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1.  Complete Genome Sequence of Mycoplasma putrefaciens Strain 9231, One of the Agents of Contagious Agalactia in Goats 
Genome Announcements  2013;1(3):e00354-13.
Mycoplasma putrefaciens is one of the etiologic agents of contagious agalactia in goats. We report herein the complete genome sequence of Mycoplasma putrefaciens strain 9231.
doi:10.1128/genomeA.00354-13
PMCID: PMC3707581  PMID: 23766410
2.  Dissimilatory Fe(III) and Mn(IV) Reduction by Shewanella putrefaciens Requires ferE, a Homolog of the pulE (gspE) Type II Protein Secretion Gene 
Journal of Bacteriology  2002;184(1):142-151.
Shewanella putrefaciens strain 200 respires anaerobically on a wide range of compounds as the sole terminal electron acceptor, including ferric iron [Fe(III)] and manganese oxide [Mn(IV)]. Previous studies demonstrated that a 23.3-kb S. putrefaciens wild-type DNA fragment conferred metal reduction capability to a set of respiratory mutants with impaired Fe(III) and Mn(IV) reduction activities (T. DiChristina and E. DeLong, J. Bacteriol. 176:1468–1474, 1994). In the present study, the smallest complementing fragment was found to contain one open reading frame (ORF) (ferE) whose translated product displayed 87% sequence similarity to Aeromonas hydrophila ExeE, a member of the PulE (GspE) family of proteins found in type II protein secretion systems. Insertional mutants E726 and E912, constructed by targeted replacement of wild-type ferE with an insertionally inactivated ferE construct, were unable to respire anaerobically on Fe(III) or Mn(IV) yet retained the ability to grow on all other terminal electron acceptors. Nucleotide sequence analysis of regions flanking ferE revealed the presence of one partial and two complete ORFs whose translated products displayed 55 to 70% sequence similarity to the PulD, -F, and -G homologs of type II secretion systems. A contiguous cluster of 12 type II secretion genes (pulC to -N homologs) was found in the unannotated genome sequence of Shewanella oneidensis (formerly S. putrefaciens) MR-1. A 91-kDa heme-containing protein involved in Fe(III) reduction was present in the peripheral proteins loosely attached to the outside face of the outer membrane of the wild-type and complemented (Fer+) B31 transconjugates yet was missing from this location in Fer mutants E912 and B31 and in uncomplemented (Fer−) B31 transconjugates. Membrane fractionation studies with the wild-type strain supported this finding: the 91-kDa heme-containing protein was detected with the outer membrane fraction and not with the inner membrane or soluble fraction. These findings provide the first genetic evidence linking dissimilatory metal reduction to type II protein secretion and provide additional biochemical evidence supporting outer membrane localization of S. putrefaciens proteins involved in anaerobic respiration on Fe(III) and Mn(IV).
doi:10.1128/JB.184.1.142-151.2002
PMCID: PMC134750  PMID: 11741854
3.  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
4.  VNTR analysis reveals unexpected genetic diversity within Mycoplasma agalactiae, the main causative agent of contagious agalactia 
BMC Microbiology  2008;8:193.
Background
Mycoplasma agalactiae is the main cause of contagious agalactia, a serious disease of sheep and goats, which has major clinical and economic impacts. Previous studies of M. agalactiae have shown it to be unusually homogeneous and there are currently no available epidemiological techniques which enable a high degree of strain differentiation.
Results
We have developed variable number tandem repeat (VNTR) analysis using the sequenced genome of the M. agalactiae type strain PG2. The PG2 genome was found to be replete with tandem repeat sequences and 4 were chosen for further analysis. VNTR 5 was located within the hypothetical protein MAG6170 a predicted lipoprotein. VNTR 14 was intergenic between the hypothetical protein MAG3350 and the hypothetical protein MAG3340. VNTR 17 was intergenic between the hypothetical protein MAG4060 and the hypothetical protein MAG4070 and VNTR 19 spanned the 5' end of the pseudogene for a lipoprotein MAG4310 and the 3' end of the hypothetical lipoprotein MAG4320.
We have investigated the genetic diversity of 88 M. agalactiae isolates of wide geographic origin using VNTR analysis and compared it with pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) analysis. Simpson's index of diversity was calculated to be 0.324 for PFGE and 0.574 for VNTR analysis. VNTR analysis revealed unexpected diversity within M. agalactiae with 9 different VNTR types discovered. Some correlation was found between geographical origin and the VNTR type of the isolates.
