Index

A. laidlawii foreign geneexpression, 247-259

a-amylase—producing coloniesselection, 252, 253

detection, 254DNA library construction,

250-253PCR, 252-254

pNZ18 cloning, 254Agarose gel electrophoresis,

M. mycoides subsp. mycoidesSC PCR detection, 170, 174,175

AIDS, mycoplasma association,7,12

Amplitaq, 147Animal mycoplasmas recovery,

37-43complications, 37, 38materials, 38method, 39-42

sample collection, 39—41sample isolation, 41, 42sample transport, 41ruminants, 37-43

Antigenic surface proteins,identification, 4

AP-PCR protocol, RAPDfingerprinting, 183

Arginine hydrolysis, mycoplasmaidentification, 74, 75

ArthritisM agalactiae, 18, 19M bovis, 19,20M. pneumoniae, 9M. synoviae, 45U urealyticum, 11

Automated solid-phase DNAsequencing, 16sRNA genes,146

Avian mycoplasmas, 20, 21, 45-51epi-immunofluorescence test, 110recovery, 45-50

materials, 46-48methods, 48, 49

media preparation, 48specimen collection, 48specimen culturing, 48, 49

Avidin-biotin complex (ABC)procedure,immunohistochemical staining,133-135

BBacteriophages, 239

characterization, 243genome structure, 244isolation, 241purification, 243replication, 242, 243

Biochemical characteristics,mycoplasma identification,69-78

substrate utilization, 95—103

319

320 Index

Bovine respiratory mycoplasmosis,19,20

C

Casein digestion, mycoplasmaidentification, 76

Cationic lipids, mycoplasmatransformation, 231

Coagulated serum liquefaction,mycoplasma identification, 76

Collection methodanimal mycoplasmas, 39-42

lung samples, 39milk samples, 39nose, eye, or ear, 39pleural fluid, 39, 41

avian mycoplasmas, 48human mycoplasmas, 25—35, 53—59ureaplasmas, 53—59

Colorimetric PCR detection,M mycoides subsp. mycoidesSC, 170,172-174

Commensal mycoplasmas, 12clinical presentation, 12history, 12

Contagious agalactia, 18, 19cause, 19economics, 19epidemiology, 19outbreaks, 19transmission, 19vaccine, 19

Contagious bovinepleuropneumonia (CPBB),17-18,37,135, 167

Contagious caprinepleuropneumonia (CCPP), 18

Crocodiles, new mycoplasma, 21

D

Desert tortoises, M. agassizi, 21

DIG DNA Labeling and DetectionKit, 190

Digitonin sensitivity, 69, 73Digoxygenin-labeled probe

preparation, insertion sequenceanalysis, 201

Direct lmmunofluorescence test,119, 120

Direct immunohistochemicalstaining, 133

Direct solid-phase DNA sequencing,149-150

Disk film inhibition test, serologicalidentification, 108

DNA binding fluorochromes(DNAFs), 217, 218, 220, 221

DNA-DNA hybridization, 189-195Dot-blot hybridization, 191-193filter disk hybridization, 192, 194hybridization rates, 189, 190materials, 191, 192methods, 192-194mollicute identification, 3

DNA extraction, 141-143, 171,181,200

guamdium thiocyanate method,142, 143

materials, 142methods, 142, 143phenol/chloroform method,

142, 143DNA-binding fluorochromes, 217,

218,220,221DNA library construction, A.

laidlawii foreign geneexpression, 250-253

DNA probes, M. genitaliumdetection, 10

DNA purification, 141-144Dot-blot hybridization

Index 321

DNA-DNA hybridization,191-193

DNA immobilization, 192DNA labeling, 192, 193hybridization membrane

immunological staining, 193Dot immunobinding on membrane

filtration (MF DOT)mycoplasma identification,

113-116advantages, 114materials, 114, 115methods, 115sensitivity, 115

Double staining, fluorescencemethods, 220-222

E

Electroporation, mycoplasmatransformation, 230

Elephants, M. elephantis, 22ELISA, MAb-based sandwich,

127-131Emerging mycoplasmas, 21, 22Energy sources, M. mycoides subsp.

mycoides SC, differentiation, 96Enzootic pneumonia

economics, 20swine, 20

Enzymatic characterization,cell collection, 83—85cell growth, 82-84cell washing, 83-85enzyme analysis, 79—91enzyme reaction rate calculation,

