TAJJLE 23

Galli-

naru1n Galli- Galli- 29 s.

Serum narU'tn narurn a. b.

Antigen.

a. b. s. a. b. s. l;nab- Galli- Galli- 29. sorbed. naru1n.

na·runt.

G allinarmn ''0" .............. 0 0 1,600 0 2 9- ''0'' ............. 0 0 1,600 0

I ' ^I

Gallinarum = gallinarurn 416 of N.C. of type cultures.

0 = less than 1 in 50.

a.b. = absorbed by.

s. = serum.

29 s. 29 s.

a.b. Una.b- 29. sorbed.

0 1,600

0 1,600

A studv was a lso made of 55 cultures obtained from

a.

number of f ew day· old chi cks suffering from an acute disease, and from the ovaries of h ens t h at gave a positive agglutination test for pullm· 1 1 .m . Against three of these, cu ltures 317, 322, and 436, antisera were prepared for absorption t ests. The results , which are given in Table 24, show that culture 317 removes all the " 0 "

a gglutinin s from pullont?n serum and from the homolo gous serum , while pullo1 ·um also compl etely exhau sts both these ser a. Similar results were also obtained with cultures 322 and 436 and p· ullorum.

When galhnM'1tm was substitute d for pulloru?n identical results were obta ined, so t hat identification of the cultures could not be made en tirely on the basis of t h e serological test- fer m enta t ion test s were necessary for the complete differenti ation b etween gallinarum and pv. llor1t1n; t h ese are given in Table 25. Unilateral absorption t ests were p erformed with the other 52 cultures and pullorum or gall1:na1·um serum , r esulting in the compl ete ab sorption of the sera. But when fermentation tests were carrieCl out, it was found that the reactions of 42 of the cultures resembled tho se of pullorum, while t h e other 10 corr esponded to gall1:narum .

163

ANTIGENIC STRUCTURE OF SALMONELLAS.

Antigen.

Pullorum " 0 " ...

317- " 0 " ...

TABLE

24.

PullonLm YullonLm Pullorurn 317

Serum Serum Serum

Absorbed Absorbed Serum

Absorbed by by Un-

absorbed. by

Pullorum. 317. Pullorum.

0 0 3,200 0

0 0 3,200 0

P1dlorurn = P1Lllorum Bb. 26.

0 = less than 1 in 50.

317 317

Serum

Absorbed Serum by Un-

absorbed.

317.

0 1,600

0 1,600

R ecent ly I h ave studied a culture of pullrYrum i solated fr om t h e spleen of a duck by a colleag-u e, Mr. Haig.

According to th e fer mentation reaction s, ther efor e, 45 of t h e cultures from f ew day old chicks and infected ovaries of ad ult h ens sh ould b e 1 ·egard ed as pullonm,, while t he other 10 cultures fall under ga llinm·u m . 'rhe dinical symptom s and lesions p resen ted by th e chicks from which prullon m iJ cultures were isolated di<l not differ m aterially fr om those whi ch yield ed cultures of gallin m 'UT/1,. A diagnosis of infection wit h eit her gall1>na rum or

p~tllorum

in very young chicks should, t herefore, not be mad e, u nless fermentation tests h ave been carried out, as well as serolog-ical tests.

The fermen tation reactions given i n Table 25 include t h e rhamnose test of Bitter, W ei gm ann and Habs (1926), the glycerin - fu chsin-broth test of Stern (1916) and the d-tartrate test of J ordan and H armon (1928). By u sing solid ag ar m edi a advised by J ord an and H armon m ore clear-cut r esults were obtained t h an wit h the :fluid media of Silber stein (1931); in posit ive reactions th e colour of the (ph en ol -red) was ch an ged yellow by th e acid formed. The t h e exten t to whi ch t h is d iscolouraticn of t h e agar occurred varied even with differen t strains of t h e sam e organism ; stab cultures wer e made and the discolo uration started f rom th e inoculum, spreading from t his p oint in all direction s. In som e cases barely a quarter of th e medium was ch anged. , while in others as mu ch as a half or three- quarter s h ad t ur ned yellow.

