Characteristics of lysophospholipase activity expressed by cytosolic phospholipase A2

Evidence has accumulated to suggest that a wide variety of mammalian cells and tissues express a cytosolic phospholipase A2 with arachidonoyl preference (cPLA2). Purified rabbit platelet-derived cPLA2, as well as the human recombinant enzyme originally identified in the monocytic leukemic cell line U937, exhibit significant lysophospholipase activity. Several series of experiments indicated that a single protein mediated both activities. Treatment of the purified enzyme with p-bromophenacylbromide or an anti-(rabbit platelet cPLA2) monoclonal antibody, RHY-5, suppressed the activity of phospholipase A2 without any appreciable effect on lysophospholipase activity, suggesting that the domain(s) required for phospholipase A2 activity may be located separately from that for lysophospholipase activity. Lysophospholipase activity was appreciably detected above the critical micellar concentration of the substrate. Lysophosphatidylcholine was also hydrolyzed efficiently when it was incorporated into liposomes made of dialkylphosphatidylcholine. The hydrolysis of lysophospholipid was dependent on the fatty acid bound at the sn1 position; the relative rates of hydrolysis of 1-oleoyllysophosphatidylcholine, 1-palmitoyllysophosphatidylcholine, and 1-stearoyllysophosphatidylcholine were 23, 8, and 1, respectively. A similar order of reactivity was observed with lysophospholipid incorporated into dialkylphosphatidylcholine liposomes. cPLA2 may function not only as an arachidonate liberation enzyme but also as an enzyme responsible for degradation of certain molecular species of lysophospholipids formed in membranes.     

Comparison of six mammalian lysophospholipases

Lysophospholipases participate in the regulation of the levels of lysophospholipid, compounds with pleiotropic biological effects. Lysophospholipases were purified from a macrophage cell line (WEHI 265.1), a myelocytic leukemia cell line (HL-60) and peripheral blood eosinophils. WEHI 265.1 cells contain three lysophospholipases 28, 27 and 110 kDa as determined by polyacrylamide gel electrophoresis. The 110 kDa lysophospholipase also exhibits phospholipase A2 activity and appears to be identical to a previously described 110 kDa phospholipase A2. Similarly, the HL-60 cells have three lysophospholipases, the largest again a 110 kDa enzyme with phospholipase A2 activity and the smaller are 20 and 21 kDa. The low molecular mass lysophospholipases have distinctive chromatographic properties and amino acid compositions. However, the two low molecular mass enzymes from a given cell type are not radically different, e.g., 15 of the 20 amino acids of the C-terminal sequences of the HL-60 enzymes are identical. A single lysophospholipase, approx. 15 kDa, is a major eosinophil protein. This enzyme is different from those described above.     

Analysis of hydrolytic products from choline-labeled host cell phospholipids during growth of Rickettsia prowazekii

A phospholipase activity has been associated with the interaction of Rickettsia prowazekii with the surface of erythrocytes and competent host cells as well as during the growth of the rickettsiae within their host cells. Both fatty acid and lysophosphatides have been found in the interaction of rickettsiae with the surface of eucaryotic cells; this finding provided strong evidence for the activity of a phospholipase A. However, fatty acids, but not lysophosphatides, were found during the growth of rickettsiae within cells in which the phospholipids had been radiolabeled with oleic acid; this observation left the type of phospholipase activity in doubt. In this study, the water-soluble components of phospholipid hydrolysis by phospholipase A plus lysophospholipase and phospholipase C were determined following the growth of rickettsiae in host cells in which the phospholipids had been radiolabeled with choline. In infected cells relative to mock-infected cells, there was a loss of phosphatidylcholine with a corresponding increase not in lysophosphatidylcholine but in the water-soluble components. There was a large increase in glycerylphosphorylcholine (185%) and a smaller increase in phosphorylcholine (16%). These results indicate that both phospholipase A activity (plus a lysophospholipase activity) and phospholipase C were increased during infection by R. prowazekii and that the former was the predominant activity.     

