Efficacy of an inactivated Mycoplasma hyorhinis vaccine in pigs.

Lameness and polyserositis in pigs caused by Mycoplasma hyorhinis are generally treated with antibiotics and may require multiple doses. The costs of these antibiotics combined with economic losses from culling and reduced feed conversion due to lameness are hardships to the swine producer. In this study we have demonstrated efficacy of an inactivated M. hyorhinis vaccine administered to three-week old caesarian-derived colostrum-deprived piglets. Three doses of vaccine (high, medium, and low) were evaluated and compared to a placebo control. Mycoplasma hyorhinis challenge occurred three weeks after vaccination. Pigs were observed for lameness and respiratory distress for three weeks following challenge. Pigs were then euthanized and a gross pathological evaluation for polyserositis and arthritis was performed. A minimum immunizing dose of vaccine was defined as containing at least 7.41 × 107 CCU of M. hyorhinis per 2.0 mL dose as represented by the medium dose vaccine. This vaccine provided significant reductions in lameness and pericarditis with preventive fractions of 0.76 (95% CI [0.26, 0.92]) and 0.58 (95% CI [0.31, 0.74]), respectively, compared to the placebo control group. A significant increase in post-challenge weight gain (P < .0001) was also achieved with this vaccine, with an average daily gain (ADG) of 0.92 lbs/day compared to 0.57 lbs/day in the placebo group.

Age susceptibility of caesarian derived colostrum deprived pigs to Mycoplasma hyorhinis challenge.

Mycoplasma hyorhinis (MHR) is a major cause of lameness, arthritis, and polyserositis among growing pigs. Reduced performance and culling due to MHR infection result in economic losses in swine production. We have previously developed an MHR challenge model in seven week-old CDCD pigs using cell-associated challenge material which results in both severe pericarditis and lameness. In this study we sequentially challenged CDCD pigs at seven, ten, thirteen, and sixteen weeks of age. Lameness was observed in >60% of the animals in the first three age groups but only 33% in the oldest age group. The number of animals with arthritis declined from 100% at seven weeks, to 56% at ten weeks and approximately 25% at both thirteen and sixteen weeks of age. Pericarditis was observed in 87% of the seven week challenge group, 28% in the ten week challenge group, 8% in the thirteen week challenge group and 4% in the sixteen week challenge group. All challenged groups showed a reduced average daily gain (ADG) compared to their age-matched non-challenged control groups. The largest disparity in ADG (1.2 lbs/day difference) was noted at thirteen weeks of age. Results of this study demonstrate that these animals were susceptible to MHR-associated lameness through sixteen weeks of age while susceptibility to MHR-associated polyserositis appeared to peak at seven weeks of age.

Signal regulatory protein alpha (SIRPalpha) cells in the adaptive response to ESAT-6/CFP-10 protein of tuberculous mycobacteria.

BACKGROUND: Early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) are co-secreted proteins of Mycobacterium tuberculosis complex mycobacteria (includes M. bovis, the zoonotic agent of bovine tuberculosis) involved in phagolysosome escape of the bacillus and, potentially, in the efficient induction of granulomas. Upon tuberculosis infection, multi-nucleate giant cells are elicited, likely as a response aimed at containing mycobacteria. In tissue culture models, signal regulatory protein (SIRP)alpha (also referred to as macrophage fusion receptor or CD172a) is essential for multi-nucleate giant cell formation. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, ESAT-6/CFP-10 complex and SIRPalpha interactions were evaluated with samples obtained from calves experimentally infected with M. bovis. Peripheral blood CD172a(+) (SIRPalpha-expressing) cells from M. bovis-infected calves proliferated upon in vitro stimulation with ESAT-6/CFP-10 (either as a fusion protein or a peptide cocktail), but not with cells from animals receiving M. bovis strains lacking ESAT-6/CFP-10 (i.e, M. bovis BCG or M. bovis DeltaRD1). Sorted CD172a(+) cells from these cultures had a dendritic cell/macrophage morphology, bound fluorescently-tagged rESAT-6:CFP-10, bound and phagocytosed live M. bovis BCG, and co-expressed CD11c, DEC-205, CD44, MHC II, CD80/86 (a subset also co-expressed CD11b or CD8alpha). Intradermal administration of rESAT-6:CFP-10 into tuberculous calves elicited a delayed type hypersensitive response consisting of CD11c(+), CD172a(+), and CD3(+) cells, including CD172a-expressing multi-nucleated giant cells. CONCLUSIONS/SIGNIFICANCE: These findings demonstrate the ability of ESAT-6/CFP-10 to specifically expand CD172a(+) cells, bind to CD172a(+) cells, and induce multi-nucleated giant cells expressing CD172a.

