of less than or equal to 1:20. Also eight type-specific bovine antisera were reacted with M agalactiae subsp. bovis and M. bovigenitalium antigens in homologous and heterologous tests. Homoogous reactions showed IHA antibody titers greater than or equal to 1:320, whereas heterologous reactions showed IHA titers of less than or equal to 1:20. This IHA test promises to be useful for the detection of bovine mycoplasma antibodies in sera from cattle infected with M. agalactiae subsp. bovis or M. bovigenitalium. Thes test is sensitive, reproducible and specific and the technique is relatively simple and rapid. The antigens were stable for at least seven months.
PMCID: PMC1277514  PMID: 1000374
10.  Incidence of Staphylococci and Streptococci During Winter in Mastitic Milk of Sahiwal Cow and Murrah Buffaloes 
Indian Journal of Microbiology  2011;52(2):153-159.
Mastitis is a serious problem in dairy sector and among various aetiological agents, the incidence of staphylococci and streptococci remains high in milking animal. The present study was focused on detection of staphylococci and streptococci in winter season. Milk samples (117) of mastitic animals were tested for presence of staphylococci and streptococci using biochemical and PCR based assays. The testing revealed majority of animals (90.6%) were infected with more than one causative agent. Amongst 117 sample, 109 and 90 comprised of staphylococci and 90 streptococci, respectively. Distribution proportion of S. aureus, S. epidermidis, S. agalactiae, S. uberis and S. dysgalactiae among the mastitic cases was found as 64.9, 7.7, 5.1, 1.7, 48.7, 65.8 and 0.8%, respectively. Streptococci and staphylococci were observed in different combinations and the frequent were S. aureus/S. agalactiae/S. uberis, S. aureus/S. uberis, S. aureus/S. agalactiae and S. agalactiae/S. uberis which were accounted for 23.9, 19.7, 5.9 and 2.6%, respectively. Approximately half of the (52.1%) cases were observed for reoccurrence of mastitis. Reoccurrence of mastitis in winter season among these cases was significantly low as compared to summer (cattle-5 cases; buffaloes-2 cases). In addition, prevalence of S. aureus, S. agalactiae, S. uberis, and S. epidermidis in reoccurring mastitic cases was 73.7, 63.9, 45.9 and 6.6%, respectively. The observations revealed mastitis causing pathogens remains in hidden phase in winter season; however, cannot be neglected. The observation might be helpful in culling or segregation of cows for mastitis reduction programmes.
doi:10.1007/s12088-011-0207-1
PMCID: PMC3386454  PMID: 23729875
Mastitis; Staphylococci; Streptococci; Winter; Reoccurrence
11.  Genome-Scale Analysis of Mycoplasma agalactiae Loci Involved in Interaction with Host Cells 
PLoS ONE  2011;6(9):e25291.
Mycoplasma agalactiae is an important pathogen of small ruminants, in which it causes contagious agalactia. It belongs to a large group of “minimal bacteria” with a small genome and reduced metabolic capacities that are dependent on their host for nutrients. Mycoplasma survival thus relies on intimate contact with host cells, but little is known about the factors involved in these interactions or in the more general infectious process. To address this issue, an assay based on goat epithelial and fibroblastic cells was used to screen a M. agalactiae knockout mutant library. Mutants with reduced growth capacities in cell culture were selected and 62 genomic loci were identified as contributing to this phenotype. As expected for minimal bacteria, “transport and metabolism” was the functional category most commonly implicated in this phenotype, but 50% of the selected mutants were disrupted in coding sequences (CDSs) with unknown functions, with surface lipoproteins being most commonly represented in this category. Since mycoplasmas lack a cell wall, lipoproteins are likely to be important in interactions with the host. A few intergenic regions were also identified that may act as regulatory sequences under co-culture conditions. Interestingly, some mutants mapped to gene clusters that are highly conserved across mycoplasma species but located in different positions. One of these clusters was found in a transcriptionally active region of the M. agalactiae chromosome, downstream of a cryptic promoter. A possible scenario for the evolution of these loci is discussed. Finally, several CDSs identified here are conserved in other important pathogenic mycoplasmas, and some were involved in horizontal gene transfer with phylogenetically distant species. These results provide a basis for further deciphering functions mediating mycoplasma-host interactions.