Phospholipid interactions affect substrate hydrolysis by bovine brain phospholipase A1

The specificity of substrate hydrolysis by bovine brain phospholipase A1 (PLA1) was examined. In the presence of Mg2+, using pH values of 7 to 9, the purified enzyme deacylated 1-palmitoyl-2-[1-14C]arachidonoyl-phosphatidylethanolamine yielding 2-[1-14C]arachidonoyl-lysophosphatidylethanolamine at a rate of 70 mumol/min per mg. In the absence of Mg2+, however, the reaction rate slowed at pH values above 7.25. In contrast, brain PLA1 slowly (3.8 mumol/min per mg) hydrolyzed 1-palmitoyl-2-[1-14C]arachidonoyl-phosphatidylcholine (PAPC) unless phosphatidylserine (PS) was included. Maximal PAPC hydrolyzing activity required a PAPC/PS molar ratio of 2.5:1, Mg2+, and a pH value of 8.5-9.5. Replacing PS with phosphatidylethanolamine (PE) or phosphatidic acid (PA), but not phosphatidylinositol (PI), produced a similar effect. Moreover, hydrolysis of either arachidonoyl-substituted or dipalmitoyl-substituted PC at pH 7.5 was enhanced by increasing the mol fraction of PE. Brain PLA1 also hydrolyzed 1-stearoyl-[1-14C]arachidonoyl-PI with high velocity, but only if the substrate was dispersed in PE vesicles. In contrast, the velocity of PS, 1-palmitoyl-lyso-PC or diacylglycerol hydrolysis was low and unaffected by PE. In summary, PLA1 hydrolyzed PE with high velocity and specificity, whereas a high rate of PC or PI hydrolysis was observed only if PS, PE, or PA was present. In addition, PLA1 activity was greatly influenced by pH and Mg2+, implying that the substrate conformation is important to the catalytic efficiency of PLA1. Finally, the high rate of PE, PC or PI hydrolysis suggests PLA1 significantly contributes to the turnover of these phospholipids in the brain.     

Study of bound phospholipase activities of fungal mycelia using an organic solvent system

It has been possible to demonstrate and characterize high phospholipase activities in mycelia of Rhizopus arrhizus and Mucor javanicus by use of a system in which substrates were dissolved in diisopropyl ether. Such activities were associated with bound enzymes and would have been difficult to detect using aqueous assay systems. In both cases, phosphatidylcholine hydrolysis was by phospholipase A1 (EC 3.1.1.32) activity followed by the action of lysophospholipase (EC 3.1.1.5). Phospholipase D (EC 3.1.4.4) activity was also detected. The methods used appear to be of general applicability for the detection and study of insoluble phospholipases.     

Sulfatases, trapping of the sulfated enzyme intermediate by substituting the active site formylglycine

Sulfatases contain an active site formylglycine residue that is generated by post-translational modification. Crystal structures of two lysosomal sulfatases revealed significant similarity to the catalytic site of alkaline phosphatase containing a serine at the position of formylglycine. To elucidate the catalytic mechanism of sulfate ester hydrolysis, the formylglycine of arylsulfatases A and B was substituted by serine. These mutants upon incubation with substrate were covalently sulfated at the introduced serine. This sulfated enzyme intermediate was stable at pH 5. At alkaline pH it was slowly hydrolyzed. These characteristics are analogous to that of alkaline phosphatase which forms a phosphoserine intermediate that is stable at pH 5, but is hydrolyzed at alkaline pH. In wild-type sulfatases the hydroxyl needed for formation of the sulfated enzyme intermediate is provided by the aldehyde hydrate of the formylglycine. The second, non-esterified hydroxyl of the aldehyde hydrate is essential for rapid desulfation of the enzyme at acidic pH, which most likely occurs by elimination. The lack of this second hydroxyl in the serine mutants explains the trapping of the sulfated enzyme intermediate. Thus, in acting as a geminal diol the formylglycine residue allows for efficient ester hydrolysis in an acidic milieu.     

Lipoproteins are substrates for human secretory group IIA phospholipase A2: preferential hydrolysis of acute phase HDL

Group IIA secretory phospholipase A2 is an acute phase enzyme, co-expressed with serum amyloid A protein. Both are present in atherosclerotic lesions. We report that human normal and acute phase high density lipoproteins and low density lipoprotein are effective substrates for human group IIA phospholipase A2. The enzyme hydrolyzed choline and ethanolamine glycerophospholipids at the sn -2 position resulting in an accumulation of the corresponding lysophospholipids, including the unhydrolyzed alkyl and alkenyl ether derivatives. The hydrolysis of acute phase high density lipoprotein was 2- to 3-fold more rapid and intensive than of normal high density lipoprotein. The hydrolysis of lipoproteins was noted at enzyme concentration as low as 0.05 microgram/mg protein, which was within the range observed in the circulation in acute and chronic inflammatory diseases. The enzyme hydrolyzed the different molecular species of the residual glycerophospholipids in proportion to their mass, showing no preference for the release of arachidonic acid. Group IIA phospholipase A2 preferentially attacked the hydroxy and hydroperoxy linoleates and possibly other oxygenated fatty acids, which were released from the glycerophospholipids at early times of incubation. There was no effect on the content or molecular species composition of the sphingomyelins or neutral lipids of the lipoproteins. In conclusion, human plasma lipoproteins are the first reported natural biological substrates for human group IIA phospholipase A2. The enhanced hydrolysis of acute phase high density lipoproteins is probably due to its association with serum amyloid A protein, which enhances the activity of the enzyme and may promote its penetration to the lipid monolayer. As sPLA2-induced hydrolysis of the lipoproteins leads to accumulation of lysophosphatidylcholine and potentially toxic oxygenated fatty acids, overexpression of this enzyme may be proatherogenic.     

