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Isolation and Identification of Mycoplasmas from the Respiratory Tract of the White Leghorn Chicken in the Sudan
Br.
vel . J
( 1982)
138, 253
ISOLATION AND IDENTIFICATION OF MYCOPLASMAS FROM THE RESPIRATORY TRACT OF THE WHITE LEGHORN CHICKEN IN THE SUDAN
Bv M.S. M . A. HAR BI, A. MUSTAFA*, M. M. SALIHt , AND S.M. M. ELAM IN*
Department of MycojJlasma, Velerinmy Research Administration, P.O. Box 8067 (El Amaral), Khartoum, Sudan, *Department of Avian M edicine, Leahurst, University of Liverpool, Live1pool, England and fFAOIWHO Collaborating Centre for Animal Mycoplasmas, Unive rsity of Aarhus, DK-8000 Aarhus C, Denmark
SU MMARY The poss ibl e importance of mycopl as m as in respiratory di seases of ch icken in the Sud a n provided th e st imu lus for thi s study. T h ree species of m yco pl as mas, .MycojJlasma gallinamm, MycojJlasma iners a nd AclwlejJlasma laidlawii, were isolated a nd ident ifi ed. The frequ ent occu rrence of Mycoplasmagallinarum, wh ich m ay be important as a seco nd ary in vader in res piratory infections of chi cken , s ugges ted that furth er study of the possible pathoge ni city of the orga nism is m erited. A sea rch for pat hoge n ic m yco pl as m as in sites other tha n the res pirato ry tract is reco mm ended. INTRODU CT ION Th ere is at prese nt worldwide interest in av ia n m yco plasmas, especia ll y associated w ith domesti c poultry (O .I.E. , 1979). Pat hogen ic a nd non-pathogeni c my co pl as m as have o ft en been iso lated from the respiratory tract of po ultry (Adl er & Ya mamoto, 1957; Chu, 1958; Yam a moto & Adl er, 1958; Yode r , 1965; J ordan, 1979) . In th e Suda n , the first publish ed ev idence o f m ycop lasmas in pou ltry was that of Khogali ( 1970), who studi ed resp iratory di seases in foreig n breeds of chi cken. Hi s six iso lates we re non-pa thoge ni c , and they were no t id entifi ed. H a rbi & Khoga li ( 1975 ) iso lated a my .:op las m a (sero type C) from th e respiratory tract of the loca l Baladi breed o f chi cken . The pat hogeni c sig nifi ca nce of se rotype C is poo rl y understood Uord a n, 1979). H a rbi , Mu sta fa & Sa lih ( 1980) isola ted MycojJlasnza gallinanmz from th e resp iratory tract of loca l Ba ladi chick ens . Mycoplasma gallinanmz is co nsidered by m a ny workers Uordan & Kul asegara m , 1968; Kl even, 1977 ;J ord a n , 1979) to be pathoge ni c wh en associa ted with ce rtain ot her agents.
254
BRITISH VETERINARY JOURNAL, 138, 3
This pa p er repo rts the isolation a nd id en tifi cat ion of mycop lasma s pec ies from the respiratory tract of norma l a nd d iseased chi ckens.
