.J
Onder·stepoort J 0 II rna I of
r
eter··inary Science and A Ill: mal Industry, Volume 13, .Vuml1er ] . htly, 1939.Printed in the Union of South Africa b~- tlw Government Printer, Pretoria.
The Antigenic Structure of Salmonellas obtained from Domestic Animals
and Birds in South Africa.
By
M. vV. HBN~ING, Section of Bacteriology, Onclerstepoort.
CONTENTS.
PAGE I.- GENERAL l:NTRODt:C'I'lO~................. . . • • . . . . . • . . . . • . • . . . 80
H.-SALMONELLA INFECTION OF CAL YES . . . . . . . . . . . . . • . . . . . • . . . . . • . . . . • • S4
(1) Introduction... . . . 84 (2) Technique Employed .......... _............ . . • . 91 (3) Serology. . . . . . 9fi IlL- SALMONELLA INFECTION OF SHEEI'. . . . . . . . . . . . • . . . . . • . . . . . • . . . . • . . 10 J
IV.- SALMONELLA INFECTION OF PIGS.. . . ..... . ... . . ... . . . .. . . . .. .. J ]()
V.- SALMONELLA INFECTIO:>r OF EQuiNES... . . . • . . . . . • . . . . . . . . . . • . . . . • . 120
VI.-SALMONELLA IN}'ECT!ON OF BIRDS . . . . •.. . . • . . . •.. " . • • . . . . . . . . . . • 127
(1) Pigeons. . . 1:2()
(2) Canaries ................................. _. 131 (3) Geese ............... _ . . . . . . 134 (4) Ducks.......... . . . . . 1:~6 (5) Turkeys . . . _ . . . • . . . ... . . ..•. . . .. . .. ... . . . (6) Fowls . . . ... . . • . . . .. . . .. .....•.. ..•. . .....
141 142
Vli.- (a) SuMMARY . . . _ . .. . . . . . . . . . . . . . . . . . • . . . . . • . . 168 (b) FERMENTATION REACTIONS. . . ..... . . ... . . ..... . . .... . . ...... ](j9 Vlll.- REFERE"'CES . . . .. . . .•... " ..•.... .. . ..•..... ..... . • . ]7:{
79
.\.,; br as possible an attempt has been lll<Hlt· to pre,;eni. tht•
results in the form of tables. 'l'hese are gi1·en on the follo"·ing- pages:-
'/'able-
~-
:1 .............. .
+ ..
.)
ti .............. . 7.
K.
!1.
10 . . . . II.
I~.
I;{ ..............•........ 14 ..
1.).
]'A(: 1-:
U7 100 100 IO:l 10:l 107 10() ]()!) J 17 lJ!) 121
1n
l :12 133 I·Hi
'l'"ble- 16A ..
16ll 17.
18 ...••...
l!l .. :WA ..
20B.
20(' ...................... . 20n.
n ..
22 ......... . 2:{ ..
24 ....... . :!i) ....•....•.
26 .... ............ .
INTRODUCTION.
1':\t: E
14(i 147 1+7 14rl lii(l I;);) 1:)6 J!il I :)\I I (i2 i(i:l lli:l l(i+
l(i!l l(i.-.
'l'he sig-Jtifi('~lld part pLtye1l hy S.nlmonelln infe,·tiun in both lll<lll :1ntl animrd and 1 hP fn·quene_y 11'i t h 1d1 ich IIH'lllOl'ls of this group of mp;anisms han• heen :lssoc·iatecl 11·ith outhreaks of fuocl-poisoJting in man, haYe led loa dehli1ecl stu.l.1· of th1·ir ;tJdig·,•ni,. l'OllljlOlH'IlL'i during l'P('cnt .~·e;l!',;. \Vith the ;tdl'!•nt of l'l'li;ih1e methoch of
;;Prological analy,;i,; it has lH'l'llln<· pu;;sihle tu l'('l'Og'llisP s<'Yl'ral ''''".
si.raiw; of ,'-;ai!IIOIIP!la and to snhcliYidP a !ll!mher of olrl<'l' forllh into d j,.,ti nd t,qlf'.s in 1·as!':i 11·h,·re gTllll ps of t ht•,;t• 1\'t'l'l' j)l'l'l·iously g·,·ottpl'd in a hapha;o:<trd f<tshio11 tiiH!t-r one ll<lllll' on l'ithl'r ,·1inil'al,
;o:uolog·i('al or ('llHural grounds. \\'h<•re pion(·er ,,·ork,., .. , haJ lo rl'l.1·
Lt rg·e1y or ~ol,·l.l· 011 tIll' fl'rllH'IIi.<d ion Jt'act ion~ uf the l.qw,.; for n rlifl'tJl'ellt.iat ion uf lht• groups, a dPst·ription of tht· org;~ni,;IIJ at prest>11l ('<Ill ht• a<·cPpt(•d only if hast·d on Tl'liahlP SProlog-i,·;li 11·ork. inYohi11p:
riH· (·onlpit-lP ""ti.!.!·<·nil' ;1n<tl.l·si,.; of the had,·riulll.
S('hulzt• (11)2(1) pointed out lh<· futility of gruuping· .'.almonl'llus 011 ('lini('al and ;o:oo1ogic·a1 grounds, and sho11·ed tJt,. Y<~1ue of f;Pro-
log-i<·<~1 11il•thods of ('hssificali<'ll. B\· 1110311,; of ah,;urptiou lPsb I1P 11·as <Jble to di1·id,· tlw lll'p:aniRms i11r:hHlecl in ihl' :-ln·tryr·l.·e group into a lllllllhe1· of h·pes. ]hn<·<· 1\'JJite (ID2fi, ·192!) a.h.), h.1· arlopling Sl'in1be's typ(•:i <1:< ;1 primar.v b:tsi,; for sluily, 1111ifie(l tlw Salmonello
taxonom~· h~- 1·ompari;;on of suC'h l'PJH'PS('lltat i1·e stTain;; ;lS he <·llu1;l find ;l\·aibh1e. JJ,. irl<•JdifiPd lhcs<• Yarious strain..- ;~nd intro<lu('l'tl
'1 sy..;t,•m of Ldw11ing for th(·ir dii1't·n'nt ;mlig·(·ni(' <·ompunt>nl:i.
Kauft'nw1111 (IIJ:!!J <~.h., 1930 a.h.c., 19:H, l!J;1-:l, l!J:V1 ,~.h.P .. , 1n:n) l·olltinul'<l <111(1 t•x[,•nd<'cl thP 1wr1; initiatt>1l ln· Jhu(·t· \\'hill' hut usPcl a difh·reJil s~·.sten1 of 1nhPiling·. LoYell ( h :t!. <l) eurreLil(•;l 1 h,, formul.te presPntetl hy thest' t11o "·orkt·rs h.1· g·i1·ing· tht· equil·:11t•llt 11111nlwrs nn<l lelter,.; useil in th<· i11·q svstemf\. In order to ohvi;~h-•
the confusion that 11·as honnd to o1·t·u;· from ll1e P:o.:islt•nce of lll'<l
~eparate system,; of antigf>ni(' l;~ht>lling tho SolmonPllo Suh-colnJni(t('P of thP Jn(Prn;tlion;il So1·iet.l· of .Jiinohiolog.1· (J(J:l-J.) ;~cloptecl Kauff- mann",; terminolog,1· for gellf'rnl use.