Conclusion
VNTR analysis represents a useful, rapid first-line test for use in molecular epidemiological analysis of M. agalactiae for outbreak tracing and control.
doi:10.1186/1471-2180-8-193
PMCID: PMC2585094  PMID: 18992155
5.  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
6.  Shewanella and Photobacterium spp. in Oysters and Seawater from the Delaware Bay▿  
Applied and Environmental Microbiology  2008;74(11):3323-3327.
Shewanella algae, S. putrefaciens, and Photobacterium damselae subsp. damselae are indigenous marine bacteria and human pathogens causing cellulitis, necrotizing fasciitis, abscesses, septicemia, and death. Infections are rare and are most often associated with the immunocompromised host. A study was performed on the microbiological flora of oysters and seawater from commercial oyster harvesting sites in the Delaware Bay, New Jersey. From 276 water and shellfish samples tested, 1,421 bacterial isolates were picked for biochemical identification and 170 (12.0%) of the isolates were presumptively identified as S. putrefaciens, 26 (1.8%) were presumptively identified as P. damselae subsp. damselae, and 665 (46.8%) could not be identified using the API 20E identification database. Sequencing of the 16S rRNA genes of 22 S. putrefaciens-like isolates identified them as S. abalonesis, S. algae, S. baltica, S. hafniensis, S. marisflavi, S. putrefaciens, Listonella anguillarum, and P. damselae. Beta-hemolysis was produced by some S. algae and P. damselae isolates, while isolates of S. baltica and L. anguillarum, species perceived as nonpathogenic, also exhibited β-hemolysis and growth at 37°C. To our knowledge, this is the first time these beta-hemolytic strains were reported from shellfish or seawater from the Delaware Bay. Pathogenic Shewanella and Photobacterium species could pose a health threat through the ingestion of contaminated seafood, by cuts or abrasions acquired in the marine environment, or by swimming and other recreational activities.
doi:10.1128/AEM.00060-08
PMCID: PMC2423050  PMID: 18378645
7.  Emergence of Atypical Mycoplasma agalactiae Strains Harboring a New Prophage and Associated with an Alpine Wild Ungulate Mortality Episode 
Applied and Environmental Microbiology  2012;78(13):4659-4668.
The bacterium Mycoplasma agalactiae is responsible for contagious agalactia (CA) in small domestic ruminants, a syndrome listed by the World Organization for Animal Health and responsible for severe damage to the dairy industry. Recently, we frequently isolated this pathogen from lung lesions of ibexes during a mortality episode in the French Alps. This situation was unusual in terms of host specificity and tissue tropism, raising the question of M. agalactiae emergence in wildlife. To address this issue, the ibex isolates were characterized using a combination of approaches that included antigenic profiles, molecular typing, optical mapping, and whole-genome sequencing. Genome analyses showed the presence of a new, large prophage containing 35 coding sequences (CDS) that was detected in most but not all ibex strains and has a homolog in Mycoplasma conjunctivae, a species causing keratoconjunctivitis in wild ungulates. This and the presence in all strains of large integrated conjugative elements suggested highly dynamic genomes. Nevertheless, M. agalactiae strains circulating in the ibex population were shown to be highly related, most likely originating from a single parental clone that has also spread to another wild ungulate species of the same geographical area, the chamois. These strains clearly differ from strains described in Europe so far, including those found nearby, before CA eradication a few years ago. While M. agalactiae pathogenicity in ibexes remains unclear, our data showed the emergence of atypical strains in Alpine wild ungulates, raising the question of a role for the wild fauna as a potential reservoir of pathogenic mycoplasmas.
doi:10.1128/AEM.00332-12
PMCID: PMC3370481  PMID: 22522685
8.  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
9.  Differentiation of Shewanella putrefaciens and Shewanella alga on the basis of whole-cell protein profiles, ribotyping, phenotypic characterization, and 16S rRNA gene sequence analysis. 
