Effects of in utero methylmercury exposure on a spatial delayed alternation task in monkeys

Mycoplasma gallisepticum and M synoviae are important avian pathogens causing respiratory diseases which result in great economic losses in poultry farming. Two oligonucleotide probes, complementary to the variable region V8 of 16S rRNA from the avian mycoplasmas M gallisepticum and M synoviae have, therefore, been designed and used in direct filter hybridisation experiments. Both probes gave strong hybridisation signals with their homologous targets, whereas no cross-hybridisations were obtained with any of the other avian mycoplasmas tested. It was possible to detect 2-3 x 10(4) mycoplasma organisms by direct filter hybridisation experiments with radiolabelled probes. The probes were also used to analyse several laboratory strains and field isolates of M gallisepticum and M synoviae with complete agreement between the probe technique and the other methods used for species determination. Atypical M gallisepticum strains also gave strong hybridisation signals with the M gallisepticum specific probe.     

Detection of Mycoplasma in avian live virus vaccines by polymerase chain reaction

The polymerase chain reaction (PCR) was evaluated to detect mycoplasma contamination of avian live virus vaccines. The specificity of the primers showed that 34 strains belonging to nine species of avian mycoplasma DNA could be detected. The sensitivity of PCR to detect mycoplasma DNA was 10(0.2) colony forming units (cfu) of Mycoplasma synoviae and 10(0.7) cfu of Mycoplasma gallisepticum. When M. synoviae and M. gallisepticum were spiked into several avian live virus vaccines, PCR gave a positive reaction except for the avian pox and the avian encephalomyelitis vaccines which were prepared from organ homogenates. Short-term incubation of avian encephalomyelitis vaccines improved the sensitivity of PCR to detect both M. synoviae and M. gallisepticum. Therefore, PCR, combined with the short-term incubation, were shown to be most effective in detecting mycoplasma contamination in all of avian live virus vaccines.     

Studies on Pasteurella multocida. III. In vitro assay for cell-mediated immunity

Peripheral blood lymphocytes (PBL) from turkeys immunized against fowl cholera with a bacterin or a live avirulent vaccine (strain CS-148) were cultured in vitro with various antigenic preparations from Pasteurella multocida (strain P-1059). The degree of lymphocyte stimulation (blastogenesis) was quantitated by measurement of the uptake of (3H) thymidine. Higher stimulation indices were obtained with immune lymphocytes rather than nonimmune lymphocytes. Stimulation was specific since PBL from turkeys immunized against P. multocida failed to react with Escherichia coli or Mycoplasma synoviae antigens. These differences were statistically significant as analyzed with the student's t-test. The lymphocyte transformation assay was emphasized as a convenient and useful in vitro indicator of cell-mediated immunity that should help define the role of cell-mediated immunity in P. multocida infections of turkeys.     

Mycoplasmas in wild turkeys living in association with domestic fowl

One hundred and nineteen Merriam's wild turkeys (Meleagris gallopavo merriami) and 31 domestic chickens coexisting on a ranch in west-central Colorado (USA) were surveyed for mycoplasmosis by serologic and cultural methods. Although no clinical signs were apparent in any wild turkeys tested, 51 (43%) had positive rapid plate agglutination (RPA) reactions for M. gallisepticum (MG) and/or M. synoviae (MS); 37% of 56 adults and 48% of 63 subadults were classified as positive reactors to MG and/or MS. No turkeys tested in 1992 (n = 61) and 17 (29%) of 58 turkeys tested in 1993 were RPA-positive for M. meleagridis (MM). Hemagglutination inhibition (HI) test results were negative for MG, MS and MM as were most enzyme-linked immunosorbent assay (ELISA) test reactions (MG = 99%, MS = 93%, MM = 87%). Immunoblotting showed mild to moderate reactivity to MG proteins in 49% of 41 samples tested. Most chickens were strongly positive for MS by RPA (81%), HI (58%) and ELISA (87%); 48% also were positive for MG by RPA but all were MG-negative by HI and ELISA. No pathogenic mycoplasmas were isolated from either group of birds. Mycoplasma gallopavonis was commonly identified from the wild turkeys, and M. gallinaceum was isolated from both the chickens and wild turkeys. In a transmission study conducted in 1994, disease-free domestic turkeys failed to seroconvert when co-housed with wild turkeys from this population that were RPA-positive for MG. Collectively, the results of this study were inconclusive regarding the status of pathogenic mycoplasmas within this wild turkey population.     