doi:10.1371/journal.pone.0025291
PMCID: PMC3179502  PMID: 21966487
12.  Phenotypic and genotypic identification of streptococci and related bacteria isolated from bovine intramammary infections 
Background
Streptococcus spp. and other Gram-positive, catalase-negative cocci (PNC) form a large group of microorganisms which can be found in the milk of cows with intramammary infection. The most frequently observed PNC mastitis pathogens (major pathogens) are Streptococcus uberis, Strep. dysgalactiae, and Strep. agalactiae. The remaining PNC include a few minor pathogens and a large nonpathogenic group. Improved methods are needed for the accurate identification and differentiation of PNC.
A total of 151 PNC were collected from cows with intramammary infection and conclusively identified by 16S rRNA sequencing as reference method. Nine phenotypic microbiological tests (alpha-hemolysis, CAMP reaction, esculin hydrolysis, growth on kanamycin esculin azide agar and on sodium chloride agar, inulin fermentation, hippurate hydrolysis, leucine aminopeptidase and pyrrolidonyl peptidase activity), multiplex PCR for the three major pathogens (target genes for Strep. uberis, Strep. dysgalactiae and Strep. agalactiae: pauA, 16S rRNA, and sklA3, respectively), and mass spectroscopy using the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS) were evaluated for the diagnosis and discrimination of the three clinically most relevant PNC.
Results
The probability that a strain of Strep. uberis, Strep. dysgalactiae and Strep. agalactiae was correctly identified by combining the results of the 9 phenotypic tests was 92%, 90%, and 100%, respectively. Applying the multiplex PCR, all strains of the three major pathogens were correctly identified and no false positive results occurred. Correct identification was observed for all strains of Strep. uberis and Strep. agalactiae using MALDI-TOF MS. In the case of Strep. dysgalactiae, some variability was observed at the subspecies level, but all strains were allocated to one single cluster.
Conclusions
The results of the present study show that reliable identification of the clinically most relevant PNC (Strep. uberis, Strep. agalactiae and Strep. dysgalactiae) can be obtained by use of a combination of colony morphology, hemolysis type and catalase reaction, and a multiplex PCR with specific primers restricted to these 3 pathogens. The MALDI-TOF MS is a fast method that shows promising results, although identification of Strep. dysgalactiae at the subspecies level is not yet satisfactory.
doi:10.1186/1751-0147-55-53
PMCID: PMC3723560  PMID: 23866930
Mastitis; Cattle; Streptococci; Identification; Mass spectroscopy
13.  Mycoplasma agalactiae, an Etiological Agent of Contagious Agalactia in Small Ruminants: A Review 
Mycoplasma agalactiae is one of the causal agents of classical contagious agalactia (CA), a serious, economically important but neglected enzootic disease of small ruminants. It occurs in many parts of the world and most notably in the Mediterranean Basin. Following the infection common complications are septicaemia, mastitis, arthritis, pleurisy, pneumonia, and keratoconjunctivitis. Primary or tentative diagnosis of the organism is based upon clinical signs. Various serological tests, namely, growth precipitation, immunofluorescence, complement fixation test, haemagglutination inhibition, agglutination, immunodiffusion, enzyme immunoassays, immunoelectrophoresis, blotting techniques, and others, are available. Molecular tools seem to be much more sensitive, specific, and faster and help to differentiate various strains. The real-time PCR, multiplex PCR, quantitative PCR, PCR-RFLP, MLST, and gene probes, complementary to segments of chromosomal DNA or 16S ribosomal RNA (rRNA), have strengthened the diagnosis of M. agalactiae. Both live attenuated and adjuvant (alum precipitated or saponified) inactivated vaccines are available with greater use of inactivated ones due to lack of side effects. The present review discusses the etiology, epidemiology, pathogenesis, and clinical signs of contagious agalactia in small ruminants along with trends and advances in its diagnosis, treatment, vaccination, prevention, and control strategies that will help in countering this disease.
doi:10.1155/2014/286752
PMCID: PMC4109668  PMID: 25097796
14.  Unexpected genetic diversity of Mycoplasma agalactiae caprine isolates from an endemic geographically restricted area of Spain 
Background
The genetic diversity of Mycoplasma agalactiae (MA) isolates collected in Spain from goats in an area with contagious agalactia (CA) was assessed using a set of validated and new molecular typing methods. Validated methods included pulsed field gel electrophoresis (PFGE), variable number of tandem repeats (VNTR) typing, and Southern blot hybridization using a set of MA DNA probes, including those for typing the vpma genes repertoire. New approaches were based on PCR and targeted genomic regions that diverged between strains as defined by in silico genomic comparisons of sequenced MA genomes.
