In recent years, mycoplasma taxonomists have found that numerous mycoplasma strains from goats are serologically indistinguishable from Mycoplasma mycoides subsp. mycoides, the causative agent of contagious bovine pleuropneumonia (CBPP), by routinely used tests, e.g. the metabolism- and growth-inhibition tests. As a result, such organisms are now openly referred to as M. mycoides subsp. mycoides. Seven of these so-called M. mycoides subsp. mycoides strains from goats were compared with two strains of M. mycoides subsp. mycoides from CBPP, and with one strain of M. mycoides subsp. capri, by means of two in-vivo tests, namely, (1) a test of the ability of each strain, injected intraperitoneally into mice, to produce mycoplasmaemia, and (2) a cross-protection test in mice. Of the seven strains, only one ('O goat') was indistinguishable from genuine M. mycoides subsp. mycoides; it also had small colonies resembling those of genuine M. mycoides subsp. mycoides. The other six were easily distinguished from genuine M. mycoides subsp. mycoides, and they produced large colonies. These six strains and others like them should no longer be given a name that fails to distinguish them from the causative agent of CBPP. Cross-protection tests showed that the seven goat strains referred to above differed from M. mycoides subsp. capri.
In vivo methods were used to study the F38-type mycoplasma in parallel with related mycoplasmas. Three of five strains of 'bovine serogroup 7' with an unknown history of subculture produced mycoplasmaemia in mice inoculated intraperitoneally. A strain of 'bovine serogroup L' also produced mycoplasmaemia, but no evidence of similar ability could be found for single strains of Mycoplasma capricolum, M. equigenitalium and M. primatum, or for two strains of the F38-type mycoplasma. In cross-immunization tests a bovine serogroup 7 strain (NCTC 10133) and a strain ('Blenheim') of the SC (small colony) type of M. mycoides subsp. mycoides were used for the purpose of challenge. Cross-protection was described as 'complete' or 'partial', depending on whether it was as great as, or less than, that produced by homologous vaccine. Although strain NCTC 10133 protected strongly, possibly completely, against Blenheim, and Blenheim gave partial protection against NCTC 10133, challenge with NCTC 10133 and Blenheim gave strikingly different results. Thus (1) F38-type strains, M equigenitalium and M. primatum all gave partial cross-protection against NCTC 10133 but not against Blenheim, (2) NCTC 10133, unlike Blenheim, was seldom susceptible to partial cross-protection by LC (large colony) strains of M. mycoides subsp. mycoides, and (3) three SC strains - which would have protected completely against Blenheim - protected only partially against NCTC 10133. NCTC 10133 and Blenheim were similar, however, in that M. capricolum and M. mycoides subsp. capri failed to cross-protect against them both.
Small colony (SC) strains of Mycoplasma mycoides subsp. mycoides from contagious bovine pleuropneumonia (CBPP) and from goats were compared with large colony (LC) strains of so-called M. mycoides subsp. mycoides from goats and sheep by means of a cross-protection test in which mice were challenged with M. mycoides subsp. capri. Of 13 LC strains, all gave partial cross-protection, and 11 were shown to be more closely related than four SC strains to subspecies capri. In a further experiment, six SC strains--three from CBPP and three from goats--all gave weak partial cross-protection against subspecies capri.
Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small-colony type (SC) is among the most serious threats for livestock producers in Africa. Glycerol metabolism-associated H2O2 production seems to play a crucial role in virulence of this mycoplasma. A wide number of attenuated strains of M. mycoides subsp. mycoides SC are currently used in Africa as live vaccines. Glycerol metabolism is not affected in these vaccine strains and therefore it does not seem to be the determinant of their attenuation. A non-synonymous single nucleotide polymorphism (SNP) in the bgl gene coding for the 6-phospho-β-glucosidase (Bgl) has been described recently. The SNP differentiates virulent African strains isolated from outbreaks with severe CBPP, which express the Bgl isoform Val204, from strains to be considered less virulent isolated from CBPP outbreaks with low mortality and vaccine strains, which express the Bgl isoform Ala204.