Seventy-six presumed Shewanella putrefaciens isolates from fish, oil drillings, and clinical specimens, the type strain of Shewanella putrefaciens (ATCC 8071), the type strain of Shewanella alga (IAM 14159), and the type strain of Shewanella hanedai (ATCC 33224) were compared by several typing methods. Numerical analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell protein and ribotyping patterns showed that the strains were separated into two distinct clusters with 56% +/- 10% and 40% +/- 14% similarity for whole-cell protein profiling and ribotyping, respectively. One cluster consisted of 26 isolates with 52 to 55 mol% G + C and included 15 human isolates, mostly clinical specimens, 8 isolates from marine waters, and the type strain of S. alga. This homogeneous cluster of mesophilic, halotolerant strains was by all analyses identical to the recently defined species S. alga (U. Simidu et al., Int. J. Syst. Bacteriol, 40:331-336, 1990). Fifty-two typically psychrotolerant strains formed the other, more heterogeneous major cluster, with 43 to 47 mol% G + C. The type strain of S. putrefaciens was included in this group. The two groups were confirmed by 16S rRNA gene sequence analysis. It is concluded that the isolates must be considered two different species, S. alga and S. putrefaciens, and that most mesophilic isolates formerly identified as S. putrefaciens belong to S. alga. The ecological role and potential pathogenicity of S. alga can be evaluated only if the organism is correctly identified.
PMCID: PMC168511  PMID: 9172338
10.  Shewanella putrefaciens mtrB Encodes an Outer Membrane Protein Required for Fe(III) and Mn(IV) Reduction 
Journal of Bacteriology  1998;180(23):6292-6297.
Iron and manganese oxides or oxyhydroxides are abundant transition metals, and in aquatic environments they serve as terminal electron acceptors for a large number of bacterial species. The molecular mechanisms of anaerobic metal reduction, however, are not understood. Shewanella putrefaciens is a facultative anaerobe that uses Fe(III) and Mn(IV) as terminal electron acceptors during anaerobic respiration. Transposon mutagenesis was used to generate mutants of S. putrefaciens, and one such mutant, SR-21, was analyzed in detail. Growth and enzyme assays indicated that the mutation in SR-21 resulted in loss of Fe(III) and Mn(IV) reduction but did not affect its ability to reduce other electron acceptors used by the wild type. This deficiency was due to Tn5 inactivation of an open reading frame (ORF) designated mtrB. mtrB encodes a protein of 679 amino acids and contains a signal sequence characteristic of secreted proteins. Analysis of membrane fractions of the mutant, SR-21, and wild-type cells indicated that MtrB is located on the outer membrane of S. putrefaciens. A 5.2-kb DNA fragment that contains mtrB was isolated and completely sequenced. A second ORF, designated mtrA, was found directly upstream of mtrB. The two ORFs appear to be arranged in an operon. mtrA encodes a putative 10-heme c-type cytochrome of 333 amino acids. The N-terminal sequence of MtrA contains a potential signal sequence for secretion across the cell membrane. The amino acid sequence of MtrA exhibited 34% identity to NrfB from Escherichia coli, which is involved in formate-dependent nitrite reduction. To our knowledge, this is the first report of genes encoding proteins involved in metal reduction.
PMCID: PMC107715  PMID: 9829939
11.  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
12.  In Vitro Susceptibilities of Mycoplasma putrefaciens Field Isolates▿  
MICs were determined for 15 antimicrobial agents against 37 Mycoplasma putrefaciens isolates. The most effective antimicrobial drug classes were the fluoroquinolones, the tetracyclines, the lincosamide lincomycin, and the macrolides. The susceptibility profile of the isolates correlated with the geographic origin. This is the first report of decreased susceptibility to the macrolides, lincomycin, and the tetracyclines in M. putrefaciens strains.
doi:10.1128/AAC.00420-07
PMCID: PMC2043204  PMID: 17638695
13.  Variable Surface Protein Vmm of Mycoplasma mycoides subsp. mycoides Small Colony Type 
Journal of Bacteriology  2002;184(13):3712-3722.