MATERIALS AND METHODS
Chickens Seventy-five White Leghorn chi ckens, aged a bo ut seven months, were obtai ned from private fa rm s in the vicin ity of the labo ratory . Of th ese, 32 showed early signs of res pirato ry di sease; the rem a ind er were appa ren tl y h ea lthy. The cl ini ca l sy mpto ms were sporad ic. All the birds were kil led and the head s, trac hea, lungs, and airsacs collected in steril e co nta in ers. Reference strains and sera The six kn-own species of avia n mycoplasmas, together with their correspond in g hyperimmune se ra, were kindl y provided by Professor E. A. Freundt, Direc tor, FAO/ WHO Collaborating Ce ntre for An im a l M yco pl as m as, Aarhus, Denm ark . Growth media Liquid a nd so lid m edi a were used : b acto hea rt infu sio n brot h a nd agar (Difco), bruce ll a broth a nd agar (Aib imi ), a nd m ycop las ma brot h a nd agar (Oxoid). H o rse se rum , 20%; yeast ex tract, 3 g/1; DNA, 0·6 g/1; penicill in, 200 000 i.u./ ml a nd l ml 10% solu tion th a lliu m aceta te were usua ll y in co rpo rated in the media . N icot in am id e adenine dinucleotide (NA D ) an d L-cysteine, which a re specifi ca ll y req uired by Mycoplasma synoviae, were a lso add ed to the medi a. Cultural methods These co mpri sed the strea king of the so lid medi a with the s uspect ma ter ia l, a nd subsequ ent sub cu ltures in liquid a nd soli d medi a. Pl ates we re put in to polythene bags to provide suffi cientl y humid condit ions witho ut ca usin g th e co nta min at io n th at has so m eti mes been enco unte red when using humid chambers co ntain ing wa ter. Liquid a nd solid m ed ia were in cu bated ae rob ical ly a t 37°C . Pl ates were exam in ed fo r th e ap pea rance of umbonate co loni es at interva ls of up to three weeks, a nd broth cu ltures checked fo r a period of two weeks. After three to five passages on medium co nta in in g antibac terial age nts, isola tes were tested fo r revers ion tO bac terial form by subject in g them tO fiv e co nsecuti ve subcu ltures in med ia without a n tib iotics (I .C.S. B. , 1979) . Biological and serological properties After cl oning, th e iso la tes were id entifi ed by the methods or Ern0 & St ipkov itz ( 1973). They were tes ted for sensiti vity to digitonin, a property eharacrerist icofste roldependent mycoplasmas (F reun dr et al., 1973). Di gitonin-se nsit ive orga ni sms were tested for phosphatase ac tivity, fermentation or glucose, hydro lys is or urea a nd arginin e, a nd redu ction of 2, 3, 5, tripheny l tetrazo li um chl ori de under ae robic con diti o ns. Produ ction of ' film a nd spots' was tested as described by Fa bri ca nt & Freundt ( 1967) on media conta inin g 20% ho rse se rum. Proteo lys is was tested by s treak ing three-day cu ltures on coagu lated horse serum sla nts. T he slants were
MYCO PLA MA
11 C HI C K E
11 T H E
DA
255
in cubated for two weeks. Three-d ay cultures were a l o examin ed for haemagglutinating activ ity. As un co nce ntra ted cultures do no t a lways giv cl arl readabl e res ults (Yoder & Hofstad 1962) , w u ed culture centrifugate . H aemol ti c activity was tested against sheep red blood ell (RBC) by mea n of a n overla technique (EI asri, 1967). Th e sol serologica l tes t used was the growth inhibiti on tes t; it was carried ou t by the paper dis c technique (C lyd e, 1964) and a l o by the meth od of Bl ack ( 1973) in whi h well s were pun ched in th e aga r. RE
LT
Of the 44 digitonin-sensitive mycopl as ma i olate , 17 were ubj ected to id entifi ca tion procedures: two isola tes from hea lthy birds a nd 14 from di seased birds proved to be Mycoplasma gallinarum. On e isolate from a hea lth y bird proved to be Mycoplasma iners a nd one digitonin-resistant iso la te from a hea lth y bird wa id entifi ed as Aclzoleplasma laidlawii. Mycoplasma iners a nd Acholeplasma laidlawii have no t prev iously been record ed as occurring in the res piratory tra t of chi ckens in th e ud a n . Mycoplasma gallinanan isola tes had a ll th e cha racteristi cs of the type stra in PG 16 (Edwa rd & Freund t, 1956). Th y hydrol y ed a rginin e a nd redu ced tetrazo lium, but fa il ed to hydrolyse urea or ferm ent glu cose. Th ey produced 'film and spo t ' (Fi g. I), fai led to digest in spissa ted serum , a nd were phosph a tase nega tive. Alpha-ha emolys is was detected after in cubation for 24 to 36 h a t 37°C; beta -h ae molys is was detec ted a fter fi ve to seven days incubation .
Fi g. I. ' Film and spo ts' produ cd b M;-coplasma gallinanan i ola t .