Strains of M. mycoides subsp. mycoides SC considered virulent and possessing the Bgl isoform Val204, but not strains with the Bgl isoform Ala204, do trigger elevated levels of damage to embryonic bovine lung (EBL) cells upon incubation with the disaccharides (i.e., β-D-glucosides) sucrose and lactose. However, strains expressing the Bgl isoform Val204 show a lower hydrolysing activity on the chromogenic substrate p-nitrophenyl-β-D-glucopyranoside (pNPbG) when compared to strains that possess the Bgl isoform Ala204. Defective activity of Bgl in M. mycoides subsp. mycoides SC does not lead to H2O2 production. Rather, the viability during addition of β-D-glucosides in medium-free buffers is higher for strains harbouring the Bgl isoform Val204 than for those with the isoform Ala204.
Our results indicate that the studied SNP in the bgl gene is one possible cause of the difference in bacterial virulence among strains of M. mycoides subsp. mycoides SC. Bgl does not act as a direct virulence factor, but strains possessing the Bgl isoform Val204 with low hydrolysing activity are more prone to survive in environments that contain high levels of β-D-glucosides, thus contributing in some extent to mycoplasmaemia.
So-called LC strains of Mycoplasma mycoides subsp. mycoides and, where appropriate, SC strains, were examined, together with M. mycoides subsp. capri strains, by growth, fermentation, and antibiotic-sensitivity tests. Growth curves in BVF-OS medium showed that, from the 6th until at least the 19th day of incubation, strain MankefÃ¥r 2833 had a viable count strikingly higher than that of any other LC strain. Its robust growth properties may explain its ability--unusual among LC strains--to produce mycoplasmaemia readily in mice. Strain 143-A66 Conn, also shown by earlier experiments in mice to possess unusual properties, lost viability more rapidly than any other LC strain between the 13th and 19th days of incubation. The viable count of a subsp. capri strain was considerably lower than that of any LC strain for much of the period between days 6 and 19. In fermentation tests with 27 substrates and sensitivity tests with 11 antibiotics the strains gave results that, in all but the following respects, were uniform. Sorbitol was fermented to varying degrees by all the LC and subsp. capri strains tested but was unaffected by the SC strains. The LC and subsp. capri strains were in general more resistant than the SC strains to streptomycin. The growth of the LC strains, much more than that of the other strains, was greatly stimulated by the presence of fermentable substrate.
Much evidence of immunogenic heterogeneity among the LC strains of Mycoplasma mycoides ssp. mycoides emerged from cross-immunization and -hyper-immunization experiments in mice in which three LC strains (Vom/Plum Island, 74/2488, and MankefÃ¥r 2833) were used for challenge purposes. All heterologous LC-strain vaccines cross-immunized against the three challenge strains, but protection was usually only 'partial', i.e. significantly less than that given by homologous vaccine. Cross-hyperimmunization with all heterologous LC but not SC strains produced protection against challenge with Vom/Plum Island that was virtually 'complete', i.e. similar to that produced by homologous vaccine. Challenge with 74/2488 gave generally similar results; but against MankefÃ¥r 2833 six heterologous LC vaccines gave complete protection and six did not. Vaccines prepared from the Smith (1423) strain of M. mycoides ssp. capri gave some protection against Vom/Plum Island but none against 74/2488 or MankefÃ¥r 2833. The cross-immunizing ability of three further M. mycoides ssp. capri strains appeared to resemble that of Smith (1423). In a cross-hyperimmunization experiment, vaccines prepared from SC strains of M. mycoides ssp. mycoides varied greatly in their ability to protect against challenge with strains 74/2488 and MankefÃ¥r 2833.
Four strains of Mycoplasma mycoides subsp. mycoides small colony type (MmmSC) isolated from recent outbreaks of contagious bovine pleuropneumonia (CBPP) in Africa have been investigated. One Botswanan strain, M375, displayed numerous and significant phenotypic differences from both contemporary field isolates and older field and vaccine strains (African, Australian, and European strains dating back to 1936). Differences include altered morphology, reduced capsular polysaccharide production, high sensitivity to MmmSC rabbit hyperimmune antisera in vitro, and unique polymorphisms following immunoblotting. While insertion sequence analysis using IS1634 clearly indicates a close evolutionary relationship to west African strains, hybridization with IS1296 shows the absence of a band present in all other strains of MmmSC examined. The data suggest that a deletion has occurred in strain M375, which may explain its altered phenotype, including poor growth in vitro and a relative inability to cause septicemia in mice. These characteristics are also exhibited by Mycoplasma capricolum subsp. capripneumoniae (causal agent of contagious caprine pleuropneumonia [CCPP]), against which M375 antiserum exhibited some activity in vitro (unique among the various MmmSC antisera tested). These findings may have evolutionary implications, since CCPP is believed to be lung specific and without a septicemic phase (unlike CBPP). Since M375 was isolated from a clinical case of CBPP, this novel biotype may be fairly widespread but not normally isolated due to difficulty of culture and/or a potentially altered disease syndrome. Bovine convalescent antisera (obtained from contemporary naturally infected cattle in Botswana) were active against strain M375 in an in vitro growth inhibition test but not against any other strains of MmmSC tested. There exists the possibility therefore, that strain M375 may possess a set of protective antigens different from those of other strains of MmmSC (including vaccine strains). These findings have implications for the control of the current CBPP epidemic in Africa.