A variable surface protein, Vmm, of the bovine pathogen Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides SC) has been identified and characterized. Vmm was specific for the SC biotype and was expressed by 68 of 69 analyzed M. mycoides SC strains. The protein was found to undergo reversible phase variation at a frequency of 9 × 10−4 to 5 × 10−5 per cell per generation. The vmm gene was present in all of the 69 tested M. mycoides SC strains and encodes a lipoprotein precursor of 59 amino acids (aa), where the mature protein was predicted to be 36 aa and was anchored to the membrane by only the lipid moiety, as no transmembrane region could be identified. DNA sequencing of the vmm gene region from ON and OFF clones showed that the expression of Vmm was regulated at the transcriptional level by dinucleotide insertions or deletions in a repetitive region of the promoter spacer. Vmm-like genes were also found in four closely related mycoplasmas, Mycoplasma capricolum subsp. capricolum, M. capricolum subsp . capripneumoniae, Mycoplasma sp. bovine serogroup 7, and Mycoplasma putrefaciens. However, Vmm could not be detected in whole-cell lysates of these species, suggesting that the proteins encoded by the vmm-like genes lack the binding epitope for the monoclonal antibody used in this study or, alternatively, that the Vmm-like proteins were not expressed.
doi:10.1128/JB.184.13.3712-3722.2002
PMCID: PMC135138  PMID: 12057968
14.  A Conserved Histidine in Cytochrome c Maturation Permease CcmB of Shewanella putrefaciens Is Required for Anaerobic Growth below a Threshold Standard Redox Potential▿ †  
Journal of Bacteriology  2006;189(3):1036-1043.
Shewanella putrefaciens strain 200 respires a wide range of compounds as terminal electron acceptor. The respiratory versatility of Shewanella is attributed in part to a set of c-type cytochromes with widely varying midpoint redox potentials (E′0). A point mutant of S. putrefaciens, originally designated Urr14 and here renamed CCMB1, was found to grow at wild-type rates on electron acceptors with high E′0 [O2, NO3−, Fe(III) citrate, MnO2, and Mn(III) pyrophosphate] yet was severely impaired for growth on electron acceptors with low E′0 [NO2−, U(VI), dimethyl sulfoxide, TMAO (trimethylamine N-oxide), fumarate, γ-FeOOH, SO32−, and S2O32−]. Genetic complementation and nucleotide sequence analyses indicated that the CCMB1 respiratory mutant phenotype was due to mutation of a conserved histidine residue (H108Y) in a protein that displayed high homology to Escherichia coli CcmB, the permease subunit of an ABC transporter involved in cytochrome c maturation. Although CCMB1 retained the ability to grow on electron acceptors with high E′0, the cytochrome content of CCMB1 was <10% of that of the wild-type strain. Periplasmic extracts of CCMB1 contained slightly greater concentrations of the thiol functional group (-SH) than did the wild-type strain, an indication that the Eh of the CCMB1 periplasm was abnormally low. A ccmB deletion mutant was unable to respire anaerobically on any electron acceptor, yet retained aerobic respiratory capability. These results suggest that the mutation of a conserved histidine residue (H108) in CCMB1 alters the redox homeostasis of the periplasm during anaerobic growth on electron acceptors with low (but not high) E′0. This is the first report of the effects of Ccm deficiencies on bacterial respiration of electron acceptors whose E′0 nearly span the entire redox continuum.
doi:10.1128/JB.01249-06
PMCID: PMC1797334  PMID: 17142390
15.  Roles of UndA and MtrC of Shewanella putrefaciens W3-18-1 in iron reduction 
BMC Microbiology  2013;13:267.
Background
The completion of genome sequencing in a number of Shewanella species, which are most renowned for their metal reduction capacity, offers a basis for comparative studies. Previous work in Shewanella oneidensis MR-1 has indicated that some genes within a cluster (mtrBAC-omcA-mtrFED) were involved in iron reduction. To explore new features of iron reduction pathways, we experimentally analyzed Shewanella putrefaciens W3-18-1 since its gene cluster is considerably different from that of MR-1 in that the gene cluster encodes only four ORFs.
Results
Among the gene cluster, two genes (mtrC and undA) were shown to encode c-type cytochromes. The ΔmtrC deletion mutant revealed significant deficiencies in reducing metals of Fe2O3, α-FeO(OH), β-FeO(OH), ferric citrate, Mn(IV) and Co(III), but not organic compounds. In contrast, no deficiency of metal reduction was observed in the ΔundA deletion mutant. Nonetheless, undA deletion resulted in progressively slower iron reduction in the absence of mtrC and fitness loss under the iron-using condition, which was indicative of a functional role of UndA in iron reduction.