80
M. W. HENNIKG.
In the stucl5· of the ~Peeifie-pha.,e-JJon-spec:ific-pha~e variation of Andrewes (1922, 1U25) the presence of two well-defined, but mutually conYertible. types of organisms was recognised within the limits of a spe(·ies. This phenomenon explainerl several of the factors conc·erned 11·ith the Cl'os:;-agglutinations observed in a number of different type!~ of S'al111onel1(/. Hut investigation~ ou the antigeni(' structure of bacteria were nctu:dl.v commenced by Smith and Heagh
(1903) when the,\' studied motile and non-motile strains of the hog-
r;holera hacillns. They were the first to describe flagellar ancl
~oma!iG agglntination as hYo distinct pro('esses and to show that the same organism may contain two agglntiuable substances, which haYe the property of proclu('ing· hYo corresponding agglutiuin:; in animab.
They found thnt animals inorulnted with motile stn1ins :vielcled a serum 11·hich ag-glutiJJatecl 1'lw homologous motile 01 ga11isms at a dilution of oYer 1: 10,000 but lwrely aftertecl the non-1notile orgnnisms at a ·1: 500 dilution. Sera prepared against non-motile fmm~
had a !itJ·e of onlY lOU to :)00 for both motile and non-motile strain;;.
They recognised t"·o types of ag-glutination, (1) lnrge, loose, rapidly- app("<uing tl.occnlent clumps of :fiag-ellater1 (motile) orgaui,;ms, and (2) smnll, compnd, dense, slowly-forming (somatic-) gTanules of non- motile organisms. The.'· nssoc:iated the agg-lutinins in the sera pre- pared ll'ith the non-motile organisms (somatie nntigen) with the bodies of the bacilli and not ,,·ith the flagella. On absorbing' the sera made '"ith the motile shain with non-motile hacteria, the somntie agg·lutinins nlone "·ere remoYed. the flagellar ag·g-lutinins rt>mai ning behinrl.
Ahont tlw ;;mne time J oos (1900) described two kimls of agglu- tinogen and t\\'o f'OJTesponding agglutinins in S. typlu. He also ohserve<l bl·o forms of dumping associated \Yith hYo diffenmt agglntinngens, npparently (·onesponding- to the flagelbr and somati(' agglutination of Smith ;nul Heagh. ~Ioreover, Joos noticed j hat heating at ()()0 to 62°
r.
de:>troye(1 the antigen ref;punsible for the large loose floccules but not the flagellar agglutinins, whil<> this temperature had no effect on the antigen forming the small g-rnnul<:"s, but destroyed the agglutinin producNl by it. Soon afterwards Be)·er and Reagh (1904), nlso "·orhng "·ith the hog-cholera bacillus, found that the fiagellnr agglutinable suhsbnce was greatl.Y <lamage<l h)·heating- nt 10° C. for more Lhao 20 minutes, ,,·hill' the somatic c;ubstnnee wns not affected; but the heating- did not destroy the agglutinogenie property of the flagellar substance. }Ir)l'eover, these worhen; showed that heating- nt 70° C. (lestroyed the somatif' hut not the Hngellnr agglutinins.
The importance of these finrlings was not fully renlisecl until '\Yeil nnd Felix (1917) observerl that Y:1riation in the grO\dh dwracters of Protew ~Y19 "·ns associaterl "·ith Yery striking- sero- lou·ir·al rlifferences. 'l'h<' one Yariant, termed by them j·he " H ,. (Hauch) form, gTe\Y as a spreading- film on age1· and gave risP ton mnrked, loose flo('(·ular ag-g-lutination with its 0\\'ll st>rum; "·hile the other Ynriant, the " 0 ., (ohne Haurh) form, gore,,- as circular
dump~ IYith is own .-;ernlll. The.\· <·alle<l the ap:glut.inahle ~ubstau('e
present in the " 0 ,. form, " 0 " reeeptors and the mnterial responKihle for the large floccules of the " H " forms, " H " reeep- tors. They showerl. that the " H " forms contained l~oth receptors,
81
.>i"TIGE~IC STitUcTl:RE OF ~.\L~!Oi"ELLAS.
while the " 0 · · forms f'onlainerl only the " 0 · · receptor. 8era of rabbits inoculated "·ith the " H ,. variant of l'mtens ~\:19 con- tained agg-lutinins fol' hoth " H " a1Hl " 0 " fonns, "·hile rabbits injectell with the " 0 ,. variant, produced agglutinins for the " 0 "
fmm alone. ,,-hen the " U ., nlrinnt wns hE'nted at 100° C. or exposed to dilute acids or to pure alcohol its agglutiuatiYe power remained unaltered, but "-hen the " H .. form wa,; similarly treated
or grown on phenol-agar it lm;t its power of agglutinating in large, loose florcult-s but retained the property of fonniug- small granules.
'l'hese re,;ulti-i showed the complete analogy beb1·een the motile
<Hid non-motile form:-; of the hog--dwlera bal'illus deserihe<l by Smith and H.eugh nnd the " H " ancl " 0 ·· forms of \'Veil and Felix. Soon aftenl·;nds Brnun and Schaeffer (1 919) clemonstrated that the " B ., antigen occurs only in cultures of motile organisms, while the " 0 " antig-Pn is present in both motile and non-motile cultures.
Later \-reil and Felix (1920) llemonstratell the presence of similar antig-enfl in o:·g-anisms o£ the typhoid-pa1·n typhoid group, au obsenation subsequently confirmed by Grusrhka (1920), 8chiff (1923), Brur·e \\'hite (1925) i:lncl others. Bruce \Yhitf' (1926) advised the u::;e of the term " H " antigen for thE' labile, floc<·ulutiug flagellar form, aucl tlle term " () ., antigen for the stable granular form; the coiTespon<ling- agglutinins he referred to as " H ,. and
" 0 " agg-lutinins respectively. It is now eonvPntional to attaeh
the label " H " to the heat-labile :flagellar antigE'JI~. and i he lnhel
" 0 ., to the he:d-stahle somatic anligPus.
A further .-ulvance with :fiagelhu- a11tl somatic agg·lutillatiou was made by Oreutt (J924a) when she wnfirmed the \York of Smith anrl Heagh (1903) by using- motile nnrl non-motile strains of the hog- cholera bacillus derin'd from a single strain, originally motile.
~he employed a suspension of fbg-elb as an agglutinogen an,l, by using rabbits, prorhwed a serum containing only flagellar 1nd no smtwtic agglutiuins. This ::;ennn agg·lutinated motile strains to a titee of 1:f>,000, hut fniletl to flo('('Ulate nm1-motile shains ai 1:40, while antisera prepared with thP washPrl bodies ag-g-lutinatE'<l both motile aud non-111oiile straiw; up to I: 1,000. Orcutt (1!)24h) nlso founrl that hPating the free flagella at jl)O f'. dr>stroyE'(I thPir agglu- tinating p011·er "·ithont materially altering their ag-glutinog·pnic propeit.y. On the other hand neither heating at 70° C. 11or at 120°
C. destroyed the ag-gh1tinatiug- and absorbing- properties of thP soma- tic nntigen. The :-;omatic agg'lutinins were partly <lesiroyed at 70°
C. and oompletely at 75° C.; but the flag-ellar ag-g·lutinins, althou~·h
unaffected at 70° C., IYere partly impaired at 7:)° C. 'l'lw 11·ork of Craigie (1931) on the distribution of the " H " ancl " 0 " <mtig·ens in the bacterial borly confirmed the rE'sults ohtaine<l b_,- Orcutt.