Results
Overall, the data showed that all typing tools yielded consistent results, with the VNTR analyses being the most rapid method to differentiate the MA isolates with a discriminatory ability comparable to that of PFGE and of a set of new PCR assays. All molecular typing approaches indicated that the Spanish isolates from the endemic area in Murcia were very diverse, with different clonal isolates probably restricted to separate, but geographically close, local areas.
Conclusions
The important genetic diversity of MA observed in infected goats from Spain contrasts with the overall homogeneity of the genomic background encountered in MA from sheep with CA in Southern France or Italy, suggesting that assessment of the disease status in endemic areas may require different approaches in sheep and in goats. A number of congruent sub-typing tools are now available for the differentiation of caprine isolates with comparable discriminatory powers.
doi:10.1186/1746-6148-8-146
PMCID: PMC3514313  PMID: 22920649
Mycoplasma agalactiae; Molecular typing; Contagious agalactia; Goats
15.  In vitro and in vivo cell invasion and systemic spreading of Mycoplasma agalactiae in the sheep infection model 
Generally regarded as extracellular pathogens, molecular mechanisms of mycoplasma persistence, chronicity and disease spread are largely unknown. Mycoplasma agalactiae, an economically important pathogen of small ruminants, causes chronic infections that are difficult to eradicate. Animals continue to shed the agent for several months and even years after the initial infection, in spite of long antibiotic treatment. However, little is known about the strategies that M. agalactiae employs to survive and spread within an immunocompetent host to cause chronic disease. Here, we demonstrate for the first time its ability to invade cultured human (HeLa) and ruminant (BEND and BLF) host cells. Presence of intracellular mycoplasmas is clearly substantiated using differential immunofluorescence technique and quantitative gentamicin invasion assays. Internalized M. agalactiae could survive and exit the cells in a viable state to repopulate the extracellular environment after complete removal of extracellular bacteria with gentamicin. Furthermore, an experimental sheep intramammary infection was carried out to evaluate its systemic spread to organs and host niches distant from the site of initial infection. Positive results obtained via PCR, culture and immunohistochemistry, especially the latter depicting the presence of M. agalactiae in the cytoplasm of mammary duct epithelium and macrophages, clearly provide the first formal proof of M. agalactiae's capability to translocate across the mammary epithelium and systemically disseminate to distant inner organs. Altogether, the findings of these in vitro and in vivo studies indicate that M. agalactiae is capable of entering host cells and this might be the strategy that it employs at a population level to ward off the host immune response and antibiotic action, and to disseminate to new and safer niches to later egress and once again proliferate upon the return of favorable conditions to cause persistent chronic infections.
doi:10.1016/j.ijmm.2014.07.011
PMCID: PMC4282308  PMID: 25129554
Mycoplasma agalactiae; Cell invasion; Systemic spreading; Persistence; Immunohistochemistry; Intracellular
16.  The effect of pre-enrichment on recovery of Streptococcus agalactiae, Staphylococcus aureus and mycoplasma from bovine milk. 
Epidemiology and Infection  1989;103(3):465-474.
The study was conducted to determine whether pre-enrichment would increase sensitivity of detecting Streptococcus (Str.) agalactiae, Staphylococcus (S.) aureus, and mycoplasma in bovine milk. Two procedures were followed, one involving direct inoculation of milk on bovine blood agar, and the other involving preenrichment in broth followed by inoculation on agar. Logistic regression was used to predict the probability of isolation as a function of culture procedure and two additional covariates, the California Mastitis Test (CMT) score of the milk and the type of sample (indicating sample storage temperature and herd mastitis status). A total of 13778 milk samples was cultured for each of the three bacteria. By using results of both direct inoculation and pre-enrichment, the probability of isolation compared to use of direct inoculation only and adjusted for effects of other variables was increased 3.6-fold for Str. agalactiae, 1.6-fold for S. aureus and 1.7-fold for mycoplasma. The probability of isolation for all three bacteria increased as the CMT score increased. For Str. agalactiae, there was a statistical interaction predicting that enrichment improved the odds of isolation more from milk with high CMT scores than from milk with low scores. Results indicate that pre-enrichment can substantially increase the sensitivity of bacteriological screening of dairy cows for mastitis caused by Str. agalactiae, S. aureus, and mycoplasma.