Immune responses to defined antigens of Mycobacterium bovis in cattle experimentally infected with Mycobacterium kansasii.

Cross-reactive responses elicited by exposure to nontuberculous mycobacteria often confound the interpretation of antemortem tests for Mycobacterium bovis infection of cattle. The use of specific proteins (e.g., ESAT-6, CFP-10, and MPB83), however, generally enhances the specificity of bovine tuberculosis tests. While genes for these proteins are absent from many nontuberculous mycobacteria, they are present in M. kansasii. Instillation of M. kansasii into the tonsillar crypts of calves elicited delayed-type hypersensitivity and in vitro gamma interferon and nitrite concentration responses of leukocytes to M. avium and M. bovis purified protein derivatives (PPDs). While the responses of M. kansasii-inoculated calves to M. avium and M. bovis PPDs were approximately equivalent, the responses of M. bovis-inoculated calves to M. bovis PPD exceeded their respective responses to M. avium PPD. The gamma interferon and nitrite responses of M. kansasii-inoculated calves to recombinant ESAT-6-CFP-10 (rESAT-6-CFP-10) exceeded corresponding responses of noninoculated calves as early as 15 and 30 days after inoculation, respectively, and persisted throughout the study. The gamma interferon and nitrite responses of M. bovis-inoculated calves to rESAT-6-CFP-10 exceeded the corresponding responses of M. kansasii-inoculated calves beginning 30 days after inoculation. By using a lipoarabinomannan-based enzyme-linked immunosorbent assay, specific serum antibodies were detected as early as 50 days after challenge with M. kansasii. By a multiantigen print immunoassay and immunoblotting, serum antibodies to MPB83, but not ESAT-6 or CFP-10, were detected in M. kansasii-inoculated calves; however, responses to MPB83 were notably weaker than those elicited by M. bovis infection. These findings indicate that M. kansasii infection of calves elicits specific responses that may confound the interpretation of bovine tuberculosis tests.

Diagnostic implications of antigen-induced gamma interferon production by blood leukocytes from Mycobacterium bovis-infected reindeer (Rangifer tarandus).

The only approved method of tuberculosis (TB) surveillance of reindeer within the United States is tuberculin skin testing; however, skin testing has an apparent lack of specificity, since numerous reindeer are classified as reactors, yet Mycobacterium bovis is not isolated from tissues upon necropsy. The objective of this study was to evaluate the ability of an in vitro assay (the Cervigam assay) to detect gamma interferon (IFN-gamma) produced by blood leukocytes in response to mycobacterial antigens from M. bovis-infected reindeer. Thirteen male reindeer approximately 9 months of age were inoculated with 10(5) CFU M. bovis in their tonsillar crypts. Stimulation of whole-blood cultures with a mitogen resulted in significant production of IFN-gamma compared to that by nonstimulated samples. Responses by infected reindeer to M. bovis purified protein derivative (PPD) were as much as 3.5-fold higher than those by noninfected reindeer (n = 4). Despite differences in responses to PPD by the two groups, reindeer within the noninfected group had responses of >0.1 change in optical density (DeltaOD) (a level generally considered positive) to PPD. Mean responses by infected reindeer to a rESAT-6-CFP-10 fusion protein (Mycobacterium tuberculosis complex specific) were as much as 20-fold higher than respective responses by noninfected reindeer at all time points. Additionally, responses by 3/4 noninfected reindeer were <0.1 DeltaOD (considered negative) at each time point. To further evaluate the specificity of the assay, samples were collected from reindeer in a TB-free herd. All reindeer had responses to mitogen; however, only 1 of 38 had a response to PPD, and none of the reindeer responded to rESAT-6-CFP-10. Together, these findings indicate that IFN-gamma-based tests may prove useful for TB surveillance of reindeer.