88extrachromosomal elements,

239-245gene nucleotide sequencing, 81,82materials, 82, 83

malate dehydrogenase (MDH)assay, 84, 87

methods, 84-88phosphoglycerate kinase (PGK)

assay, 83, 86pyruvate kinase (PK) assay, 83,

86,87spectrophotometry, 85

Epilumination, 120Epimmunofluorescence test, 110Erythromycin,

M. hominis, 12M. pneumoniae, 9U. urealyhcum, 11

Eschenchia coliM. gallisepticum, 45mycoplasma gene expression,

259-264

mating, transposonmutagenesis, 236, 237

European Molecular BiologyLaboratory data bank,146,162

Extrachromosomal elementdemonstration, 239-245

bacteriophage characterization,243

bacteriophage isolation, 241bacteriophage purification, 243bacteriophage replication,242,243genome content, 243, 244genome structure, 244materials, 240megaplaque assay, 241methods, 241-244PFU assay, 241,242plaque assays, 241, 242plaque staining, 242

322 Index

Extrachromosomal elements,enzymatic characterization,239-245

FF38, see also

M. capricolum subsp.capripneumoniae, 18

Film production, mycoplasmaidentification, 75, 105, 106

Filter disk hybridization,DNA-DNA hybridization,192, 194

Fixed tissues, immunohistochemicalstaining, 133—139

Fluorescemated antibodies, 217,218,220,221

Fluorescence methods, mycoplasmadetection, 217-225

Fluorometric enzyme analysis, 82Foreign gene expression,

A. laidlawii, 247-259Functional multienzyme proteins,81

G

Geese, M. anseris, 20GenBank, 146, 150, 162Gene expression

foreign, A. laidlawii, 247-259mollicutes, 248mycoplasma, E. coh, 259-264

Gene nucleotide sequencing,enzyme analysis, 81,82

Genetic methods, mollicuteidentification, 3

Genetics Data Environment (GDE),150

Genital samples,human mycoplasmas recovery,

29ureaplasmas, 53-59

Genomic library screening,mycoplasma gene expression,E coh, 261,262

Genotypic classification, 189Glucose fermentation, mycoplasma

identification, 74Glycerol oxidation, M. mycoides

subsp. mycoides SC, Africanvs European isolates, 95, 96

GoatsCCPP, 18contagious agalactia, 19M. agalactiae, 18M. mycoides subsp. mycoides LC,

18M. putrefaciens, 18

Gray seals, M phocidae, 21Growth inhibition, human

mycoplasmas recovery, 32Growth and metabolic inhibition

tests, serological identification,105-110

Guanidium thiocyanate method,DNA extraction, 142, 143

HHaemophilus somnus, 20Harbor seals, M. phocidae, 21Harp seals, M. phocidae, 21Hemadsorption, mycoplasma

identification, 76, 77HIV, see AIDS, 12House finches, M. gallisepticum, 46Human mycoplasmas

biochemical and biologicalproperties, 31

growth characteristics, 30medical significance, 7-12ureaplasmas, 53-59

Human mycoplasmas recovery,25-34

Index 323

biochemical and biologicalreactions, 30-32

culture examination, 28, 29erythrocyte hemasdsorption, 32erythrocyte hemolysis, 32genital samples, 28, 29growth inhibition, 32identification, 30-32immunofluorescent staining, 32materials, 25-27methods, 27-33respiratory samples, 27, 29staining, 30ureaplasmas, 53-59

I

Immunoelectron microscopy,M. hominis, 315

Immunofluorescence testsadvantages, 120direct vs indirect, 119, 120mycoplasma identification,

119-124serological identification,

119-124Immunofluorescent staining, human

mycoplasmas recovery, 32Immunogold double labeling,

molhcute surface antigens,313-315

Immunogold staining, M. hominis,314,316

Immunogold staining andtransmission electronmicroscopy

mollicute surface antigens,309-317

immunogold double labeling,313-315

immunogold staining, 312, 313

methods, 312-317microorganism cultivation,

312negative staining, 312

Immunohistochemical stainingABC procedure, 133-135advantages, 135fixed tissues, 133-139

materials, 136, 137methods, 137-139paraffin section preparation, 137staining procedure, 137, 138