Conclusions
These results provide physiological and biochemical evidences that UndA and MtrC of Shewanella putrefaciens W3-18-1 are involved in iron reduction.
doi:10.1186/1471-2180-13-267
PMCID: PMC4222724  PMID: 24274142
Shewanella putrefaciens W3-18-1; Iron reduction; c-type cytochrome
16.  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
17.  Genome Sequence of Mycoplasma capricolum subsp. capripneumoniae Strain M1601 
Journal of Bacteriology  2011;193(21):6098-6099.
Mycoplasma capricolum subsp. capripneumoniae is the causative agent of contagious caprine pleuropneumonia, a devastating disease of goats listed by the World Organization for Animal Health. Here we report the first complete genome sequence of this organism (strain M1601, a clinically isolated strain from China).
doi:10.1128/JB.05980-11
PMCID: PMC3194924  PMID: 21994928
18.  Mycoplasmoses of ruminants in France: recent data from the national surveillance network 
Background
Ruminant mycoplasmoses are important diseases worldwide and several are listed by the World Organization for Animal Health to be of major economic significance. In France the distribution of mycoplasmal species isolated from clinical samples collected from diseased animals upon veterinary request, is monitored by a network known as VIGIMYC (for VIGIlance to MYCoplasmoses of ruminants). The veterinary diagnostic laboratories collaborating with VIGIMYC are responsible for isolating the mycoplasmas while identification of the isolates is centralized by the French Food Safety Agency (AFSSA) in Lyon. The VIGIMYC framework can also be used for specific surveys and one example, on the prevalence of M. bovis in bovine respiratory diseases, is presented here.
Results
Between 2003 and 2008, 34 laboratories were involved in the network and 1904 mycoplasma isolates, originating from the main ruminant-breeding areas, were identified. For cattle, the high prevalence of M. bovis in bronchopneumonia, notably in young animals, was confirmed by VIGIMYC and an associated specific survey, whereas the non-emergence of species such as M. alkalescens and M. canis was also demonstrated. The etiological agent of bovine contagious pleuropneumonia was never isolated. The principal mycoplasmosis in goats was contagious agalactia with M. mycoides subsp. capri as main agent. Ovine mycoplasmoses, most of which were associated with pneumonia in lambs, were infrequently reported. One exception was ovine contagious agalactia (due to M. agalactiae) that has recently re-emerged in the Pyrénées where it had been endemic for years and was also reported in Corsica, which was previously considered free.
Conclusions
Although VIGIMYC is a passive network and somewhat biased as regards sample collection and processing, it has provided, in this study, an overview of the main mycoplasmoses of ruminants in France. The French epidemiological situation is compared to those existing elsewhere in the world.
doi:10.1186/1746-6148-6-32
PMCID: PMC2892444  PMID: 20525406
19.  Mycoplasma mycoides, from "mycoides Small Colony" to "capri". A microevolutionary perspective 
BMC Genomics  2011;12:114.
Background
The Mycoplasma mycoides cluster consists of five species or subspecies that are ruminant pathogens. One subspecies, Mycoplasma mycoides subspecies mycoides Small Colony (MmmSC), is the causative agent of contagious bovine pleuropneumonia. Its very close relative, Mycoplasma mycoides subsp. capri (Mmc), is a more ubiquitous pathogen in small ruminants causing mastitis, arthritis, keratitis, pneumonia and septicaemia and is also found as saprophyte in the ear canal. To understand the genetics underlying these phenotypic differences, we compared the MmmSC PG1 type strain genome, which was already available, with the genome of an Mmc field strain (95010) that was sequenced in this study. We also compared the 95010 genome with the recently published genome of another Mmc strain (GM12) to evaluate Mmc strain diversity.
Results
The MmmSC PG1 genome is 1,212 kbp and that of Mmc 95010 is ca. 58 kbp shorter. Most of the sequences present in PG1 but not 95010 are highly repeated Insertion Sequences (three types of IS) and large duplicated DNA fragments. The 95010 genome contains five types of IS, present in fewer copies than in PG1, and two copies of an integrative conjugative element. These mobile genetic elements have played a key role in genome plasticity, leading to inversions of large DNA fragments. Comparison of the two genomes suggested a marked decay of the PG1 genome that seems to be correlated with a greater number of IS. The repertoire of gene families encoding surface proteins is smaller in PG1. Several genes involved in polysaccharide metabolism and protein degradation are also absent from, or degraded in, PG1.
