Serodiagnosis and monitoring of contagious bovine pleuropneumonia (CBPP) with an indirect ELISA based on the specific lipoprotein LppQ of Mycoplasma mycoides subsp. mycoides SC.

An indirect ELISA, based on the specific and strongly antigenic recombinant peptide of the N'-terminal half of the lipoprotein LppQ from Mycoplasma mycoides subsp. mycoides small colony type (SC) was developed for the detection of antibodies to M. mycoides subsp. mycoides SC. It was evaluated for its suitability for serodiagnosis and monitoring of contagious bovine pleuropneumonia (CBPP). The recombinant peptide containing poly-histidine residue tails was expressed in Escherichia coli and subsequently purified by Ni(2+) chelate affinity chromatography to be used as antigen to coat microtiter ELISA plates. The specificity of the antigen was tested against rabbit hyperimmune sera directed against related Mycoplasmas of the M. mycoides cluster and with sera from cattle that were either free of CBPP, but suffered from other mycoplasmal infections such as M. bovis, or showed cross-reactions in the complement fixation test. The sensitivity of the ELISA was assessed with sera from artificially infected animals and with sera from cattle originating from areas where CBPP was endemic at the time of blood sampling. The study revealed that the ELISA was both specific and sensitive for CBPP positive bovine sera and was shown also to be robust to harsh climatic conditions.     

A Serological Investigation into Contagious Caprine Pleuropneumonia (CCPP) in Ethiopia

For a comparison of serological tests for CCPP, sera from 767 goats were examined. They were subjected to three tests: complement fixation test (CFT) with Mycoplasma capricolum subspecies capripneumoniae antigen; blocking ELISA (B-ELISA) with Mycoplasma capricolum subspecies capripneumoniae antigen; and CFT with Mycoplasma mycoides subspecies mycoides small colony type antigen. Antibodies were detected by these three tests in 23%, 2% and 12%, respectively, of sera from districts in which CCPP had not been reported, and in 60%, 83% and 87%, respectively, in sera from areas in which CCPP had been reported. The specificity of the tests is discussed. The use of the B-ELISA test for the diagnosis and for epidemiological studies of CCPP is strongly recommended.     

Studies of the origin of false positive reactions in the serodiagnosis of contagious bovine pleuropneumonia

Four groups of cattle were experimentally immunised by four mycoplasma species of "mycoides-like" group, Mycoplasma (M) capricolum, M. mycoides subsp. mycoides (LC), M. mycoides subsp. capri and M. species group 7 of LEACH (PG50). They were then bled weekly during 2 months to establish antibodies kinetics against homologous and heterologous antigens. The standard method of complement fixation test (CFT) used in Europe and a new ELISA test for diagnosis of contagious bovine pleuropneumonia were performed in comparison with passive haemagglutination test (PHA) against antigens used for experimental immunisation. Cross reactions obtained are rather equal to the degree of similitude between these mycoplasma species. With CFT-cross reactions are transitory and occur only while homologous titers are very high, particularly with "PG50" and the two caprine mycoides strains. ELISA results using a threshold of positivity of optical density of 0.20, were similar to that obtained with CFT except ELISA specificity is not so different from CFT one. This experimental model could explain some natural situations.     

Genetic and antigenic characterisation of elongation factor Tu from Mycoplasma mycoides subsp. mycoides SC