PAP procedure, 133-135Immunological methods, mollicute

identification, 3Immunoperoxidase tests,

advantages, 120Indirect fluorescent antibody test,

mycoplasma identification,121-123

Indirect immunofluorescence test,120

Indirect immunohistochemicalstaining, 133

Insertion sequence analysis,194-204

digoxygenin-labeled probepreparation, 201

DNA purification, 200, 201IS element identification,

198, 199materials, 199, 200methods, 200-203results interpretation, 203Southern-blot analysis, 201, 202

Insertion sequences, defined, 197L

LipofectACE, 228Lipofection, 228

324 Index

Lipoprotein radiolabelmg,membrane proteincharacterization, 280, 285, 286

Lrposome-encapsulated DNA,mycoplasma transformation,228,231

MM. agalactiae, 18, 19M. agassm, desert tortoises, 21M. ansens, 20M. bovigenitalium, pneumonia, 20M. bovirhinis, pneumonia, 20M. bovis, 19,20

MAb-based sandwich ELISA,128

M buteonis, 21M. capricolum subsp. capricolum

16S rRNA sequence, 146M. capricolum subsp.

capripneumoniaeCCPP, 18growth media, 38polymorphisms, 151recovery, 37, 38

M. corogypsi, isolation, 21M. dispar

growth media, 38pneumonia, 20

M elephantis, 22M.falconis, 21M.fermentan,

immunohistochemical staining, 135isolation, 7

M. flocculare, 20M. gallinaceum,

immunohistochemical staining,135

M. gallinarum,immunohistochemical staining,135

M. gallisepticumchickens, 20, 45E coli, 45house finches, 46immunostaining, 290trypsm treatment, 284turkeys, 20, 45

M. genitalium, 9, 10clinical presentation, 10history, 9, 10isolation, 10sequencing, 2therapy, 10vs M. pneumoniae, 10

M. gypis, 21M hominis, 11, 12

clinical presentation, 11, 12epidemiology, 11, 12history, 11immunogold staining, 314isolation, 11therapy, 12transmission, 11, 12

M. hyopneumomae, 20M, hyorhinis, 20

immunohistochemical staining,135

M. iowae, 20M. meleagridis, 20M mycoides subsp. mycoides LC 18

contagious agalactia, 18M. mycoides subsp. mycoides SC

CBPP, 17, 18detection, 17European vs African strains, 17glycerol oxidization, European vs

African strains, 95, 96immunohistochemical staining,

135,136pathogenicity, 17

Index 325

PCR detection, 167, 177agarose gel electrophoresis,

170, 174, 175colorimetric detection, 170,

172-174DNA amplification, 169, 170,

172materials, 168-170restriction enzyme digestion,

170,175sample preparation, 169,

171,172specimen collection, 168, 169

recovery, 37, 38M. mycoides cluster,

members, 18MAb-based sandwich ELISA,

128identification, MAb, 119

M. orale detection, 12M. penetrans, isolation, 7M. phocacerebrale, isolation, 21M. phocarhims, isolation, 21M. phocidae, harbor seals, 21M. pirum, isolation, 7M. pneumoniae, 7-9

clinical presentation, 8, 9complications, 9direct detection, 26epidemiology, 9history, 7, 8isolation, 7outbreaks, 9sequencing, 2serological tests, 26subtypes, 9symptoms, 8therapy, 9transmission, 9vs M. genitalium, 10

M. pnmatum, isolation, 7M. salivarium, detection, 12M. spermatophilum

characteristics, 2, 3isolation, 7

M. synoviae, 20chickens, 45gene expression library, 292turkeys, 45

MacrolidesM. genitalium detection, 10M. pneumoniae, 9

Malate dehydrogenase (MDH)activity, 87classification, 87enzyme analysis, 84, 87forms, 87

Mastitis,M. agalactiae, 18, 19M bovis, 19

Maxam-Gilbert procedure, 146Megaplaque assay, 239, 240

extrachromosomal elementdemonstration, 241

Membrane protein characterizationcolony lifts immunoblotting, 281,

289, 290immunological and biochemical,

279-295library screening, 281-283,

290-293lipoprotein radiolabeling, 280,

285,286materials, 280-283monospecific polyclonal anatisera

production, 281, 287-289mycoplasma strains, 280triton X-l 14 phase partitioning,

280,284, 285trypsin, 280, 283, 384

326 Index

Membrane proteins, sequencecharacteristics, 293, 294

Metabolic inhibition test,serological identification, 106,108, 109

Metabolism detection, substrateutilization rate determination,97,98-100

Mollicutes, see mycoplasmaMonoclonal antibody (MAb)-based

sandwich ELISAdiagnostic application, 127-131

MAb biotinylation, 128, 129MAb purification, 128, 129materials, 128, 129methods, 129-131reagent optimization, 129, 130

