Transmissibility of Mycoplasma mycoides subsp. mycoides infection from experimentally inoculated goats to other goats and calves was studied. Eight goats and six calves were housed in an 18 m2 room. Six of the goats were inoculated endobronchially with strain D44 isolated from a natural case of polyarthritis in Ontario. These six goats died within a week of Mycoplasma septicemia. The two contact goats or the six calves never showed signs of disease and M. mycoides subsp. mycoides was not recovered from these animals. The contact goats and four calves were killed 25 days after exposure. They were all seronegative, M. mycoides subsp. mycoides was not recovered at necropsy and none had pathomorphological changes attributable to this Mycoplasma. The two remaining calves were inoculated endobronchially with 10(9) CFU of strain D44 and observed for 20 days. They never showed signs of disease and did not have significant lesions at necropsy. Both developed a significant serological response to M. mycoides subsp. mycoides, although this organism was not recovered during the experimental period or at necropsy. This study did not provide evidence for transmission of M. mycoides subsp. mycoides from endobronchially inoculated goats to contact goats or calves and endobronchially inoculated calves did not develop pneumonia. This would suggest that the infection of the goat population in Canada with this pathogen would not be a significant threat to the cattle population.
Two unidentified mycoplasmas, N3 and N11, isolated from the respiratory tract of horses, were found to cross-react with strains of M. mycoides subsp. mycoides in indirect immunofluorescence tests, growth-inhibition tests carried out by the running drop/agar-well method, and in complement-fixation and double immunodiffusion tests. Serologically, the equine mycoplasmas were not completely identical with any of the reference strains of M. mycoides with which they were compared. Their cultural characteristics, ability to digest coagulated serum and casein, and survival at 45 degrees C, however, suggested that they were more closely related to strains of M. mycoides subsp. mycoides, such as Y-goat, which are found in goats, than to strains of that subspecies which are pathogenic for cattle.
Association of Mycoplasma mycoides subspecies capri (Mmc) with natural goat mastitis has been studied earlier largely by detecting the Mmc DNA using molecular methods. However, report on detection of cultivable Mmc isolates from natural goat-mastitis milk is still very rare. In this study, Mmc was isolated from milk samples (n = 171) of goats with or without clinical signs of mastitis. Mmc isolates were further characterized by biochemical and species-specific PCR methods. Intra species strain variation was also studied by 16S amplified rDNA restriction analysis (16S ARDRA). The study recovered a total of 6 Mmc isolates (3.5%). Three types of intraspecies variants among the recovered Mmc isolates were found by 16S ARDRA. The study concluded that Mmc may be an etiological agent of mycoplasmal mastitis in Indian goat herds.
The electrophoretic patterns of cell proteins in polyacrylamide gels were used for the study of several taxonomic problems in the Mycoplasmatales. The patterns of five Mycoplasma hominis strains showed marked differences that corresponded with their known serological and nucleic acid heterogeneity. The patterns of three M. mycoides var. mycoides strains isolated in different countries were essentially identical. The electrophoretic patterns of several caprine strains resembled those of M. mycoides var. mycoides, supporting their classification as M. mycoides var. capri. Strain B3, a swine isolate, accordingly was tentatively identified as M. mycoides var. capri. The bovine mastitis strain M. agalactiae var. bovis possessed a pattern basically similar to that of the goat mastitis strain M. agalactiae, supporting the inclusion of both strains in one species. Three M. pulmonis strains isolated from rats or tissue cultures showed nearly identical patterns. The pattern of the toxigenic M. neurolyticum (Sabin A) strain resembled but was not identical with that of the nontoxigenic PG28 strain. The avian Mycoplasma species, M. gallisepticum, M. meleagridis, M. synoviae, M. gallinarum, and M. iners showed easily distinguishable and specific patterns, supporting their present classification in different species. Several improvements in the electrophoretic technique are described, and its advantages and limitations as a taxonomic tool are discussed.