Hae maggluti na tion of chi cken RB C was poo r a nd consid ered t be non- pecifi . Th stra in were inhibit d by hyperimmun e era to th e type tra in of Mycoplasmagallinarum. Th train id entifi ed as Mycoplasma iners had th e properti e of th e type stra in PG 30 (Edwa rd & K a na rek, 1960). It did no t ferm ent glu co e but hyd ro! sed arginin e. It wa urea nega tive, ph sph a tase nega ti ve a nd fa il ed to redu ce tetrazolium . o ' film a nd spo t ' were observed on solid medi a a nd oag ul a ted serum rema in ed undiges ted .
256
BRITISH VET ERI NARY .JO U RNAL , 138, 3
Alph a-h ae mo lys is was o bse rve d , bu t ch ic ke n RB C were no t hae m agg lutin a ted . Growth was strong ly inhibi ted by a nti se ra to the ty pe st ra in . AcholejJlasma laidlawii was id ent ifi ed on th e g ro und s th a t it was nega ti ve to dig itonin a nd a cl ea r zo ne o f inhibitio n was prod uced by hy perimmun e se rum aga in st th e ty pe s tra in PG8 (Ed w a rd & Freundt , 19 73 ) in th e g row th inhibi tion tes t. Th e zo ne o f inhibiti o n prod uced by th e well m eth od was es pecia ll y p ro no un ced .
DI SCUSS I ON
In thi s s tud y MycojJlasma gaLLinamm was th e most co mm o nl y occ urrin g s pec ies. I t was iso lated from va ri o us sites, es pec ia ll y th e sinu ses a nd trac heas . M a ny a uth o rs co nsid e r this species to be no n-path oge ni c for chi ckens (Yod er & H ofs ta d , 196 4·; Yod e r, 1978 ; J o rd a n , 1979). H owever , J ord a n ( 19 79) r eported th a t it could be pa th oge ni c wh e n occurrin g toge th er wi th o th er avia n pa th oge ns. Syn erg ism between m yco p las m as a nd o th er a vi a n pathogen s (e.g . N ewcas tle di sease viru s a nd infec ti o us bron chiti s viru s) has been repo rted U01·d a n & Kul asega ra m , 1968 ; Crostve t & Sadl er, 1966). K leve n ( 19 77) studi ed th e poss ibl e importa nce o f M)'cojJLasma gaLLinanan in mu ltip le in fec ti o n. H e reprodu ced a irsacculiti s ex perimenta ll y in yo un g chi cke ns by a dm ini s terin g Mycoplasma ga LLinarum a nd th e viru s of infec ti o us bro nchi tis. N eith er the viru s no r th e m yco pl as m a a lo ne co u ld produ ce infecti o n . !11ycojJLasma iners, wh ose ty pe s tra in was orig in a ll y deriv ed from th e res pira tory tract o f a chi cke n (Ba rb er & Fa bri ca nt, 197 1), a ppea rs to be no n-pat hoge ni c U o rd a n , 197 9 ). Thi s is s uppo rted by th e prese nt s tud y, in whi ch the orga ni sms were isola ted from a bird with no c lini ca l di sease. W e iso la ted AchoLejJLasma Laidla wii on on ly o ne occas io n : th e so urce was a hea lth y bird. Thi s o rga ni sm ~ as a lready bee n repo rted in hea lth y chi ckens (Amin , 1977). AclwlejJLasma LaidLawii is often found as a comm ensa l in va ri o us a na to mi ca l s ites, es pec ia ll y th e res p ira tory trac t. I t was ori g in a ll y iso la ted fro m sewage (La id la w & Elfo rd , 1936). In thi s S(Ud y, th e hi g h preva lence of MJ'COfJ Lasma gaLLinarum in chi ckens with sy mpto ms of res pira tory di sea se may indi ca te th e ex is tence of a viru s o r o th er co ncomita nt pa thoge n . Syn ergy with pa th oge ni c m ycopl as m as , s uch as Mycoplasma galh:sejJticum and MycojJLasma SJIIIO viae, ca nn o t be rul ed o ut. Th eir prese nce co uld con ce iva bly ha ve bee n masked by th e ra pidly g rowing non-path oge ni c M)lcOjJLasma gaLlinarum . As th e egg is consid e red impo rta nt in th e tra nsmi ss io n o f MycojJLasma