\'Veil, Felix and ~Iitzenm:1cher (19]8), "-hile 1nn·king IYith typhoid and paratyphoid org-ani;;ms, found Loth " H .. and " 0 "
ag-glutinins iu the sera of patients as well ns in Hte :;era of rabbits inondatecl with "-holE' bacilli. \\Then ba.ctPrial snspensio11S lwatecl at 100° C. were inoculated into rabbits agglutinius wer<' formed which caused small granular flocculation of the " 0 ,. forms. These ohsPrvations 11·ere subsequent]~· confirmed hy Hnwe \Yhiir> (192G).
82
.M. \\ .. llE~NHiG.
Anclrewes (1922) found that the same culture of a pure growth o£ a motile Salmonella often contained two sets of indiYirlual bacilli with entirely different " H ., antigen~, the one sper:ific for the particular race, or for a fe,y races, ,,·hile the other had wide affini- ties for a whole group of alliell race~ of Salmonella. By picking a number of siugle colonies from un agar plate he succeeded in ::;el)arating these bYo Yariants, ''"hich he referred to as the specific anr1 the gToup phases; but on sub-c1dtivation, especially in fluid media, he found that each of the hYo phases usually mutated rapidly into organism;; of both types. For the purpose of examining this phenomenon Amlre1Ye:s (1925) adviserl the use of sz1ec1Jic ancl gro11,p sera, prepared h:v absorbing the ngglntinins not required from a serum which r;ontain<->d both. ·w hite (1 H2:)) shrmed that these phases were concerner1 purely with change,- in the flagellar antigen, the somatic antigen being the sal1le in both phases. Scott (1926a) showed that a strain of thompson occuning in a quasi-group phase could be changed into a type (specific) plwse. In on1er to suppress the exeess of group antigen he grew the strain in a po"·erful group ::;ernm, viz. media eontaining 15 parts nutrient broth and 1 part of a strong group serum. After 2-t hours the supernatant fiuid in the tube became clear, \Yhile a thi<'k deposit rollected at the bottom. After ('entrifuging the c·ulture, another tube with group- f'enun-bi·oth was inoculated and a drop was plater1 fm individuail eolonies. The procedure \Yas repeated after every G hours, plating a drop at ead1 time. After a few pas;;ages a pure culture with a new phase wa" ohtainecl anrl the cleposit \Yas no longer formed in the tube. By employing Wassen's (1D35) morlifirati.on of Scott's method Rrune1· ;md Edwards (1938 11 and /1) antl Ech ·arrls and Bruner (19;39) were able to demonstrate nrlclitional plw~f's in a number of organisms tl1at \Yere ]JreYiou;;ly reganler1 as lll<Jnopllasie.
Schutze (1922). Bruce White (1925, 1926. 1!J2!J), Kauffmann (192Ga, 19:10a,h,(' 19;:!Ja, 1D35b, 1!);{5c, 1D:n ete.) and others have pointed out th<lt thf' SOIIIatic as \Yell a,; the flagellar antigen of Srtlrnonellas may b~~ multiple, the somatic antigen being gene- rall:s·: reg a nh·d as tlw r·onnedin g- 1iu k behn•f'n difft>l·ent raceR of ,.;peues.
Ficker (1~)0:~) and Dreyer (1908) us<->d broth nlltures extensiYely as agglutinating ::;uspensions for routinf' ding·nosis. But the aggluti- Jtation obtainec1 shoulcl be regarded as ~~n "H "-agglut.iuation be('ause liquid ('Ultures generaHy contain bar·terin "·hid1 are more motile and bet·ter supplied with tlng-ella tor "'H "-agglut;,tntion than solid eultures. }foreover, Dreyer nclviserl tlw use of dead cultures killed by the aclcli tion of 0 · 1 per cent. formalin anr1 exposure
;It ;11° C. for sou1e clays. Pyper (1923). un the other hand, found that bacterial suspension~; containing formalin are unsuitable for purposes of routiu e diagnosis -he succeedPd in detecting many more positive eases of typhoid fever with the f'Omplement fixation test than with a vViclal test in which he was using formalised suspensions.
Later Felix and Olitsk.'' (1928) showed that for somatir agglutina- tion the antigen must he kept free from formalin and carbolic acid as either of these inhibit somatic agglutination in the presenre of
"H "-antigen. Thus, by using a formalised antigen foT his test, 83
.1:\TIGE.I\"IC STitCCITI\E OF S.·IL~lO:\I·:LL.\S.
Pyper :;uc·ceeded in deteding mtl.,,· those ~.:ases in ,drich ihe serum conh.tint>d '· 11 " n:.q.rlniini11~. 'l'lto,;e t·:tses c·ontaining· .. 0 ,. ag·glu- tinin:;, hut no · · H '·, failed to rend.
Bien ::utd Sonntag (1917) suc~.:eedetl in killing the motile form~
aud in flcstroying the ffagelLt by l1eatiug the organi,;mc; in 30 per cent. alcohol at 37° C.: thn,; le::~Ying au almost· pure '' 0 " suspensiou.
Braun and 81'ltaefft·r (J!Jl9) found that the afldition of 0·1 per cent.
phPnol to agar supJn·e,;~etl the deYelop111ent of flagella :lllcl, iherefore, the· protluction of the t;O!l'e.-;ponding Lthile :tntigt'n.
At present tlw ag·p:httination tP~t i,; genPr:tlly rep::tnlE'tl a,; a Yer;.·
reliable :1i.d to thP I'E'I'ogniiion and dn~~ification of pathog<'tti<"
bacteria. Vor das,;ifil':tt ion hoth ,;om:d iC' :1tH1 flagt•ll:tJ' ant igPnc; tlllt:-d bE' Pmploye<l, :tltltong·b tltP~e :tnJ not cqnally intporlant in nll familie.-;.
In /Jocillus flrofl'll-', f:11 ex:ttnplP, tht> flag·t·llar :tntigt"n <"<Ill IJe tt.-ied for tlisiittgui~-;lting· lalgT grottp-;, ,,·hilt· the ;-;olllati(' :1nt-igc•n i:-; fat·
more sp<'r·i::tli.-;ecl. In Snlmonell:t;-;, on thE' oiher hnnd, some of the SOJtl:l.tie :tntigPnf\ :m• Yt'r\· "·idd;.· disirihuted in the group, 11·hilP the flngPll::tr :mtig·pn i~ lllmh mon· ~JH'<·ifi1· ( \\~ eil :1nrl Feli:-;, J 020, Brure
·white 102G).
Both S('ltutze (lf)22) and Bruce \\'hit<' (J!J20. UJ2G) nJUintaitJ that no reliahlP and snf<' antigeJtit· rt?l:Jtion~ltip (':lll lw arrived a(. by means nf ah.-;orptinn tt'~h. nnle;-;~ !'ro~;-;-:tb;-;m ption and t·ro~,;-:qzgluti
nation mPthotl~ arp nuriecl nut "·it It hot It shnin~ of hn<"IPria and their sent emplny<'d in i he IPst. i .P .. uttle;-;.~ th!' r·omplE'te minor trcd i-;
perforntE'rl. Hntr·p \Yltit~" oldaint·d a numlwr of so-r-~tlled " Srh01t- muller ., ~tl:1ins, i:-;ol::t!Pd from cahes :1nd dPscrihecl hy Cluistinnsen
(1914) a.-; Pmotyzd,1n-n (SchothnullPI'). B;.· menns of propPr ah:-;orp-
iion te-;1.-; Hrut·e \\'lti!!' (10'2fi) fnuntl ilte;-;c• org:tni~u1.-i to lw typi<":11 S. typhi-n1111'i11m..
