A. R.C. Institute for Research on Animal Diseases, Compton, near Newbury, Berkshire RG16 ONN

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1 J. MED. MICROBI0L.VOL. (198) The Pthologicl Society df Gret Britin nd Irelnd 006 /8/ $0.00 THE PTHOGENICITY OF MYCOBCTERIUM VIUM ND RELTED MYCOBCTERI FOR EXPERIMENTL NIMLS P. COLLINS, P. R. J. MTTHEWS,. MCDIRMID ND NNE BROWN. R.C. Institute for Reserch on niml Diseses, Compton, ner Newbury, Berkshire RG ONN SUMMRY. The pthogenicity of 40 strins of Mycobcterium vium, M. prtuberculosis, M. intrcellulre nd M. lepremurium ws * investigted in chickens, rbbits, guinepigs, mice nd clves. Mycobctin dependence nd serologicl type were lso determined. There ws no evidence tht mycobctin dependence ws relted to pthogenicity. ntigenic similrities were demonstrted between M. vium nd M. prtuberculosis, nd one isolte hd the pthogenic chrcteristics of both species. INTRODUCTION Pthogenicity studies on Mycobcterium vium nd M. prtuberculosis hve been difficult to interpret in the pst becuse of vritions in the dose injected, expressed s weight of wet orgnisms. Mtthews nd Collins (1978) described method of obtining stndrd inocul for pthogenicity tests. This method hs been used in the present study to compre the pthogenicity of 40 selected strins of mycobcteri. The orgnisms comprised typicl nd typicl (mycobctindependent) strins of M. vium from wild nd domestic nimls (McDirmid, 1948, 196; Mtthews, 1969; Mtthews nd McDirmid, 1979), nd strins of the relted mycobcteri M. prtuberculosis, M. intrcellulre nd M. lepremurium (see Stnford, 197). ll strins were exmined serologiclly nd for mycobctin dependence, s well s for pthogenicity. MTERILS ND METHODS Culture methoh. Sturt s (1965) medium with mycobctin pg/ml (Mtthews nd Collins, 1978) ws used for the mintennce of stock cultures, the preprtion of ntigens nd inocul, nd the isoltion of orgnisms from tissue. Before culture, tissues were treted with oxlic cid 5% (w/v) (Mtthews, 1969); feces were pretreted with benzlkonium chloride 0.6% in trisodium phosphte 10% (w/v) (Mtthews, Collins nd Jones, 1976). Egg yolk emulsion 5% (w/v) nd mphotericin B 50 pg/ml were dded to the medium used for fecl cultures. Strins of mycobcteri. Forty isoltes of M. vium (including typicl mycobctindependent strins), M. prtuberculosis, M. intrcellulre nd M. lepremurium (tble 11) were used. They were subcultured not more thn three times nd stored t 70 C in liquid medium. Mycobctin dependence. This ws ssessed s described by Mtthews et f. (1978). Tenfold dilutions of ech strin were inoculted on to medium with nd without mycobctin, nd the bility of ech strin to grow without mycobctin t ech dilution ws scertined. Received Nov. 1981; revised version ccepted 10 ug Downloded from by IP: On: St, 08 Sep :8:4

2 8 P. COLLINS ET L. Serotyping. Polyvlent nd typespecific ntiser (Mtthews, Brown nd Collins, 1979) were used. The clssifiction of strins followed the scheme proposed by Wolinsky nd Schefer (197). Pthogenicity experiments. Stndrd suspensions of ech strin were prepred by the method of Mtthews nd Collins (1978). Ech suspension ws stored in liquid medium t 70 C nd the vible count ssyed shortly before use. These suspensions were then djusted by dilution to obtin the required dose for niml injection. The nimls used, doses dministered, routes of injection, nd durtion of ech experiment re shown in tble I. M. Iepremurium ws injected s 10% (w/v) homogente of mouse tht contined > lo7 orgnisms/g of tissue. t postmortem exmintion, cultures were mde from tissues showing mcroscopic bnormlities; the mycobcteri recovered were identified. The virulence of different strins ws compred in C57 mice. The comprisons were bsed on the spleen weight to body weight rtio expressed s percentge (SBR) in mice killed 100 dys fter the intrperitonel injection of (14.5) x lo8 vible units (Mtthews et l., 198). The norml SBR for C57 mice is 0.