Disintegration of lysosomes mediated by GTPgammaS-treated cytosol: possible involvement of phospholipases

We showed previously that cytosol treated with guanosine 5'-O-(3-thiotriphosphate) (GTP-gammaS) disintegrated lysosomes in vitro [Sai, Y. et al. (1994) Biochem. Biophys. Res. Commun. 198, 869-877] in time-, temperature-, and dose-dependent manners. This also requires ATP, however, the latter can be substituted with deoxy-ATP, ADP, or ATPgammaS, suggesting no requirement of ATP hydrolysis. The lysis was inhibited by several chemical modifiers, including N-ethylmaleimide, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, and by various phospholipase inhibitors (trifluoperazine, p-bromophenacyl bromide, nordihydroguaiaretic acid, W-7, primaquine, compound 48/80, neomycin, and gentamicin), but not by ONO-RS-082, an inhibitor of phospholipase A2. The reaction was also inhibited by phospholipids (phosphatidylinositol, phosphatidylserine, phosphatidic acid, and phosphatidylcholine) and diacylglycerol. Among the phospholipase A2 hydrolysis products of phospholipids, unsaturated fatty acids (oleate, linoleate, and arachidonate) and lysophospholipid (lysophosphatidylcholine) by themselves broke lysosomes down directly, whereas saturated fatty acids (palmitate and stearate) had little effect. We found that GTPgammaS-stimulated cytosolic phospholipase A2 activity was highly sensitive to ONO-RS-082. These results suggest the participation of phospholipase(s), though not cytosolic phospholipase A2, in the GTPgammaS-dependent lysis of lysosomes.     

The biochemical prerequisites for preventing pathogenic lysolecithin activity in the human gallbladder

Human gallbladder epithelium was disintegrated to complete loss of microscopic structure and incubated at 37 degree C together with unlabelled lysolecithin and 14C-lysolecithin. During each incubation lysolecithin was degraded and stoichiometrically equivalent amounts of free fatty acids formed. The maximum rate of degradation was obtained at pH 7.0 and at 200 muM lysolecithin. With increasing amounts of gallbladder epithelial cell constituents the reaction became faster. After heating the epithelial components at 70 degrees C for 10 min the reaction was inhibited. The results suggest the presence of a heat labile lysophospholipase (phospholipase B) activity in the human gallbladder epithelium. This activity may operate to protect the gallbladder epithelium against potentially pathogenic lysolecithin activity. Its presence in the gallbladder epithelium meets the prerequisites for a local anti-inflammatory mechanism and lends further support to the hypothesis of lysolecithin as a mediator of cholecystitis.     

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.     

Fractionation of gliadin hydrolysates in water-ethanol by ultrafiltration with modified or unmodified membranes

Ultrafiltration was applied to the fractionation of neutral vs. charged peptides of similar size. The peptides, produced from gliadins, a major fraction of wheat storage proteins, were obtained by limited hydrolysis with alpha-chymotrypsin in water-ethanol 80/20 (v/v). Peptides, according to their elution by RP-HPLC, were quasineutral (repetitive peptides) irrespective of pH, or positively charged (nonrepetitive peptides) at pH below 5. The transmission through the membranes of the nonrepetitive peptides was less (until sevenfold) than that of the repetitive ones, because of the role of electrostatic repulsion involved in the retention of charged solutes. The difference of transmission was more efficient at acidic pH (3) and low ionic strength with inorganic membranes and in a wider range of pH and ionic strength with membranes modified by coating of positively charged polymers (polyvinylimidazole PVI, polyethyleneimine PEI). A continuous diafiltration process using an inorganic membrane of low molecular cut-off permitted the selective enrichment of the retentate in nonrepetitive peptides (up to 80%) and of the permeate in repetitive peptides (up to 80%) from hydrolysate feed containing about 60/40% of repetitive and nonrepetitive peptides, respectively, with a diafiltration volume of 4.     

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.     

