Under:stepourt Joumol of l"etennary Science and Animal 1 ndvstty, rolume i, .rumuer 1, ld .t;, 193G.
Pasteurellosis : An Outbreak Among st Sheep.
Hy )I. \\T_ JTEXXIXU .~xu :JI. H. Y. BHO\Y~, Sediou,; of Bad e1·iology :mll Pathology, 0 llller~tepoort.
INTRODUCTION AND BRIEF SUMMARY OF LITERATURE.
(!_·\SJ<:S of pa~teurellosi~ or i11fedion "·ith l1ipol::n- orgallli:illl~ Ill
clomestic a11imab anti hinb are h equPnHy reported from diftPI'l'llt parts of South Africa; but, ap:n·t h-om the llesl:riptioll of thl' <li~<':t;;e in ::;lwep giw u ],y Maybin (19Jl) uncl the :ll:l:Ollllt of F<ml Cholc>r<l
hy !Ienning· :uHI ColPs (HJ;l:J), ihe inforwation funtished n•ganlinp;
the illl:idell('e of pasleurellosi,; in this ('Ountr.v is lllll'elialde :tllll illl:Omplete. A('('onling to ::;·ome of thP muii11e p<~t lwlogical :iJleciJnPns recei1·ecl :d ()mlPrstepoort, ho\YE'Yer, it "·oul<l :l]lpPar Uwt p<~stPurc>l
losis in sht'Pp is far. lllOI'E' "·icle;;preall tha11 is gv11erall,v rel·ogniseLl.
)byhin foun<l tl1e clisE':Ise exheJllPly prPYalPnt in South-\Ye~t Ah i('a,
"·here it aftedecl goal,; as \Yell as ,-arious hrPells ·of ,;hpep, the highe:-;t mortalit~- n<·cuni ug in K.a ra kul sheep and thP i r nos'Ps; hom i he pathologi('al spP('imens re<·PiYe<l at ()nller~tPpom·t it nppear~ tlwi in the F nion l'ersian shel-']1 arP mo~n_,- afl'c-decl, )Ierino sht>ep not haYing made ~my sip:nihcaut c.antri bution.
fn Eur·ope haemonhag-ic; septicaemia of she<'p wac; firsi descril,e<l in l.S89 (GaltiPr) ancl sinl:e then it has heen l'Pcogn ised 0\·er "·ide areas of ilw con tin en t. 'l'he relation, ho\YPYer, bet"·een Europe~m haemorrhag·ic septicaemi:~ of sheep an<l tlw conditwn hpre clescrilwcl is not dPar. Hutyra anll )LnPk (l92G) descriLe per:lcutP, Ul·ute,
;-:uh:~<:ut.e ancl c;hronit, fonns of the disease whcn-'aS i11 this -outlli·Pak the~e form~ coulcl 11ot hP llifi'Pl'PJdi:decl- apart fron1 OIH' m· t11·o :H·lltP C':\~E's in thP E'XperimPntal <lllllllal_.; onl.1· j-IJp ('lll'oni(' form 11·iU1 the deYelojJ!llent of pneumonia :~nd plrm·it is \1'<1S seen.
Le~·shon·::; (Ul3:2) clb l'l'iiJliou of .. An ()yine Affel:tion ,. l-oiucilles lllOJ'l-' doselv "·itl1 thl· conlliho11 1·econlecl l1ere. He obSPI'Yed c::lt;es of ;;ostnu1~1la infE'l'tion 111 slwep ,,-]JerE' the ani1u:ils liYPtl from t\\·eln' hour;; to tine<-' or four rb_,-,.,, and shm1·erl :1t :ntiop,;,\· pne11!l!Ollia
"·ith IIUliJ<•rou..; hipobr org-anisms [)l'<'~l'DL Ilis ,lesniption agrees brg·Pl.Y "·itlt Dung-all's (HJ:ll) al'l'Ol.lllt of a t·onL1gions pnrumoni:1 of shePp oc<;ttrring- iu l('ela1Hl heh1een CktolH>r a11d .June; h om some of ilwse l'<lsPs hi polar org-ani~m~ pathogpn il· to sherp \Yere isolated.
Laikipia hmg clisea,;p ()Iettam, UJ::lO) appe:us io he :lnothPr form of i his l'OJHl iiiou. Cnrac;s·on :nul Di,lier (l£):32) record henn· mortalitY in sheep, espe('iall.1· amongst :--·oung- nnin1;1J:;, clue to a J!Osfeurello i n'fec- tinn. It i:s interesti11g to note ihat these \Y·orkers usecl a Yac<;ine ltlade fJ u111 a fonnoli,E'rl ('lllt1lrP which "·a,; clainwfJ to he E'fi'Pd iYe in reduC'ing mortality.
113
PASTEuRELLOSIS : AN OuTBREAK A:YIOKGST SHEEP.
K umerous other w·orkers including M:iessner and Schern, Frohner und Zwick, \:Viemann J., and others describe ovine pasteurella infections and allied conditions. A few years ago Schutze (1929) gave a comprehensive review of pasteurellosis.