PMCID: PMC2249543  PMID: 2691266
17.  The Novel Fibrinogen-Binding Protein FbsB Promotes Streptococcus agalactiae Invasion into Epithelial Cells  
Infection and Immunity  2004;72(6):3495-3504.
Streptococcus agalactiae is a major cause of bacterial sepsis and meningitis in human newborns. The interaction of S. agalactiae with host proteins and the entry into host cells thereby represent important virulence traits of these bacteria. The present report describes the identification of the fbsB gene, encoding a novel fibrinogen-binding protein that plays a crucial role in the invasion of S. agalactiae into human cells. In Western blots and enzyme-linked immunosorbent assay (ELISA) experiments, the FbsB protein was demonstrated to interact with soluble and immobilized fibrinogen. Binding studies showed the N-terminal 388 residues of FbsB and the Aα-subunit of human fibrinogen to recognize each other. By reverse transcription (RT)-PCR, the fbsB gene was shown to be cotranscribed with the gbs0851 gene in S. agalactiae. Deletion of the fbsB gene in the genome of S. agalactiae did not influence the binding of the bacteria to fibrinogen, suggesting that FbsB does not participate in the attachment of S. agalactiae to fibrinogen. In tissue culture experiments, however, the fbsB deletion mutant was severely impaired in its invasion into lung epithelial cells. Bacterial invasion could be reestablished by introducing the fbsB gene on a shuttle plasmid into the fbsB deletion mutant. Furthermore, treatment of lung epithelial cells with FbsB fusion protein blocked S. agalactiae invasion of epithelial cells in a dose-dependent fashion. These results suggest an important role of the FbsB protein in the overall process of host cell entry by S. agalactiae.
doi:10.1128/IAI.72.6.3495-3504.2004
PMCID: PMC415667  PMID: 15155657
18.  Temporal Characterization of Carrot Broth-Enhanced Real-Time PCR as an Alternative Means for Rapid Detection of Streptococcus agalactiae from Prenatal Anorectal and Vaginal Screenings ▿  
Journal of Clinical Microbiology  2010;48(12):4495-4500.
Analysis of overnight carrot broth culture using the BD GeneOhm StrepB assay (carrot broth-enhanced PCR) yields increased sensitivity compared to that of carrot broth culture alone for the detection of Streptococcus agalactiae. We investigated the prospect of reducing the carrot broth incubation time prior to PCR performance. In vitro experimentation demonstrated that carrot broth-enhanced PCR nominally detected 10 CFU S. agalactiae after 4 h of carrot broth incubation with competitive flora. Detection rates improved with inocula of 100 and 1,000 CFU S. agalactiae, with the majority of these aliquots demonstrating detection after 2 h of carrot broth incubation. Carrot broth was prospectively inoculated with clinical vaginal/anorectal swabs, with 500-μl aliquots collected. Early aliquots from 227 specimens were subjected to carrot broth-enhanced PCR (early-aliquot carrot broth-enhanced PCR) in instances of subsequent positive carrot broth culture or positive overnight clinical carrot broth-enhanced PCR. The S. agalactiae detection rate by early-aliquot carrot broth-enhanced PCR (66.1%) exceeded that observed for 227 remnant swabs retrospectively tested by direct swab PCR (56.4%; P = 0.03). Early-aliquot carrot broth-enhanced PCR detection rate differences were most pronounced in aliquots from 83 carrot broth aliquots collected after 6 h (84.3%) compared to detection rates from either direct swab PCR of these samples (51.8%; P < 0.0002) or early-aliquot carrot broth-enhanced PCR of 144 carrot broth aliquots collected after fewer than 6 h of incubation (55.6%; P < 0.0002). Enhanced sensitivity of early-aliquot carrot broth-enhanced PCR versus direct swab PCR suggests that this assay could serve as a surrogate rapid detection method facilitating the prevention of group B streptococcal disease.
doi:10.1128/JCM.01734-10
PMCID: PMC3008479  PMID: 20980578
19.  Xer1-Mediated Site-Specific DNA Inversions and Excisions in Mycoplasma agalactiae▿ ‡  
Journal of Bacteriology  2010;192(17):4462-4473.