Use of recombinant ESAT-6:CFP-10 fusion protein for differentiation of infections of cattle by Mycobacterium bovis and by M. avium subsp. avium and M. avium subsp. paratuberculosis.

Immunological diagnosis of Mycobacterium bovis infection of cattle is often confounded by cross-reactive responses resulting from exposure to other mycobacterial species, especially Mycobacterium avium. Early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) are dominant gamma interferon (IFN-gamma)-inducing antigens of tuberculous mycobacteria, and they are absent from many environmental nontuberculous mycobacteria. Because M. avium exposure is the primary confounding factor in the diagnosis of M. bovis-infected animals, in vitro responses to a recombinant ESAT-6:CFP-10 (rESAT-6:CFP-10) fusion protein by blood leukocytes from cattle naturally exposed to M. avium or experimentally challenged with Mycobacterium avium subsp. avium or Mycobacterium avium subsp. paratuberculosis were compared to responses by M. bovis-infected cattle. Responses to heterogeneous mycobacterial antigens (i.e., purified protein derivatives [PPDs] and whole-cell sonicates [WCSs]) were also evaluated. Tumor necrosis factor alpha (TNF-alpha), IFN-gamma, and nitric oxide responses by M. bovis-infected cattle to rESAT-6:CFP-10 exceeded (P < 0.05) the corresponding responses by cattle naturally sensitized to M. avium. Experimental infection with M. bovis, M. avium, or M. avium subsp. paratuberculosis induced significant (P < 0.05) IFN-gamma and nitric oxide production to WCS and PPD antigens, regardless of the mycobacterial species used for the preparation of the antigen. Responses to homologous crude antigens generally exceeded responses to heterologous antigens. Nitric oxide and IFN-gamma responses to rESAT-6:CFP-10 by blood leukocytes from M. bovis-infected calves exceeded (P < 0.05) the corresponding responses of noninfected, M. avium-infected, and M. avium subsp. paratuberculosis-infected calves. Despite the reported potential for secretion of immunogenic ESAT-6 and CFP-10 proteins by M. avium and M. avium subsp. paratuberculosis, it appears that use of the rESAT-6:CFP-10 fusion protein will be useful for the detection of tuberculous cattle in herds with pre-existing sensitization to M. avium and/or M. avium subsp. paratuberculosis.

Cloning of a beta-hemolysin gene of Brachyspira (Serpulina) hyodysenteriae and its expression in Escherichia coli.

Brachyspira (Serpulina) hyodysenteriae induces a mucohemorrhagic diarrheal disease in pigs. The production of a beta-hemolysin has been considered a major virulence attribute of this organism. Previous reports have failed to correlate a specific cloned gene sequence with a purified beta-hemolytic protein sequence. Thus, questions still remain concerning the structural gene sequence of the hemolysin. To answer this question unequivocally, the beta-hemolytic toxin was purified from extracts of log-phase spirochetes, and the N-terminal amino acid sequence was determined (K-D-V-V-A-N-Q-L-N-I-S-D-K) and compared with the translated sequences of previously cloned genes, tlyA to tlyC. The lack of homology between tlyA to tlyC translated sequences and the purified beta-hemolytic toxin sequence resulted in the study that is reported here. A degenerate probe was designed based on the N-terminal amino acid sequence of the purified beta-hemolysin and used to screen a B. hyodysenteriae genomic library. Three overlapping clones were identified, and one was sequenced to reveal an open reading frame coding for a putative 8.93-kDa polypeptide containing the N-terminal sequence of the purified beta-hemolysin. To distinguish this gene from the tlyA to tlyC genes, it has been designated hlyA. A hemolysis-negative Escherichia coli strains containing hlyA was beta-hemolytic on blood agar media. Also, the hemolytic activity of the recombinant protein had identical protease and lipase sensitivities and electrophoretic mobility to those of native B. hyodysenteriae beta-hemolysin. Based on sequence analysis, the translated protein had a pI of 4.3, an alpha-helical structure, and a phosphopantetheine binding motif. Hybridization analysis of genomic DNA indicated that the hlyA gene was present in B. hyodysenteriae and B. intermedia but was not detected in B. innocens, B. pilosicoli, or B. murdochii under high-stringency conditions. The location of hlyA on the chromosomal map was distinct from the locations of tlyA, tlyB, and tlyC.