Th!' n bsorption of :tgglu1 in in;-; front ~era "'<tS fir.~t employetl b.1·
Dortlet (1~0!l), Ei,,<'ltherg· ancl Yalk (l!lfl2), ('astellani (1002) :m<l .loo,;
(1 f)(H). SuhsPquPntly ihis mdhod "·:1;-; E'Xi<'nsiYel~· usrrl hy seYeral 11·orker;; for tltr antigenic analy~i;-; of diff<·rrnt gTOttp,.; of h:1f'!C'ri:1.
Bo.HoH (190G) 11·:1'i OttE' of thP fir;-;t workl'r,; to rlistingui~lt heh1·l'l'll Pam./yplw.ms Jj nntlllol'/ . . ·le!lr,itd·c h1· ih u~P; H:linhridg·t) (HJtl!J) and O'HriPn (1910) aLo :ulopterl it for thr tliffert'nti:ttion of p:tt:t-
(~·plwid org-:tni~Jtls, IYitilr Sl'itutzt' (]!J20. 1022) and Hrnc·t• \YititP (J!J2;\ lfl2(i, 1!)29 :t and h) Jll:lllP p:-;(pn;-;i,·p tJst• of alJsot·ption trsts for thP da,.;,.;ifi(':Jtion of Sulnwnellos. Tor thP .-ihttly of the antig-0nic nnalysiR of h:tde1·ia ah-;orption tests 11011· pl:l\' a JJJost inl[Wrl:tnt. :JtHl indispt>ns:1 hlP p:nt. 'Jforr rlt't :1 ilPtl i 11 forttt:t! ion on t hr ,-;uhojP<·t of
:1.Q'~J;lutinnfion ,,·ill h<' fotiJHl i11 a l'P\·ie"· g·i,·vlt hy Ark,,·1·ighi ("1011).
SALMONELLA INFECTION OF CALVES.
(1) I :o;TJWJJC('TIO:\.
Iu l~urope, es]wci:tll~· in lrolbntl. ])pnmark :tn<l fterm:tn;,·. Solmonl'lla infection in c·attlP has assulllecl t·on~idPrahlc> proportions in <"erhtin lo<"uliiiPs, "·here it tenrlc; to J·eclll' Yt'Ul' affpr vc>::tr in an E'n;.moti(' fontt, cau~inp: Yery heaY.1· losse~ ::nno;1g .1·oung· ~lock. The inc:iclen<·t• of Salnwncllo infN·tion in nrlult, is genrrnll_1· reg-anle<l aR
84
:bl. W. HENNING.
~poradic. lJuct. enteriti£i1's of Uae1tner itl the orgamsm commonly incriminated a:; the cau:;e of calf mortality, but as this labelling frequently includes a number of closely allied serological types most o£ the record,; referring to it are incomplete and u1neli able. lVIore- o,·er, the member:> of this group cau,;e di:;ease in JDan as ,,·ell as in animals, but it is seldom pos~i ble to recognise the exact type of organism involved, a~; a reliable antigenic description of the orga11ism i:> hardly eve1· available. ·when Smith and Scott. (1930) stuuied some of the organi:;m,; isolated from cases of tal£ diarrhoea and labelled Bact. entetitidis GuertneT, they iouud that these belonged to the dttblin type.
According to Jensen (1913) a form of calf diarrhoea (Kulberruhr) has been known in Europe for more than a century. Obich (1865)
\\'aS probably the first to regard the Uisease a:; infectious, but it was left to Franck (187Ci) to prove it. The first bacteriological study.
however, was maue by Jensen (1891) when he in>estigated a seriou~
outbreak of Kulbenuhr in Denmark; but he could not completely Jistinguish the bacteria obtained from the normal intestinal contentR of healthy calves from those of Ktilberruhr. Later 'rhomassen (1897) described an outbreak of calf diarrhoea in Hollull(l associated with a bacteraemia and caused by org-anisms which were called "pseu(lo- typhoid bacilli ". Soon afterwards .Poels (1899) :;tudieu a t1isease iu calves which he ascribed to psetldocollbacill£. He (hstinguished pseudocolilwcllli from ordinary virulent JJ. coli by virtue of their higher vi1ulence for small animals, their greater motility and thei1·
inability to ferlllent lacto~e. Later Jensen (1903, 1913) described diseases in y01mg- cattle and in cah·es under the term " paracoli- bacillosis ". The org-anisms recovered frorn the tissues of affected animals fermented g-lucose, llulcite, mannite, maltose, xylose.
rhamnose and sorbite, but not lactose or saccharose. Jensen stateu that cases of meat-poisoning as well as some outbreaks of paratyphoid in man could be traced to the consumption of the meat of calves suffering froru this uisease. He divide<l the organisms into three serological groups (1) those which corre::;pond to Gaertner's bacillus and which comprise the majority of sb·ains, (2) those which resemble pm'atyzJhi-B and (3) a few strains which resemble neither Gaertner nor paratyphi-B.
Mohler auu Buckley (1902) reported a ,;pontaneous enzootic in cattle due to a bacillus of the entwriti(Us group. They obtained the causal organism in pure culture from the internal organs of affected animals. Schmitt (1908) isola ted Gaertner-like bacilli from calve~
affected with septicaemia, dianhoea and pneumonia. He regarded this disease (cal£ paratyphoid) as probably identical with pseudo- lwcillosis of Poels and pamcol-ibacillosis of Jensen. Soon afterwards Luxwolda (1913), vVarnecke (1914) and Douma (1916) described cases of enteritidis Gaertner infection in Holland, Christiansen (1915) regarded pm'acoli bacilli as identical with bacteria of the ente1·it1:d:is- paratyzJhus-B g-1oup. Nfeyer, Traum and Roadhouse (1916) investi- gated an outbreak of infectious diarrhoea among a group of hand- reared calves, from 1 to 4 days old. They incriminated as the cause Bact. enteritidis isolated from the blood and internal organs of the affected calves.
85
A~TI0ENJC STH~CT~RE 0~ SALhlUXELLAS .
.Miessuer and Kohlstock (1812), Lutje (HJ2G) :wd Lehr (1D:21) tlescribed outbreak,; of parat~·phoid disease in ~Hlult cattle. Two children became infected after receiYing milk from a sick cow studied by Lutje. Lehr noticed that the agglutinalion titre of the ;;em of infected animals varied hom 1:100 to 1: 20,000; hom the faeces of some animals, the ;;era of which hncl a titre of I: 100 to 1:200, he isolated Gaertner lwcilh and he found the milk of a cow that excreted Gaertne1' bacilli in the faeces to be infected.