Fifty of th e strains lab elled du blin gave n egativ e tests wit h Bitter 's rhamnose and Stern's glycerin-fu chsin -broth ; with t h e d-tartrate test of J ord an and H armon t he indicat or was ch an ged yellow ab out h alf-way down t h e t ub es ( + +) i n 14 cultures, and about one quarter down t h e tube ( +) in the rem aining 36 cultures- 47 of t h e cultures were n ot test ed wit h these m edia . Out of 97 cultures, 76 were arabinose negative and 21 were positive after 5 days incubation; 79 wer e rhamnose posit ive am:l 18 wer e n egative after 48 h ours incubation . But t h e rhamnose was gen erally ferm en ted after 4 or 5 d ays in th e incubator. Th e an t igeni c structu re of all the 97 st rains was id en ti cal with t h at of S . enteritidis var.

dub lin.

164

TABLE

26. Swnnwry of r es ' ults obt(/ 'ined w it h the antig e n ' ic analys1;s of 3 18

st/'lll;us

o f Sa l mone ll as i so lat e d from dom es ti c am>nals and !J1:tds 'in South Afr , ;ca .

Type of Salrnonella. Enteritidis var, dublin, ..... Enteritidis, . , ... , . , , , 'l'yphi .... '. ' ... '.' I-' Gallinarurn ... ,

g;

Pullorum ... , ... , . , .. Abortus-equi ... , .. , . , . , . , Typhi-murium ... . 'l'yphi-mu,rium var. Copen- hagen (Storrs) Cholerae-suis (Kunzendorf), Bovis-rnorbificans .... , ... . Anatum .. , ........ . Onder·stepoort ... , ... . A rnersjoort , ... , ... , ... .

No. of Strains Studied. 07* 3 1 149 45

It

lO 6 l l l 2

Origin of Organisms. " 0 "-Antigen. 06 Calves and 1 human ... , .. LX, (XII) ... ., .... . Calves ... , . , . , . , . , . , , .... , ... . IX, (XII) ... ., . Chick .. ,, ... ,., .... ,.,,.,.,.,.,.,. IX, (XII) ... .,. 139 Adult fowls, lO few day old chicks.,. IX, (XII) ... ., . 42 Chicks, 3 ovaries of hens .. , , . , , .. , .. . IX, (XII)., ... . Adult horse (tendo-vaginitis) ... , .. , .. , .. . IV, (XII).,.,, .... . 2 Calves, 2 canaries, l finches, l canary-IV, V (XII)., .. ., , food, l pig, 1 chickens, 1 sheep, 1 rabbits Foal (purulent arthritis).,, ... ,.,.,.,. IV, (XII) ... ,,. Pigs ... ,,.,.,., ... ,., .. ,.,., .. . Pork .. ,.,.,., ... ,.,., .. ,,, .... , .... . VI, VII (XII). , ... VI, VIII. ... .,. Fowls,, .. , ... ,, ... ,., .... , .... ,. X, IlL . ., ... . Sheep ... , . , ... , ... , , . , .. . 1 Chickens, 1 adult fowls ... , . , . , .. . XIV ... . VI, VII, (XII)., .. ,

*

Two of these strains were non-motile and were, therefore, devoid of H-specific factors gp.

'' H ''-ANTIGEN. Type (Specilic). a-Phase. gp gorn d r eh eh d

~-Phase. (j ?) (enx.) enx 'f The one strain studied had lost its motility and, therefore, did not contain an H-antigen, factors enx.

Group (non- specific). 1, 2, 3. l, 2, 3, l, 3, 4, 5. 1, 3, 4, 5. l, 4, 6. l, 4, 5. 0-factor (XII) has been recently added by Kauffmann (l935b) and is supposed to occur in a number of different species that are not related. Since the completion of this paper 10 more strains of dublin have been isolated from calves and 17 more strains of gallinarum have been obtained from outbreaks of fowl typhoid. In their antigenic structure and biochemical reactions the dublin strains correspond to those dublin strains described above and the reactions of the gallinarurn strains resembled those of the gallinarurn cultures given above. The total number of dublin strains therefore, should be 107 and of gallina,rum 166, and the total number of Salmonellas 345.

1;::

:8

p:: M ~ ~

....