The ‘Mycoplasma mycoides cluster’ comprises the ruminant pathogens Mycoplasma mycoides subsp. mycoides the causative agent of contagious bovine pleuropneumonia (CBPP), Mycoplasma capricolum subsp. capripneumoniae the agent of contagious caprine pleuropneumonia (CCPP), Mycoplasma capricolum subsp. capricolum, Mycoplasma leachii and Mycoplasma mycoides subsp. capri. CBPP and CCPP are major livestock diseases and impact the agricultural sector especially in developing countries through reduced food-supply and international trade restrictions. In addition, these diseases are a threat to disease-free countries. We used a multilocus sequence typing (MLST) approach to gain insights into the demographic history of and phylogenetic relationships among the members of the ‘M. mycoides cluster’. We collected partial sequences from seven housekeeping genes representing a total of 3,816 base pairs from 118 strains within this cluster, and five strains isolated from wild Caprinae. Strikingly, the origin of the ‘M. mycoides cluster’ dates to about 10,000 years ago, suggesting that the establishment and spread of the cluster coincided with livestock domestication. In addition, we show that hybridization and recombination may be important factors in the evolutionary history of the cluster.
A latex agglutination test (LAT) has been developed for the diagnosis of contagious bovine pleuropneumonia (CBPP). The latex microspheres were coated with MmmSC polyclonal immunoglobulin G antiserum and detected MmmSC antigen in the serum of cattle infected with CBPP and in growth medium containing MmmSC. The specific antigen recognizsed by this test appeared to be the capsular polysaccharide (CPS). The LAT recognized all 23 strains of MmmSC examined in this study, with a sensitivity level of 2 ng of CPS, or the equivalent of 5 × 103 CFU, in a reaction volume of 0.03 ml. Therefore, rapid identification of MmmSC cultures should be possible. Agglutination was also observed with the related goat pathogens and “Mycoplasma mycoides” cluster members Mycoplasma mycoides subsp. mycoides large colony biotype (four of six strains positive) and Mycoplasma mycoides subsp. capri (three of six strains positive), in agreement with the suggestion that these latter two mycoplasmas may in fact represent a single species (although collectively exhibiting two capsular serotypes). Comparisons in diagnosis with the complement fixation test (CFT) were made by using African field sera from CBPP-infected cattle. After 2 (or 3) min of incubation, the test detected 55% (or 61%) of CFT-positive sera and 29% (or 40%) of CFT-negative sera, with an overall correlation in diagnosis of 62% (or 61%). The rates for false-positive diagnoses made by using “known” CBPP-negative sera from the United Kingdom were 3 or 13% after 2 or 3 min of incubation, respectively. The data agree with previous findings that some CBPP CFT-negative misdiagnoses may occur due to “antibody eclipsing” by excess circulating antigen. The LAT combines low cost and high specificity with ease of application in the field, without the need for any specialist training or equipment.
The genes encoding the 62-kDa lipoproteins from the
Mycoplasma mycoides subsp. mycoides
large-colony type (LC) strain Y-goat and the M. mycoides
subsp. capri strain PG3 were cloned and analyzed by
sequencing. These two lipoproteins have been named LppA[MmymyLC] and
LppA[Mmyca], and their corresponding genes have been named
lppA[MmymyLC] and lppA[Mmyca],
respectively. The nucleotide and deduced amino acid sequences of these
two lipoproteins showed a very high degree of similarity between these
two mycoplasmas. Given the sequence data, LppA seems to fulfill the
same structural functions as the previously described major
lipoproteins P72 of M. mycoides subsp. mycoides
small-colony type and P67 of the Mycoplasma species bovine
group 7. Based on lppA gene sequences of M.
mycoides subsp. mycoides LC and M.