%. Comprtive tuberculin tests with vin nd mmmlin PPD tuberculins (Centrl Veterinry Lbortory, Weybridge, Surrey) were performed on guinepigs 6 nd 1 weeks fter injection, nd in clves t intervls of 8 weeks fter injection. The rections were recorded fter 48 h in guinepigs, nd fter 7 h in clves. Feces smples from clves were cultured t monthly intervls for mycobcteri. RESULTS Mycobctin dependence The minimum number of vible units of ech test strin tht produced observble growth on medium without mycobctin is shown in tble 11. The 1 strins of M. vium vried considerbly in mycobctin dependence. The six strins of M. prtuberculosis were ll strongly mycobctindependent. The 1 strins of M. intrcellulre were uniformly independent of mycobctin. On medium contining mycobctin pg/ml the minimum number of vible units tht produced observble growth ws < lo for ll the mycobcteril strins tested. Serologicl typing Tble I1 shows the results of gglutintion tests with polyvlent nd typespecific ntiser. With the exception of strin 4, ll the nonmycobctindependent strins of TBLE I Detils of pthogenicity experiments Number of nimls ge or Mximum given ech weight t Volume Number of durtion of mycobcteril time of of vible units Route of experiment nimls strin injection inoculum injected injection (months) Clves or 4 weeks 10.0 ml (1.7 x )(5.7 x 1O'O) Intrvenous 6 Mice ((57 strin) 10 0 g 0.5 ml (1.05) x lo8 Intrperitonel 5 Rbbits 500 g 1.0 ml (1.05) x lo7 Intrvenous 6 Guinepigs 50 g 0.5 ml (14.5) x lo7 Intrmusculr Chickens 1 months 1.0 ml.(1.&.5) x lo6 Intrvenous 6 Downloded from by IP: On: St, 08 Sep :8:4

3 M. VIUM ND RELTED MYCOBCTERI 9 TBLE I1 The mycobctin dependence nd serologicl rections of 40 isoltes of M. vium nd reltedspecies Minimum number of vible units Strin gglutinted with necessry for \ growth on polyvlent specific M ycobcteril medium without ntiserum ntiserum for species Strin no. Source m ycobc tin pool serotype no. M. vium (nonmycobctindependen:$) I, M. vium (mycobctindependen!,),$ M. prttfxrculosis M. intrc+dre, 9, M. lepremurium *I NCTC8559 * NCTC856 Got Deer (Dum dum) Deer (Dum h ) plumbus L) Woodpigeon (Columb plumbus L) < 10 1 o6 < 10 N < lo o6 104 Phesnt (Phsinus < lo colchicus) 9 Phesnt (Phsinus 104 colchicus) *10 Mn < Woodduck (ix spons) < 10 1 Crow (Corvus cornix) < 10 1 Woodpigeon (Columb < lo *8 *9 *0 * 1 * * *4 *5 *6 *7 *8 *9 t40 Deer (Dum &m) Deer (Dum h ) Got Got Cttle Cttle Cttle Cttle Cttle Cttle TCC 1950 Woodduck (ix spons) Mn Mn Mouse 1 o o6 1 o < 10 < 10 < loz < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 < 10 N N N N N N N N D N C C E C B F D Not determined N = not gglutinted by ny test serum. * Supplied by Dr J. Stnford, Middlesex Hospitl Medicl School, London. t Supplied by Dr P. Drper, MRC, Mill Hill, London. $ The mycobctin requirements stted in the first column re those found t primry culture. 8 See Mtthews et l., N 1 N N N N N N N N N N N N 1 nd 9 nd 9 7nd 1 N Downloded from by IP: c On: St, 08 Sep :8:4