!Z Cl

.-\XTIGEKIC ST.Il1JC1TRE OF SAL,.lOSELLAS.

l<'or compar ison fiv e :>tock st rains of

d'Ubl~n

wer e studied . 0£

t h ese P esch 25G, Cambridge 1 and 'ropley w ere positive with Stern' s

glycerin-fu~:;chin-broth,

while

d'Ublin

(Knox) and

Famcoli

(Savage) 250 gave a negative reaction. All five Jeacted negatively with Ditter's rhamnose and posit ively 11·ith Jordan and H armon 's d-tartrate . On comparing t h e antigenic strudure of Cambridge l a nd ' l'opley wit h that of

dvbl-ln

(Knox) by m eans of agglutination anrl absorption test s the three cultures " ·ere foun d to b e identical;

both Cambridge 1 and 'l'opl ey com pletel y exhau. ,;tecl

dvhlin

(Knox) senun.

Of the t hree stra in s 11·hi ch corresponded antigen ically to

S.

enteritid·is

a ll ga1·e a pm;it i,·e reaction

to

Hi tter's rh;tmnose, two (cultures 290 and 418) r eacted positiv el y with Stem's fuch sin-brot h an<l one (c ulture 2J(i) fail ecl to change it. All th ree

cult~tn•s

were po;,itive wit h J orcl an an· d Harmon's m edium. Tho three ;,tock strains, M.7. and D.5, and vVt:ybridge gav e a positive reaction wit h Bi1ter's rha mnose, Ste rn's fuc-h sin-broth a nd :Jordan and Harmon 's d-tartrate.

Of t h e 13 strains 1rhi eh \H're a ntigenically identical with

typlu:- nmriu?n

(Glasgow) all were Stern a nd d-tartrate positive, twelve were B itter positiYe and one (eulture 357) \\·as Bittt>r negative.

Eig-ht of t he cultures were inositr positiYe a nd t "·o (strains 357 and 1)02) wer e n egative; nin e wer e positiY e and 011 e (si rain 357) was neg-ativ e with rhamnosf:. while a ll the ten testecl ferm ented arabinose . Cul ture 307 was both rlwmnose and i nosite negative.

~foreover,

a complete mirror absorptio n test p erformer! with c ultu re 357 (Table 20c) sho11·ecl 11· itlt out doubt that it is a strain of

typhi- m111·iv111.

Of th e

typlii-mw·imn

stock strains t ·ested a ll five were Bitter and d-tartrate positive , three (:Mutton 74, Glasgow and IV eyhridge) w ere Stt-:m posiliYe,

~~-hile

h ·o (Binns ancl Bre,;la11) IYere Stern n egatiYe .

The on e strain of

/;yphi-rruurium

Yar.

Cnprmlwgen-Stons-

( culture 4 7 8) studied g ave p osi. tiYe Bitter , Stern and d-tartrate reactio11 s. It fermented b oth inosite and malt- ose, hut not a rabin ose:

while the stock strain

StorTs

19500 fernw n ted arabin ose and inosite.

but n ot m altose; it wa;, also Bitter an(l d-t:u-tn1te p osit ive,

hut

Stem negative. r rhe two st rains of

CoJienlla!JPn (6~'>9

aml llJ7) both fe rmented m a lt ose and ara1>inose but not · in o;,ite; both IYere Bitter, Stern and rl-tartrate positive. Antigeni rally, bmnYer,

Copenhagen (Storrs)

478 ,

Stons

19500 aocl t he t1vo strain s of

Copenha.r;en

w ere identical (Tabl e 12).

1 <'or comparison 5

::dock

strain s of

JHUtlf!JJihi-B >~·ere

ioclud<>il in the t est. Of these three (D.<'. ,

Gre~'

all(] Oden se) wPre Stern

po~itive

a nd two (Sch ottmnller a n d Lo,,·esto H ) 11·ere n egat ive; only one (Grey) was Bi tter positive, the oth er four ,,-er e negative . All fi 1 ·e reacted n eg-;ltivel.v '"ith Jordan and Hannon 's cl-ta rtrate .

' l'he six strains which resembled

cholcrae-.m1:s

(K11nxe nd or f) a u tig-eni call.\· were all Bitter a n-3 cl-hutrate positin• hut Stern negative .

' l'here was only one c ulture (strain 391) wh ich h<tcl t h e f-ame a ntigenic structur e as

bo1Jis-morbijicans.

Lik e the original

c~ulture

16G

:M:. \\'. I-IENNJ I\" G.

of Dasenau it

w<~s

Ditter, S tern and d-tartrate positive, but the strain of Sladden an<l Scott differed from it on account of its n E-gative Bitter reaction.

