Identification ofa SecondHemolysin (HlyII) inActinobacillus pleuropneumoniae Serotype 1 andExpression ofthe GeneinEscherichia coli

Hemolysin genes ofthereference strains ofActinobaciUlus pkuropneumoniae serotypes 1 and2 were identified, cloned, andexpressed inEscherichia coli byusing polymerase chainreaction amplification with oligonucleotides derived fromtheDNA sequenceofthecorresponding appAgenefromA.pleuropneumoniae serotype 5.Thethree genesfromserotypes 1,2,and5haveidentical restriction maps andappear toencode a hemolysin which was previously identified inserotype 2anddesignated HlyII. GeneappAisdifferent from hlylA encoding themajorhemolysin typeI(HlyI) which was identified earlier inserotype 1.Polymerase chain reaction amplification witholigonucleotides derived fromtheDNA sequenceofhIyIAofserotype 1showed thatthegeneencoding HlyIispresent inserotype 1 butnotinserotype 2,incontrast tothegene encoding HlyIIthat was present inbothserotypes. Thiswas confirmed byWestern blot(immunoblot) experiments usingmonoclonal antibodies specific foreither recombinant HlyIor recombinant HlyII, whichshowed thatA.pleuropneumoniae serotype 1strain 4074produces bothHlyIandHlyII, whereas serotype 2strain S1536produces onlyHlyII. Theexpression ofbothhemolysins was investigated inallserotypes bythe useofmonoclonal antibodies. HlyI was showntobeexpressed bythereference strains ofserotypes 1,5a, Sb,9,10,and11,whereas HlyII was showntobeexpressed bythereference strains ofall12serotypes tested except serotype 10.A.pleuropneumoniae serotype 1strain 4074isthefirst bacterium whichhasbeenshownto contain twodifferent actively expressed RTXtoxin genes.Comparison ofourdatawiththose fromother groups showsthattheoriginally described strongly hemolytic hemolysin typeI(HlyI) corresponds tocytolysin I(Clyl) whichwas recently described byothers, whiletheweakly hemolytic hemolysin typeII(HlyII) seems tobe identical toClylI andAppA. Thehemolysins ofActinobacillus pleuropneumoniae, the