Asymmetry of the phospholipid bilayer of rat liver endoplasmic reticulum

The phospholipids of intact microsomal membranes were hydrolysed 50% by phospholipase C of Clostridium welchii, without loss of the secretory protein contents of the vesicle, which are therefore not permeable to the phospholipase. Phospholipids extracted from microsomes and dispersed by sonication were hydrolysed rapidly by phospholipase C-Cl. welchii with the exception of phosphatidylinositol. Assuming that only the phospholipids of the outside of the bilayer of the microsomal membrane are hydrolysed in intact vesicles, the composition of this leaflet was calculated as 84% phosphatidylcholine, 8% phosphatidylethanolamine, 9% sphingomyelin and 4% phosphatidylserine, and that of the inner leaflet 28% phosphatidylcholine, 37% phosphatidylethanolamine, 6% phosphatidylserine and 5% sphingomyelin. Microsomal vesicles were opened and their contents released in part by incubation with deoxycholate (0.098%) lysophosphatidycholine (0.005%) or treatment with the French pressure cell. Under these conditions, hydrolysis of the phospholipids by phospholipase C-Cl. welchii was increased and this was mainly due to increased hydrolysis of those phospholipids assigned to the inner leaflet of the bilayer, phosphatidylethanolamine and phosphatidylserine. Phospholipase A2 of bee venom and phospholipase C of Bacillus cereus caused rapid loss of vesicle contents and complete hydrolysis of the membrane phospholipids, with the exception of sphinogomyelin which is not hydrolysed by the former enzyme.     

Hydrolysis of hexose pentaacetate esters in rat pancreatic islets

The pentaacetate esters of selected hexoses were recently found to stimulate insulin release. The kinetics of their hydrolysis was now investigated in both rat pancreatic islet homogenates and intact islets. In islet homogenates, the hydrolysis of alpha-d-glucose pentaacetate, as judged from the measurement of acetate production, displayed a pH optimum of 7.4 and a Km for the ester of 0.95 mM. At pH 7.4, the reaction velocity was about 5 times higher than the rate of alpha-d-glucose pentaacetate hydrolysis by intact islets, as judged from the ester-induced increase in the acetate content of both the islet and surrounding incubation medium. Comparable results were obtained in intact islets exposed to either beta-l-glucose pentaacetate or beta-d-galactose pentaacetate. The ester content of the islets after 120 min incubation was close to 0.1 nmol/islet, yielding an apparent intracellular concentration at least one order of magnitude higher than the extracellular concentration (1.7 mM). These findings indicate that hexose esters that either stimulate insulin release or fail to do so are equally well taken up and hydrolyzed by islet cells. They are compatible, therefore, with the view that the insulinotropic action of some of these esters may be favored by the catabolism of their hexose moiety, although some other mechanisms for stimulation of insulin release must be operative in the case of beta-l-glucose pentaacetate.     

Heat-induced elevation of ceramide in Saccharomyces cerevisiae via de novo synthesis

Sphingolipid-related metabolites have been implicated as potential signaling molecules in many studies with mammalian cells as well as in some studies with yeast. Our previous work showed that sphingolipid-deficient strains of Saccharomyces cerevisiae are unable to resist a heat shock, indicating that sphingolipids are necessary for surviving heat stress. Recent evidence suggests that one role for the sphingolipid intermediate ceramide may be to act as a second messenger to signal accumulation of the thermoprotectant trehalose. We examine here the mechanism for generating the severalfold increase in ceramide observed during heat shock. As judged by compositional analysis and mass spectrometry, the major ceramides produced during heat shock are similar to those found in complex sphingolipids, a mixture of N-hydroxyhexacosanoyl C18 and C20 phytosphingosines. Since the most studied mechanism for ceramide generation in animal cells is via a phospholipase C-type sphingomyelin hydrolysis, we examined S. cerevisiae for an analogous enzyme. Using [3H]phytosphingosine and [3H]inositol-labeled yeast sphingolipids, a novel membrane-associated phospholipase C-type activity that generated ceramide from inositol-P-ceramide, mannosylinositol-P-ceramide, and mannose(inositol-P)2-ceramide was demonstrated. The sphingolipid head groups were concomitantly liberated with the expected stoichiometry. However, other data demonstrate that the ceramide generated during heat shock is not likely to be derived by breakdown of complex sphingolipids. For example, the water-soluble fraction of heat-shocked cells showed no increase in any of the sphingolipid head groups, which is inconsistent with complex sphingolipid hydrolysis. Rather, we find that de novo ceramide synthesis involving ceramide synthase appears to be responsible for heat-induced ceramide elevation. In support of this hypothesis, we find that the potent ceramide synthase inhibitor, australifungin, completely inhibits both the heat-induced increase in incorporation of [3H]sphinganine into ceramide as well as the heat-induced increase in ceramide as measured by mass. Thus, heat-induced ceramide most likely arises by temperature activation of the enzymes that generate ceramide precursors, activation of ceramide synthase itself, or both.     