Specific serodiagnosis of contagious bovine pleuropneumonia (CBPP) is hampered by the low antibody titers against Mycoplasma mycoides subsp. mycoides small-colony type (MmmSC) antigens in calf serum due to persistent infections and by the existence of cross-reactions among the members of the mycoides cluster. In order to identify potential diagnostic antigens, we have constructed a genomic library from MmmSC which was screened with antibodies from naturally-infected animals. Using this strategy, a genome fragment has been isolated and characterised. The complete nucleotide sequence of this fragment revealed the presence of several open reading frames, including that of translation elongation factor Tu (EF-Tu), whose product was responsible of the positive reaction observed when expressed in E. coli. The organisation of this MmmSC genome region differed from that of other Mycoplasma species whose complete genome sequences are known, but was similar, by PCR amplification analysis of genomic DNA, to other members of the mycoides cluster, such as Mycoplasma capricolum subsp. capricolum (Mcc). Nevertheless, the MmmSC and Mcc amplicons could be distinguished by digestion with restriction enzymes AseI or HindIII, strategy that could be used as a tool for differential diagnosis of infections caused by members of the mycoides cluster. The full recombinant EF-Tu was produced in E. coli, after correction of an unusual tryptophan codon by site-directed mutagenesis, and used to investigate anti-EF-Tu circulating antibodies in bovine sera.     

Confirmation of the presence of Mycoplasma bovis in Hungarian cattle with pneumonia resembling pleuropneumonia.

Cattle from several farms in Hungary were investigated for the presence of mycoplasmal infections after the discovery of pulmonary lesions in some animals at slaughter. The pneumonic lesions, which resembled those of contagious bovine pleuropneumonia (CBPP) macroscopically and histologically were found to be caused by Mycoplasma bovis and not Mycoplasma mycoides subspecies mycoides (MmmSC) which is the causative agent of CBPP. No other bacterial pathogens were isolated. Negative results in complement fixation tests also showed that there was no serological evidence of CBPP. PCR tests for the detection of the M mycoides cluster and specifically for MmmSC were also negative. However, PCR and bacteriological culture detected cases of M bovis and the pneumonias may therefore be attributed to this mycoplasma.     

Screening of Hungarian cattle herds for Mycoplasma mycoides subspecies mycoides small colony infection with negative results

At abattoirs and farms, 1248 sera were collected from animals representing 121 farms, and examined by complement fixation test using Mycoplasma mycoides subspecies mycoides small colony type (MmmSC) antigen. All sera were negative except seven from four farms, giving ++ reactions in the serum dilution of 1:10. On retesting, these sera and additional 30 sera collected repeatedly in both farms gave negative results. In isolation attempts, 953 lung samples collected from slaughtered cattle at the same abattoirs, and 326 nasal swabs collected from 11 herds proved to be negative for the presence of MmmSC, but M. bovis was isolated frequently. In the small farms 23.95% of the animals had pleurisy and/or pneumonia while in the large herds 34.69% had lesions. DNA extracted from 50 nasal swabs and 430 lung samples was examined by polymerase chain reaction (PCR) using M. mycoides cluster-specific primers. DNA from further 325 lung samples was tested by the more specific M. mycoides subspecies mycoides small colony/large colony/capri specific primers and 196 samples by nested PCR specific for MmmSC. All gave negative results. The detection level of cluster-specific primers and the more specific primers was 33.4 pg of DNA, whereas that of nested PCR was 0.33 pg.     

Antigen heterogeneity among Mycoplasma mycoides subsp. mycoides SC isolates: discrimination of major surface proteins

The protein and antigen profiles of 60 isolates, strains and the type strain PG1 of Mycoplasma mycoides subsp. mycoides SC were compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblot analysis. Analysis using contagious bovine pleuropneumonia antisera and hyperimmune rabbit sera against several representative strains revealed some differences in protein profiles and variability in antigens among strains from different geographic regions. The most common antigenic bands had the molecular masses of 110, 95, 80, 69, 62, 60, 48, 44, 39 and 38 kDa. There were differences among European strains, where a larger group coming from Italy lacked the p98 antigen, thus, with one exception, distinguishing the Italian strains from Portuguese, French and Spanish strains. African, Australian and PG1 strains showed heterogenic profiles, with quantitative differences and in a few strains some antigenic bands were absent. The group constituting African, Australian and PG1 strains was characterised by the presence of 71.5/70 kDa antigens, which were not detected in European strains. Mycoplasma mycoides subsp. mycoides SC membrane proteins were characterised by Triton X-114 partitioning and p110, p98, p95, p62/60 and p48 were identified as immunogenic antigens. The simultaneous presence of these five antigens was common to all the sera examined and, therefore, indicates the diagnostic potential of immunoblotting. Most immunodominant antigens are surface-exposed proteins as determined by the trypsin treatment.     