Surface antigen variation in Mycoplasma agalactiae, the etiologic agent of contagious agalactia in sheep and goats, is governed by site-specific recombination within the vpma multigene locus encoding the Vpma family of variable surface lipoproteins. This high-frequency Vpma phase switching was previously shown to be mediated by a Xer1 recombinase encoded adjacent to the vpma locus. In this study, it was demonstrated in Escherichia coli that the Xer1 recombinase is responsible for catalyzing vpma gene inversions between recombination sites (RS) located in the 5′-untranslated region (UTR) in all six vpma genes, causing cleavage and strand exchange within a 21-bp conserved region that serves as a recognition sequence. It was further shown that the outcome of the site-specific recombination event depends on the orientation of the two vpma RS, as direct or inverted repeats. While recombination between inverted vpma RS led to inversions, recombination between direct repeat vpma RS led to excisions. Using a newly developed excision assay based on the lacZ reporter system, we were able to successfully demonstrate under native conditions that such Xer1-mediated excisions can indeed also occur in the M. agalactiae type strain PG2, whereas they were not observed in the control xer1-disrupted VpmaY phase-locked mutant (PLMY), which lacks Xer1 recombinase. Unless there are specific regulatory mechanisms preventing such excisions, this might be the cost that the pathogen has to render at the population level for maintaining this high-frequency phase variation machinery.
doi:10.1128/JB.01537-09
PMCID: PMC2937384  PMID: 20562305
20.  The Transcriptional Regulator RovS Controls the Attachment of Streptococcus agalactiae to Human Epithelial Cells and the Expression of Virulence Genes  
Infection and Immunity  2006;74(10):5625-5635.
Streptococcus agalactiae is part of the normal flora of the human gastrointestinal tract and also the leading cause of bacterial infections in human newborns and immunocompromised adults. The colonization and infection of different regions within the human host require a regulatory network in S. agalactiae that senses environmental stimuli and controls the formation of specific virulence factors. In the present study, we characterized an Rgg-like transcriptional regulator, designated RovS (regulator of virulence in Streptococcus agalactiae). Deletion of the rovS gene in the genome of S. agalactiae resulted in strain 6313 ΔrovS, which exhibited an increased attachment to immobilized fibrinogen and a significant increase in adherence to the eukaryotic lung epithelial cell line A549. Quantification of expression levels of known and putative S. agalactiae virulence genes by real-time PCR revealed that RovS influences the expression of fbsA, gbs0230, sodA, rogB, and the cyl operon. The altered gene expression in mutant 6313 ΔrovS was restored by plasmid-mediated expression of rovS, confirming the RovS deficiency as the cause for the observed changes in virulence gene expression in S. agalactiae. DNA electrophoretic mobility shift assays showed that RovS specifically binds to the promoter regions of fbsA, gbs0230, sodA, and the cyl operon, indicating that RovS directly regulates their expression. Deletion and mutation studies in the promoter region of fbsA, encoding the main fibrinogen receptor in S. agalactiae, identified a RovS DNA motif. Similar motifs were also found in the promoter regions of gbs0230, sodA, and the cyl operon, and alignments allowed us to propose a consensus sequence for the DNA-binding site of RovS.
doi:10.1128/IAI.00667-06
PMCID: PMC1594887  PMID: 16988238
21.  Relevance of Peptide Uptake Systems to the Physiology and Virulence of Streptococcus agalactiae 
Journal of Bacteriology  2004;186(5):1398-1408.