Sometimes there exists a definite relationship behYeen the disease in auult cattle anJ cah-es. Bour·mer and ])oetsch ( 1 U28) de,;criberl several cases of Gaertner infection in both co1n; and cah-e,;. A number of adult animals excreted the bacilli 11·ith !he faeces, and the milk of one particular ro,,· that !tad to he emergency slaughtered caused infection in man. They also described an outbreak of para- typhoid involving more than SO people "·ho had partaken of cheese preparerl from the milk of au apparently health~· eow which was discharging Gae1'tner bacilli with her faeces. .Kinloch, Smith and Taylor (1926) described a widespread outbreak of acute e11 teri tis affecting 497 persons in Aberdeen. )'[ilk was found to be the cause of the disease and the source of infection \Ht:; traced to a cow with un indurated udder "·hich later developed septicaemia. Uaertner ba(·illi were isolated from the faeces and the vomit of a !lumber of paticnt.s, from the infected milk and from the udder and flesh of the cow. Tn order to determine 11·hether Gae1·tne1' bac£lli are excreted with the milk Standfuss and \Yilken (1933) carefully examined the milk of two cows that were discharging large numbers of Gae1'tner bal'1"lh in the faeces. The results were entirely negatiYe and these workers came to the condusion that when paratyphoid bacilli occur in thE' rnilk it is due entirely to contamination.
Ri111pau (1937) studied au outbreak of acute gastro-enteritis in 80 persons of an inf>titntion dne to typlu:-111Utiu111 (JJreslau). The vehicle of infection was ice-eream, and it was found that the ere am used originated from a herd in which there \Ya~ a calf discharging typhi-mrurium.
A detailed study of the incidence of paratyphoid in calves ani!
adult cattle \\"as made by Proscholdt (1931). Calves were found to be far more susceptible to infection than adults and Gan·tner IHwilli were considered to he the most important pathog-en for 1·ahes, wh.ile adult cattle infected with this mganism were 1·egarde<l as the prin- cipal source of meat-poisoning-. Proscholdt <leseribe<l two outbreaks of Gaertne1· infection spreading from adult ea1tle to calYes. Ag-gluti- nation tests carried out ~ometillles revealed a titre of 1: 1.00 in Jwalthy animals, a tihe of 1:200 being regarded as suspicious, and one ~f 1:400 as positive. Out of 465 cases tested by Pr(jscholdt, 404 were positive for Go elf ne1' and only 61 to typl1'£-mv7 imn. Rievel (1 g:n ) kept 4 infected carriers under observation for 1.8 months. In :;ome animals the agglutination titres were as high as l: 3,200. PeriodicallY the organisms could not be detected in the faeces, and the presence of Gaertner bacilli could not be demonstrated in the milk at any time. Pallaske and Lommatzsch (1933) were unable to recover Gae1'fner bac1:Zl1: from the org·ans of more than 47 out of 79 cases which showed patholog-ical c·hanges of paratyphoid..
n:v
cultivatin~· sus-pecte<1 material for long· periods in enrichment me dill they were a hle 86
(p de!t-d hac·teria in a b1·1·('e nun1her of ('a~c·;;. (Jut pf -t:l outhreab of p~tratyphoid in c·atlle, Franc·kc·, Standfuss aJJd \Yilken (l!J:l-+) found :!:-) elm· in d~tldill, 11 to l.IJJJI,i-1111/l'tltlll :111d n fe11· tu ms/'ol'l.·. ln I tal,1·
Slrozzi (UJ:l4) fouud S. l'llfl'l'lilllls Yar. d11IJ!in a~ 1hP nlo::>t in1portanl c-:IIL'nl ~1gent of c·alf parat_qJiwicl. h:tr~ten (1!):)3) ~dso nwdP a c·onl- pnrntivc• :study of thP inciclt>llc·e of Uuettller i11fed ioJ1 i11 adult c·:lttlc•
a11d c·:ilYe::>. ]II' l'onsiclc-rPcl lhai adult l'altlt>. unlike l'ah-e:;, l'ould di:scha rg-e para t yphu itl h~will i '" i tit t Itt> LH•ce:-; for long periods, allll ht> emphasised t hP dangl'r of ,udt discharg-er-; to all an imab l hal e·onll' in C'untae·t ll'ith thPnl. ('LnPnburg (19:!:!), on inn·,;tig-nting ~'"
outb,·c•:ik of paratyphoid infc•dion, found apparentl_v health.\· c·alve' and ecm,.; di:;charging· Gaedm•1· bal'illi in t lw f<lpc·es. On ~laug-hlPring
thc· c·al1·es threc• II'PI'ks latPr he· disc·OYI'rP<l h·pic·:\l ue•c·Jotic· fol'i in tlw liYe'r and kichw_ys, hut failed 10 i~ohtl' the· org·ani::>nls frolll lhe inle:;- tinal l'ontenls or intc•mnl on~·ans: hul the· titn• of the• st-'n"'' at first lll-'g:ttii·P 11·a,.; nu11· found to.ht' I :2()(1 and I :-+00. \\'ith r<"g-ard to JllPaL in,;pt:·dion, Clarenhurg (1~):14) shoii'Pd tl~;d il is c•xtrf'liiPly diHi- c:ult to obtain Uocrlner {)({c-il/i hom the· lllU:i('IPs of 'OlliE' aninl<~l, in which org-a11 c:ttlt-urc·~ lwn• yielrlc·cl po~iti1·p n·sult~. llc• f'Ollsicfpn•d
!lw u,,. of .-nri..lll11Pllt lllt>dia (e.g·. lPtrathioJiate broth) p,;,;pnti:1l for i!H-' 1'1'('0\'ery or (,'{{.f'rflll'l' IJI/('ilh rmm the lllUSdes.
\\"pl.Jer (J~J;I{j) n•ganJs tlte• w~dJs or till' t;(UlllHI'lt :111d iule::>tinc•s "' rrPdilPdi.on sitE's for S. {'1/il'rit{(/is, and tlterPfnn· ;Jfhll'lte:; ('()Jl:iiclc>r- :ti.Ic· inqwrtancT to t l1e h:tdPriolog-ical PX<Illlin~il io11 of lltP nltH·osa of ::Ill sn,;pedt>d casps; he dai"'" h1 h:11·p ,;uc·c·ePclecl i11 clc·ter-!illg 1nally
carrier~ by placing ;-;craping·s fro11t tltt> i11testinal muc·o,;a in Pnrich- ment n1edia, ll'hE'n lhe c·nltiY:ilioll of faec·P:i yit>lclecl IlP)..(':ltiYe result,;.
He• also noticed that Unerflll'r /J(u·i11i are frequent l.Y pxndc•d inter- llliHent [_,- in lhP f:lc-•ce~ and I hat :1 lH'gatiYe "'·rnlogi(':il tt>:;l lll<~\- lw obtained even 11·ht>n an ani111al disc·hargp,; h~1('illi ll'itlt the faPcf's.
Klimmeck (19:J(j), ou studying a liUlllher of herds of c·~dtle for para- typhoid c~lrrier;-;, found -+I nclnlt, a1ul :19 c·ah·es posiliYP: of the adults 2:1 disc:hargecl Gan-tner nne! :2:1 iyphi-lllurinm, ,,·hill-' :1:3 cail-e' ex('rett>d G'oPI'flll'l' ancl oni1· --l liffllll.-lllllrtu/11. A Yer_, .. 'illlall]H~rcen(ag·l'
of t-lw aclnlt cnrriPrs o·ai'P :1 posifiYc' ~wo·lut ination n•:tdion 11·hilc•
tl11~ proportion in c·;ih':'P.' ,,-;" _,t.ill .'illl:dl~r. T\nolh (I!J:)(i) /ll;HlP :1 elifl'c•rPnlinl stucl,· of :)(il slr:1ill' of Ua('J'/ner hor·illi ohhtinecl frOlll slaug·hlc·r ~~nim:1i,.;, 11.~ing ar:dlillosl' nnd rh~illlllOSP hmtlt. Bittt->r's ,,-hPY and Sinn's g-l_n;erine-fur·hsill hrolh for his icle·ntificatiou. Jlc·
i.nduc!Pcl 1 .-;tr~lill (0·2 J!Pr t'Pilt.) i11 the .iPna t.YJJf', 12 (2·1 per f'rnL.) in thc· Hostol'l' ty]W, 20 !0·fi prr C'Pnt.) in thP lbtin typE' ancl !12R
\CJ-+·1 pPr c·Pnt.) in tht> T\it>l t.I'JW.