Abattoir-based survey of contagious bovine pleuropneumonia in cattle in Turkey

Blood samples collected from 945 cattle at four local abattoirs in Turkey were examined for contagious bovine pleuropneumonia (CBPP) by the complement fixation test (CFT) and competitive ELISA (cELISA). In addition, the carcases of the animals were examined macroscopically at the abattoirs and 62 lung samples which had lesions suggestive of CBPP were collected for bacteriological culture. To identify suspicious isolates the PCR was used in addition to the routine biochemical tests. By the CFT, two of the 945 serum samples were seropositive, and by the cELISA, four of them were seropositive. In the bacteriological culture of the lungs, growth was observed in 18 (29 per cent) of the samples by the observation of turbidity in the broths. However, when these broths were inoculated into an agar base, growth was observed in only three (4.8 per cent) samples. These isolates were identified as Mycoplasma species on the basis of biochemical tests. In the PCR analysis of DNA extracted from the broths, none of the isolates was identified as Mycoplasma mycoides subspecies mycoides small colony or one of the members of the M mycoides cluster, but amplification was obtained in only eight (44.4 per cent) of 18 samples, using Mycoplasma-genus specific primers. These DNA samples were examined further with primers specific to 16S rRNA and were then sequenced and compared with the databanks; DNA homologies at different levels were observed in five samples, with Mycoplasma alkalescens, Mycoplasma canadense, Mycoplasma bovis and Mycoplasma bovigenitalium.     

Indirect infection of cattle with contagious bovine pleuropneumonia.

Hay infected with Mycoplasma mycoides sub-species mycoides (M mycoides) was fed to six animals on three occasions. Five animals developed complement-fixing antibody in their sera and gave a positive reaction to the comparative intradermal allergic test. No animal died from contagious bovine pleuropneumonia (CBPP) but when they were killed, three had unequivocal lesions of the disease. M mycoides was isolated from the animals with these lesions but not from the others. CBPP was transmitted to two animals put in contact with those animals showing disease, and M mycoides was recovered from lesions in both animals. It is suggested that when unexplained outbreaks of CBPP occur, the possibility of indirect transmission should be considered.     

Antigenic and genetic characterisation of lipoprotein lppC from Mycoplasma mycoides subsp. mycoides SC

Lipoprotein lppC, an immunodominant antigen, and its corresponding gene lppC were characterised in Mycoplasma mycoides subspecies mycoides small colony (SC) type, the etiological agent of contagious bovine pleuropneumonia (CBPP). The lppC gene was found in the type strain of M. mycoides subsp. mycoides SC and in field strains isolated in Europe, Africa, and Australia, as well as in vaccine strains. Southern blot analysis indicated the presence of at least four copies of lppC in the genome of M. mycoides subsp. mycoides SC, of which only one seems to be functional. Genes homologous to lppC have also been detected in closely related mycoplasmas such as M. mycoides subsp. mycoides large colony (LC) type and in M. sp. bovine group 7. lppC is encoded as a precursor with a consensus sequence for a prokaryotic signal peptidase II. The amino acid sequence of lppC and its precursor showed similarity to both LppB (at the N-terminal domain) and LppQ (at the C-terminal domain), two lipoproteins described previously in M. mycoides subsp. mycoides SC. The N-terminal domain of the mature lppC seems to be surface exposed. The C-terminal domain presented an integral membrane structure made up of five repeated units, rich in hydrophobic and aromatic amino acids, which may have pore forming potential in the mycoplasmal membrane. A recombinant peptide representing the N-terminal half of lppC was obtained following cloning in vector pETHIS-1 and expression in Escherichia coli hosts. The recombinant protein was used on immunoblots for serological analysis of sera from cattle that were naturally or experimentally infected with M. mycoides subsp. mycoides SC.     