R1 region of P97 mediates adherence of Mycoplasma hyopneumoniae to swine cilia.

Adherence of Mycoplasma hyopneumoniae to the swine respiratory tract is mediated by the membrane protein P97. This protein is located on the outer membrane surface, and its role in adherence has been firmly established. The general region of P97 that mediates adherence to swine cilia is thought to be the R1 region near the carboxy terminus of the protein, but it was not clear if this region could mediate adherence to swine cilia independently of other P97 sequences. To examine this in more detail, a series of R1 repeat sequences containing different numbers of repeating units cloned in frame with lacZ was used to produce R1-beta-galactosidase fusion proteins. These proteins were then tested for adherence to swine cilia and for reactivity to the adherence-blocking monoclonal antibody F2G5 and convalescent-phase swine sera. In this way it was possible to accurately define the cilium binding epitope of P97 and the minimal epitope recognized by antibody. Our results indicate that eight R1 repeating units are required for cilium binding and that three repeating units are needed for antibody recognition. These results could lead to more effective therapeutic measures against this important swine pathogen.

Cloning of mnuA, a membrane nuclease gene of Mycoplasma pulmonis, and analysis of its expression in Escherichia coli.

Membrane nucleases of mycoplasmas are believed to play important roles in growth and pathogenesis, although no clear evidence for their importance has yet been obtained. As a first step in defining the function of this unusual membrane activity, studies were undertaken to clone and analyze one of the membrane nuclease genes from Mycoplasma pulmonis. A novel screening strategy was used to identify a recombinant lambda phage expressing nuclease activity, and its cloned fragment was analyzed. Transposon mutagenesis was used to identify an open reading frame of 1,410 bp, which coded for nuclease activity in Escherichia coli. This gene coded for a 470-amino-acid polypeptide of 53,739 Da and was designated mnuA (for "membrane nuclease"). The MnuA protein contained a prolipoprotein signal peptidase II recognition sequence along with an extensive hydrophobic region near the amino terminus, suggesting that the protein may be lipid modified or that it is anchored in the membrane by this membrane-spanning region. Antisera raised against two MnuA peptide sequences identified an M. pulmonis membrane protein of approximately 42 kDa by immunoblotting, which corresponded to a trypsin-sensitive nucleolytic band of the same size. Maxicell experiments with E. coli confirmed that mnuA coded for a nuclease of unknown specificity. Hybridization studies showed that mnuA sequences are found in few Mycoplasma species, suggesting that mycoplasma membrane nucleases display significant sequence variation within the genus Mycoplasma.

Molecular analysis of the P97 cilium adhesin operon of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae causes an economically significant respiratory disease of swine called Enzootic Pneumonia. The disease process is initiated by adherence of M. hyopneumoniae to the cilia of swine respiratory epithelium through an interaction involving P97, a surface-associated protein, and cilia-specific receptors. Binding specificity is associated with a repeat region located near the C-terminus of the P97 protein. Further analysis of the DNA sequences surrounding the P97 structural gene revealed an operon composed of two ORFs, P97 and one coding for a 102.3-kDa protein designated P102. Hybridization analysis and subcloning experiments showed that the P97 adhesin-encoding gene was present as a single copy in the M. hyopneumoniae chromosome. P102 sequences, however, were found on four distinct chromosomal fragments, suggesting that multiple copies of P102 were present in the chromosome. One of these clones was identified by screening the genomic library with swine convalescent sera showing that P102 is expressed in vivo during M. hyopneumoniae infections. All copies of P102 were mapped to a single chromosomal region comprising approximately 13% of the genome (140kb), although the exact distance between the copies is not known. The function of P102 is also not known, but the translated sequence shows a prominent transmembrane domain, suggesting that it may be a surface protein.