gaLLisejJticum a nd, to a lesse r extent , o f Mycoplasma s_ynoviae (Yod e r, 1969; Kl eve n & And erson , 1975), it would ha ve been prefera b le to sea rch for th ese o rga ni sm s in o th er sites as well as in th e res pira to ry trac t. Th e ov idu ct o f th e fem a le a nd th e se m en of the m a le mi g ht ha ve bee n ofint erest (Yod er & H o fs tad , 1964 ). White Leghorn s a rc usua ll y impo rt ed for egg produ cti o n as d ay-o ld chi cks. \A/ e d o not sugges t th a t th e infec ti on was co nge nit a l; it was m o re prob a bl y acquired in th e Sud a n . !ltfycojJLasma gaLlinarum a nd ce rta in o th er m yco pl as mas have a lrea d y bee n recogni zed in nativ e breed s (H a rbi & Kh oga li , 19 75; H a rbi et aL., 1980). M)'cOjJLasma iners a nd AchoLepLasma Laidlawii, o n th e oth er ha nd , have no t bee n repo rted in chi ckens in the Sudan prev io usl y; we isol a ted th e m fro m bird s with no cl ini ca l di sease. Th e sys tem of m a nage m ent in th e Sud a n m ay be a facto r in perpe tu a tin g m ycopl as ma res pira tory infec ti on. Th e hi g h preva lence o f infect ion (60 %) s ugges ts th a t m ycopl a sm as m ay pl ay a ro le in ca usin g di sease a nd loss of produ ctio n .
!'viYCOP LASMAS IN C HI CKENS IN T H E SUDAN
257
1\CKNOViLEDCEM ENTS
Th e a uth ors a re g rate ful to the Directo r, V eterin a ry R esea rch Admini st ra tion for encourage ment , a nd to Dr G. R. Smith , H ead of D epart m ent of I nfectious Di seases, Zoologica l Society of London, fo r revisi ng the manusc ript. W e also tha nk Professo r E . A. fr eundt a nd hi s co ll abo rato rs for the un fa iling help a nd advi ce to our D epartm ent of ifyco pl as m a, whi ch is hi gh ly a pprecia ted. Thi s pape r is publish ed through th e kind permi ss ion oft he P .U .S . Anim a l R eso urces, MAfNR, Kh a rtoum , Sud a n . REFERENCES
AULER, H. £. & YAMAMOTO , R. ( 195 7). Jlme1-ican J oumal of Veterinary Research 18 (69), 655. AMI N, M . M . (1977) . PhD thesis, Univers ity of Liverpool. BAR BER, T. L. & fABRI CANT,.J . ( 1971) . Jl via n Diseases 15, 12.1. BLACK, f . T . ( 1973). !ljJjJlied Microbiology 25, 528. CHU, H . P. ( 1958) . Veterinary Record 70, 55 . CLYDE, \'ll A . ( 1964). J ournal of Immunology 92, 958. CORSTVET, R. E. & SADLF.R, W . W. ( 1966). American J ournal of VelerinG':)' Research 27, 172 1. EDWARD, D . G. FF. & fR EUN DT, E. A. ( 1956). J oumal of General J\1/icrobiolog;• 14, 197. EDWARD, D. G . FF. & FREUND'!', E. A. ( 1973). lntemalionalJoumal of Systematic Bacteriology• 22, 55. EDWARD, D. G. FF. & K ANARCK, A. B. ( 1960). !lnnals of the New York AcademJ' of Science 79, 696. EL NASR I, M . ( 1967). Cited by J. R. H ucl so n ( 197 1). FJI 0 !lgricullural Studies No. 8, p. 3. ER N0, H . & ST JPK OV ITZ, L. ( 1973). Acta vete rinaria scandina vicn 14, 450. r ABR ICi\NT, .J. & FREU NDT, E. A. ( 1967) . Annals of the New York Academy of Science 143, 50. tRE UNDT, E. A., ANDRF.WS, B. E., ERN0, H ., K UNZ E, M. & Bi..'ICK, F. T. ( 1973) . Zen lralblat!Jiir Baktieriolog ie, Parasilenkunde, lnfektionrkrankheilen und HJ•giene Jib!. Orig . 11 . 225, I 04 . HARB I, M.S. M . A., M UST;\FA , A. & SAI.IJ-1, M. M . ( 1980). Sudanese .Journal of VeterinGI)' Research 1, 5 1. H ARB I, M.S. M.A. & KH OC.