mycoides subsp. capri type strains, a specific PCR
assay was developed so that it amplified this gene in all field strains
of the two species analyzed in this study but not in the other members
of the M. mycoides cluster. Analysis of the PCR-amplified
lppA genes with frequently cutting restriction enzymes
showed a certain degree of genetic variability which, however, did not
cluster the two subspecies. This PCR therefore allows a rapid
identification of M. mycoides subsp. mycoides
LC and M. mycoides subsp. capri but does not
distinguish between these two closely related subspecies. LppA was
expressed in Escherichia coli K-12 and used for the
production of polyclonal mouse antiserum. Antibodies against
recombinant LppA[MmymyLC] reacted with a 62-kDa protein in all
M. mycoides subsp. mycoides LC and M.
mycoides subsp. capri type strains and field strains
tested but not with the other members of the M. mycoides
cluster, thus showing the antigenic specificity of LppA and further
supporting the concept that a close relationship exists between these
Specific humoral immune responses in a clinical trial on cattle for vaccines against contagious bovine pleuropneumonia (CBPP) were investigated. The trial included a subunit vaccine consisting of five recombinant putative variable surface proteins of the infectious agent Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides SC) compared to the currently approved attenuated vaccine strain T1/44 and untreated controls. Humoral immune responses to 65 individual recombinant surface proteins of M. mycoides SC were monitored by a recently developed bead-based array assay. Responses to the subunit vaccine components were found to be weak. Animals vaccinated with this vaccine were not protected and had CBPP lesions similar to those of the untreated controls. In correlating protein-specific humoral responses to T1/44-induced immunity, five proteins associated with a protective immune response were identified by statistical evaluation, namely, MSC_1046 (LppQ), MSC_0271, MSC_0136, MSC_0079, and MSC_0431. These five proteins may be important candidates in the development of a novel subunit vaccine against CBPP.
A recombinant antigen cocktail enzyme-linked immunosorbent assay (ELISA) for diagnosis of contagious bovine pleuropneumonia (CBPP) was developed after careful selection of antigens among one-third of the surface proteome proteins of the infectious agent Mycoplasma mycoides subsp. mycoides small colony (M. mycoides SC). First, a miniaturized and parallelized assay system employing antigen suspension bead array technology was used to screen 97 bovine sera for humoral immune responses toward 61 recombinant surface proteins from M. mycoides SC. Statistical analysis of the data resulted in selection of eight proteins that showed strong serologic responses in CBPP-affected sera and minimal reactivity in negative control sera, with P values of <10−6. Only minor cross-reactivity to hyperimmune sera against other mycoplasmas was observed. When applied in an ELISA, the cocktail of eight recombinant antigens allowed a fivefold signal separation between 24 CBPP-affected and 23 CBPP-free sera from different geographical origins. No false-positive results and only two false-negative results were obtained. In conclusion, the selected recombinant mycoplasma antigens qualified as highly specific markers for CBPP and could be employed in both a suspension bead array platform and a cocktail ELISA setting. This set of proteins and technologies therefore offers a powerful combination to drive and further improve serological assays toward reliable, simple, and cost-effective diagnosis of CBPP.
Contagious Bovine Pleuropneumonia (CBPP) is the most important chronic pulmonary disease of cattle on the African continent causing severe economic losses. The disease, caused by infection with Mycoplasma mycoides subsp. mycoides is transmitted by animal contact and develops slowly into a chronic form preventing an early clinical diagnosis. Because available vaccines confer a low protection rate and short-lived immunity, the rapid diagnosis of infected animals combined with traditional curbing measures is seen as the best way to control the disease. While traditional labour-intensive bacteriological methods for the detection of M. mycoides subsp. mycoides have been replaced by molecular genetic techniques in the last two decades, these latter approaches require well-equipped laboratories and specialized personnel for the diagnosis. This is a handicap in areas where CBPP is endemic and early diagnosis is essential.
We present a rapid, sensitive and specific diagnostic tool for M. mycoides subsp. mycoides detection based on isothermal loop-mediated amplification (LAMP) that is applicable to field conditions. The primer set developed is highly specific and sensitive enough to diagnose clinical cases without prior cultivation of the organism. The LAMP assay detects M. mycoides subsp. mycoides DNA directly from crude samples of pulmonary/pleural fluids and serum/plasma within an hour using a simple dilution protocol. A photometric detection of LAMP products allows the real-time visualisation of the amplification curve and the application of a melting curve/re-association analysis presents a means of quality assurance based on the predetermined strand-inherent temperature profile supporting the diagnosis.