Detection of antibody against Mycoplasma mycoides subsp mycoides in cattle by an enzyme-linked immunosorbent assay.

The results of an enzyme-linked immunosorbent assay (ELISA) For the detection of antibody against Mycoplasma mycoides subsp mycoides are presented. Antibody was detected in the sera of cattle at least 19 months after recovery from an infection and at least 23 months after vaccination. Almost half the sera of some animals in an area of Nigera where contagious bovine pleuropneumonia is enzootic contained antibody. Antibody was rarely detected when the same sera were examined by other established serological tests, emphasising the sensitivity of the ELISA.     

The effect of antibody and complement on the expression of herpesvirus of bovine malignant catarrhal fever in cultured rabbit lymphocytes

Using indirect immunofluorescence with a hyperimmune calf serum, a virus-induced antigen was demonstrated on the surface of lymphocytes expressing intracellular malignant catarrhal fever virus antigens. Antibody to the antigen was also detected in terminal sera of both cattle and rabbits. Antisera did not restrict virus expression in explanted lymph nodes unless they were supplemented with two to four units of lytic complement per ml culture. While human, bovine and guinea pig complements caused immune lysis of infected lymphocytes, rabbit complement was ineffective. The relevance of the findings in the pathogenesis of the lymphoid proliferation caused by MCFV is discussed.     

Development of a bead-based multiplex PCR assay for the simultaneous detection of multiple Mycoplasma species

We describe the development and analytical validation of a 7-plex polymerase chain reaction assay coupled to a bead-based liquid suspension array for detection of multiple ruminant Mycoplasma spp. The assay employs a combination of newly designed and previously validated primer-probe sets that target genetic loci specific for Mycoplasma bovis, Mycoplasma mycoides cluster, Mycoplasma mycoides subsp. mycoides SC (MmmSC) and Mycoplasma capricolum subspecies capripneumoniae (Mccp). Analytical sensitivity for the targeted Mycoplasma species ranged from 10 fg to 1 pg of purified gDNA extracted from broth cultures (approximately 8-800 MmmSC genome equivalents). In silico comparison of primers and probes, and analytical assessment with a range of near-neighbor Mycoplasma species and multiple bacterial respiratory pathogens demonstrated 100% analytical specificity of the assay. To assess assay performance and diagnostic specificity, 192 bovine respiratory samples were analyzed by incorporating a high throughput DNA extraction platform. The assay correctly classified all samples as negative for MmmSC or Mccp. All 33 field samples confirmed as positive for M. bovis by sequencing the uvrC gene were positive in the assay. The results from this study indicate that the bead-based liquid suspension array will provide a reliable, analytically sensitive and specific platform to simultaneously interrogate ruminant respiratory samples for multiple Mycoplasma species, including M. mycoides cluster organisms that are exotic to the United States. Sequential addition of primer-probe sets to the assay did not significantly impact analytical sensitivity of individual primer-probe combinations, suggesting that expanding the assay to include more Mycoplasma species will not compromise overall performance.     

Characterization of antigens from mycoplasmas of animal origin

Alcholeplasma laidlawii, Mycoplasma gallisepticum, M mycoides subsp mycoides, M agalactiae, M bovirhinis, mycoplasmal strain ST-6, and culture medium were compared with M bovis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme-linked immunosorbent assay (ELISA), and gel electrophoresis-derived ELISA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated there were areas of homology and areas of heterology among the species tested. Sera from rabbits hyperimmunized with the mycoplasma organisms and noninoculated culture medium demonstrated ELISA reactivity with M bovis antigens immobilized on polystyrene. Absorption of the serum from a rabbit hyperimmunized with M bovis reduced 65.9% of its reactivity with culture medium, 29.7% to 32.7% of its reactivity with the heterologous species, and 21.1% of its reactivity with the homologous species. Gel electrophoresis-derived ELISA performed on immobilized M bovis antigens separated by molecular weight, using sera from rabbits hyperimmunized with the mycoplasmal species under study and noninoculated culture medium revealed antigenic components which are shared among species or with the culture medium and several components which may be unique to M bovis.     