Identification of the cilium binding epitope of the Mycoplasma hyopneumoniae P97 adhesin.

Mycoplasma hyopneumoniae colonizes the swine respiratory tract at the level of ciliated cells by attaching specifically to the cilium membrane. This interaction involves an adhesin called P97; the cilium binding activity of this protein was localized to the carboxy terminus, which included two repeat regions, R1 and R2 (T. Hsu, S. Artiushin, and F. C. Minion, J. Bacteriol. 179:1317-1323, 1997). To further delineate the molecular mechanisms of M. hyopneumoniae interactions with ciliated epithelium, we used a bank of transposon inserts in the cloned P97 gene to identify the site for cilium binding by testing the truncated gene products in an in vitro microtiter plate adherence assay. These studies showed that the cilium binding site was located in the AAKPV(E) repeat sequence of P97, referred to as the R1 repeat. For functional binding, at least seven AAKPV(E) repeats were required. The adherence-blocking monoclonal antibody F1B6 also recognized this region but required fewer AAKPV(E) repeats for recognition. We then constructed R1 region-lacZ gene fusions and used the resulting R1 repeat-beta-galactosidase fusion proteins in an in vitro assay to confirm the role of R1 in cilium binding. A comparison of the R1 regions of M. hyopneumoniae strains displaying variation in cilium adherence failed to identify changes that could account for the differences in adherence shown by the strains. Thus, we concluded that other proteins, in addition to P97, must be involved in cilium adherence, possibly in combination with P97.

Cloning and functional analysis of the P97 swine cilium adhesin gene of Mycoplasma hyopneumoniae.

Colonization of the swine respiratory tract by Mycoplasma hyopneumoniae is accomplished by specific binding to the cilia of the mucosal epithelial cells. Previous studies have implicated a 97-kDa outer membrane-associated protein, P97, that appeared to mediate this interaction. In order to further define the role of P97 in adherence to porcine cilia, the structural gene was cloned and sequenced, and the recombinant products were analyzed. Monoclonal antibodies were used to identify recombinant clones in a genomic library expressed in an opal suppressor host because of alternate codon usage by mycoplasmas. The gene coding for P97 was then identified by Tn1000 mutagenesis of recombinant clones. DNA sequence analysis revealed an open reading frame coding for a 124.9-kDa protein with a hydrophobic transmembrane spanning domain. The N-terminal sequence of purified P97 mapped at amino acid position 195 of the translated sequence, indicating that a processing event had occurred in M. hyopneumoniae. Both recombinant P97 protein expressed in an Escherichia coli opal suppressor host and M. hyopneumoniae bound specifically to swine cilia, and the binding was inhibited by heparin and fucoidan, thus supporting the hypothesis that P97 was actively involved in binding to swine cilia in vivo.

Arbitrarily primed PCR analysis of Mycoplasma hyopneumoniae field isolates demonstrates genetic heterogeneity.

Mycoplasma hyopneumoniae is the primary agent of mycoplasmal pneumonia in swine. In this study we performed an arbitrarily primed PCR (AP-PCR) analysis, in which low-stringency amplification with a single primer was used, to investigate genetic variability in M. hyopneumoniae strains and field isolates. We performed preliminary experiments to examine the efficacy of 40 different 10-mer oligonucleotides for priming an AP-PCR with M. hyopneumoniae JT (T = type strain) chromosomal DNA. On the basis of our results, we selected primers OPA-3, OPA-17, and OPB-10 for use in an analysis performed with 23 field isolates. The most informative results were obtained with primer OPA-3. A total of 21 of 23 clinical isolates produced multiband patterns with this primer, while 2 isolates failed to produce any detectable bands. Our data show that M. hyopneumoniae is genetically diverse and that M. hyopneumoniae strains can be divided into at least six epidemiological subgroups on the basis of AP-PCR results.

Use of an enhanced Escherichia coli opal suppressor strain to screen a Mycoplasma hyopneumoniae library.