The CBPP LAMP developed in a robust kit format can be run on a battery-driven mobile device to rapidly detect M. mycoides subsp. mycoides infections from clinical or post mortem samples. The stringent innate quality control allows a conclusive on-site diagnosis of CBPP such as during farm or slaughter house inspections.
CBPP Mycoplasma mycoides; Isothermal; Loop-mediated amplification LAMP; Molecular diagnostic; Field test
Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides, is an important livestock disease in Africa. The current control measures rely on a vaccine with limited efficacy and occasional severe side effects. Knowledge of the protective arms of immunity involved in this disease will be beneficial for the development of an improved vaccine. In previous studies on cattle infected with M. mycoides subsp. mycoides, a correlation was detected between the levels of mycoplasma-specific IFN-γ-secreting CD4+ T lymphocytes and reduced clinical signs. However, no cause and effect has been established, and the role of such cells and of protective responses acquired during a primary infection is not known.
We investigated the role of CD4+ T lymphocytes in CBPP by comparing disease patterns and post mortem findings between CD4+ T cell depleted and non-depleted cattle. The depletion was carried out using several injections of BoCD4 specific murine monoclonal antibody on day 6 after experimental endotracheal infection with the strain Afadé. All cattle were monitored clinically daily and sacrificed 28-30 days post-infection. Statistically significant but small differences were observed in the mortality rate between the depleted and non-depleted animals. However, no differences in clinical parameters (fever, signs of respiratory distress) and pathological lesions were observed, despite elimination of CD4+ T cells for more than a week. The slightly higher mortality in the depleted group suggests a minor role of CD4+ T cells in control of CBPP.
The phylogenetically related Mycoplasma capricolum subsp. capricolum and M. mycoides subsp. mycoides biotype Large Colony are two small-ruminant pathogens involved in contagious agalactia. Their respective contributions to clinical outbreaks are not well documented, because they are difficult to differentiate with the current diagnostic techniques. In order to identify DNA sequences specific to one taxon or the other, a suppression-subtractive hybridization approach was developed. DNA fragments resulting from the reciprocal subtraction of the type strains were used as probes on a panel of M. capricolum subsp. capricolum and M. mycoides subsp. mycoides biotype Large Colony strains to assess their intrataxon specificity. Due to a high intrataxon polymorphism and important cross-reactions between taxa, a single DNA fragment was shown to be specific for M. capricolum subsp. capricolum and to be present in all M. capricolum subsp. capricolum field isolates tested in this study. A PCR assay targeting the corresponding gene (simpA51) was designed that resulted in a 560-bp amplification only in M. capricolum subsp. capricolum and in M. capricolum subsp. capripneumoniae (the etiological agent of contagious caprine pleuropneumonia). simpA51 was further improved to generate a multiplex PCR (multA51) that allows the differentiation of M. capricolum subsp. capripneumoniae from M. capricolum subsp. capricolum. Both the simpA51 and multA51 assays accurately identify M. capricolum subsp. capricolum among other mycoplasmas, including all members of the M. mycoides cluster. simpA51 and multA51 PCRs are proposed as sensitive and robust tools for the specific identification of M. capricolum subsp. capricolum and M. capricolum subsp. capripneumoniae.
In two trials the efficacy of inactivated vaccines against contagious bovine pleuropneumonia was tested by exposing vaccinated cattle to droplet infection provided by close contact with experimentally infected 'donors'. Complete protection was given by an extreme form of vaccination in which a heavy suspension of killed Mycoplasma mycoides subsp. mycoides emulsified with Freund's complete adjuvant was given in two large doses. 'Mouse-protective antibody' (MPA) was also produced, i.e. serum transferred to mice 2-4 h before intraperitoneal challenge prevented the development of mycoplasmaemia. However, the study did not answer the question 'Is MPA protective for cattle?'. No protection was given by a milder form of vaccination in which a lighter suspension of killed mycoplasmas emulsified with Freund's incomplete adjuvant was given in a comparatively small dose on a single occasion.