Conclusions
The genome of MmmSC PG1 is larger than that of Mmc 95010, its very close relative, but has less coding capacity. This is the result of large genetic rearrangements due to mobile elements that have also led to marked gene decay. This is consistent with a non-adaptative genomic complexity theory, allowing duplications or pseudogenes to be maintained in the absence of adaptive selection that would lead to purifying selection and genome streamlining over longer evolutionary times. These findings also suggest that MmmSC only recently adapted to its bovine host.
doi:10.1186/1471-2164-12-114
PMCID: PMC3053259  PMID: 21324191
20.  Identification of a Plasmid-Encoded Gene from Haemophilus ducreyi Which Confers NAD Independence 
Journal of Bacteriology  2001;183(4):1168-1174.
Members of the family Pasteurellaceae are classified in part by whether or not they require an NAD supplement for growth on laboratory media. In this study, we demonstrate that this phenotype can be determined by a single gene, nadV, whose presence allows NAD-independent growth of Haemophilus influenzae and Actinobacillus pleuropneumoniae. This gene was cloned from a 5.2-kb plasmid which was previously shown to be responsible for NAD independence in Haemophilus ducreyi. When transformed into A. pleuropneumoniae, this cloned gene allowed NAD-independent growth on complex media and allowed the utilization of nicotinamide in place of NAD on defined media. Sequence analysis revealed an open reading frame of 1,482 bp that is predicted to encode a protein with a molecular mass of 55,619 Da. Compared with the sequence databases, NadV was found to have significant sequence homology to the human pre-B-cell colony-enhancing factor PBEF and to predicted proteins of unknown function identified in the bacterial species Mycoplasma genitalium, Mycoplasma pneumoniae, Shewanella putrefaciens, Synechocystis sp., Deinococcus radiodurans, Pasteurella multocida, and Actinobacillus actinomycetemcomitans. P. multocida and A. actinomycetemcomitans are among the NAD-independent members of the Pasteurellaceae. Homologues of NadV were not found in the sequenced genome of H. influenzae, an NAD-dependent member of the Pasteurellaceae, or in species known to utilize a different pathway for synthesis of NAD, such as Escherichia coli. Sequence alignment of these nine homologues revealed regions and residues of complete conservation that may be directly involved in the enzymatic activity. Identification of a function for this gene in the Pasteurellaceae should help to elucidate the role of its homologues in other species.
doi:10.1128/JB.183.4.1168-1174.2001
PMCID: PMC94989  PMID: 11157928
21.  Characterization of the 16S rRNA genes from Mycoplasma sp. strain F38 and development of an identification system based on PCR. 
Journal of Bacteriology  1994;176(9):2577-2586.
Mycoplasma sp. (strain F38) is the causative agent of contagious caprine pleuropneumonia, which is a goat disease of great global concern. Strain F38 belongs to the so-called "Mycoplasma mycoides cluster," and the members of this cluster have many biochemical and serological properties in common, which makes it difficult to differentiate between them by conventional methods. Their phylogenetic interrelationship are thus uncertain. The 16S rRNA gene of the rrnB operon from strain F38 was cloned and sequenced. The sequence was compared with the 16S rRNA sequences of related mycoplasmas, and phylogenetic trees were constructed by parsimony analysis. A three-way ambiguity among strain F38, Mycoplasma capricolum, and Mycoplasma sp. strain PG50 was observed in the trees. This observation is in agreement with a recent proposal to reclassify strain F38 and M. capricolum. A primer set was designed for in vitro amplification by PCR of a fragment of the 16S rRNA genes from the M. mycoides cluster. The amplimers of strain F38 could be distinguished easily from the corresponding amplimers from other members of the M. mycoides cluster by restriction enzyme analysis with PstI. This observation was utilized to design an identification system for strain F38. Part of the 16S rRNA gene of the rrnA operon from strain F38 was also cloned, and several sequence differences between the two rRNA operons were discovered, revealing microheterogeneity between the two 16S rRNA genes of this organism.
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PMCID: PMC205395  PMID: 8169205
22.  The cymA Gene, Encoding a Tetraheme c-Type Cytochrome, Is Required for Arsenate Respiration in Shewanella Species▿  
Journal of Bacteriology  2007;189(6):2283-2290.