Multigene families encoding the major hemagglutinins in phylogenetically distinct mycoplasmas

Mycoplasma synoviae has two major membrane antigens, MSPA and MSPB, both of which are phase variable and which may be coordinately involved in adhesion of the organism to erythrocytes. A single gene (vlhA) from M. synoviae was characterized, and polypeptides were expressed from nonoverlapping 5' and 3' regions in Escherichia coli. The expression product of the vlhA 5' region reacted with specific reagents against MSPB, while that of the 3' region reacted with specific reagents against MSPA. Analysis of the predicted amino acid sequence showed a characteristic signal peptidase II cleavage site, and the presence of the acylation site was confirmed by identification of a lipid-associated membrane protein, similar in molecular mass to MSPB, in [3H]palmitate-labelled membrane proteins. Further sequence analysis of the vlhA gene revealed a high identity with the Mycoplasma gallisepticum pMGA1.7 gene, a member of a large translated family. The vlhA gene was shown to hybridize to multiple restriction fragments of the M. synoviae genome, suggesting that it was also a member of a multigene family. These findings indicate that coordinate phase variation of the two major surface antigens of M. synoviae WVU may be due to their expression from the same gene and that homologous gene families encode the major hemagglutinins of two phylogenetically distinct mycoplasmas. The presence of homologous multigene families in such phylogenetically distinct species, but not in the genomes of more closely related species, suggests that the families may have been transferred horizontally.     

Differentiation of two strains of Mycoplasma gallisepticum with monoclonal antibodies and flow cytometry

Identification of infecting Mycoplasma spp. is difficult and not routine for strain. This paper describes a procedure for the rapid identification of the strain of M. gallisepticum. Monoclonal antibodies were prepared against M. gallisepticum F and M. gallisepticum S6. Aliquots of 24-hour broth cultures of these organisms were incubated briefly with either of the monoclonal antibodies. A second incubation was made with anti-mouse immunoglobulin conjugated to fluorescein isothiocyanate. Fluorescent intensity associated with the organisms was measured with a flow cytometer. The criterion for identification was a comparative increase in fluorescent intensity when the strain and monoclonal antibody were homologous. The procedure correctly differentiated the F and S6 strains of M. gallisepticum in a blind study.     

Experimental studies on the growth of cultured epithelium and endothelium of rabbit cornea exposed to low temperature

Three strains of Mycoplasma synoviae (MS) (Olson-WVU 1853, reported in 1956; and Massachusetts 9895 and 5044, respectively isolated in 1957 and 1972) were used for experimental inoculation of chickens to evaluate the various mycoplasma serologic tests. The MS plate agglutination and hemagglutination-inhibition (HI) tests were highly sensitive for detection of infection; reactions persisted for test periods extending to 63 weeks. Correlation between MS HI-tube and micro methods was good. HI titers were highest in sera from birds inoculated with strain 9895. All three strains produced cross-reactions with M. Gallisepticum (MG) plate antigens, which were detectable for 2 to 12 weeks following MS inoculation; strain 5044 produced the lowest percentage of cross-reactions. MG HI titers of 16 were observed occasionally in sera from all three group inoculated with strain 1853. It appears from these limited serologic studies in chickens that there may be antigenic differences among the three MS strains.     

Further observations on the use of a bivalent bacterin against Haemophilus gallinarum

Chickens vaccinated with two doses of a bivalent Haemophilus gallinarum bacterin were protected against seven strains of the organism. Vaccinated and unvaccinated birds with antibody to Mycoplasma gallisepticum had increased HI titers when challenged with H. gallinarum. Birds positive for antibody to Mycoplasma gallisepticum which were not challenged showed no increase in HI titer.     

Evaluation of the efficacy of zwitterionic dodecyl carboxybetaine surfactants for the extraction and the separation of mycoplasma membrane protein antigens