Many mycoplasma genes contain internal UGA (opal) codons because of their use as tryptophan coding codons. This results in a lack of expression of many cloned mycoplasma antigenic epitopes in Escherichia coli. It has been shown that opal suppressors can be used to enhance expression of defined mycoplasma gene sequences, but no studies have been published using E. coli suppressor strains to screen mycoplasma gene libraries for immunoreactive epitopes. The E. coli suppressor strain ISM612 was used to screen a Mycoplasma hyopneumoniae Lambda gene library. This strain contained an inducible opal suppressor, trpT, as well as the release factor 2 mutation prfB3. Strain ISM612 was shown to enhance antibody recognition of cloned mycoplasmal gene sequences.

The effect of thiol-active compounds and sterols on the membrane-associated hemolysin of Mycoplasma pulmonis.

Previous studies had shown that Mycoplasma pulmonis contained a bovine serum albumin-dependent, membrane-associated hemolysin. Biochemical analyses were performed to further characterize this activity. The membrane-associated hemolytic activity could be activated by dithiothreitol and beta-mercaptoethanol, and inactivated by oxidizing compounds, a sulfhydryl inhibitor and heat treatment. Cholesterol and other sterols were inhibitory in a stereo-specific manner, but they did not interfere with adherence of M. pulmonis to red blood cells. These results indicated that once attached, the M. pulmonis hemolysin recognized cholesterol in the opposing membrane leading to red cell lysis. Because of the unique location of this toxin and its sensitivity to cholesterol, the mycoplasma membrane hemolysins may belong to a unique class of bacterial toxins.

Phylogenetic analysis of mycoplasma strain ISM1499 and its assignment to the Acholeplasma oculi strain cluster.

A mycoplasma strain designated ISM1499 was used to develop a mycoplasma genetic system (G. G. Mahairas and F. C. Minion, J. Bacteriol. 171:1775-1780, 1989; G. G. Mahairas, C. Jian, and F. C. Minion, Gene 93:61-65, 1990), but phenotypic inconsistencies led to the conclusion that this organism had been classified incorrectly as a member of the species Mycoplasma pulmonis. Studies were initiated to determine the proper taxonomic position of ISM1499, and on the basis of the results of our genetic analysis, this strain was assigned to the Acholeplasma oculi strain cluster. The base composition of strain ISM1499 was identical to the base composition of A. oculi 19L, but not to the base composition of Acholeplasma laidlawii PG8 (28.3 and 30.7 mol% G+C, respectively). The taxonomic position of ISM1499 was examined by performing a parsimony analysis with 16S ribosomal DNA sequence data, and the results were compared with previous phylogenetic reconstructions. Our results indicated that ISM1499 is more closely related phylogenetically to A. oculi 19L than to A. laidlawii PG8 and JA1. Heterogeneity in the 16S ribosomal DNA sequences of A. oculi 19L and ISM1499 and in the 16S ribosomal DNA sequences of A. laidlawii PG8 and JA1 may indicate that unusual dissimilarities occur in the 16S ribosomal DNA sequences of members of the genus Acholeplasma.

Transformation of Mollicutes with single-stranded Tn4001 DNA.

Acholeplasma oculi ISM1499 and Mycoplasma gallisepticum were transformed with single-stranded and double-stranded plasmids containing Tn4001. The transposon mobilized to the chromosome using both single-stranded and double-stranded DNA at the same frequency. M. gallisepticum transformed at a 2 log lower frequency than did A. oculi ISM1499. Restriction enzyme digestion of single-stranded DNA indicated homologous base pairing in the inverted repeat regions, which could account for the transpositional activity of single-stranded DNA.

Transformation of Mycoplasma gallisepticum with Tn916, Tn4001, and integrative plasmid vectors.

Mycoplasma gallisepticum causes respiratory disease in avian species, but little is known about its mechanism(s) of pathogenesis. These studies were undertaken in order to develop genetic systems for analysis of potential virulence factors. M. gallisepticum was transformed with plasmids containing one of the gram-positive transposons Tn916 or Tn4001, which inserted randomly into the mycoplasmal chromosome. Plasmids containing cloned chromosomal DNA were also constructed and tested for integration into regions of DNA homology derived either from chromosomal fragments or from the gentamicin resistance marker from Tn4001. These studies demonstrate that M. gallisepticum is amenable to transformation with both transposons and integrative vectors.