In Ea~t Afric·a D:tnhnc•1· (1!)27) inYc-•slig·ate•cl :1 Yt·n· clt>~truc·tiYc'
forlll of calf paratyphoid a,·sol'iatt>d 11·ith h{ng- lt>sion,, .nel'l'otic: foc·i in the liYf'r, hac•molThag-il' Pnteritis, tumor spleni,; and hal'leral-'lni;l.
11c· oht:1inc·d org-anisms of tlu-• .\olmrmello eulcritirlis 11-JW fron1 thc•
bloorl. intt>rnal organs ~tnd be('p,; of nfft>ded e~thes. In lncli<t Shirln11·
(1!)~!5) inYPstig·atecl a hig-hly fatal clisease in l'~dYE's l'allsed h~- a lllPmlwr of the So1monP17rt Clllentid,s g-roup. (';~h-t>:-J rang·ing- from 4 to !20 clays old wen• affedt>d. J\ tentatiYc• di<tg-nosis of S. t<lltl'l'itidis
11·as nt:Hle purt>l.1· on hioc.:hrlnic·al gTouncl,_ TT~·gienil' fac·tor,; \l't-'l'P
rrg·~nclerl to play an imporb nt ri'.IP in tlw g·eJH•sis of thP disease.
Si
AXTIGENIC STil CClTllE 01' SALi\IOXELLAS.
In domestic mannnals calf paratyphoid is by far the most serious and most common form of Salrrwnella infection. Of the ] 04 calf ,;trains studied by me, 2 were found to be tyzJhi-mu rinm., 3 ente1'it·idis awl ninety-nine enteritidis var. du.Dlin- tbe latter desCI"ibed first by Bruce ·white (1929). The strain described by Bruce ""White was isolated by Biggar from a mnn affectell "·ith septicaemia following an operation on his kidney. Smith aud Scott (1930) recognised this organism as the cause of th1·ee case;; of continued fever in man studied by them. They considered that several of the cases of so-called
" Gaertner septicaemia " encountered in man ,,·ere iu reality due to infection 11·ith the dublin type of orgaui.~m. Some old laboratory strains obtained from outbreaks of food-poisoning, septicaemia and meningitis, and labelled Bact. entnitidis, were examined by them and found to be of the d·ublin type. Six strains isolated from outbreaks of calf dysentery in Denmark which were included iu the paracolon group (H. Jlrt'racol i) of .J en::;en (1910) 11·ere al;;o ret:ognised as belong- ing to the dublin type. Smith and Scott pointed out that in the majority of cases where this organism had been isolated from man, milk was incrin1inated as the cause; they regarded it a::; having a special association with bovine animals and concluded that cows' milk was the common vehicle of human infection. Bosworth and J_,ovell (1931) described three outbreaks of dul1lin infection in calves in Great Britain, where Salmonella infection is generally regarded to be very rare. A little later Smith (1934) recorded bvo fatal cases in chiluren due to infection with dubliu. The organisms were obtained from the blood, cerebro-spinal fluid, throat ~wabs and pus hom the pleural sac of one or other of the patients.
In South Africa, Huteheon (1893) referred to a disease of calves in the Eastern Province of the Cape that can probably be identified with " lewersiekte " of Otto Henning (1894). Hutcheon believeil that the infection was spread from farm to farm by means of the faeces of infected calves.
Otto Henning (1894) described the disease under the name of
" yellow liver " or " lewersiektf' ". Subsequently calf diarrhoea wa~
reported from different part~ of t.he country. In 1920 I (Hf'nning.
1932) investigated an outbreak near Estcourt in Natall and found lesions of necrotic foci in the liver and acute enteritis, but the etiology remained obscure until Viljoen and Martinaglia (1926, 1928) and Martinaglia ( 1929) incriminated Salmonella enteritid,is, obtained from the organs of affected calves as the cause of the malady. They regarded this organism as a frequent secondary invader affecting 1nostly calves whose vitality had been lowered by factors such ai>
improper feeding, bad hygiene, piroplasmosis, and anaplasmosis.
Martinaglia (1929) described outbreaks of Salmonella infection in horses, fowls and canaries as well as in calves. He discussed the bacteriology, symptomatology, pathology and diagnosis of the disease caused by a number of different strains, and classified the organism;;;
almost entirely on their biochemical characters, no attempt being- made to give the antigenic structure of the bacteria described. As a result of the work of Andrewes, Schutze, Bruce White, Kauffman and others, reliable analytic methods of serological comparison are now available so that I have been able to devote my time largely to the study of the antigenic stntcture of different strains of Salmn·nel111
88
M. W. HE 'NINU.
isolated from domestic animals in this country. But, for the sake of (oomparison, the hiochemir!al characters of the organisms are also given. (Table 25.)
During the last three years no less than 102 outbreaks of calf puratyphoill were recorlled in South Africa and in the majority o£
these the lo,;ses were considerable; from these outbreaks I have obtained 102 different strains of Salmonella. Accorlling to informa- tion received from different parts of the eountry it is quite evident that outbreaks occur which are never reported. In many cases the farmer inoculates his calves with paratyphoid vaccine as soon as he suspects the disease, and the inoculation frequently protects the auimals against infection. In other instances the vnccination hail little or no effect in protecting calves that are exposed in grossly infected areas or in premises hnrbouring a particularly virulent strain of the organism. At one time it was thought that these apparent breakdowns in immunity occurred only when the vaccine was pre- pared from a stock strain of Salmonella (rl1tblin), but it was subse- quently found that even vaccines prepared from local strains could not produce an immunity strong enough to resist n natural infection. The disease is always most severe in very young calves, but it may affect calves up to 4 months old. All affecterl cnh es discharge large numbers of bacilli with their faeces resulting in their wholesa]e dissemination. The scourge usually commences on a farm with a few cases of a(;ute diarrhoea, and during the ensuing years the inci- dence of the disease may increase to an alarming extent, depending upon the conditions under which the animals are kept. In some out- breaks the infection becomes so severe that the majority of the calves reared on the place succumb to the disease. With the increase in the number of cases of paratyphoid the locality becomes more and more heavily infected resulting in the creation of a vicious eircle. Farms '"hich r-ontain the greatest number- of cattle are generally the ''"orst infected.