4 0 P. COLLINS ET L. M. vium rected with polyvlent ntiserum nd typespecific ntiserum 1, or. Five of six M. prtuberculosis strins were gglutinted by the polyvlent serum nd strins nd 6 were lso gglutinted by typespecific ntiserum. Seven of 1 M. intrcellulre strins rected with polyvlent ntiserum (B, C, D, E, or F) nd with one specific ntiserum. One strin (8) gve specific serologicl rection with serum 4 nd could therefore be clssified s M. scrofulceum. Three M. intrcellulre strins rected with ntiser to two serotypes. Strins 4, 1, 7 nd 9 were not gglutinted by ny polyvlent or typespecific ntiserum. Pthogenicity for chickens Seventeen of the strins were lethl for chickens within 1 dys (tble 111). Lesions ssocited with mycobcteri were present in the, nd orgnisms of the sme serotype s tht injected were invribly recovered. Of the five nonlethl strins of M. viurn, two becme lethl fter one pssge through chickens; the other three still filed to kill even fter pssge, but they could be recovered from the of infected birds s lte s 180 dys fter injection. Six of seven typicl (mycobctindependent) M. vium strins killed chickens nd produced extensive lesions in the. Like the infecting strins, the mycobcteri recovered were not gglutinted by polyvlent or typespecific ntiser. Strin 0 could not be recovered from chickens killed 180 dys fter injection (tble 111), nd it remined nonpthogenic fter one pssge. M. lepremurium (strin 40) did not produce ny pthogenic effect in chickens. Pthogenicity for rbbits Tble I11 shows the effect of injecting 7 strins of mycobcteri into rbbits. Four strins (, 6, 7 nd 9) were lethl in 48 weeks nd produced lesions in the lung nd, from which mycobcteri of the sme serotypes s those injected were recovered. Rbbits tht hd been given injections of the remining strins were killed fter 180 dys, nd mycobcteri were commonly isolted from the lung, nd joints. Pthogenicity for guinepigs ll 40 strins sensitised guinepigs to vin tuberculin. Erythemic rections 101 mm in dimeter were obtined with vin tuberculin fter infection with 7 of the strins. The remining 1 strins produced rections of c 10 mm dimeter. Three strins (, 1 nd 4) produced miniml rection of c 5 mm dimeter with mmmlin tuberculin. No deths occurred. Lesions were not observed in the or spleen when the nimls were killed 1 weeks fter infection. However, slight enlrgement of the lymph nodes in the lumbr region occurred in some nimls. Seven of the 40 strins, including five M. vium strins (tble 111), were recovered from lymph nodes. Pthogenicity for C57 mice Tble I11 shows the SBRs for groups of mice injected with the 40 strins. Of strins, 1 produced SBR of >.5%. Three M. intrcellulre strins (9,7 nd 8) Downloded from by IP: On: St, 08 Sep :8:4

5 M. VIUM ND RELTED MYCOBCTERI 1 TBLE I11 Results in chickens, rbbits, guinepigs nd mice of injection of 40 isoltes of M. vium nd relted species Injected strin Result of Spleen weight recovered from tuberculin to body weight M ycobcteril / \ test in rtio (%) in species Strin no. chicken rbbit guinepigs C57 mice M. boium (nonmyco bctindependent) 1, M. vium (m ycobctindependent) 9, M. prtubyulosis M. lepremurium lung, joint lung lung, lung, joint lung, joint lung, lung, lung, lung,, joint* lung, lung,, joint lung,, joint, joint, joint, joint, joint lung,, joint lung, lung, joint lung * =niml died. t = Mycobcteri isolted from lymph nodes. =vin tuberculin rection 1&1 mm dimeter; = < 10 mm. b = slight rection ( < 5 mm dimeter) occurred with bovine tuberculin. =No lesions produced nd no mycobcteri recovered. =Not tested. b t t t t t t b b * * Downloded from by IP: On: St, 08 Sep :8:4