L-α-glycerophosphate oxidase (GlpO) plays a central role in virulence of Mycoplasma mycoides subsp. mycoides SC, a severe bacterial pathogen causing contagious bovine pleuropneumonia (CBPP). It is involved in production and translocation of toxic H2O2 into the host cell, causing inflammation and cell death. The binding site on GlpO for the cofactor flavin adenine dinucleotide (FAD) has been identified as Gly
17. Recombinant GlpO lacking these six amino acids (GlpOΔFAD) was unable to bind FAD and was also devoid of glycerophosphate oxidase activity, in contrast to non-modified recombinant GlpO that binds FAD and is enzymatically active. Polyclonal monospecific antibodies directed against GlpOΔFAD, similarly to anti-GlpO antibodies, neutralised H2O2 production of M. mycoides subsp. mycoides SC grown in the presence of glycerol, as well as cytotoxicity towards embryonic calf nasal epithelial (ECaNEp) cells. The FAD-binding site of GlpO is therefore suggested as a valuable target site for the future construction of deletion mutants to yield attenuated live vaccines of M. mycoides subsp. mycoides SC necessary to efficiently combat CBPP.
CBPP; L-α-glycerophosphate oxidase; FAD-binding site; targeted deletion mutant; neutralizing antibody
The ELISA and an immunoblotting technique were used to study F38-type mycoplasmas - an important cause of contagious caprine pleuropneumonia - and a number of related mycoplasma species, subspecies, types or serogroups. Two-way ELISA cross-reactivity was demonstrated between five mycoplasmas, namely strain F38, Mycoplasma mycoides subsp. mycoides (LC strain), M. equigenitalium, M. primatum and bovine serogroup 7. In addition one-way cross-reactivity was demonstrated between F38 and each of the following mycoplasmas: M. mycoides subsp. mycoides (two SC strains), M. mycoides subsp. capri, and bovine serogroup L. F38 and M. capricolum did not cross-react. Immunoblot analysis, unlike ELISA, revealed that F38 and M. capricolum were closely related. At least four major protein antigens were shared between F38, M. mycoides subsp. mycoides (SC and LC strains), M. mycoides subsp. capri and bovine serogroup 7. The ELISA cross-reactions (above) shown by M. equigenitalium and M. primatum with each other, with F38 and with other mycoplasmas were not apparent by immunoblotting.
Mycoplasma mycoides subsp. mycoides SC is the pathogenic agent of contagious bovine pleuropneumonia (CBPP), the most important disease of cattle in Africa causing significant economic losses. The re-emergence of CBPP in Europe in the 1980s and 1990s illustrates that it is still a threat also to countries that have successfully eradicated the disease in the past. Nowadays, probe-based real-time PCR techniques are among the most advanced tools for a reliable identification and a sensitive detection of many pathogens, but only few protocols have been published so far for CBPP diagnosis. Therefore we developed a novel TaqMan®-based real-time PCR assay comprising the amplification of two independent targets (MSC_0136 and MSC_1046) and an internal exogenous amplification control in a multiplex reaction and evaluated its diagnostic performance with clinical samples.
The assays detected 49 MmmSC strains from diverse temporal and geographical origin, but did not amplify DNA from 82 isolates of 20 non-target species confirming a specificity of 100%. The detection limit was determined to be 10 fg DNA per reaction for the MSC_0136 assay and 100 fg per reaction for the MSC_1046 assay corresponding to 8 and 80 genome equivalents, respectively. The diagnostic performance of the assay was evaluated with clinical samples from 19 experimentally infected cattle and from 20 cattle without CBPP and compared to those of cultivation and a conventional PCR protocol. The two rt-PCR tests proved to be the most sensitive methods and identified all 19 infected animals. The different sample types used were not equally suitable for MmmSC detection. While 94.7% of lung samples from the infected cohort were positively tested in the MSC_0136 assay, only 81% of pulmonal lymph nodes, 31% of mediastinal lymph nodes and 25% of pleural fluid samples gave a positive result.
The developed multiplex rt-PCR assay is recommended as an efficient tool for rapid confirmation of a presumptive CBPP diagnosis in a well-equipped laboratory environment.
Contagious bovine pleuropneumonia (CBPP) is the most serious cattle disease in Africa, caused by Mycoplasma mycoides subsp. mycoides small-colony type (SC). CBPP control strategies currently rely on vaccination with a vaccine based on live attenuated strains of the organism. Recently, an lppQ− mutant of the existing vaccine strain T1/44 has been developed (Janis et al., 2008). This T1lppQ− mutant strain is devoid of lipoprotein LppQ, a potential virulence attribute of M. mycoides subsp. mycoides SC. It is designated as a potential live DIVA (Differentiating Infected from Vaccinated Animals) vaccine strain allowing both serological and etiological differentiation. The present paper reports on the validation of a control strategy for CBPP in cattle, whereby a TaqMan real-time PCR based on the lppQ gene has been developed for the direct detection of M. mycoides subsp. mycoides SC in ex vivo bronchoalveolar lavage fluids of cows and for the discrimination of wild type strains from the lppQ− mutant vaccine strain.