Protein kinase C regulation of ATP-induced phosphoinositide hydrolysis in bovine aorta endothelial cells

This study investigated the mechanism of protein kinase C-mediated inhibition of ATP-induced phospholipase C activation in cultured bovine aorta endothelial cells (BAEC). In BAEC labeled with 3H-inositol, phorbol myristate acetate (PMA) prevented ATP-induced inositol bisphosphate and inositol trisphosphate formation. In membranes prepared from these PMA-treated cells, Ca(2+)-, sodium fluoride-, GTP gamma S-, and ATP plus GTP gamma S-stimulated inositol bisphosphate, but not inositol trisphosphate, formation was inhibited. Inositol trisphosphate phosphatase activity was not altered in membranes from PMA-treated BAEC. These results suggest that 1) protein kinase C inhibits ATP-induced phospholipase C activation in BAEC through interference with the coupling of phospholipase C with a G-protein and through an effect on phospholipase C itself, and 2) different mechanisms are responsible for the inhibition by protein kinase C of the phospholipase C-mediated hydrolysis of phosphatidylinositol bisphosphate and phosphatidyl-inositol phosphate.     

Generation by limited proteolysis of a catalytically active 39-kDa protein from the 115-kDa form of phosphatidylinositol-glycan-specific phospholipase D from bovine serum

It has been suggested previously that small amounts of the mature 115-kDa form of phosphatidylinositol (PtdIns)-glycan-specific phospholipase D from bovine serum may exist as a 47-kDa form which can also be generated in vitro by treatment with proteases. In this study, we investigated the possible proteolytic processing by trypsin of partially purified PtdIns-glycan- specific phospholipase D from bovine serum and found that tryptic digestion caused an apparent activation of the enzyme when assayed in the presence of 0.1% (mass/vol.) Triton X-100. Trypsin cleaved the 115-kDa form of PtdIns-glycan-specific phospholipase D into three major polypeptides with molecular masses of 33, 39, and 47 kDa. Under non-denaturing conditions, the polypeptides remained tightly but noncovalently associated with each other. However, in the presence of 6 M urea, the polypeptides could be separated by anion-exchange chromatography. After renaturation, PtdIns-glycan-specific phospholipase D activity was found to be associated with a 39-kDa fragment. Based on its size and its amino acid sequence, the active-site-containing fragment consisted of approximately 275 residues of the N-terminal region of PtdIns-glycan-specific phospholipase D. The active 39-kDa fragment hydrolyzed the PtdIns-glycan-anchors of solubilized acetylcholinesterase from bovine erythrocytes and variant surface glycoprotein from blood stream trypanosomes. However, this fragment was inactive on membrane-associated acetylcholinesterase and PtdIns.     

Mechanism of pancreatic lipase action. 1. Interfacial activation of pancreatic lipase

Hydrolysis of dissolved p-nitrophenyl acetate by pancreatic lipase follows the classical acyl enzyme pathway already proposed for other esterases. Kinetic parameters of the hydrolysis have been determined. The turnover rate of the reaction is many orders of magnitude slower than that for the natural emulsified substrates. Nevertheless, several arguments are in favor of the specificity of this hydrolysis: (1) triacetin, which resembles the usual substrates for the enzyme, is also hydrolyzed very slowly in solution; (2) dissolved triacetin and tripropionin are competitive inhibitors for the p-nitrophenyl acetate hydrolysis; (3) the same chemical structural features which are required in the case of emulsified substrates are also necessary to promote hydrolysis of dissolved p-nitrophenyl esters. This suggests that the same active site (or part of the same active site) is responsible for hydrolysis of both p-nitrophenyy acetate and specific emulsified substrates. Since deacylation is the rate-limiting step in the catalysis of p-nitrophenyl acetate, the intermediate acetyl enzyme can be isolated by trapping it at pH 5.0. Kinetic competence of this intermediate has been demonstrated. Hydrolysis by pancreatic lipase of dissolved monomeric p-nitrophenyl acetate and triacetin is considerably enhanced (100- to 500-fold) by various interfaces. This suggests that at least the deacylation step, which is rate limiting in absence of interface, is accelerated by the presence of inert interfaces. Siliconized glass beads were directly shown to accelerate the deacylation of isolated [3H]acetyl lipase by at least a hundred times. This step does not directly involve the ester substrate.Thus, it is suggested that a part of the activation of lipase at interfaces may be due to a conformational change resulting from adsorption.     

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.