6 P. COLLINS ET L. TBLE IV Results in clves of injection of isoltes of M. vium nd relted species Mximum rection Recovery Tissues Number Dose ge t to vin tuberculin of strins from which Mycobcteril Strin of (vible necropsy in mm (months from strins were swies no. clves units) (months) fter infection) feces recovered M. vium (non.5 x 10" [we mycobctin 1. [ide dependyt).5 x x 1o'O 18 bcde [lbcde 6.4 x 10" 1.5* [lbcde * blue 1.7 x b[c]de 1 b[cie M. vium (mycobctindependent),, 1, 0 M. prtuberculosis 4,, 7 M. intrcellulre , 8 9 M. lepremurium x x 10'0 1.7 x lo8 1.7 x 1.6 x x x x 5. x 1o'O 1.7 x 10" 5.7 x 1o'O 6.0 x x 10" 1.0 x 10' It b bcde bd b[c]de de b[c]de b bcde bcde [lbcde [lbcde e [l [l = Lung; b = medistinl node; c = smll intestine; d = mesenteric node; e = ileocecl node; [ ] = lesions present; =not tested. *niml died;?niml died of cuses unrelted to experiment. de bd Downloded from by IP: On: St, 08 Sep :8:4

7 M. VIUM ND RELTED MYCOBCTERI produced SBRs of 1.,1 nd 5.% respectively; the other nine strins of this species produced vlues of < 0.8%. Pthogenicity for clves Of the strins injected into clves, five (,5,6,9 nd 7) were lethl in t lest one clf within months t the highest dose given (tble IV). The most obvious pthogenic effects were consolidted nd discoloured picl lobes of the lungs, from which mycobcteri were invribly isolted. One clf given strin 5 survived nd when killed fter 18 months showed no obvious lesions, lthough the strin ws recovered from vrious tissues. Strins nd 9 were injected t lower doses of.5 x lo7 nd 8. x lo7 vible units respectively nd by 8 weeks fter injection two of the three clves from ech group hd infected feces. No further isoltions could be mde from feces, nd when the nimls were killed fter 1 1 months mycobcteri could not be recovered from the tissues. Clves given strins (from woodpigeon; Mtthews nd McDirmid, 1979),, 4 nd 7 ll developed the clinicl signs of Johne s disese (prtuberculosis). Intermittent excretion of mycobcteri in the feces occurred from months fter injection. t postmortem exmintion the smll intestinl mucos ws inflmed nd thickened, nd the mesenteric nodes were lrge nd oedemtous. Tble IV shows the isoltion of mycobcteri from tissues nd the intrderml rection of clves to vin tuberculin, which vried considerbly between strins nd individul clves. Eight strins (1 6,,0, 1, 4, 8, 9 nd 40), the results for which re shown in tble IV, did not produce clinicl signs of infection, nd lesions were not observed t necropsy. However, mycobcteri were recovered from the lymph nodes of clves given strins,, 0, 1 nd 9. DISCUSSION The r6le of mycobctin s growth fctor for M. prtuberculosis is well documented (Frncis et l., 195; Merkl nd Currn, 1974). typicl (mycobctindependent) strins of M. vium hve frequently been isolted in this lbortory. Their growth hs often been slower thn tht of the typicl (nonmycobctindependent) strins. However, this study nd previous work (Mtthews et l., 1978) hve shown tht some typicl isoltes of M. vium my require mycobctin for growth from smll inocul. The vrying requirement for mycobctin ws not relted to pthogenicity. Myco bctindependence hs not been demonstrted in M. intrcellulre. ntigenic similrities were demonstrted between typicl isoltes of M. vium nd M. prtuberculosis; most of the ltter rected with the polyvlent ntiserum (Mtthews et l., 1979). This polyvlent ntiserum contins ntibodies to serotypes 1,, nd 18 of the M. viumintrcellulrescrofulceum complex of orgnisms. Strins nd 6, moreover, rected specificlly with M. vium type ntiserum. The typicl mycobctindependent strins of M. vium were not gglutinted by ny of the ntiser used; this suggests tht they form t lest one new serotype. Most of the typicl nd typicl M. vium isoltes killed chickens within 100 dys. Downloded from by IP: On: St, 08 Sep :8:4