Mycoplasma mycoides subsp. mycoides SC; TaqMan real-time PCR; Bronchoalveolar lavage fluids; lppQ− mutant vaccine strain; DIVA
A new strategy for rapidly selecting and testing genetic vaccines has been developed, in which a whole genome library is cloned into a bacteriophage λ ZAP Express vector which contains both prokaryotic (Plac) and eukaryotic (PCMV) promoters upstream of the insertion site. The phage library is plated on Escherichia coli cells, immunoblotted, and probed with hyperimmune and/or convalescent-phase antiserum to rapidly identify vaccine candidates. These are then plaque purified and grown as liquid lysates, and whole bacteriophage particles are then used directly to immunize the host, following which PCMV-driven expression of the candidate vaccine gene occurs. In the example given here, a semirandom genome library of the bovine pathogen Mycoplasma mycoides subsp. mycoides small colony (SC) biotype was cloned into λ ZAP Express, and two strongly immunodominant clones, λ-A8 and λ-B1, were identified and subsequently tested for vaccine potential against M. mycoides subsp. mycoides SC biotype-induced mycoplasmemia. Sequencing and immunoblotting indicated that clone λ-A8 expressed an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible M. mycoides subsp. mycoides SC biotype protein with a 28-kDa apparent molecular mass, identified as a previously uncharacterized putative lipoprotein (MSC_0397). Clone λ-B1 contained several full-length genes from the M. mycoides subsp. mycoides SC biotype pyruvate dehydrogenase region, and two IPTG-independent polypeptides, of 29 kDa and 57 kDa, were identified on immunoblots. Following vaccination, significant anti-M. mycoides subsp. mycoides SC biotype responses were observed in mice vaccinated with clones λ-A8 and λ-B1. A significant stimulation index was observed following incubation of splenocytes from mice vaccinated with clone λ-A8 with whole live M. mycoides subsp. mycoides SC biotype cells, indicating cellular proliferation. After challenge, mice vaccinated with clone λ-A8 also exhibited a reduced level of mycoplasmemia compared to controls, suggesting that the MSC_0397 lipoprotein has a protective effect in the mouse model when delivered as a bacteriophage DNA vaccine. Bacteriophage-mediated immunoscreening using an appropriate vector system offers a rapid and simple technique for the identification and immediate testing of putative candidate vaccines from a variety of pathogens.
As contagious bovine pleuropneumonia (CBPP) is spreading fast in many African countries, there is an increasing demand for rapid and sensitive diagnostic methods that can be used to confirm the initial diagnosis based on clinical symptoms or pathological findings. Two PCR-based diagnostic systems for identification of the infectious agent, Mycoplasma mycoides subsp. mycoides SC (M. mycoides SC), in various samples are presented. Both systems involve group-specific amplification of the two 16S rRNA genes from mycoplasmas of the M. mycoides cluster. The laser-induced fluorescence assay is based on a unique sequence length difference between the two 16S rRNA genes in M. mycoides SC. This region was amplified by PCR, and the products were separated by polyacrylamide gel electrophoresis in a DNA sequencer. The resulting electropherogram showed two peaks for strains of M. mycoides SC and one peak for all other members of the M. mycoides cluster. The second system was based on restriction endonuclease analysis and agarose gel electrophoresis. Restriction of amplicons from a region containing a polymorphism, which is found in M. mycoides SC only, resulted in an extra band on the agarose gel because an AluI site is lacking in the rrnA operon. Specimens from cows with postmortem signs of CBPP were analyzed with the two PCR systems. M. mycoides SC was clearly identified in pleural fluid and lung tissue, and the methods were found to be robust and rapid. The results were in agreement with those obtained by conventional diagnostic techniques.
•Cattle were vaccinated with Mycoplasma mycoides mycoides (Mmm) recombinant l-α-glycerol-3-phosphate oxidase.•A mouse mAb to l-α-glycerol-3-phosphate oxidase was generated.•Mouse mAb blocked H2O2 release by Mmm, but cattle antisera did not.•Cattle were not protected after challenge.