The habit of kraaling calves, or of lcranling the cows while the calves a1·e admitted during the milking, or any procedure which permits calves under conrlitions where they have to rome in eontaet with infected manure, favours infection. It is not kno·wn how long the manure in infected premises will remain infective; all the avail- nble evidence suggests that the infection persists for a matter of years. In 1934 I inoculated a young bovine with a virulent culture of Salm.onella dublin (strain 154). After a severe reaction the animal recovered, but remained a carrier and discharged the organisms in its faeces for several months aftenvards. Some of the infected faeces 11·ere collected, spread in a thin layer over a Petri rlish and dried in the incubator for 48 hours; the dried manure was scraped out, bottlerl and placed on a shelf in the laboratory. Periodically this manure was test~(] for the presence of d1tbhn; this was clone by inoculating some mnnure in an enrichment medium, like tetrathionate broth, and by spreading some of the growth obtained on MacConkey's lactose bile-salt ngar. After 1,069 days the last test was made and the manure wns found to be as badly infected as nt the first test. Whether the organisms will survive for as long a period under natural condi- tions in the kraal or stable manure remains to he proverl, but the
5
89
.\XTIGI-:X[(' STHLTITIU: OF S.\L~IoXI·:f.L.\S.
fact that, under ('ertain ('undition."', duulin bacilli ('an n·main uli'"e itt the manure for Jit•arh thrPt· \·ears is an irHlication that tlwy an·
YPI'\ rPsistarrt and that ~lr\' m:lrl.ure from infP..tecl pre1nises Jlll;:it he reganl<><l a . ..; wry cbng-erou~. The ]Jussibility of cah-Ps obtaining- tiH·
infection from 1ht• lll:lnun· ur1<ler nal11ral condition,.; . ..;hould, tlwrefore. be elllplwsisPd. JiorPOYer. \\·ltpn ('0\\..; are milkt·<l in stahlf':i or kra:tb with the floors t·oyerPtl \Yith nwr1Urf'. drY or 1noi~t. t·onbrnin;~iion of the milk \\'ith rnanure maY lf':Hl to the dissemin;~tion of rl1tlilw through thf' milk: a nun1ht~r ol' l~:uropean \Yorkerc< (st>e ;~bow~) baYP shown ibal the milk of CO'o\'S dist'harg-ing- paratyphoid ha('illi with thP fat>ct>s mny be t·ontan1innted \\·ith these har·illi. ;~ncl that whrn milk is infPderi, the infection 1s ;~h\·;1_\·s ohtainPd fmm tht· f:H•r·<>s and not
l'rorn i hE' ucltler .
. \[y ohsen·:tLiuns ;~gTet• \\·itlr lho:.;t• of JbuhnP\' (I!J~7) and \'iljot'II and M:ntin;~g-lia (19:2.'-\), Yiz .. i lr:t1 <·xposurP of t'aln·..; to unfaYour:tidP t·onditions nr1d to disPa~e,; likt· piropLISnroc;is ;lncl ar1:1.plasr1tosis prt•- cli::;po;;<• Lh<•m to inft>t·iion. I ha\·t· ,..;iudit>d Sf'Yf'ral outhrpaks in \\·hil'i1 the t'aiY<'..; \\'PI'(' so h:1clh inh·.·dt•tl \\·itlt iit'b th;ll tlwir r·f'sistaJit'P r1111sL h:1Ye hf't'll IO\\'Prf'd ~ttn:;idt•r;~hl\·. Sorllf'tirllt':i t ht•rp \\'<IS no tlouht that iht• rt1orL1Iiiy t·otild he att;·ihuif'd eilhPr to piropLt~IIIO..;i..;, ana]Jhsrnosis, g-orldt•rio:-<is. or ht•;trf\\·;rfe>r, hut. in orllt'l' Gases the (';llYP:i
\\'CrP infet·tt•tl \Yith rfufJlin ;t,.; \\·pJl ;1..; 011(' OJ' rllo!'e of thf' ti('k-hOl'IH' cli:-;easp,.;, The rf1le played lJy 11If' l;~tter in predi . ..;po,.;ing· c·aln•,.; to paratyphoid inh•dion must, tht·r·pfnrP, lw c·oH..;iclf'r<~hle. In solliP of tltc outbreak:; :;(udiecl b)· n1e 1 t·onsidPr parat_,·phoicl ;ts a disPa..;f' JII'J
se. but io many l reg·ard tht• lit·l;-horllf' diseasP as ihf' primary t·aust•
of illnP;;s and t ltf' rlublin i11ft•d ion ;t,; ,.;prondarY. :Jfm·eoYPI', t l11·
h_yg-icnit· •·ondition~ undpr \\·hid1 tht· :lnirnal..; nr~ kt•pt abo jtlay :1 very irnportani pent in thP g·erw-;i.-; nl' thf' ,]ise;l..;e- p:ldic·ularl\· \dwn they <II'P fn·qnPr1tly f'XJlO.-;t'd to t·ttndition,.; that bring- them iu c·oniat'l with infet'll'tl lll:lntll'e.
J>roph_,·Lit·lit· lliPasnrc.-; fur t·Onlha(ing· cnlf p:1mt_\·phoicl, then'- fore, should Pnlail thP sysiPm:llit· f'l':ldic:rlion of tit·b as well as lht•
appli('ation 11f rigomu...; h)·giPIIic· lllt':1Slll'f'S 111 all prPmJ;;e,; \\·lwrP t'aln•s arP r:li,.:t•d .
. \II c•xn d:1 :1ntl inferi<•d t·:1n·asPs .-;hould lw plopt·rly disposPd of. ar1d ht·:t!th.v cal\'t'S ,..;hould h<' I'PitiOY!'tl horn tl11· inft·.-!Pd Jlrernisp,.; In dt'<lll ..;ul'l'olrll:ling-:-<. \':lt'f'in:tlion. :lliltong-11 <I useful nrt>lhod of pro- phylnxis, t'annot be n-•liPd 11pon .-;olt·l_,.: it . ..; Yalue i;.; gn-':IIPst when it i.' usc·d in t'on.iundion \l'ith th,, :1pplit·:1tion of ..;niLtlt!e II\·g-icnie and ti,·k <·ratlic·atinn IIH'<t . ..;nres. But. ;b \';H·c·inalion ag·:1insi calf p:tr;l- iyphoid for·111:i tilt· suhjN:(. nl' ;lnolh!'r Jl<ljH'I' \\·hic·h is lwir1g· prPp;lrPd in t'Oihbo1·:1lion \\·ith nthf'r \\·orkPr.-; nl Ondc•r . ..;tf'pom·t, it \\·ill not ht•
,] i scu ss<'cl lw re.
J.'rom lhP~<' ru·ords it i.~ c·lf'ar that ,'.rriii/0/IC!Ias :ll'l' <·orllllltlll pathog-<•ns u[ cain .. ::; ir1 cli.fl't•rPnt part~ nf the \Yorld. g-f!nPrall)· ~Pit.ing
up syrnpton1...; of sPptic:wrni:l. :1c·uiP cliarrhof'<l. piiPUrlronia. and liiPtling-iti.~ \\·ith lf'.-;ious of lweruorrhng-i(' eniPriti,.;, bront'ho-pneHrnoni;~.
tumor spleni~. Jl<'n otir· foc·i in ill!' 1iYPr' ancl kidne)·..;. :lri!l lllf'lling·ih..-. In the YflSi majority of ouihreak..; dt>.-;nibe<l, S. r•Jife,itidis is illnimi- n:ded as tht' e~tU:it' of the di~PnsP: hut, ap:1rt fr-orn thP \York of HnH'P
\\'hilt• (19:2!J), Sn1ith and S<·o(t ( 18:1()), Hos,,·orth a11d LO\·Pll (1931 ), 90
Smith (1934), Kauftmatm (19051, J930c) ancl a few othe.r:;, the identi- fication of the org:1uism was not bnsecl on it~ serologica1l charaden;.