-\LI , A. R. ( 1975). Veterinao• Record 97, 11 8. I.C.S. B. ( 1979). I ntern a ti ona l Comm ittee of Systemat ie Bac teri ology. lnlemalional J oumal of Systematic Bacteriology 29 (2), 17 2. J ORDAN, F. T . W . ( 1979). Th e MycojJiasmas, Vol. I I. Lond on: Acad emi c Press I ne . .J OR DAN , F. T. \A/ . & KULASECA RAM , P. ( 1968) .Joumal of HJ•giene, Cambridge 66 , 249. KH OCAI.I, A. R. ( 1970). tv1VSc th es is, University of Kh a rt oum. KLF.VF. N, S. H . ( 1977) . Cited by F. T . W . .J ord an ( 1979). KL EVEN, S. H . & ANDERSON, D. P. ( 1975). Proceedings of the West em Poulll)' Diseases Conference, 21th Poulll)• Health SymjJosium, p. 69 . Davis: Un iversity of Californ ia. I. .-\IDJ...\11', P. P. & ELFORD , W . .J. ( 1936). Proceedingsof the RoJ•aiSociei)•, LondnnSer. /3 . 120, 292. 0. 1. E. ( 1979) . Offi ce l nter11 a ti onal d 'Epizootic. Report No. 309. Yi\M.-\M OTO, R. & ADLER , J-1 . E. (1958).Joumal of hifeclious Diseases 102, 143. YODER , H. W ., .J R ( 1965). Jlmes. Iowa Stale University Press, 5th edn, p. 4 11 . YODER, H . \•V., .J R ( 1969). Ci ted by F. T. W . .J ord a n ( 1979). YODER, H . W ., .J R ( 1978). Jlmes. Iowa Stale Universil)• Press, 7th ed n, p. 233 . YODER , H. W., .J R & HOFSTA D, M .S. ( 1962) . Avia n Diseases (692), 146. YOillcR, J-1 . W ., .J R & HOFSTAD , M .S. ( 1964). Avian Diseases 8, 48 1. (Accepted fo r publication 8 Ortober 198/)
vel . J
( 1982)
138, 253
ISOLATION AND IDENTIFICATION OF MYCOPLASMAS FROM THE RESPIRATORY TRACT OF THE WHITE LEGHORN CHICKEN IN THE SUDAN
Bv M.S. M . A. HAR BI, A. MUSTAFA*, M. M. SALIHt , AND S.M. M. ELAM IN*
Department of MycojJlasma, Velerinmy Research Administration, P.O. Box 8067 (El Amaral), Khartoum, Sudan, *Department of Avian M edicine, Leahurst, University of Liverpool, Live1pool, England and fFAOIWHO Collaborating Centre for Animal Mycoplasmas, Unive rsity of Aarhus, DK-8000 Aarhus C, Denmark
SU MMARY The poss ibl e importance of mycopl as m as in respiratory di seases of ch icken in the Sud a n provided th e st imu lus for thi s study. T h ree species of m yco pl as mas, .MycojJlasma gallinamm, MycojJlasma iners a nd AclwlejJlasma laidlawii, were isolated a nd ident ifi ed. The frequ ent occu rrence of Mycoplasmagallinarum, wh ich m ay be important as a seco nd ary in vader in res piratory infections of chi cken , s ugges ted that furth er study of the possible pathoge ni city of the orga nism is m erited. A sea rch for pat hoge n ic m yco pl as m as in sites other tha n the res pirato ry tract is reco mm ended. INTRODU CT ION Th ere is at prese nt worldwide interest in av ia n m yco plasmas, especia ll y associated w ith domesti c poultry (O .I.E. , 1979). Pat hogen ic a nd non-pathogeni c my co pl as m as have o ft en been iso lated from the respiratory tract of po ultry (Adl er & Ya mamoto, 1957; Chu, 1958; Yam a moto & Adl er, 1958; Yode r , 1965; J ordan, 1979) . In th e Suda n , the first publish ed ev idence o f m ycop lasmas in pou ltry was that of Khogali ( 1970), who studi ed resp iratory di seases in foreig n breeds of chi cken. Hi s six iso lates we re non-pa thoge ni c , and they were no t id entifi ed. H a rbi & Khoga li ( 1975 ) iso lated a my .:op las m a (sero type C) from th e respiratory tract of the loca l Baladi breed o f chi cken . The pat hogeni c sig nifi ca nce of se rotype C is poo rl y understood Uord a n, 1979). H a rbi , Mu sta fa & Sa lih ( 1980) isola ted MycojJlasnza gallinanmz from th e resp iratory tract of loca l Ba ladi chick ens . Mycoplasma gallinanmz is co nsidered by m a ny workers Uordan & Kul asegara m , 1968; Kl even, 1977 ;J ord a n , 1979) to be pathoge ni c wh en associa ted with ce rtain ot her agents.