The membrane-associated enzyme l-α-glycerol-3-phosphate oxidase (GlpO) of Mycoplasma mycoides subs. mycoides (Mmm), the causal agent of contagious bovine pleuropneumonia (CBPP) has been identified as a virulence factor responsible for the release of toxic by-products such as H2O2 that mediate host cell injury. Since CBPP pathogenesis is based on host inflammatory reactions, we have determined the capacity of recombinant GlpO to generate in vivo protective responses against challenge in immunized cattle. We also investigated whether sera raised against recombinant GlpO in cattle and mice inhibit production of H2O2 by Mmm. Immunization of cattle with recombinant GlpO did not protect against challenge with a virulent strain of Mmm. Further, although both murine and bovine antisera raised against recombinant GlpO detected recombinant and native forms of GlpO in immunoblot assays with similar titres, only murine antibodies could neutralize GlpO enzymatic function. The data raise the possibility that Mmm has adapted to evade potential detrimental antibody responses in its definitive host.
Mycoplasma; Antibody; Vaccination; Cattle; Evolution; l-α-glycerol-3-phosphate oxidase
Contagious bovine pleuropneumonia (CBPP) is a mycoplasmal disease caused by Mycoplasma mycoides subsp. mycoides SC (MmmSC). Since the disease is a serious problem that can affect cattle production in parts of Africa, there is a need for an effective and economical vaccine. Identifying which of the causative agent's proteins trigger potentially protective immune responses is an important step towards developing a subunit vaccine. Accordingly, the purpose of this study was to determine whether phage display combined with bioinformatics could be used to narrow the search for genes that code for potentially immunogenic proteins of MmmSC. Since the production of IgG2 and IgA are associated with a Th1 cellular immune response which is implicated in protection against CBPP, antigens which elicit these immunoglobulin subclasses may be useful in developing a subunit vaccine.
A filamentous phage library displaying a repertoire of peptides expressed by fragments of the genome of MmmSC was constructed. It was subjected to selection using antibodies from naturally- and experimentally-infected cattle. Mycoplasmal genes were identified by matching the nucleotide sequences of DNA from immunoselected phage particles with the mycoplasmal genome. This allowed a catalogue of genes coding for the proteins that elicited an immune response to be compiled. Using this method together with computer algorithms designed to score parameters that influence surface accessibility and hence potential antigenicity, five genes (abc, gapN, glpO, lppB and ptsG) were chosen to be expressed in Escherichia coli. After appropriate site-directed mutagenesis, polypeptides representing portions of each of these proteins were tested for immunoreactivity. Of these five, polypeptides representing expression products of abc and lppB were recognised on immunoblots by sera obtained from cattle during a natural outbreak of the disease.
Since phage display physically couples phenotype with genotype, it was used to compile a list of sequences that code for MmmSC proteins bearing epitopes which were recognised by antibodies in the serum of infected animals. Together with the appropriate bioinformatic analyses, this approach provided several potentially useful vaccine or diagnostic leads. The phage display step empirically identified sequences by their interaction with antibodies which accordingly reduced the number of ORFs that had to be expressed for testing. This is a particular advantage when working with MmmSC since the mycoplasmal codon for tryptophan needs to be mutated to prevent it from being translated as a stop in E. coli.
The cytotoxicities of various strains of Mycoplasma mycoides subsp. mycoides small colony type (SC), the agent of contagious bovine pleuropneumonia (CBPP), were measured in vitro using embryonic calf nasal epithelial (ECaNEp) cells. Strains isolated from acute cases of CBPP induced high cytotoxicity in the presence of glycerol, concomitant with the release of large amounts of toxic H2O2 that were found to be translocated into the cytoplasms of the host cells by close contact of the Mycoplasma strains with the host cells. Currently used vaccine strains also showed high cytotoxicity and high H2O2 release, indicating that they are attenuated in another virulence attribute. Strains isolated from recent European outbreaks of CBPP with mild clinical signs, which are characterized by a defect in the glycerol uptake system, released small amounts of H2O2 and showed low cytotoxicity to ECaNEp cells. M. mycoides subsp. mycoides SC strain PG1 released large amounts of H2O2 but was only slightly cytotoxic. PG1 was found to have a reduced capacity to bind to ECaNEp cells and was unable to translocate H2O2 into the bovine cells, in contrast to virulent strains that release large amounts of H2O2. Thus, an efficient translocation of H2O2 into host cells is a prerequisite for the cytotoxic effect and requires an intact adhesion mechanism to ensure a close contact between mycoplasmas and host cells.