Anna Kanci

A novel mechanism for control of antigenic variation in the haemagglutinin gene family of Mycoplasma synoviae

High‐frequency phase and antigenic variation of homologous lipoprotein haemagglutinins has been seen in both the major avian mycoplasma pathogens, Mycoplasma synoviae and Mycoplasma gallisepticum. The expression and, hence, antigenic variation of the pMGA gene family (encoding these lipoproteins in M. gallisepticum) is controlled by variation in the length of a trinucleotide repeat motif 5′ to the promoter of each gene. However, such a mechanism was not detected in preliminary observations on M. synoviae. Thus, the basis for control of variation in the vlhA gene family (which encodes the homologous haemagglutinin in M. synoviae) was investigated to enable comparison with its homologue in M. gallisepticum and with other lipoprotein gene families in mycoplasmas. The start point of transcription was identified 119 bp upstream of the initiation codon, but features associated with control of transcription in other mycoplasma lipoprotein genes were not seen. Comparison of three copies of vlhA revealed considerable sequence divergence at the 3′ end of the gene, but conservation of the 5′ end. Southern blot analysis of M. synoviae genomic DNA revealed that the promoter region and part of the conserved 5′ coding sequence occurred as a single copy, whereas the remainder of the coding sequence occurred as multiple copies. A 9.7 kb fragment of the genome was found to contain eight tandemly repeated regions partially homologous to vlhA, all lacking the putative promoter region and the single‐copy 5′ end of vlhA, but extending over one of four distinct overlapping regions of the 3′ coding sequence. Examination of sequential clones of M. synoviae established that unidirectional recombination occurs between the pseudogenes and the expressed vlhA, with duplication of pseudogene sequence and loss of the corresponding region previously seen in the expressed gene. Expression of the 5′ end of two variants of the vlhA gene showed that they differed in their reaction with monoclonal antibodies specific for this region. These data suggest that the control of vlhA antigenic variation in M. synoviae is achieved by multiple gene conversion events using a repertoire of coding sequences to generate a chimeric expressed gene, with the greatest potential for variation generated in the region encoding the haemagglutinin. Thus, completely distinct mechanisms have been adopted to control antigenic variation in homologous gene families.

Development of a Recombinant Protein-Based Enzyme-Linked Immunosorbent Assay for Diagnosis of Mycoplasma bovis Infection in Cattle

ABSTRACT Mycoplasma bovis causes a range of diseases in cattle, including mastitis, arthritis, and pneumonia. However, accurate serological diagnosis of infection remains problematic. The studies described here aimed to identify an antigen that might be used to develop a more specific and sensitive diagnostic assay. A 226-kDa immunogenic protein was consistently detected in Western blots by antibodies in sera from calves experimentally infected with M. bovis. This protein was shown to be a membrane protein with lipase activity and was named mycoplasma immunogenic lipase A (MilA). Different regions of MilA were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins and recombinant products from the amino-terminal end shown to have strong immunoreactivity with M. bovis-specific bovine sera. The most immunoreactive fusion protein, GST-MilA-ab, was used to develop indirect IgM and IgG enzyme-linked immunosorbent assays (ELISAs). The IgM ELISA detected M. bovis-specific IgM antibody 2 weeks after infection with 97.1% sensitivity and had a specificity of 63.3%, while the IgG ELISA detected M. bovis-specific IgG 3 weeks after infection with 92.86% sensitivity and had a specificity of 98.7%, demonstrating that the IgG ELISA has potential for use as a sensitive and specific assay for detecting infection in cattle.

Development and immunogenicity of recombinant GapA+Mycoplasma gallisepticum vaccine strain ts-11 expressing infectious bronchitis virus-S1 glycoprotein and chicken interleukin-6

Mycoplasma gallisepticum (MG) is a major pathogen of poultry that causes chronic respiratory disease in chickens and infectious sinusitis in turkeys. A live attenuated vaccine, ts-11, has been used for the control of MG in several countries. The efficacy of this vaccine is highly dose dependent and the flock antibody response is weak. To improve the functionality of the vaccine and investigate its potential as a delivery vector for foreign antigens and immunomodulatory proteins, we developed a derivative of ts-11 expressing infectious bronchitis virus-S1 glycoprotein (IBV-S1) and releasing chicken interleukin-6 into the extracellular milieu (MG ts-11 C3 (+CS)) using a transposon-based delivery vector. Following administration of MG ts-11 C3 (+CS) to chickens by eye-drop, an antibody response to MG and IBV-S1, as determined by the rapid serum agglutination test (RSA) and Western blotting, respectively, could be detected. Birds inoculated with the recombinant vaccine had significantly enhanced weight gain and were partially protected against damage by pathogenic IBV. These results indicate that the ChIL-6 released by MG ts-11 C3 (+CS) may have had a non-specific effect on growth rate. They also suggest that ts-11 is a promising vaccine vector, capable of delivering heterologous protective antigens, and may also provide non-specific benefits when engineered to express immunomodulatory proteins. With some improvements in the expression system, it could be used to induce a targeted immune response against specific mucosal pathogens, and co-expression of several antigens would allow development of a novel multivalent vaccine.