254
BRITISH VETERINARY JOURNAL, 138, 3
This pa p er repo rts the isolation a nd id en tifi cat ion of mycop lasma s pec ies from the respiratory tract of norma l a nd d iseased chi ckens.
MATERIALS AND METHODS
Chickens Seventy-five White Leghorn chi ckens, aged a bo ut seven months, were obtai ned from private fa rm s in the vicin ity of the labo ratory . Of th ese, 32 showed early signs of res pirato ry di sease; the rem a ind er were appa ren tl y h ea lthy. The cl ini ca l sy mpto ms were sporad ic. All the birds were kil led and the head s, trac hea, lungs, and airsacs collected in steril e co nta in ers. Reference strains and sera The six kn-own species of avia n mycoplasmas, together with their correspond in g hyperimmune se ra, were kindl y provided by Professor E. A. Freundt, Direc tor, FAO/ WHO Collaborating Ce ntre for An im a l M yco pl as m as, Aarhus, Denm ark . Growth media Liquid a nd so lid m edi a were used : b acto hea rt infu sio n brot h a nd agar (Difco), bruce ll a broth a nd agar (Aib imi ), a nd m ycop las ma brot h a nd agar (Oxoid). H o rse se rum , 20%; yeast ex tract, 3 g/1; DNA, 0·6 g/1; penicill in, 200 000 i.u./ ml a nd l ml 10% solu tion th a lliu m aceta te were usua ll y in co rpo rated in the media . N icot in am id e adenine dinucleotide (NA D ) an d L-cysteine, which a re specifi ca ll y req uired by Mycoplasma synoviae, were a lso add ed to the medi a. Cultural methods These co mpri sed the strea king of the so lid medi a with the s uspect ma ter ia l, a nd subsequ ent sub cu ltures in liquid a nd soli d medi a. Pl ates we re put in to polythene bags to provide suffi cientl y humid condit ions witho ut ca usin g th e co nta min at io n th at has so m eti mes been enco unte red when using humid chambers co ntain ing wa ter. Liquid a nd solid m ed ia were in cu bated ae rob ical ly a t 37°C . Pl ates were exam in ed fo r th e ap pea rance of umbonate co loni es at interva ls of up to three weeks, a nd broth cu ltures checked fo r a period of two weeks. After three to five passages on medium co nta in in g antibac terial age nts, isola tes were tested fo r revers ion tO bac terial form by subject in g them tO fiv e co nsecuti ve subcu ltures in med ia without a n tib iotics (I .C.S. B. , 1979) . Biological and serological properties After cl oning, th e iso la tes were id entifi ed by the methods or Ern0 & St ipkov itz ( 1973). They were tes ted for sensiti vity to digitonin, a property eharacrerist icofste roldependent mycoplasmas (F reun dr et al., 1973). Di gitonin-se nsit ive orga ni sms were tested for phosphatase ac tivity, fermentation or glucose, hydro lys is or urea a nd arginin e, a nd redu ction of 2, 3, 5, tripheny l tetrazo li um chl ori de under ae robic con diti o ns. Produ ction of ' film a nd spots' was tested as described by Fa bri ca nt & Freundt ( 1967) on media conta inin g 20% ho rse se rum. Proteo lys is was tested by s treak ing three-day cu ltures on coagu lated horse serum sla nts. T he slants were