8 4 P. COLLINS ET L. The M. intrcellulre strins were not pthogenic. Becuse only one of three M. uiurn serotype strins killed chickens it seems possible tht this serotype is of low virulence. Only one of the M. prtuberculosis strins (6) used in the study ws lethl for chickens; orgnisms were recovered from those given strins 5 nd 6. Lrson nd Moon (197) hve shown tht chickens re susceptible to some strins, but lesions do not usully develop for 91 months. Virulent strins of M. uiurn re usully lethl for rbbits within 50 dys; thus the poor response in rbbits in this study ws unexpected. The dose of (I0.5)~ lo7 vible units normlly would hve produced cute infection (Engbek nd Jespersen, 1966); most of our strins filed to kill within 180 dys nd often produced chronic rthritis s described by Engbek (1961). It is possible tht the rbbits were less susceptible thn those used by other workers, or tht higher chllenge dose would hve produced different results. The resistnce of guinepigs to M. uiurn is confirmed by this study. ll test strins induced sensitivity to vin tuberculin, but the rections induced by three of six M. prtuberculosis strins nd most M. intrcellulre strins were slight. The mjority of the typicl nd typicl M. viurn isoltes tht were lethl in chickens produced SBRs of > % in mice. Strins tht were less virulent for chickens usully but not lwys produced SBRs in mice of <%. The only strin of M. prtuberculosis tht ws lethl for chickens (strin 6) corresponded serologiclly with M. viurn serotype, despite its slow growth on culture medi. It is difficult to decide whether such strins should be designted M. prtuberculosis or M. viurn. None of the strins of M. intrcellulre ws lethl for chickens, nd ll except strins 9, 7 nd 8 produced SBRs of < 1% in mice. Strin 8, which hd the norml growth chrcteristics of M. intrcellulre nd ws not pigmented, rected specificlly with M. scrofulceurn ntiserum type 4. It ws lso exceptionlly virulent for C57 miceunlike most typicl strins of this serotype. M. intrcellulre strins 9 nd 7 were of porcine origin nd when injected into clves produced lethl infection; this ws surprising s they were nonpthogenic for chickens. The susceptibility of clves to both nturl nd experimentl M. viurn infection hs been recognised for long time (Feldmn, 198). Tylor (195) showed tht it ws necessry to inject M. prtuberculosis into clves t n erly ge to produce clssicl Johne s disese. From the experimentl infection of clves in this study it seems tht one of the following three types of infection my occur: subclinicl infection, the orgnisms being recoverble from vrious tissues t necropsy; n cute nd rpidly lethl infection, with deth in 68 weeks; nd slow progressive infection, lmost lwys ftl, seen typiclly with M. prtuberculosis. In order to estblish tht low doses of rpidly lethl orgnisms of M. viurn or M. intrcellulre do not produce chronic infection, strins nd 9 were injected in reduced doses. The orgnism did not become estblished nd ws not recovered from the tissues. Therefore, it seems unlikely tht smll doses of lethl M. viurn or M. intrcellulre strins could produce clinicl Johne s disese. Clves tht survived the initil period of months, during which deths sometimes occurred, ll becme sensitised to vin tuberculin. The time t which the mximum tuberculin response occurred vried with the strin nd with the individul niml nd bore no reltion to the development of disese. Despite the vried ssortment of M. uiurn strins used, no significnt cross rections to bovine tuberculin Downloded from by IP: On: St, 08 Sep :8:4