The ability to extract mycoplasma membrane protein antigens using the alkyl carboxybetaine surfactants (N-dodecyl-N,N-dimethylammonio)butyrate (DDMAB, CMC = 4.3 mM) and (N-dodecyl-N,N-dimethylammonio)undecanoate (DDMAU, CMC = 0.13 mM) was assessed by protein titration and SDS-PAGE analysis. The maximum yields of membrane protein solubilization ranged from 20 to 90%, depending upon both the mycoplasma membrane investigated and the surfactant used. In five of six cases, the extraction was optimal for surfactant concentrations of ca. 25 mM. DDMAB displayed a higher efficiency in membrane protein extraction. The order of efficiency for both surfactants was Spiroplasma melliferum > Acholaplasma laidlawii > Mycoplasma gallisepticum. In contrast, DDMAU proved much more selective. The order of selectivity was M. gallisepticum > S. melliferum > A. laidlawii. The highest selectivity was recorded for the major proteins p67 and spiralin of M. gallisepticum and S. melliferum, respectively. For p67, notably, DDMAU proved superior to 10 other surfactants. Dot immunobinding and crossed immunoelectrophoresis analyses showed that both dodecyl carboxybetaines were suitable as membrane protein-solubilizing agents in immunological techniques. Furthermore, these surfactants did not exhibit effects adverse to the activity of A. laidlawii membrane NADH oxidase. One promising application of DDMAU is the separation of membrane proteins by ion-exchange HPLC as illustrated by the good resolution of M. gallisepticum membrane proteins and purification of p67 to almost homogeneity. These data show that dodecyl carboxybetaine surfactants are useful for the extraction of mycoplasma membrane antigens under mild conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular biology of Mycoplasma

Mycoplasmas are the smallest free living microorganisms with the smallest genome. The G+C content is in general low (25-33%) and the coding capacity is about 600 proteins. Mycoplasma species are phylogenetically related, they use the genetic codon UGA for tryptophan, and show rapid evolution, with a high rate of divergence. The genomes of Mycoplasma genitalium and Mycoplasma pneumoniae have been fully sequenced. Striking features of the M. genitalium sequencing project are the presence of a high number of membrane proteins with no resemblance to previously sequenced genes and the presence of repeated fragments of the gene encoding the tip-localized 140 kDa adhesin (MgPa). Many Mycoplasma species display a high frequency of antigenic variation, both as phase and size variation of individual antigens. Mycoplasma hominis isolates are known to be antigenic heterogeneous, as reflected in the reactivity with monoclonal antibodies (MAbs). The genetics of the antigenic variation has been studied for three different surface exposed antigens: P120, Lmp, and P50/Vaa. The gene encoding P120 had a hyper-variable region in the N-terminal region. In addition, a second gene with homology to p120 was identified. The gene encoding Lmp, a 135 kDa protein is repeated and both genes are translated and both contain internal repeated sequences. Deletion mutants in the lmp gene were obtained by cultivation of M. hominis PG21 with MAb 552 specific for the repeated part of Lmp. One of the lmp genes had deletions of from four to eight repeats. The other gene was left unaltered. The genes encoding P50/Vaa show a different form of variability where domains of the genes seem to be exchangeable. The genomic maps of five M. hominis strains showed that even though the size of the genomes varied the position of the different genes were in general conserved.     

Genomic, protein homogeneity and antigenic variability of Mycoplasma agalactiae

Eleven strains of Mycoplasma agalactiae differing in pathogenicity, animal species origin and geographic localisation, showed similar chromosome restriction profiles with four endonucleases. However the international reference strain PG2 showed a unique profile. The protein and antigenic variabilities of 31 strains of M. agalactiae were investigated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting performed with naturally infected animal sera and purified antibodies against the 29 kDa protein. Protein profiles were similar but antigenic profiles could be separated into two main groups according to geographic origin: (i) strains isolated in south-west France and (ii) strains from north-east France. Some differences also occurred from strain to strain within each group. The antigenic profile variability found in immunoblotting, originated in two different phenomena: (i) some epitopes were expressed only in strains of one profile type and (ii) some other epitopes were common to all strains but located on several proteins which differed in number and molecular mass from one strain to another. The presence of epitopes which undergo phase variation in the same lineage of clones from a single cell is discussed.     

A filter immunobinding technique for the rapid detection and simultaneous identification of avian and bovine mycoplasmas

A filter immunobinding (FIB) method was developed for the detection and identification of mycoplasmas. Type strains of a total of 18 avian and bovine mycoplasma species propagated in broth media were diluted and immobilized on a nitrocellulose membrane as antigens for investigating the specificity with rabbit hyperimmune sera. Non-specific FIB reactions were easily eliminated by the procedure of absorbing rabbit hyperimmune sera in the broth. Absorbed rabbit hyperimmune sera exhibited clear species-specificity with mycoplasma antigens by the FIB. These specific reactions always agreed with the results of identification by tests of biochemical properties and growth inhibition for the isolates of M. bovirhinis, M. bovis, M. columbinum, M. columborale, M. gallisepticum and M. synoviae. Some bovine mycoplasma species, which were impossible to identify by growth inhibition test, because of their strong production of film and spots on the agar, specifically reacted with absorbed rabbit hyperimmune sera against M. bovis in the FIB. The detection limit of mycoplasmas by this method was about 10(4)-10(5) colony-forming units/ml, which is lower than that of colony determination on agar. The FIB seems to be a useful technique for rapid detection and simultaneous identification of mycoplasmas.     