The strai n of anatvm (c ulture 414) studied gav e a posit iYe reaction with Bitter, Stern and d-tartrat e, resem bling, ther,efore , the two stoc k strains (3701 and 3702).

Like abortus-equi W.H .2, the one strain (culture 219) studied reacted positively to Bitter and d-tartrate, bu t n egatively

tr1

Stern' s glycerin-fuchsin-broth. It was foun· d to be anaerogenic.

Both strains of amwrsfom·t and the one of ondeTst epoort were positive with Stern, Bitte r and d-tartrate.

All the forty gallinarum strain s tested gave a n egative readion with Bitter and Stern, but a positive on e with J orclan and R armon 's d-tartrate. Out of 139 strains 119 fermented arabinose ,

whi~e

:20 failed to do so after 4 days. All the cultures fermented rhamnose, but in the case of 17 of the s trains the fermentation was d el ayed.

All were dulci te and maltose positiYe. · 'l'h e fifteen strains of J 7ullo rum test e< l all fail ed to rea ct

<Ja

Bitter , :::ltern or d-ta rtrate . Thirty-seven of the 45 strain s ferm ente d arabinose, but 8 failed to do so. All t h e 45 strains fermented rhamnose, but in ] 0 the r eaction was delayed. All we re dulcite a nd maltose negative , and all 45

~trains

were anaerogenic. (Rece ntly a fe,v ae rogenic strains were isola ted.)

For the differentiation of S . galhnarum and S. p' ullormn the cysteine-gel atin medium of

Hinsha~~·

and Hettger (1936) was t ried , but the organisms failed to gTo\1· in the m e(lium so t hat no change could be · Jetected in the tubes. ' rhe cysteine u se d was freshly prepared.

Only one strain of S. tvph i \Yas studie d. The original c ulture, obtained from n c hicken by :M:artinaglia, was foun d to b e composed o£ a mixture of two organisms when I r eceived it; the one non- motile and the other motile . After " sing le-c. elling " , the H on- motil e culture was labell ed " stra in 207 " and t h e mo tile one

"strain 208 " . 'l'h e former resembled pvllo1·um and .gall£na1'1n n antigenically ('l'able 22) and pnllorum bioch emically; while culture 208 wa s found to have the same antigenic

strt~cl

ure as typh1: (Table 21). T he £erment·ation reactions of the latter, howev er, did n ot altogeth er corr espond to those of t he stoc k strains of typhi e mployed . Like typiP' , culture 208 -was Bitter and Stern negati ve, and d-tartrate positive. Four year s ag-o c ulture 208 f ermentec l dulc ite slowly, ancl maltose within 24 hours, but at present it fa ils to ferm e nt clul cite and the fermentation of m altose is delayecl for about five clays. All the stock strain s of typh£ u sed fe rmented maltose within 24 hours, but fai leJ to ferment chtlcite. The ferme ntation reactions of c ulture 208 are, therefore, not quite typical of typhi, but its antigen ic structur e is identical wit h t hat of this ba cterium. 'When i t wa,;

found t hat a chang-e had occurred in the fermentation reactions of culture 208 after four years, t he serological tests were repeated . The r esults of these t ests sh ow that the aniigenic structure of culture 208 l1as remained una ltered , a ncl t h at i t · still resembl es typh, i.

· 167

ANTIGENIC STRUCTURE OF SALMONELLAS.

When the biochemical reactions of the different types of Salmonellas studied are compared with their serological reactions very marked differences may be exhibited by several strains belong- ing to the same serological t ype. For example, some striking variations in their fermentation reactions were manifested by the four strains of typh· i-mu1·ium vaT . starTs (Copenhagen) studied; the one strain 478 · .iescribed by me and the other 3 stock strains. All four gave positive Bitter and d-tartrate tests; three (StoTrs 478 and Copenhagen 659 and 1147) were Stern positive and fermented maltose, while one (Sto1'1'S 19500) . gave a negative reaction to these tests. ' 1\>o (Stons 478 and 19500) were inosite positive and two (Copenhagen 659 and 1147) were n egative. All four were antigeni- cally identical ('l'a ble 12).

Analogous variations have been observed in t he case of typhi-

muP?:um, paratyphi-B, ente1'itidis, entePitid· is var. dublin, bovis-

moTbificans and , to a lesser degree with typhi, pullorum and

gallinanlm .