Effect of the caprine variant of Mycoplasma mycoides subsp mycoides on endothelium, monocytes, and complement of guinea pig, calf, sheep, and goat serum

The caprine variant of Mycoplasma mycoides subsp mycoides causes septicemia with coagulopathy in goats. Pathogenetic mechanisms that might explain the coagulopathy, the ability of the Mycoplasma to persist in the blood, and its specificity for goats were studied. Severe endothelial damage was seen by electron microscopy of goat aorta tissue exposed in vitro to 10(7) colony-forming units of mycoplasmas. The Mycoplasma did not damage 51Cr-labeled adherent cells from peripheral blood of goats. The hemolytic complement titer was reduced by 94%, 50%, 50%, and 25% in guinea pig, calf, sheep, and goat serum, respectively, 30 minutes after treatment with 8 X 10(9) colony-forming units of the Mycoplasma. Freshly prepared serum from these animal species killed the Mycoplasma. Heat-inactivated serum was not mycoplasmacidal. Complement from these 4 animal species was activated by the Mycoplasma through the classical pathway, because ethyleneglycoltetraacetic acid precipitation of serum Ca2+ inhibited activation. Proof that the classical pathway was functional in goats was not conclusive because Ca2+ supplementation of ethyleneglycoltetraacetic acid-treated serum did not restore complement activity. Endothelial damage and complement activation may explain the coagulopathy. The function that complement activation may have in the inflammatory response of this disease is not known. Difference in susceptibility of calves, sheep, and goats to M mycoides septicemia cannot be explained by species variation in complement mycoplasmacidal activity.     

Identification of bovine herpesvirus-1 polypeptides involved in serum neutralization

Bovine herpesvirus (infectious bovine rhinotracheitis virus)-infected cell antigens were solubilized with Nonidet P-40. The crude antigen extract was separated by reaction with bovine hyperimmune serum in line immunoelectrophoresis; individual immunoprecipitates were used to immunize rabbits. Rabbit sera possessing serum neutralizing activity were analyzed by reaction with crude antigen extract in immunoprecipitation sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Four virus-specified glycopeptides, with molecular weights of 69-75K, 77-81K, 82-92K and 108-115K, appeared to be involved in inducing serum neutralizing antibody.     

Immunodiffusion test antigen from bluetongue virus-infected newborn mouse brains

Three methods of extracting bluetongue virus (BTV)-infected newborn mouse brains to prepare immunodiffusion (ID) test antigen were used. The most readily readable and reproducible results were obtained with fluorocarbon-extracted brains homogenized in 8.5% sucrose. Mouse brain- and reference cell culture-derived antigens gave a line of identity with anti-BTV serum. Extracts of noninfected brains were nonreactive. ID tests on field-collected bovine sera, comparing the two types of antigen, resulted in only 73% agreement due to a greater sensitivity of cell culture-derived antigen. A 70.5% agreement resulted when comparing mouse brain-derived antigen in ID tests with complement fixation tests, the latter being least sensitive. ID test results with sera from experimental sheep gave 95.9% agreement between cell culture- and mouse brain-derived antigens. Between ID, which detects antibody to the BTV common or group antigen, and virus neutralization, which detects type-specific antibody, the agreement was 71.4% with postchallenge sera. Data from pre- and postinjection sera, however, indicate the possible activity in Texas of viruses other than International BTV Types 10, 11, 13, and 17.     

Mycoplasma mycoides subsp. mycoides SC identification by PCR in sperm of seminal vesiculitis-affected bulls.