Lysophospholipase-catalyzed hydrolysis of lysophospholipids in Mycoplasma gallisepticum membranes

Mycoplasma gallisepticum strains have a membrane-bound lysophospholipase which hydrolyzes lysophospholipid generated in these membranes by treatment with an external phospholipase. This paper studies the hydrolysis of the membranous lysophospholipids by an enzyme residing in the same membrane (intramembrane utilization) or in adjacent membranes (intermembrane utilization). To study intermembrane hydrolysis, the phospholipids of M. gallisepticum were labeled with [3H]oleic acid. Membranes were prepared, heated at 65 degrees C, and subsequently treated with pancreatic phospholipase A2. This resulted in membranes whose enzyme was heat inactivated, but which contained lysophospholipid. When these membranes were mixed with M. gallisepticum cells or membranes, the lysophospholipid was hydrolyzed by the membranous lysophospholipase. To study intramembrane hydrolysis, [3H]oleyl-labeled membranes of M. gallisepticum were treated with pancreatic phospholipase A2 at pH 5.0. At this pH, lysophospholipid was generated but not hydrolyzed. Adjustment of the pH to 7.4 resulted in hydrolysis of the lysophospholipid by the membranous lysophospholipase. These procedures permitted measuring the initial rates of intramembrane and intermembrane hydrolysis of the lysophospholipid, showing that the time course and dependence on endogenous substrate concentration were different in the intramembrane and intermembrane modes of utilization. They also permitted calculation of the molar concentration of the lysophospholipid in the membrane and its rate of hydrolysis, expressed as moles per minute per cell or per square centimeter of cell surface.     

The effects of F strain Mycoplasma gallisepticum, Mycoplasma synoviae, and the dual infection in commercial layer hens over a 44-week laying cycle when challenged before beginning of lay. I. Egg production and selected egg quality parameters

In each of two trials, 160 commercial pullets were separated into four treatments with four replicates of 10 chickens in each treatment. Forty pullets were designated as controls and received no inoculation; 40 other pullets received F strain Mycoplasma gallisepticum (FMG); an additional 40 pullets received Mycoplasma synoviae (MS); and the final 40 pullets were inoculated with both FMG and MS (dual). Hen-day egg production, egg weight, eggshell strength, Haugh unit score, pimpling incidence, and blood/meat spot incidence were monitored and recorded in each trial through an entire laying cycle. No significant difference was observed among the treatments for hen-day egg production, egg weight, eggshell strength, or Haugh unit scores. Significant differences were observed for pimpling incidence among controls (1.63%), Mycoplasma gallisepticum (MG)-infected (2.09%), and dual-infected hens (2.41%). A significant difference in blood/meat spot incidence was observed between MG-infected hens (0.27%) and dual-infected hens (0.45%). Histopathologic examination of the ovary and all segments of the oviduct revealed no significant differences among the treatments. These results suggest that the majority of the hen reproductive tract functions similarly in FMG-vaccinated, MS-infected, or dual-infected hens as compared with Mycoplasma-clean hens.     

Safety of a temperature-sensitive clone of Mycoplasma synoviae as a live vaccine

A temperature-sensitive (ts+) clone derived from the Australian Mycoplasma synoviae (MS) field isolate 86079/7NS was produced by chemical mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and assessed for safety as a live vaccine. This clone, designated MS-H, was assessed for pathogenicity in three different models with air sac lesions as the criterion. No air sac lesions were observed when MS-H was administered to specific-pathogen-free hybrid white leghorn (HWL) chickens by eyedrop at 10 times the normal dose or directly into the thoracic air sacs or as an aerosol administered to specific-pathogen-free Webster white leghorn chickens with concurrent intratracheal T-strain infectious bronchitis virus (IBV). MS-H did not revert to virulence or lose the ts+ phenotype when passaged through five in vivo and 10 in vitro passages. No adverse effects were seen when HWL chickens were vaccinated concurrently with MS-H and combinations of Mycoplasma gallisepticum ts-11 vaccine, IBV vaccine, and infectious laryngotracheitis virus vaccine. Lateral transmission of MS-H was found to occur when vaccinated HWL chickens were mixed with unvaccinated chickens 2 wk after vaccination. At 1 wk after mixing, one out of two unvaccinated chickens had seroconverted to MS and was culture positive for MS. At 2 wk after mixing, both contact chickens were positive for MS by culture and serology.     