Few serological tests are available for detecting antibodies against Mycoplasma capricolum subsp. capripneumoniae, the causal agent of contagious caprine pleuropneumonia (CCPP). The complement fixation test, the test prescribed for international trade purposes, uses a crude antigen that cross-reacts with all the other mycoplasma species of the “mycoides cluster” frequently infecting goat herds. The lack of a more specific test has been a real obstacle to the evaluation of the prevalence and economic impact of CCPP worldwide. A new competitive ELISA kit for CCPP, based on a previous blocking ELISA, was formatted at CIRAD and used to evaluate the prevalence of CCPP in some regions of Kenya, Ethiopia, Mauritius, Tajikistan and Pakistan in an international collaborative study.
The strict specificity of the test was confirmed in CCPP-free goat herds exposed to other mycoplasma species of the “mycoides cluster”. Prevalence studies were performed across the enzootic range of the disease in Africa and Asia. Seroprevalence was estimated at 14.6% in the Afar region of Ethiopia, whereas all the herds presented for CCPP vaccination in Kenya tested positive (individual seroprevalence varied from 6 to 90% within each herd). In Mauritius, where CCPP emerged in 2009, nine of 62 herds tested positive. In Central Asia, where the disease was confirmed only recently, no positive animals were detected in the Wakhan District of Afghanistan or across the border in neighboring areas of Tajikistan, whereas seroprevalence varied between 2.7% and 44.2% in the other districts investigated and in northern Pakistan. The test was also used to monitor seroconversion in vaccinated animals.
This newly formatted CCPP cELISA kit has retained the high specificity of the original kit. It can therefore be used to evaluate the prevalence of CCPP in countries or regions without vaccination programs. It could also be used to monitor the efficacy of vaccination campaigns as high-quality vaccines induce high rates of seroconversion.
Contagious caprine pleuropneumonia; Competitive ELISA; Seroprevalence; Kenya; Ethiopia; Mauritius; Tajikistan; Afghanistan; Pakistan; Vaccine quality control
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.
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.
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
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
Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia (CBPP), a respiratory disease of cattle, whereas the closely related Mycoplasma mycoides subsp. capri (Mmc) is a goat pathogen. Cyto-adherence is a crucial step in host colonization by mycoplasmas and subsequent pathogenesis. The aim of this study was to investigate the interactions between Mmm and mammalian host cells by establishing a cyto-adherence flow cytometric assay and comparing tissue and species specificity of Mmm and Mmc strains.
There were little significant differences in the adherence patterns of eight different Mmm strains to adult bovine lung epithelial cells. However, there was statistically significant variation in binding to different host cells types. Highest binding was observed with lung epithelial cells, intermediate binding with endothelial cells and very low binding with fibroblasts, suggesting the presence of effective adherence of Mmm on cells lining the airways of the lung, which is the target organ for this pathogen, possibly by high expression of a specific receptor. However, binding to bovine fetal lung epithelial cells was comparably low; suggesting that the lack of severe pulmonary disease seen in many infected young calves can be explained by reduced expression of a specific receptor.
Mmm bound with high efficiency to adult bovine lung cells and less efficiently to calves or goat lung cells. The data show that cyto-adherence of Mmm is species- and tissue- specific confirming its role in colonization of the target host and subsequent infection and development of CBPP.
Electronic supplementary material
The online version of this article (doi:10.1186/s12917-015-0347-3) contains supplementary material, which is available to authorized users.
Mycoplasma mycoides subsp. mycoides; Contagious bovine pleuropneumonia; Cyto-adherence; Epithelial cells
Capripox viruses are economically important pathogens in goat and sheep producing areas of the world, with specific focus on goat pox virus (GTPV), sheep pox virus (SPPV) and the Lumpy Skin Disease virus (LSDV). Clinically, sheep pox and goat pox have the same symptoms and cannot be distinguished serologically. This presents a real need for a rapid, inexpensive, and easy to operate and maintain genotyping tool to facilitate accurate disease diagnosis and surveillance for better management of Capripox outbreaks.