MYCO PLA MA
11 C HI C K E
11 T H E
DA
255
in cubated for two weeks. Three-d ay cultures were a l o examin ed for haemagglutinating activ ity. As un co nce ntra ted cultures do no t a lways giv cl arl readabl e res ults (Yoder & Hofstad 1962) , w u ed culture centrifugate . H aemol ti c activity was tested against sheep red blood ell (RBC) by mea n of a n overla technique (EI asri, 1967). Th e sol serologica l tes t used was the growth inhibiti on tes t; it was carried ou t by the paper dis c technique (C lyd e, 1964) and a l o by the meth od of Bl ack ( 1973) in whi h well s were pun ched in th e aga r. RE
LT
Of the 44 digitonin-sensitive mycopl as ma i olate , 17 were ubj ected to id entifi ca tion procedures: two isola tes from hea lthy birds a nd 14 from di seased birds proved to be Mycoplasma gallinarum. On e isolate from a hea lth y bird proved to be Mycoplasma iners a nd one digitonin-resistant iso la te from a hea lth y bird wa id entifi ed as Aclzoleplasma laidlawii. Mycoplasma iners a nd Acholeplasma laidlawii have no t prev iously been record ed as occurring in the res piratory tra t of chi ckens in th e ud a n . Mycoplasma gallinanan isola tes had a ll th e cha racteristi cs of the type stra in PG 16 (Edwa rd & Freund t, 1956). Th y hydrol y ed a rginin e a nd redu ced tetrazo lium, but fa il ed to hydrolyse urea or ferm ent glu cose. Th ey produced 'film and spo t ' (Fi g. I), fai led to digest in spissa ted serum , a nd were phosph a tase nega tive. Alpha-ha emolys is was detected after in cubation for 24 to 36 h a t 37°C; beta -h ae molys is was detec ted a fter fi ve to seven days incubation .
Fi g. I. ' Film and spo ts' produ cd b M;-coplasma gallinanan i ola t .
Hae maggluti na tion of chi cken RB C was poo r a nd consid ered t be non- pecifi . Th stra in were inhibit d by hyperimmun e era to th e type tra in of Mycoplasmagallinarum. Th train id entifi ed as Mycoplasma iners had th e properti e of th e type stra in PG 30 (Edwa rd & K a na rek, 1960). It did no t ferm ent glu co e but hyd ro! sed arginin e. It wa urea nega tive, ph sph a tase nega ti ve a nd fa il ed to redu ce tetrazolium . o ' film a nd spo t ' were observed on solid medi a a nd oag ul a ted serum rema in ed undiges ted .
256
BRITISH VET ERI NARY .JO U RNAL , 138, 3
Alph a-h ae mo lys is was o bse rve d , bu t ch ic ke n RB C were no t hae m agg lutin a ted . Growth was strong ly inhibi ted by a nti se ra to the ty pe st ra in . AcholejJlasma laidlawii was id ent ifi ed on th e g ro und s th a t it was nega ti ve to dig itonin a nd a cl ea r zo ne o f inhibitio n was prod uced by hy perimmun e se rum aga in st th e ty pe s tra in PG8 (Ed w a rd & Freundt , 19 73 ) in th e g row th inhibi tion tes t. Th e zo ne o f inhibiti o n prod uced by th e well m eth od was es pecia ll y p ro no un ced .