MalF is essential for persistence of Mycoplasma gallisepticum in vivo.

There is limited understanding of the molecular basis of virulence in the important avian pathogen Mycoplasma gallisepticum. To define genes that may be involved in colonization of chickens, a collection of mutants of the virulent Ap3AS strain of M. gallisepticum were generated by signature-tagged transposon mutagenesis. The collection included mutants with single insertions in the genes encoding the adhesin GapA and the cytadherence-related protein CrmA, and Western blotting confirmed that these mutants did not express these proteins. In two separate in vivo screenings, two GapA-deficient mutants (ST mutants 02-1 and 06-1) were occasionally recovered from birds, suggesting that GapA expression may not always be essential for persistence of strain Ap3AS. CrmA-deficient ST mutant 33-1 colonized birds poorly and had reduced virulence, indicating that CrmA was a significant virulence factor, but was not absolutely essential for colonization. ST mutant 04-1 contained a single transposon insertion in malF, a predicted ABC sugar transport permease, and could not be reisolated even when inoculated by itself into a group of birds, suggesting that expression of MalF was essential for persistence of M. galliseptium strain Ap3AS in infected birds.

GapA+Mycoplasma gallisepticum ts-11 has improved vaccine characteristics

Mycoplasma gallisepticum (MG) is an important poultry pathogen that causes respiratory disease and loss of production worldwide, and is currently controlled with live attenuated vaccines. These vaccines have limitations as they vary in their pathogenicity, the protection afforded and their transmissibility, but have been shown to effectively reduce losses associated with challenge in the field. A live attenuated vaccine, ts-11, has been used for the control of M. gallisepticum in several countries. This vaccine is highly dose-dependent and the flock antibody response is weak. GapA is the primary cytadherence molecule in M. gallisepticum, and the absence of GapA expression has been observed in the vast majority of cells in the ts-11 vaccine strain. In this study the immunogenicity of a GapA(+) M. gallisepticum ts-11 vaccine was investigated in specific-pathogen-free chickens. Birds vaccinated with GapA(+) M. gallisepticum ts-11 were protected against clinical signs of disease following challenge with virulent M. gallisepticum, and GapA(+) M. gallisepticum ts-11 was shown to be non-pathogenic and more immunogenic at a lower dose than the currently available M. gallisepticum ts-11 vaccine. Thus, GapA(+) M. gallisepticum ts-11 appears to have improved potential as a vaccine candidate.

Evaluation of an IgG Enzyme-Linked Immunosorbent Assay as a Serological Assay for Detection of Mycoplasma bovis Infection in Feedlot Cattle

ABSTRACT Mycoplasma bovis is a pathogen of emerging significance in cattle throughout the world that is causing a range of diseases, including mastitis, arthritis, and pneumonia. The limited availability and efficacy of current diagnostic and prophylactic tools for its control and its increasing antimicrobial resistance are contributing to its increasing importance in beef and dairy cattle. We have developed an indirect IgG enzyme-linked immunosorbent assay (ELISA) based on a recombinant fragment of the MilA protein and have shown its potential as an effective diagnostic tool. To more comprehensively estimate the diagnostic sensitivity and specificity of this IgG ELISA for detection of infection with M. bovis in cattle and to define a suitable cutoff for use in the field, we further assessed its performance in experimentally infected calves in a closed beef herd and by applying Bayesian latent class modeling to laboratory testing results from 7,448 cattle entering Australian feedlots. The most effective cutoff points were estimated to be 68.6 antibody units (AU) for experimentally infected calves and to be 58.7 AU for a closed adult herd. Under field conditions, in feedlot cattle the globally optimal cutoff was estimated to be 105 AU. At this cutoff, the diagnostic sensitivity was 94.3% (95% probability interval [PI], 89.9% to 99.6%) with a diagnostic specificity of 94.4% (95% PI, 90.3% to 99.6%). Applying this 105 AU cutoff, 13.1% of cattle were seropositive for infection with M. bovis on entry into feedlots, and 73.5% were seropositive when followed up approximately 6 weeks later suggesting a high risk of infection shortly after entry into feedlots.