In this study, by using the polymerase chain reaction (PCR) diagnosis for the detection and identification of Mycoplasma, we investigated mycoplasmas contaminating the semen of yearling bulls affected by seminal vesiculitis. The bulls presented neither subclinical nor clinical contagious bovine pleuropneumonia signs and the complement fixation test for specific antibodies was negative. Furthermore, we have investigated mycoplasmas isolated from semen of healthy breeding bulls of several breeds and origins, which routinely underwent breeding soundness examinations and presented no clinical signs of seminal vesiculitis. We were able to demonstrate mycoplasma infection in all tested samples by i) growth on mycoplasma-specific media and ii) a PCR-based method using a mycoplasma-specific MGSO/GPO1 primer set to amplify the 16S fragment rDNA. In addition, the identification of Mycoplasma species was made by PCR using the MSC1/MSC2 primer set that specifically amplifies M. mycoides subsp. mycoides SC or the MM450/MM451 primer set followed by AsnI digestion analysis in order to identify M. mycoides subsp. mycoides LC. The data presented herein clearly show that M. mycoides subsp. mycoides SC infection was associated with seminal vesiculitis while M. mycoides subsp. mycoides LC was only found in bull semen from healthy control animals. Our findings confirm that the M. mycoides subsp. mycoides SC is shed in the sperm making the ejaculate a valuable biological sample for the isolation of these bacteria from serologically negative animals. Although the pathogenic role of M. bovigenitalium in bull seminal vesiculitis has been established, our clinical findings, semen characteristics, microbiological and bacterial genomic analysis strongly suggest that M. mycoides subsp. mycoides SC may contribute to induce vesicular adenitis in the bull.     

A radial growth precipitation test and its comparison with certain other serological tests for the detection of antibody in bovine sera to Mycoplasma agalactiae subsp. bovis

A radial growth precipitation test is described for measuring antibody to Mycoplasma agalactiae subsp. bovis. The test is quantitative and appears to depend on the production of soluble antigen by growing organisms. When compared with indirect haemagglutination, complement fixation and inhibition of film production, for measuring antibody to M. agalactiae subsp. bovis in bovine sera, it was found to have a sensitivity comparable to that of the complement fixation test.     

Serological cross-reactivity of porcine reference antisera to Mycoplasma hyopneumoniae, M. flocculare, M. hyorhinis and M. hyosynoviae indicated by the enzyme-linked immunosorbent assay, complement fixation and indirect hemagglutination tests.

The enzyme-linked immunosorbent assay (ELISA) indicated significant cross-reactivity between the antigens of Mycoplasma hyopneumoniae ( HyoP ) and M. flocculare (Floc), another porcine mycoplasma of wide distribution but uncertain pathogenic significance, when porcine antisera of each specificity were tested against HyoP antigen. The titers of the anti-Floc sera ranged from threefold to 13-fold less than the titer of the anti- HyoP reference serum at different times after immunization. These values ranged from onefold less than to fourfold greater than the minimal positive titer of 80. The antisera to the other porcine mycoplasmal antigens [i.e. M. hyorhinis ( HyoR ) and M. hyosynoviae ( HyoS )] reacted less strongly to HyoP antigen but titers only slightly less than to slightly greater than the minimal positive titer were noted for some sera. Cross-reactivity was also detected by the complement fixation test, although the titers for this test were generally lower than for the ELISA, presumably reflecting lower sensitivity of the complement fixation test. Positive indirect hemagglutination titers to HyoP antigen were also observed for both anti-Floc sera obtained at one or more times during the immune response. With two exceptions (one anti- HyoR serum with a complement fixation titer of 16 and one anti- HyoR serum with an indirect hemagglutination titer of 10), none of the anti- HyoR or anti- HyoS sera had detectable indirect hemagglutination or complement fixation titers to HyoP antigen at any time after immunization. The levels of cross-reactivity detected by the complement fixation test and indirect hemagglutination and, especially, the ELISA would be of significance for the development of any practical sero-diagnostic test for mycoplasmal pneumonia of swine.