HisTORY OF THE PRESENT OuTBREAIL
In December 1931 the experimental farm of the U ni.-ersity of Pretoria imported two Ryeland rams from England, and a year later five mYes of the same breed. The imported sheep were kept with a number ·of ~ferinos on the University farm. All were pastured under identical conditions in the same eamps, fed from the same food and housed in the same buildings. During the "·inter of 1933 ·one ·of the rams (ram ~o. 1) beeame unthrifty and developed a short dry cough. It "·as treated for bronchitis and kept unJer observation.
It improYed slightly ancl served a fe,Y e"·es, but in March, 193ol:, it suddenly developed acute tympanites associated with intense pulmonary distress from which it died within twenty-four hours.
An autops.'· reveale(l marked lesions of necr·otic fibrinous pleuro- pneumonia, hydrothorax and hydro peri card imn, acnte catarrhal gastro-e!ltri ti:>, tracheitis, pharyngitis and laryngitis, enlarge- ment of the liver and spleen, and degenerative changes in the kidneys.
Cultures were made from the heart-bl·ood and pneumonic areas.
Ko grmYth resulted in the tubes inoculated with heart-blood, but several ::;mooth whitish-grey, moist-looking, translucent colonies of different ,.;izes appeared ·on the serum-agar slants inoculated with material from the lungs. A few single large colonies were picked, su be ultured and studied. The organisms of the different colonies appeared to be much alike so that the grmdh obtained from only m1e colony \Yas studied in detail. This culture \Yas designated paste1trella 182 and is described below.
A \Yeek after the death of ram No. 1 the second ram (ram No. 2) also (leYeloped syrnptoms of acute pneumonia and died about fifteen hours after it was first noticed sick. An autopsy again reveale(l lesions of fibrinous pleuro-pneumonia, hydrothorax, hydroperi- carcli um, and inflammation of the pharynx, larynx and trachea. Both lungs were involved and there were fibrinous adhesions between the pulmonary and oostal pleura. There was gastro-enteritis, degeneration of the kidneys, and enlargement of the spleen and liver.
Cultures were again made from the heart blnod and lung lesions and as in the case of ram X o. 1 several greyish-white, moist, smooth, translucent colonies of different sizes developed on the me(lia inoculated with the material from the lungs, hut the tubes seeded with heart b loocl remained sterile. )forpb ologically the organ isms from the (lifferent colonies "·ere indistinguishable, and finally a culture obtained from one of the smaller colonies was kept and studied in rletail, the others being discanled. 'l'his culture was called pasteurella 181 and is described helow.
As a result of the death of both these animals the Universitv was left without any male breeding stock and it was hoped that 'a purebred ram lamb (ram No. 3) the progeny of ram 2 now a bout five months old would soon be available for breeding purposes. As
114
M. vV. HEKNING AXD )I. H. Y. DlW\YK.
the five Hyeland e\ves and their progeny, including ram 3, did not thrive so \Yell as the locally bred Merinos it was thoug-ht ach·i;;able to inoculate them \vith a vaccine made from culture 18:2, the vaccine used being a formalised saline emulsi-on of a h'•enty-four hours old serum-agar growth. All the ·heep "·e1·e inocula ted onc·e only. Soon afterwards all the ewes improved remarkably in corHlition, but ram 3, although showing a slight improYement, remained unthrifty.
Early in August it suddenly became worse aucl <leYeloped acute pneumonia from which it <liecl the follmving <lay. 1'he most out- standing lesions found on autopsy "·ere fibrinous pleuro-pneumonia, h:ydroperical'(lium, hydr·othorax, fatty degeneration ·of the li\'("r ancl ki<lneys, gastro-enteritis and iumor splenis.
Cultures \Yere ma<le hom the heart-bloo<l, liver, spleen, lung lesions and pleuritic flu i <l. K umerous smooth translucent, shiny, greyish-white, moist colonies of Y<ll'~· ing sizes \Yere o bsenecl on media inoculated \vith material from the lungs; the liver and pleuritic fluid also yielded growths. A number of the larger and S·ome of the s1naller colonies were picked, subcultured and studied. In morpholog,,· aJJ<l staining characteristies the orgaui~ms hom the large and ~mall
coloni_es were in<lishnguishable, but in virulence and bio-chemical reactions t he:v sho,ved noticeable differences. The organisms from the large colonies ,,·ere apparently identical and a culture from only one (zJasteurella 247) "·as kept f.m further study; great diftereneefi, however, \vere observed between the cultures obtained hom two of the small colonie,.; and beh,·een them and those obtaine<l from the large colonies. 1'he two small colonies studied ,-ieldecl ('ultures 24 Ia and 247e. The colonies fonn<l on the media seeded with liver were similar to the large eolonies producell on the serum-agar by the lung material; the culture obtained from the pleuritic fluid became con- taminated \vith B. subtil1:s and was discarded.
BACTERIOLOGY.
(1) PASTEUliELL:~ 182.
Morphology and Stainin.r; Cl,aracteristirg.