DI SCUSS I ON
In thi s s tud y MycojJlasma gaLLinamm was th e most co mm o nl y occ urrin g s pec ies. I t was iso lated from va ri o us sites, es pec ia ll y th e sinu ses a nd trac heas . M a ny a uth o rs co nsid e r this species to be no n-path oge ni c for chi ckens (Yod er & H ofs ta d , 196 4·; Yod e r, 1978 ; J o rd a n , 1979). H owever , J ord a n ( 19 79) r eported th a t it could be pa th oge ni c wh e n occurrin g toge th er wi th o th er avia n pa th oge ns. Syn erg ism between m yco p las m as a nd o th er a vi a n pathogen s (e.g . N ewcas tle di sease viru s a nd infec ti o us bron chiti s viru s) has been repo rted U01·d a n & Kul asega ra m , 1968 ; Crostve t & Sadl er, 1966). K leve n ( 19 77) studi ed th e poss ibl e importa nce o f M)'cojJLasma gaLLinanan in mu ltip le in fec ti o n. H e reprodu ced a irsacculiti s ex perimenta ll y in yo un g chi cke ns by a dm ini s terin g Mycoplasma ga LLinarum a nd th e viru s of infec ti o us bro nchi tis. N eith er the viru s no r th e m yco pl as m a a lo ne co u ld produ ce infecti o n . !11ycojJLasma iners, wh ose ty pe s tra in was orig in a ll y deriv ed from th e res pira tory tract o f a chi cke n (Ba rb er & Fa bri ca nt, 197 1), a ppea rs to be no n-pat hoge ni c U o rd a n , 197 9 ). Thi s is s uppo rted by th e prese nt s tud y, in whi ch the orga ni sms were isola ted from a bird with no c lini ca l di sease. W e iso la ted AchoLejJLasma Laidla wii on on ly o ne occas io n : th e so urce was a hea lth y bird. Thi s o rga ni sm ~ as a lready bee n repo rted in hea lth y chi ckens (Amin , 1977). AclwlejJLasma LaidLawii is often found as a comm ensa l in va ri o us a na to mi ca l s ites, es pec ia ll y th e res p ira tory trac t. I t was ori g in a ll y iso la ted fro m sewage (La id la w & Elfo rd , 1936). In thi s S(Ud y, th e hi g h preva lence of MJ'COfJ Lasma gaLLinarum in chi ckens with sy mpto ms of res pira tory di sea se may indi ca te th e ex is tence of a viru s o r o th er co ncomita nt pa thoge n . Syn ergy with pa th oge ni c m ycopl as m as , s uch as Mycoplasma galh:sejJticum and MycojJLasma SJIIIO viae, ca nn o t be rul ed o ut. Th eir prese nce co uld con ce iva bly ha ve bee n masked by th e ra pidly g rowing non-path oge ni c M)lcOjJLasma gaLlinarum . As th e egg is consid e red impo rta nt in th e tra nsmi ss io n o f MycojJLasma gaLLisejJticum a nd, to a lesse r extent , o f Mycoplasma s_ynoviae (Yod e r, 1969; Kl eve n & And erson , 1975), it would ha ve been prefera b le to sea rch for th ese o rga ni sm s in o th er sites as well as in th e res pira to ry trac t. Th e ov idu ct o f th e fem a le a nd th e se m en of the m a le mi g ht ha ve bee n ofint erest (Yod er & H o fs tad , 1964 ). White Leghorn s a rc usua ll y impo rt ed for egg produ cti o n as d ay-o ld chi cks. \A/ e d o not sugges t th a t th e infec ti on was co nge nit a l; it was m o re prob a bl y acquired in th e Sud a n . !ltfycojJLasma gaLlinarum a nd ce rta in o th er m yco pl as mas have a lrea d y bee n recogni zed in nativ e breed s (H a rbi & Kh oga li , 19 75; H a rbi et aL., 1980). M)'cOjJLasma iners a nd AchoLepLasma Laidlawii, o n th e oth er ha nd , have no t bee n repo rted in chi ckens in the Sudan prev io usl y; we isol a ted th e m fro m bird s with no cl ini ca l di sease. Th e sys tem of m a nage m ent in th e Sud a n m ay be a facto r in perpe tu a tin g m ycopl as ma res pira tory infec ti on. Th e hi g h preva lence o f infect ion (60 %) s ugges ts th a t m ycopl a sm as m ay pl ay a ro le in ca usin g di sease a nd loss of produ ctio n .
!'viYCOP LASMAS IN C HI CKENS IN T H E SUDAN
257
1\CKNOViLEDCEM ENTS
Th e a uth ors a re g rate ful to the Directo r, V eterin a ry R esea rch Admini st ra tion for encourage ment , a nd to Dr G. R. Smith , H ead of D epart m ent of I nfectious Di seases, Zoologica l Society of London, fo r revisi ng the manusc ript. W e also tha nk Professo r E . A. fr eundt a nd hi s co ll abo rato rs for the un fa iling help a nd advi ce to our D epartm ent of ifyco pl as m a, whi ch is hi gh ly a pprecia ted. Thi s pape r is publish ed through th e kind permi ss ion oft he P .U .S . Anim a l R eso urces, MAfNR, Kh a rtoum , Sud a n . REFERENCES
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