Streptococcus agalactiae is a major cause of invasive infections in human newborns. To satisfy its growth requirements, S. agalactiae takes up 9 of the 20 proteinogenic amino acids from the environment. Defined S. agalactiae mutants in one or several of four putative peptide permease systems were constructed and tested for peptide uptake, growth in various media, and expression of virulence traits. Oligopeptide uptake by S. agalactiae was shown to be mediated by the ABC transporter OppA1-F, which possesses two substrate-binding proteins (OppA1 and OppA2) with overlapping substrate specificities. Dipeptides were found to be taken up in parallel by the oligopeptide permease OppA1-F, by the dipeptide ABC transporter DppA-E, and by the dipeptide symporter DpsA. Reverse transcription-PCR analysis revealed a polycistronic organization of the genes oppA1-F and dppA-E and a monocistronic organization of dpsA in S. agalactiae. The results of quantitative real-time PCR revealed a medium-dependent expression of the operons dppA-E and oppA1-F in S. agalactiae. Growth of S. agalactiae in human amniotic fluid was shown to require an intact dpsA gene, indicating an important role of DpsA during the infection of the amniotic cavity by S. agalactiae. Deletion of the oppB gene reduced the adherence of S. agalactiae to epithelial cells by 26%, impaired its adherence to fibrinogen and fibronectin by 42 and 33%, respectively, and caused a 35% reduction in expression of the fbsA gene, which encodes a fibrinogen-binding protein in S. agalactiae. These data indicate that the oligopeptide permease is involved in modulating virulence traits and virulence gene expression in S. agalactiae.
doi:10.1128/JB.186.5.1398-1408.2004
PMCID: PMC344423  PMID: 14973032
22.  Comparison of different sampling techniques and of different culture methods for detection of group B streptococcus carriage in pregnant women 
BMC Infectious Diseases  2010;10:285.
Background
Streptococcus agalactiae (group B streptococcus; GBS) is a significant cause of perinatal and neonatal infections worldwide. To detect GBS colonization in pregnant women, the CDC recommends isolation of the bacterium from vaginal and anorectal swab samples by growth in a selective enrichment medium, such as Lim broth (Todd-Hewitt broth supplemented with selective antibiotics), followed by subculture on sheep blood agar. However, this procedure may require 48 h to complete. We compared different sampling and culture techniques for the detection of GBS.
Methods
A total of 300 swabs was taken from 100 pregnant women at 35-37 weeks of gestation. For each subject, one rectovaginal, one vaginal and one rectal ESwab were collected. Plating onto Columbia CNA agar (CNA), group B streptococcus differential agar (GBSDA) (Granada Medium) and chromID Strepto B agar (CA), with and without Lim broth enrichment, were compared. The isolates were confirmed as S. agalactiae using the CAMP test on blood agar and by molecular identification with tDNA-PCR or by 16S rRNA gene sequence determination.
Results
The overall GBS colonization rate was 22%. GBS positivity for rectovaginal sampling (100%) was significantly higher than detection on the basis of vaginal sampling (50%), but not significantly higher than for rectal sampling (82%). Direct plating of the rectovaginal swab on CNA, GBSDA and CA resulted in detection of 59, 91 and 95% of the carriers, respectively, whereas subculturing of Lim broth yielded 77, 95 and 100% positivity, respectively. Lim broth enrichment enabled the detection of only one additional GBS positive subject. There was no significant difference between GBSDA and CA, whereas both were more sensitive than CNA. Direct culture onto GBSDA or CA (91 and 95%) detected more carriers than Lim broth enrichment and subculture onto CNA (77%). One false negative isolate was observed on GBSDA, and three false positives on CA.
Conclusions
In conclusion, rectovaginal sampling increased the number GBS positive women detected, compared to vaginal and/or rectal sampling. Direct plating on CA and/or GBSDA provided rapid detection of GBS that was at least as sensitive and specific as the CDC recommended method of Lim broth subcultured onto non chromogenic agar.
doi:10.1186/1471-2334-10-285
PMCID: PMC2956727  PMID: 20920213
23.  Prevalence of contagious mastitis pathogens in bulk tank milk in Prince Edward Island 
The Canadian Veterinary Journal  2006;47(6):567-572.