Homologue of Macrophage-Activating Lipoprotein in Mycoplasma gallisepticum Is Not Essential for Growth and Pathogenicity in Tracheal Organ Cultures

While the genomes of a number of Mycoplasma species have been fully determined, there has been limited characterization of which genes are essential. The surface protein (p47) identified by monoclonal antibody B3 is the basis for an enzyme-linked immunosorbent assay for serological detection of Mycoplasma gallisepticum infection and appears to be constitutively expressed. Its gene was cloned, and the DNA sequence was determined. Subsequent analysis of the p47 amino acid sequence and searches of DNA databases found homologous gene sequences in the genomes of M. pneumoniae and M. genitalium and identity with a gene family in Ureaplasma urealyticum and genes in M. agalactiae and M. fermentans. The proteins encoded by these genes were found to belong to a family of basic membrane proteins (BMP) that are found in a wide range of bacteria, including a number of pathogens. Several of the BMP family members, including p47, contain selective lipoprotein-associated motifs that are found in macrophage-activating lipoprotein 404 of M. fermentans and lipoprotein P48 of M. agalactiae. The p47 gene was predicted to encode a 59-kDa peptide, but affinity-purified p47 had a molecular mass of approximately 47 kDa, as determined by polyacrylamide gel analysis. Analysis of native and recombinant p47 by mass peptide fingerprinting revealed the absence of the carboxyl end of the protein encoded by the p47 gene in native p47, which would account for the difference seen in the predicted and measured molecular weights and indicated posttranslational cleavage of the lipoprotein at its carboxyl end. A DNA construct containing the p47 gene interrupted by the gene encoding tetracycline resistance was used to transform M. gallisepticum cells. A tetracycline-resistant mycoplasma clone, P2, contained the construct inserted within the genomic p47 gene, with crossovers occurring between 73 bp upstream and 304 bp downstream of the inserted tetracycline resistance gene. The absence of p47 protein in clone P2 was determined by the lack of reactivity with rabbit anti-p47 sera or monoclonal antibody B3 in Western blots of whole-cell proteins. There was no difference between the p47(-) mutant and wild-type M. gallisepticum in pathogenicity in chicken tracheal organ cultures. Thus, p47, although homologous to genes that occur in many prokaryotes, is not essential for growth in vitro or for attachment and the initial stages of pathogenesis in chickens.

Induction of enzootic pneumonia in pigs by the administration of an aerosol of in vitro-cultured Mycoplasma hyopneumoniae.

The aim of this study was to establish whether enzootic pneumonia could be induced reliably in piglets by administering an aerosolised culture of Mycoplasma hyopneumoniae. Groups of five M hyopneumoniae-free Landrace × Large White piglets weaned at 11 to 14 days of age were exposed to aerosols of in vitro cultures of a virulent strain of M hyopneumoniae. In three separate trials, 14 of 15 pigs exposed to the bacteria developed pneumonia, but pigs exposed to the culture medium alone did not develop the disease. Lung pathology, both gross and histological, indicated acute disease. Ten of the pigs were tested for seroconversion by Western blot and they were all positive. The growth rates of the infected pigs were significantly reduced and the water consumption of the infected groups was also depressed. M hyopneumoniae was recovered from eight of the 15 infected pigs.

Histochemical and morphometric characterization of broncho- pneumonia in calves caused by infection with Mycoplasma bovis

The aim of this study was to identify morphometric histological features of pneumonia caused by Mycoplasma bovis in calves. Eight three-month-old calves were infected with M. bovis and samples of their lung tissue, three weeks after exposure, compared to samples from four uninfected calves. In the uninfected animals the goblet cells were clustered in the crypt area of the epithelial folds, while in the infected calves they had migrated towards the tips of the folds and were distributed evenly throughout the folds. In infected lung tissue there was goblet cell hyperplasia and metaplasia in the bronchioles and an increased epithelial height. Goblet cell mucin in uninfected calves was acidic, but in infected calves most goblet cells contained neutral mucins. These morphometric and histochemical bronco-epithelial changes may be able to be used as markers of the severity of bovine respiratory mycoplasmosis.

Development and immunogenicity of recombinant Mycoplasma gallisepticum vaccine strain ts-11 expressing chicken IFN-γ

Mycoplasma gallisepticum (MG) is a poultry pathogen that causes respiratory disease and loss of egg production worldwide. A live attenuated vaccine, ts-11, has been used for the control of MG in several countries. To improve the functionality of the vaccine and investigate its potential as a delivery vector for host immune molecules and foreign antigens, we have developed ts-11 as a vector to express and secrete chicken IFN-gamma (ts-11 C3) using a transposon-based delivery vector. Following administration of ts-11 C3 in chickens by eye drop, up to 2 weeks post-vaccination, neither significant systemic IFN-gamma expression nor an antibody response as determined by the rapid serum agglutination (RSA) could be detected, while moderate RSA scores were detected in birds vaccinated with ts-11. However, the MG-specific IFN-gamma response in spleen cultures was significantly enhanced in ts-11 C3 vaccinated chickens and, more interestingly, significant heterophil infiltration was detected in the tracheal epithelium in ts-11 C3 vaccinated birds, but not in ts-11 vaccinated birds. These results indicate that the IFN-gamma expressed by ts-11 C3 enhanced host cellular immunity rather than humoral immunity and may also have stimulated mucosal heterophil infiltration. These results also suggest that ts-11 is a promising vector for protective antigens of other chicken respiratory pathogens.