A LAMP method was developed for the specific differential detection of GTPV and SPPV using three sets of LAMP primers designed on the basis of ITR sequences. Reactions were performed at 62°C for either 45 or 60 min, and specificity confirmed by successful differential detection of several GTPV and SPPV isolates. No cross reactivity with Orf virus, foot-and-mouth disease virus (FMDV), A. marginale Lushi isolate, Mycoplasma mycoides subsp. capri, Chlamydophila psittaci, Theileria ovis, T. luwenshuni, T. uilenbergi or Babesia sp was noted. RFLP-PCR analysis of 135 preserved epidemic materials revealed 48 samples infected with goat pox and 87 infected with sheep pox, with LAMP test results showing a positive detection for all samples. When utilizing GTPV and SPPV genomic DNA, the universal LAMP primers (GSPV) and GTPV LAMP primers displayed a 100% detection rate; while the SPPV LAMP detection rate was 98.8%, consistent with the laboratory tested results.
In summary, the three sets of LAMP primers when combined provide an analytically robust method able to fully distinguish between GTPV and SPPV. The presented LAMP method provides a specific, sensitive and rapid diagnostic tool for the distinction of GTPV and SPPV infections, with the potential to be standardized as a detection method for Capripox viruses in endemic areas.
Goat pox virus (GTPV); Sheep pox virus (SPPV); Inverted terminal repeat (ITR) regions; Loop-mediated isothermal amplification (LAMP); Differential diagnosis
Contagious caprine pleuropneumonia (CCPP), one of the most serious and dramatic diseases of goats, is caused by Mycoplasma capricolum subsp. capripneumoniae (M. capripneumoniae). This organism is very difficult to isolate and to correctly identify. In a previous report we described a method for the rapid detection and identification of M. capripneumoniae. This method is based on a PCR system by which a segment of the 16S rRNA gene from all mycoplasmas of the M. mycoides cluster can be amplified. The PCR product is then analyzed by restriction enzyme cleavage for the identification of M. capripneumoniae DNA. This system has now been further evaluated with respect to specificity and diagnostic efficacy for the identification and direct detection of the organism in clinical material. Identification by restriction enzyme analysis of amplified DNA from mycoplasmas of the M. mycoides cluster was verified for 55 strains, among which were 15 strains of M. capripneumoniae. The PCR was applied to clinical samples from the nose, ear, pharynx, pleural fluid, and lung tissue containing M. capripneumoniae or other mycoplasmas. As expected, mycoplasmas belonging to the M. mycoides cluster could be detected by the PCR. Restriction enzyme analysis of the PCR products could then be applied for the identification of M. capripneumoniae. Clinical samples and cultures containing M. capripneumoniae were dried on filter paper, to try an easier sample transport method, and were tested by PCR. M. capripneumoniae DNA could be detected in the dried specimens, but the sensitivity of the PCR test was reduced.
Infection by Mycoplasma bovis (M. bovis) can induce diseases, such as pneumonia and otitis media in young calves and mastitis and arthritis in older animals. Here, we report the finished and annotated genome sequence of M. bovis strain Hubei-1, a strain isolated in 2008 that caused calf pneumonia on a Chinese farm. The genome of M. bovis strain Hubei-1 contains a single circular chromosome of 953,114 bp with a 29.37% GC content. We identified 803 open reading frames (ORFs) that occupy 89.5% of the genome. While 34 ORFs were Hubei-1 specific, 662 ORFs had orthologs in the M. bovis type strain PG45 genome. Genome analysis validated lateral gene transfer between M. bovis and the Mycoplasma mycoides subspecies mycoides, while phylogenetic analysis found that the closest M. bovis neighbor is Mycoplasma agalactiae. Glycerol may be the main carbon and energy source of M. bovis, and most of the biosynthesis pathways were incomplete. We report that 47 lipoproteins, 12 extracellular proteins and 18 transmembrane proteins are phase-variable and may help M. bovis escape the immune response. Besides lipoproteins and phase-variable proteins, genomic analysis found two possible pathogenicity islands, which consist of four genes and 11 genes each, and several other virulence factors including hemolysin, lipoate protein ligase, dihydrolipoamide dehydrogenase, extracellular cysteine protease and 5′-nucleotidase.