Mycoides cluster, members, 17, 18Mycoplasma adhesin detection,

299-306materials, 302, 303tissue-culture plates adherence

assay, 303-305Western-blot adherence assay,

302-304Mycoplasma characterization

biochemical methods, 69—78enzyme analysis, 79—91membrane proteins, 279-295PCR and sequence analysis,

universal 16S rRNAprimers, 145-163

RAPD fingerprinting, 179-186substrate utilization, 95-103universal 16S rRNA primers

biotinylated PCR productsequence determination,152-155

direct solid-phase sequencedetermination, 149, 150

materials, 147—151mycoplasma cultivation, 147,

151phylogenetic tree construction,

150,151seminested PCR amplification,

147-149, 151, 152sequence data evaluation, 155,

158, 159Mycoplasma detection in cell

culturescultural methods, 207-214fluorescence methods, 217—225

DNA binding stain, 218DNAF staining, 220double staining, 220, 221Evans blue counterstain, 219fixatives, 218fluorescemated antibodies, 218indicator cell inoculation,

219,220indicator cell preparation, 219materials, 218, 219methods, 219-224microscopy, 221mounting solution, 219results interpretation, 221—224

Mycoplasma Experience, 38Mycoplasma gene expression

E. coh, 259-264genomic library screening,

261,262materials, 260, 261methods, 261-263plasmid vectors, 263recombinant phage screening,

262, 263Mycoplasma identification

avian, 46,47biochemical characteristics, 69-78

Index 327

arginine hydrolysis, 74, 75casein digestion, 76coagulated serum liquefaction,

76digitonin sensitivity, 73film production, 75glucose fermentation, 74growth media cell adaptation,

72,73hemadsorption, 76, 77materials, 70—72methods, 73—76phosphatase activity, 75spot production, 75tetrazohum reduction, 15,16urea hydrolysis, 73, 74

immunofluorescence, 119-124materials, 121methods, 121-123

indirect fluorescent antibody test,121-123

MFDot, 113-116materials, 114, 115methods, 115

serological, 105—113growth and metabolic

inhibition tests, 105-110substrate utilization, 95-103

Mycoplasma medium, qualitycontrol testing, 61-67

Mycoplasma transformation,227-232

cationic lipids, 231electroporation, 230liposome-encapsulated DNA,

231materials, 228, 229methods, 229-232PEG transformation, 229, 230rationale, 227

NNBT reduction, substrate

metabolism detection, 97, 100Negative staining, mollicute surface

antigens, 312Neonatal ureaplasmal infection,

erythromycin, 11

OOrphan enzymes, 80, 81Osmotic lysis, enzyme analysis, 89Oxygen uptake, substrate

metabolism detection, 97,98,99

P

PAGE, whole cell proteinseparation, 267—277

Pasteurella haemolytica, 20Pasteurella multicoda, 20PCR

A. laidlawu foreign geneexpression, 252—254

contamination, 168M. genitalium isolation, 10M. hyopneumoniae, 20M. mycoides subsp. mycoides SC,

17mollicute identification, 3detection,

M. mycoides subsp. mycoidesSC, 167-174

sequence analysis,mycoplasma characterization,

universal 16S rRNAprimers, 145-163

PEG transformation, 229, 230Peroxidase-antiperoxidase (PAP)