Evaluation of polymerase chain reaction for detection of Mycoplasma meleagridis infection in turkeys

Different methods were compared for the detection of the turkey pathogen Mycoplasma meleagridis in a turkey field flock before and after antibiotic (oxytetracycline) treatment. They included culture, serology (detection of antibodies by ELISA) and a polymerase chain reaction (PCR) based assay developed by Boyle et al. [Boyle, J.S., Good, R., Morrow, C.J., 1995. Detection of the turkey pathogens Mycoplasma meleagridis and M. iowae by amplification of genes coding for rRNA. J. Clin. Microbiol. 33, 1335-1338]. Culture and PCR assay with tracheal swab samples were much more sensitive than ELISA with serum samples. Percentages of infected birds detected by culture or PCR for samples collected prior to antibiotic treatment were almost identical but the percentage of positive samples detected after antibiotic treatment was much higher with the PCR test.     

The hemagglutination-positive phenotype of Mycoplasma synoviae induces experimental infectious synovitis in chickens more frequently than does the hemagglutination-negative phenotype

Inoculation with hemagglutination-positive (HA+) cultures of Mycoplasma synoviae AAY-4 induced acute synovitis significantly more frequently (P = 0.001) in chicken tibiotarsal-tarsometatarsal joints than did inoculation with HA-negative (HA-) cultures derived from the same clone of AAY-4. Immunoblotting analyses showed that HA+ cultures abundantly expressed two phase-variable hemadherence-associated surface membrane proteins of 53 kDa and 48 to 50 kDa defined by monoclonal antibodies. HA- cultures lacked the 53-kDa proteins and synthesized truncated 27- to 30-kDa forms of the 48- to 50-kDa proteins. Inoculation of cyclosporin A (CsA) into infected joints significantly decreased the frequency of acute synovitis (P = 0.001). Moreover, repeated intra-articular inoculation of CsA (three doses of 1 mg at 2-day intervals) significantly reduced the local antibody response to M. synoviae in the joints treated with CsA.     

The Numbing Scale: psychometric properties, a preliminary report

Twenty-one antimicrobial agents were incorporated individually into Frey's agar to evaluate their inhibitory activities against 86 isolates of avian mycoplasmas recently detected in Taiwan. Among them, 45 and 37 isolates were found positive with Mycoplasma gallisepticum and Mycoplasma synoviae fluorescent antibody conjugate, respectively. Twenty-one other isolates were unable to be identified by the above 2 conjugates. All of the field isolates were highly sensitive (with MIC50 < 1 microgram/ml) to enrofloxacin, gentamicin, myplabin, tiamutin and tylosin. However, those field isolates were highly resistant (with MIC50 > 32 micrograms/ml) to apramycin, chlortetracycline (CTC), erythromycin (ER), flumequine (FI), nalidixic acid (NA), oxolinic acid (OA), oxytetracycline (OTC) and spiramycin (SP). The inhibitory activities of the antibiotics which possessed an MIC90 of 50 micrograms/ml or less against local isolates were, in decreasing order, enrofloxacin (< 0.004 microgram/ml), gentamicin (1.53 micrograms/ml), tiamutin (1.81 micrograms/ml), tylosin (3.2 micrograms/ml), streptomycin (SM; 12.0 micrograms/ml), colistin (13.1 micrograms/ml), chloramphenicol (14.0 micrograms/ml), spectinomycin (15.0 micrograms/ml), myplabin (16.0 micrograms/ml), spiramycin (30.0 micrograms/ml), minocycline (32.0 micrograms/ml). The MIC90 of OA, CTC, SM, FI, SP, OTC, ER or NA was greater than 50 micrograms/ml; which work poorly in the control of mycoplasmoses. Since the antibiotic control policy is quite loose in Taiwan, many antimicrobial agents are often freely used in clinics, with a resulting gradual decrease in the inhibitory activity to the avian mycoplasmas.