On the basis of :1 sPries of ngglulinatiou absorption tests carriell out with all the strains of Salmonella ohlaine<l hom L·aln•s iu South Africa, I haYe been able to rPeognise the org:1nism 1e;;ponsible for eaeh oulbreal;. Th!:' l'P:>ttlts of tlwsp iPsts arr :.6n•n in 'l'ablps ], 2 ancl ;1.
(2) 'l'IIE TECT-1:\lQU: E~fi'LOYEJJ.
'l'he matc•rial studiecl 11·:1::; obtailH•d from different varts of thE' country. ln mo;.;t eases it 11·as ,-ompo,;ecl of org·nn sperimens (liYer and spleen) ,;cnt tu thE' lnborator)· in 50 per cent. gl_n·rrine; some- times fresh fae('t'S or faeees ::;t•nt in gl:yeerine ,,-eJ·e submitted for t"xamination. Occ·a:;ionalh a sick animal "·as aYail::dJlP for inYesti- gation. Iu addition s~Yeral cultm PS made hom fo,ds by Mr.
J. D. ,V. A. ColPs, Chief of thP DepadiuPut of Poultry Dise:He;;
at Onderstepoort, 11·en' studiNl. 'l'hPst' are describer! in Sedion VI.
Most of the material \\·as obtaiut>d from places 100 to 800 miles away from tilE' laboTator.v so that n was not J!Ossible to ,-isit more than one or hnl infectPd fa nne;_ As routine p1 eYcntivP inot·ubtion of all cahes in area,; iufeded 11·ith paraf_\·phoid "·as g-enerally canied out, it was not possible to obiaiu ~ick ('HIYcs for oh;erYation that had nol been prHiously inoeulatecl with pnratyphoiJ Yacr·ine. For the identification of Salmunella type;; the te('hnic111e advised by Scott (1934) <lllcl modifiPcl by me \\·as ususally emplt)\'etl. Speei- mens of suspected matt•rial (bloo<l, livPT, spleen, faPt!:'s) 11·ere ,.;prend directly on J\Iad'onkey's lat·lose hile-s;llt a.g<lt' i11 )la::;on tubeR (Mason 1933)- Scott u::;ed Pelri platc:s. Generally it is advisable to dilute some of the material in saline m· broth before it is spread on the ilfacUoukey. In this 11·ay j,.;olaled colonies 11ill be obtaineJ more easily. In <ldllit.ion material (especially fae('e;;) is inoculated into an enriehment medium, e.g. tetrathiouatP hrolh m· 1 per cent.
peptone water containing brillia.nt g-rl'<'ll (1 iu 150,000). After 18 to 24 hours incubation the :Mason tubes are examinr·d and the en- riched cultures are spread on clr)· .Jbc-C'onke)' ag-nr. The characterjs- tic pale, finely stnwtured Sal rnonello colonie6 are pie ked from the tube which is frequently ero11'(led ''"ith rolonies of lactose-ferment- ing B. coli; sometime::; colonie,; of late lactose fermenting or non- lactose fermenting- 73. coli, B. pyrwyaneu.s and B. JHoteus are see!l- these should be aYoided curd should not he eonfusell 11·ith Sal1nonellas.
'l'he suspectecl Salmonella colonies are uo"· subjeeled to ;Jn agglutina- tion iest. A portion of a suspE>cted l'olony is pil'kerl and emulsifiecl in a loopful of g-roup ~erllln (e.g-. Enropeun r-lwleroe-s'uis senm1) and in a loopful of type serum (e.g. entt>ritirlis serum) on :1 gln~s
slide, the dilution of the serum depeuding on the titre- aboui 1 in 50 if the litre is 1 : :J,OOO. A number <;f the ,;u,;pected l'olunie~
:•re emulsifietl ea('h in two sepnrate loopfuls of diluted sera (g-roup ancl type); the amount of serum earried OYer from the o!le to the other chop is too small to t{)nfust~ the 1·eac-tion.
Some of the ('olonie::; may agglutinate with one o1· other of Hw h\·o drops of o;erum; whde others may fail to agglntirwtl-· with either, or may exhibit a utere tra('e of agglutination. Org-anisms 11·hich
91
A='<TIGENIC STRCCTT:RE O:F SAL110);ELLAS.
occur in the specific phase will react with their own type sera, while those that happen to be in the non-<Opecific phase will agglutinate with a group serum. 'Vhen a reaction occurs <t characteristic flocculation is seen whi<.:h is readily distinguished from non-specific salt agglutination of Hough variants. M:oreoYer, in a positive test flocculation ''".ill occur only in the one drop and not in the other, whereas in the case of salt agglutination clumping will be observed in both. A good band lens and a dissecting microscope are Yery useful during the fishing for colonies as well as for the study of the reaction. Colonies that have given a posi ti ,.e readion are picked, subcultured and studietl further.
" Pure " type-specific sera can be prepa1·ed by inoculating rabbits with G to 8-hours old broth eultures of the lHganism in thr specific phase. But as these sera always contain a certain amount of group agglutinin, pn'li.lllinary absorption of the latter "·ith an- other Salmonella containing tbe same group phase, but another type phase, is recommended. If typhi-murium, serum, for example i, nbsorl;ed with a mixtm·e .of paraf!JJllti-11 and r·holeu£-.wis, the group agglutinins "·ill he remoYed leaving a "pure,. t,rve serum-dilution.
If the orgauisms used for the absorption contain the same somatic antigen (!:'.g. zxu·atypl,i-B and typhi-1wurium) the " 0 " agglutinins will also be remoYell; thus preYentiug them fro1n interfering ,,·ith the reaction. The " pure " type serum will contain ouly type agglu- tinins, hut neither " 0 " nor group agglutinins. For routine diag- nosis a set o{ Tepresenta t i Ye type-spPcific sera Rhoulrl be available, e.g. zJaratyph1:-JJ, typhi-murium, cholera-suis, neU"fJOTt, tl10mp::;on, potsdam, bovis-mo,·bi.fi.cans, typhi, ente?'itidis and L2 sera. If a suspected colony g-iYes a l·harnderistic reaction with only one of these sera, a preliminary d i a g·n osis is made a rHl the culture obta inerl from it is studied further by mean,; of agglutination absorption tests. If group serum is use(l, colonies occurring in the group phase will be detected. Occasionally more than one type Salmonella is present in the culture (mixed infection), but tb P second organism is not likely to be missed as long a::< a reasonahlP numbt'r of colonies 1s examined.
Sometimes, "·hen clipha,;ic Salmonellas are sh.ulie~d, there may be smne difficulty in demonstrating the existence of specific-phase colonies, if colonies in the group phase predominate. On repeated sub-c·ult i.Ya ti on of the la tte1·, ho,Ye,·er, an occasiona 1 1 ·olon:v occurring in the specific phase may he detected. But in cases like European cholnaesuis, where the orgaui~m occurs permauently ( ?) in the group phase, phase dissociation will Hot readily take place.
For the acceleration of phase dissot;ia tion Scott (1934) recommcn1ls the use of broth containing approximately 15 per cent.
group serum. Group colonies cultured in this medium yield n culture "·ith a dear supernatant fluid and a dense rlepo,;i t after 18 hours' in(;ubation. On repeated sub-cultiYahon .in group serum- hrDth, a turbid supernatant fluid may ultitndely be obhlined. If this turbid culture is no''" plated, most of the co"l.ouies resulting ·will be in the specific phase. Sometimes as many as 10 or 12 passages may be necessary before the phase dissociation becomes apparent.
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