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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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