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.
Contagious caprine pleuropneumonia (CCPP) is a major threat to goat farming in parts of Africa and Asia. It classically causes acute high morbidity and mortality early in infection, but little is known of its long term epizootiology and course. In this study, 10 goats were inoculated with Mycoplasma capricolum subsp. capripneumoniae (M. capripneumoniae) and then mixed with 15 goats for contact transmission. The disease course was monitored in each goat for 56–105 days, whereafter the goats were killed and necropsied. Varying features signifying infection occurred in altogether 17 goats (7 inoculated, 10 in-contact). Clinical signs were severe in 8 goats but no fatalities occurred. Only 6 goats had serum antibody titres against M. capripneumoniae in ELISA. Fourteen goats (5 inoculated, 9 in-contact) had chronic pleuropulmonary lesions compatible with CCPP at necropsy and 7 of those showed M. capripneumoniae antigen in the lung by immunohistochemistry. Neither cultivation nor PCR tests were positive for the agent in any goat. The results indicate that the clinical course of CCPP in a flock may be comparatively mild, M. capripneumoniae-associated lung lesions may be present at a late stage of infection, and chronic infection may occur without a significant serological response.
goat; Mycoplasma; contagious pleuropneumonia; ELISA; immunohistochemistry; serology; pathology.
Acrylamide gel electrophoresis was used to show the similarities and differences in the membrane proteins of two vaccine and two virulent strains of Mycoplasma mycoides var. mycoides. Immunoelectrophoretic (IEP) analysis was also used to partially characterize the associated antigens. Antibody spectra to the antigens of M. mycoides differ in rabbit, pig, and cattle sera. Rabbits produce better precipitating antibody against the anodic migrating protein mycoplasma antigens than cattle and pigs as seen in IEP. However, rabbit anti-M. mycoides serum did not show precipitating antibody against the heat-stable carbohydrate antigen. As judged by IEP, the major carbohydrate antigen extracted from the media, or boiled whole organism, is similar to that present in the sera-infected cattle and knee joints of calves. This carbohydrate antigen has a cathodic migration in IEP at pH 8.6. Periodate oxidation, classically used to destroy carbohydrate, also destroys most of the protein antigens. Heating the antigens to 56 C for 10 min destroys many of the noncarbohydrate antigens and 100 C eliminates all but the carbohydrate antigen. Extraction of M. mycoides with chloroform-methanol, phenol, ethanol, or ethanol-acetone reduced or eliminated most of the protein antigens. Some of the isolated antigenic fractions of M. mycoides were tested to determine their activity in the diagnostic complement fixation test for contagious bovine pleuropneumonia and their inhibitory effect in this test by using bovine anti-M. mycoides antisera having precipitating antibody and circulating antigen. The complement fixation antigen is not the galactan, cannot be extracted by chloroform-methanol, but is stable to boiling at 100 C and may be extracted by phenol and partially precipitated by ethanol-acetone.
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.
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.
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.
Contagious Bovine Pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides, is widespread in sub-Saharan Africa. The current live vaccine T1/44 has limited efficacy and occasionally leads to severe side effects in the animals. A better understanding of the immune responses triggered by Mycoplasma mycoides subsp. mycoides and their role in disease progression will help to facilitate the design of a rational vaccine. Currently, knowledge of cytokines involved in immunity and immunopathology in CBPP is rather limited. The aim of this study was to characterize the in vivo plasma concentrations of the cytokines TNF-α, IFN-γ, IL-4, IL-10 and the overall role of CD4+ T cells in the development of cytokine levels during a primary infection. Plasma cytokine concentrations in two groups of cattle (CD4+ T cell-depleted and non-depleted cattle) experimentally infected with Mycoplasma mycoides subsp. mycoides were measured and their relationship to the clinical outcomes was investigated.