In Shewanella sp. strain ANA-3, utilization of arsenate as a terminal electron acceptor is conferred by a two-gene operon, arrAB, which lacks a gene encoding a membrane-anchoring subunit for the soluble ArrAB protein complex. Analysis of the genome sequence of Shewanella putrefaciens strain CN-32 showed that it also contained the same arrAB operon with 100% nucleotide identity. Here, we report that CN-32 respires arsenate and that this metabolism is dependent on arrA and an additional gene encoding a membrane-associated tetraheme c-type cytochrome, cymA. Deletion of cymA in ANA-3 also eliminated growth on and reduction of arsenate. The ΔcymA strains of CN-32 and ANA-3 negatively affected the reduction of Fe(III) and Mn(IV) but not growth on nitrate. Unlike the CN-32 ΔcymA strain, growth on fumarate was absent in the ΔcymA strain of ANA-3. Both homologous and heterologous complementation of cymA in trans restored growth on arsenate in ΔcymA strains of both CN-32 and ANA-3. Transcription patterns of cymA showed that it was induced under anaerobic conditions in the presence of fumarate and arsenate. Nitrate-grown cells exhibited the greatest level of cymA expression in both wild-type strains. Lastly, site-directed mutagenesis of the first Cys to Ser in each of the four CXXCH c-heme binding motifs of the CN-32 CymA nearly eliminated growth on and reduction of arsenate. Together, these results indicate that the biochemical mechanism of arsenate respiration and reduction requires the interactions of ArrAB with a membrane-associated tetraheme cytochrome, which in the non-arsenate-respiring Shewanella species Shewanella oneidensis strain MR-1, has pleiotropic effects on Fe(III), Mn(IV), dimethyl sulfoxide, nitrate, nitrite, and fumarate respiration.
doi:10.1128/JB.01698-06
PMCID: PMC1899399  PMID: 17209025
23.  Siderophore-Mediated Iron Sequestering by Shewanella putrefaciens 
The iron-sequestering abilities of 51 strains of Shewanella putrefaciens isolated from different sources (fish, water, and warm-blooded animals) were assessed. Thirty strains (60%) produced siderophores in heat-sterilized fish juice as determined by the chrome-azurol-S assay. All cultures were negative for the catechol-type siderophore, whereas 24 of the 30 siderophore-producing strains tested positive in the Csáky test, indicating the production of siderophores of the hydroxamate type. Siderophore-producing S. putrefaciens could to some degree cross-feed on the siderophores of other S. putrefaciens strains and on compounds produced by an Aeromonas salmonicida strain under iron-limited conditions. The siderophores of S. putrefaciens were not sufficiently strong to inhibit growth of other bacteria under iron-restricted conditions. However, siderophore-producing Pseudomonas bacteria were always inhibitory to S. putrefaciens under iron-limited conditions. Growth of siderophore-producing strains under iron-limited conditions induced the formation of one major new outer membrane protein of approximately 72 kDa. Two outer membrane proteins of approximately 53 and 23 kDa were not seen when iron was restricted.
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PMCID: PMC201611  PMID: 16349298
24.  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
25.  Genes Found Essential in Other Mycoplasmas Are Dispensable in Mycoplasma bovis 
PLoS ONE  2014;9(6):e97100.
Mycoplasmas are regarded to be useful models for studying the minimum genetic complement required for independent survival of an organism. Mycoplasma bovis is a globally distributed pathogen causing pneumonia, mastitis, arthritis, otitis media and reproductive tract disease, and genome sequences of three strains, the type strain PG45 and two strains isolated in China, have been published. In this study, several Tn4001 based transposon constructs were generated and used to create a M. bovis PG45 insertional mutant library. Direct genome sequencing of 319 independent insertions detected disruptions in 129 genes in M. bovis, 48 of which had homologues in Mycoplasma mycoides subspecies mycoides SC and 99 of which had homologues in Mycoplasma agalactiae. Sixteen genes found to be essential in previous studies on other mycoplasma species were found to be dispensable. Five of these genes have previously been predicted to be part of the core set of 153 essential genes in mycoplasmas. Thus this study has extended the list of non-essential genes of mycoplasmas from that previously generated by studies in other species.
doi:10.1371/journal.pone.0097100
PMCID: PMC4045577  PMID: 24897538

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