complex procedure,immunohistochemical staining,133-135

328 Index

PFU assay, extrachromosomalelement demonstration,241, 242

Phosphoglycerate kinase (PGK)assay, enzyme analysis, 83, 86

pH change, substrate metabolismdetection, 97, 99, 100

Phenol/chloroform method, DNAextraction, 142, 143

Phosphatase activitymycoplasma identification, 75ureaplasmas, 70

Phosphoglycerate kinase (PGK),enzyme analysis, 83, 86

Phylogenetic analysis usingParsimony, 151

Phylogenetic groups, mycoplasmas,146

Phylogenetic tree, 16S rRNAsequences, 150

Plaque assays, extrachromosomalelement demonstration,241,242

Plasmids, 239restriction maps, 248, 249

Plasmid vectors, mycoplasma geneexpression, E. coli, 263

PleuroTRAP assay, 172Polyacrylamide gel electrophoresis,

whole cell protein separation,267-277

Polymorphisms, M capricolumsubsp. capripneumoniae,151

Primary atypical pneumonia (PAP),7,8

Protein separationPAGE, 267-277

materials, 268-271mycoplasma strains, 268

SDS polyacrylamide gels, 268,269,271-273

two-dimensional gelelectrophoresis, 270, 271,275,276

Western-blot probes withantibodies, 269, 270, 274

Western-blottingpolyacrylamide gels, 269,273,274

Proteolytic activity, ureaplasmas, 70Pyruvate kinase (PK), enzyme

analysis, 83, 86, 87

QQuality control testing

of media, 61-67materials, 63media incubation, 66media inoculation, 65media inspection, 66methods, 63-67QC test assessment, 66, 67test set selection, 65test strain dilution, 64, 65test strain preparation, 63, 64

R

RAPD fingerprintingamplification parameters

optimization, 182, 183AP-PCR protocol, 183DNA purification, 181materials, 180, 181methods, 181-183mollicute identification, 3mycoplasma characterization,

179-186primer set assessment, 181, 182

Red blood cells, mollicutesinteraction, 70

Index 329

Restriction enzyme digestion, M.mycoides subsp. mycoides SCPCR detection, 170, 175

Ribotyping, 197rRNA, phylogenetic studies, 145rRNA sequence analysis, molhcute

identification, 3rRNA sequences, data banks, 146

S

Sandwich ELISAfalse-positive results, 131MAb-based, 127-131

SDS-polyacrylamide gels, wholecell protein separation, PAGE,268,269,271-273

Sea mammal mycoplasmas, 21Seminested PCR, universal PCR

primers, 16S rRNA genes,148

Seminested PCR amplification, 16SrRNA, 147-149, 151, 152

Sequence characteristics, membraneproteins, 293, 294

Serological identification, seeMycoplasma identification

Sodium polyethanol sulfonate,mollicutes identification, 69

Southern-blot analysis, insertionsequence analysis, 201, 202

Spiroplasma, MI test, 106Spot production, mycoplasma

identification, 75Staining, immunohistochemical

staining, fixed tissues,137,138

Sterol requirement, mollicutesidentification, 69

Substrate utilization ratedetermination, 95-103

Surface antigens, mollicute,characterization, 279-295

Swine mycoplasmas, 20epi-immunofluorescence test, 110

TTetrazolium reduction,

mycoplasma identification, 75, 76substrate utilization, 95-103

Tissue-culture cell adherence assay,mycoplasma adhesin detection,303-305

Transmission electron microscopyand immunogold staining,mollicute surface antigens,309-317

Transport mediaanimal mycoplasmas recovery, 38ureaplasmas cultivation, 54

Transposon mutagenesis, 235—237E. faecahs/mycoplasma mating,

236, 237materials, 236methods, 236, 237tetracychne concentration

determination, 236T (tiny) strain mycoplasmas,

10,53Turkeys, 20, 21

M. gallisepticum, 20, 45M. meleagridis, 20, 45M. synoviae, 45

Two-dimensional gelelectrophoresis, whole cellprotein separation, PAGE,270,271,275

U

U. urealyticum, 10, 11clinical presentation, 11detection, 10

330 Index

epidemiology, 10, 11history, 10MI test, 106sequencing, 2therapy, 11

Universal 16S rRNA primers,mycoplasma characterization,PCR and sequence analysis,145-163

Universal PCR primers, 16S rRNAgenes, seminested PCR, 148

Universal sequencing primers, 16SrRNA genes, 149

Urea hydrolysis, mycoplasmaidentification, 73, 74

Ureaplasmal infection, neonatal,erythromycin, 11

Ureaplasmas cultivation, 53-59growth media, 54, 55materials, 54, 55methods, 55-57

agar plate inoculation, 56isolation, 56sample storage, 55, 56sample transport, 55, 56stock culture preparation, 56,

57viable cell number estimation,

56,57specimens, 54transport media, 54transport media selection, 53

Urease activitymycoplasma identification, 74ureaplasmas, 70

WWestern-blot adherence assay,

mycoplasma adhesin detection,302-304

Western-blotting polyacrylamidegels, whole cell proteinseparation, PAGE, 269, 273, 274