Plasma cytokine concentrations varied between animals in each group. Depletion of CD4+ T cells did not induce significant changes in plasma levels of TNF-α, IL-4, and IL-10, suggesting a minor role of CD4+ T cells in regulation or production of the three cytokines during the time window of depletion (1-2 weeks post depletion). Unexpectedly, the IFN-γ concentrations were slightly, but statistically significantly higher in the depleted group (p < 0.05) between week three and four post infection. Three CD4+ T cell-depleted animals that experienced severe disease, had high levels of TNF-α and IFN-γ. Only one severely diseased non-depleted animal showed a high serum concentration of IL-4 post infection.
Comparison of most severely diseased animals, which had to be euthanized prior to the expected date, versus less severe diseased animals, irrespective of the depletion status, suggested that high TNF-α levels are correlated with more severe pathology in concomitance with high IFN-γ levels.
Contagious bovine pleuropneumonia; Mycoplasma mycoides subsp. mycoides; Cytokines; TNF-α; IFN-γ; IL-4; IL-10
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.
Nine caprine and ovine mycoplasma strains, said to be indistinguishable serologically from Mycoplasma mycoides subsp. mycoides (the causative organism of contagious bovine pleuropneumonia; CBPP) were examined in mice by (1) a mycoplasmaemia test, and (2) a cross-protection test. Of the nine strains, two from goats belonged to a small colony (SC) type; four caprine and three ovine strains belonged to a large colony (LC) type.
The two SC strains — like a single SC strain examined in an earlier study — were indistinguishable from genuine M. mycoides subsp. mycoides as isolated from CBPP. They produced mycoplasmaemia readily. In a cross-protection test, the two SC strains and a CBPP strain immunized completely against each other.
Of the seven LC strains, six — like six LC strains examined in an earlier study — were easily distinguished from genuine M. mycoides subsp. mycoides; except for one that was not tested, all were shown to lack the ability to produce mycoplasmaemia readily. In cross-protection tests all six strains immunized partially but not completely against a CBPP strain.
The seventh LC strain (Mankefår 2833) was exceptional: it produced mycoplasmaemia readily, resembling the SC strains in this respect. Like other LC strains, in cross-protection tests it protected only partially against a CBPP strain. Strain Mankefår 2833 was isolated in ca. 1965 by Brack from a Barbary sheep (Ammotragus lervia) in a German zoo.
The ability of Mankefår 2833 to produce mycoplasmaemia enabled it to be used as a challenge strain in cross-protection tests. For the purpose of such tests the collection of nine mycoplasma strains referred to above was augmented with six LC strains from an earlier study. Partial but not complete protection against Mankefår 2833 was produced by two caprine SC strains, one CBPP strain, and nine LC strains. Three further LC strains gave protection that may have been as strong as that produced by the homologous strain, but confirmatory experiments are needed. A strain of M. mycoides subsp. capri gave no protection against Mankefår 2833.
Lipoprotein LppQ, a predominant 48-kDa antigen, and its corresponding gene, lppQ, were characterized in Mycoplasma mycoides subsp. mycoides SC, the etiological agent of contagious bovine pleuropneumonia. The lppQ gene is specific to M. mycoides subsp. mycoides SC and was found in the type strain and in field strains isolated in Europe, Africa, and Australia, as well as in vaccinal strains. LppQ is encoded as a precursor with a consensus sequence for prokaryotic signal peptidase II and a lipid attachment site. The leader sequence shows significant prominent transmembrane helix structure with a predicted outside-to-inside helix formation capacity. The N-terminal domain of the mature LppQ was shown to be surface exposed. It induced a strong, specific, early, and persistent immune response in naturally and experimentally infected animals. The C-terminal domain of LppQ possesses an integral membrane structure built up of repeated units, rich in hydrophobic and aromatic amino acids, which have a pore formation potential. A recombinant peptide representing the N-terminal domain of LppQ was obtained by site-directed mutagenesis of nine Mycoplasma-specific TGA (Trp) codons into universal TGG (Trp) codons and expression in Escherichia coli hosts. It was used for serodetection of cattle infected with M. mycoides subsp. mycoides SC, in which it was detected postinfection for significantly longer than conventional serological test reactions.