9 M. VIUM ND RELTED MYCOBCTERI 5 were found in the infected clves. Mtthews nd McDirmid (1979) hve previously reported on strin, mycobcterium isolted from woodpigeon, which behved like M. vium in chickens nd M. prtuberculosis in clves. These chrcteristics hve since been found in other isoltes from pigeons. Their significnce will be the subject of lter publiction. REFERENCES ENGBEK, H. C Pthogenicity nd virulence of typicl mycobcteri for experimentl nimls, with prticulr reference to pthologicl processes in the joints nd tendon sheths of rbbits. ct Tuberculose Scndinvic, 40, 550. ENGBEK, H. C. ND JESPERSEN, Effect of BCG vccintion on experimentl infection with M. vium. ct Pthologic et Microbiologic Scndinvic, 67, FELDMN, W. H vin tuberculosis infections. Willims nd Wilkins Co., Bltimore. FRNCIS, J., MCTURK, H. M., MDINVEITI, J. ND SNOW, G Mycobctin, growth fctor for Mycobcterium johnei. I. Isoltion from Mycobcterium phlei. Biochemicl Journl, 55, LRSEN,. B. ND MOON, H. W Experimentl Mycobcteriumprtuberculosis infection in chickens. mericn Journl of Veterinry Reserch,, MCDIRMID, The occurrence of tuberculosis in the wild woodpigeon. Journl of Comprtive Pthology, 58, 181. MCDIRMID, Diseses of free living wild nimls. F0 griculturl studies, No. 57, Rome, p 10. MTTHEWS, P. R. J The use of culture medium contining mycobctin for the isoltion of cidfst orgnisms from pig hed lymph nodes. Reserch in Veterinry Science, 10, MTTHEWS, P. R. J., BROWN,. ND COLLINS, P The use of polyvlent ser for the serotyping of mycobcteri within the Mycobcterium viumm. intrcellulrem. scrofulceum complex. Journl of pplied Bcteriology, 46,4540. MTTHEWS, P. R. J. ND COLLINS, P Stndrdistion of inocul for ssessing the virulence of strins of viumintrcellulre mycobcteri in chickens. Journl of Medicl Microbiology, 11, MTTHEWS, P. R. J., COLLINS, P., BUNCH, K. J. ND MCDIRMID, The virulence of Mycobcterium vium nd relted mycobcteri for C57 mice. Journl of Comprtive Pthology, 9, 1. MTTHEWS, P. R. J., COLLINS, P. ND JONES, P. W Isoltion of mycobcteri from diry cremery effluent sludge. Journl of Hygiene, Cmbridge, 76, MTTHEWS, P. R. J. ND MCDIRMID, The production in bovine clves of disese resembling prtuberculosis with Mycobcterium sp. isolted from woodpigeon (Columb plumbus L). Veterinry Record, 104, 86. MTTHEWS, P. R. J., MCDIRMID,., COLLINS, P. ND BROWN, The dependence of some strins of Mycobcterium vium on mycobctin for initil nd subsequent growth. Journl of Medicl Microbiology, 11, 557. MERKL, R. S. ND CURRN, B. J Growth nd metbolic chrcteristics of Mycobcterium prtuberculosis. pplied Microbiology, 8, STNFORD, J n immunodiffusion nlysis of Mycobcterium lepremurium Mrchoux nd Sorel. Journl of Medicl Microbiology, 6,4549. STURT, P Vccintion ginst Johne s disese in cttle exposed to experimentl infection. British Veterinry Journl, 11, TYLOR,. W Experimentl Johne s disese in cttle. Journl of Comprtive Pthology, 6, WOLINSKY, E. ND SCHEFER, W. B Proposed numbering scheme for mycobcteril serotypes by gglutintion. Interntionl Journl of Systemtic Bcteriology,, Downloded from by IP: On: St, 08 Sep :8:4