Abstract
The purpose of this study was to 1) estimate the herd prevalence of contagious mastitis pathogens in bulk milk from Prince Edward Island (PEI) dairy farms, 2) determine the association between bulk milk culture results and mean bulk milk somatic cell count (BMSCC), and 3) investigate the agreement of repeated bulk milk cultures. Three consecutive bulk milk samples were obtained at weekly intervals from all 258 PEI dairy herds and were cultured using routine laboratory methods. Cumulative prevalence of Staphylococcus aureus, Streptococcus agalactiae, and Mycoplasma spp. (M. bovis and M. alkalescens) was 74%, 1.6%, and 1.9%, respectively. Bulk milk somatic cell count of Staph. aureus-positive herds was higher than that of negative herds. Agreement for Staph. aureus isolation between 3 consecutive tests was moderate (kappa = 0.46). Mycoplasma bovis and M. alkalescens in bulk milk are being reported for the 1st time in PEI ever and in Canada since 1972.
PMCID: PMC1461414  PMID: 16808229
24.  Rapid Diagnosis of Bacterial Meningitis by Real-Time PCR and Fluorescence In Situ Hybridization 
Journal of Clinical Microbiology  2005;43(7):3390-3397.
Real-time PCR and fluorescence in situ hybridization (FISH) were evaluated as rapid methods for the diagnosis of bacterial meningitis and compared to standard diagnostic procedures. For PCR, a LightCycler approach was chosen, implementing eubacterial and specific PCR assays for the most relevant bacteria. For FISH, a similar probe set containing eubacterial and specific probes was composed of published and newly designed probes. Both methods were evaluated by use of cerebrospinal fluid (CSF) samples from patients with suspected bacterial meningitis. For all microscopy- and culture-positive samples (n = 28), the eubacterial PCR was positive. In addition, all identifiable pathogens were detected with specific PCR assays, according to an algorithm based on the Gram stain. The FISH method detected the pathogen in 13 of 18 positive samples. While the FISH method remained negative for all microscopy- and culture-negative samples (n = 113), the eubacterial PCR was positive for five of these samples. Sequencing of the amplicon revealed the presence of Neisseria meningitidis, Streptococcus agalactiae, and Haemophilus influenzae in three of these five samples. In addition, samples with discordant results by culture and microscopy were successfully investigated by PCR (10 samples) and FISH (5 samples). In conclusion, PCR is a highly sensitive tool for rapid diagnosis of bacterial meningitis. FISH is less sensitive but is useful for the identification of CSF samples showing bacteria in the Gram stain. Based on our results, an approach for laboratory diagnosis of meningitis including PCR and FISH is discussed.
doi:10.1128/JCM.43.7.3390-3397.2005
PMCID: PMC1169125  PMID: 16000464
25.  Characterization of P40, a Cytadhesin of Mycoplasma agalactiae  
Infection and Immunity  2002;70(10):5612-5621.
An immunodominant protein, P40, of Mycoplasma agalactiae was analyzed genetically and functionally. The gene encoding P40 was cloned from type strain PG2, sequenced, submitted to point mutagenesis in order to convert mycoplasma-specific TGATrp codon to the universal TGGTrp codon, and subsequently expressed in Escherichia coli. Nucleotide sequence-derived amino acid sequence comparisons revealed a similarity of P40 to the adhesin P50 of Mycoplasma hominis and to protein P89 of Spiroplasma citri, which is expected to be involved in adhesion. The amino acid sequence of P40 revealed a recognition site for a signal peptidase and strong antigenic and hydrophilic motifs in the C-terminal domain. Triton X-114 phase partitioning confirmed that P40 is a membrane protein. Fab fragments of antibodies directed against recombinant purified P40 significantly inhibited adherence of M. agalactiae strains PG2 to lamb joint synovial cells LSM 192. Sera taken sequentially from sheep infected with PG2 revealed that P40 induced a strong and persistent immune response that gave strong signals on immunoblots containing recombinant P40 even 3 months after infection. The gene encoding P40 was present in a single copy in all of the 26 field strains of M. agalactiae analyzed and was not detected in closely related mycoplasma species. P40 was expressed as a protein with an apparent molecular mass of 37 kDa on sodium dodecyl sulfate-acrylamide gels by all M. agalactiae strains except for serotype C strains, which showed nonsense mutations in their p40 genes.
doi:10.1128/IAI.70.10.5612-5621.2002
PMCID: PMC128363  PMID: 12228289

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