The growth and pathogenicity of Mycoplasma mycoides var. capri were studied in chicken embryo tracheal rings in rolled tubes. In these organ cultures, M. mycoides var. capri attained titers of 107 to 108 color-changing units/ml of Eagle's medium and there was inhibition of ciliary activity. The rapidity of inhibition was directly related to the number of organisms inoculated. Growth of organisms was closely associated with the tracheal tissue because multiplication was not demonstrated in “conditioned medium,” that is Eagle's medium from which the rings had been removed. Mycoplasma growth occurred when the cultures were incubated at 37 C, 33 C, and room temperature, but the cilia-stopping effect (CSE) was most rapid at 37 C and was not demonstrable at room temperature. Furthermore, there was no CSE when cultures were maintained in medium in which M. mycoides var. capri had been grown but was either filtered to remove the organisms or treated with tetracycline to stop their multiplication. This indicated that the CSE of M. mycoides var. capri was dependent upon a close association of multiplying organisms with the tracheal rings and was not due to toxic products persisting in the medium. Chicken tracheal epithelial cells did not adsorb to colonies of M. mycoides var. capri but HeLa cells did. Although their adsorption was unaffected by prior neuraminidase treatment, the addition of neuraminidase to the tracheal organ culture system delayed the CSE of M. mycoides var. capri. Peroxide production was demonstrated in M. mycoides var. capri-infected tracheal rings but not in uninfected cultures. The addition of glucose to the organ cultures delayed the CSE of M. mycoides var. capri, possibly because of the stimulation of peroxidase activity. Similarily, catalase protected the cilia from the usual damaging effect of M. mycoides var. capri. This protective effect was partially reversed by the addition of 3-amino-1, 2, 4,-triazole and it did not occur if the catalase had been inactivated by boiling. The addition of hydrogen peroxide to tracheal cultures also resulted in loss of ciliary activity. The present results show that the liberation of peroxide is an important factor in the pathogenesis of M. mycoides var. capri infection of chicken tracheal organ cultures. It is speculated that this also may be so in the natural host.
In the winter of 2002, an outbreak of mycoplasma infection in Vaal rhebok (Pelea capreolus) originating from South Africa occurred 15 weeks after their arrival in San Diego, Calif. Three rhebok developed inappetence, weight loss, lethargy, signs related to pulmonary or arthral dysfunction, and sepsis. All three rhebok died or were euthanized. Primary postmortem findings were erosive tracheitis, pleuropneumonia, regional cellulitis, and necrotizing lymphadenitis. Mycoplasmas were detected in numerous tissues by electron microscopy, immunohistochemistry, and PCR. The three deceased rhebok were coinfected with ovine herpesvirus-2, and two animals additionally had a novel gammaherpesvirus. However, no lesions indicative of herpesvirus were seen microscopically in any animal. The rheboks' mycoplasmas were characterized at the level of the 16S rRNA gene, the 16S-23S intergenic spacer region, and the fructose biphosphate aldolase gene. Denaturing gradient gel electrophoresis was carried out to address the possibility of infection with multiple strains. Two of the deceased rhebok were infected with a single strain of Mycoplasma capricolum subsp. capricolum, and the third animal had a single, unique strain most closely related to Mycoplasma mycoides subsp. mycoides large-colony. A PCR survey of DNA samples from 46 other ruminant species demonstrated the presence of several species of mycoplasmas in the mycoides cluster, including a strain of M. capricolum subsp. capricolum identical to that found in two of the rhebok. These findings demonstrate the pervasiveness of mycoplasmas in the mycoides cluster in small ruminants and the potential for interspecies transmission and disease when different animal taxa come in contact.