1'he organisms \vere generally small, ovoid and irregularly arranged in smears, sometimes pleomorphic, some being ,;hort bacil- lary (ovoid) in form while others were long and filament ou,;. 1'hey were non-motile. Bipolar staining was common and gaYe the bacilli a characteristic appearance, espec-ially in Yery young ('1.\ltures and in blood smears made from eases of septieaemia. In some C'ultnres, however, the bipolar staining \vas not apparent. 'l'he organisms were Gram-negati,·e.
C1lltural Charactm··istics.
There was a mo<lerate grmdh on most ·of the onlimuy laboratory me<lia. Freshly isolate<l cultures grew fairly well on m~trient agar, hut after repeated sub('ultivation the growth became pomer and pomer. On serum-agar a much more abundant growth was obtained.
Single colonies were raised, greyish-'"hi te, elear, translucent, moist, smooth, and spread peripherally during the course of a few days' gro"·th. Fresh cultures could be washed off readily "·ith saline,
115
PASTEUHET,LOSIS: AI\ OuTBREAK AMO~GST SHEEP.
lmt older· ones became dull and viscous, aud tended to adhere to the metlium. Saline emulsions, even when made from fresh cultures,
\Yer·e generally flocculent. On solid media a maximum growth was usually .obtained after two to three cbys incubation.
In broth a uniform turbidity was formed after twenty-four hours and a "·hite, po"·dery, sometimes floceulent deposit eollected at the l1ottom of the tube. The growth eontinued in broth for \Yeeks so that in the course of a fortnight, or so-oner, a thick fi.occulent deposit was ±ounrl at the bottom, leaYing the supernntant fluid almost clear.
'l'he tlPposit disintegrated with difficulty and it reguir·e<l a great deal Qf shaking before a uniform emulsion could he obtained. Sometimes a pelliele f.onned on the smface of the fluitl. On blood agar t.here waR no haemo]y;;is arul gelatin was not liquified.
B1.och emical Reactions.
Acid without gas \Y:ls formecl in glucose, sacchm·ose, mannite
<:Hul maltose. Litmus milk was not altered, the methyl-red and
Yoge~-Pro~kauer reactions "·ere negatiYe, while the meth~·lf'Ile blue reductase ancl catalase testR were positive. ::\fit1 ates "·er·e reclueed, ammonia aucl a small nmount .of h;vclroge11 sulphi<le as well as ill<lol
"·ere formed.
(a) F'm Gu1:nea-zn:.!Js.
Guinea-pigs 1 to 14 were inoculate<! as follmYs:-
Gui noa-pigs.
l and ~-.... .
;~ and J ..... .
<'i and 6 ..... . 7 ::tnd 8 ............. .
!.), 10, 11, 1~ ......... .
l:l ftnd l+ ... .
Method. Material.
Intraperitoneal I 1/"20 of a 24-hours old serum agar shLnt.
1(200 of a 24-hours old ser-um af!ar slant.
l/2000 of a 24-hours old scmm agar slant.
l/20000 of a 24-hours old serum agar slant.
l/200000 of a 24-hours old serum agar slant.
0·05 c.c. of a 24-honrs old broth culture.
GuinPa-pip:,; 1, 2, ~l, 4, 0, G, 7, 13, and 14 IYere dea<l ''"ithin hYeut_v- four hours. Guinea-pigs S an<l 9 died after forty-eight hours, ''"hile 10, 11, an<l ] 2 surviYecl for a ,,-eek "-hen they were reinoculated with the ;;ame dose of organisms as before. vVithin forty-eight hours all "·ere <lead. All the dead guiuea-pigs shmnd lesions o0£ severe :fibr·iuou:; peritonitis, tumor splenis and enteritis, ''"hile some sho1Yed in addition hydrothornx an<l hydroperieardium. P11steu'!'Plla in pure culture ''"ere obtained in media i 11 ocula ted with heart-bl noel from guinen-pigs 5, 7, 8, 9, JO, 11, ancl 13. Ko cultures ''"ere ma<le from tlw other;;.
Subsequently two mme guinea-pigs \\·ere inoOculatecl irdra11eri- toneally ,,-ith 1/200,000 of a twenty-four hours old serum-agar culturP. Both die<l "·ithin forty-eig·ht homs sho,Ying typieal lesions of pasteurellosis and both yielrlecl pure cultures of paste1tn1la in merlia inoculaterl with heart-bloorl.
116
(6) Fo1' Rabbits.
Rabbits 1 to 5 were inocula ted as follows : -
Rabbits.
·-'-~--- Metho~---~---
Material.l. ............ . t ............. . ,:J •.. ' ..••....•.••
4 ... '. ' ......... . 5 ......... ' ' .•...
1
Intravenous ......· I I
c.c. of a 24-hours old broth culture.I
, . . . . . . .
2 c.c. of a 24-hours old broth culture.,, . . . 1/100 of a 24-hours old serum agar culture. , . . . 1/1000 of a 24-hours old serum agar culture.
, . . . . . . . I
1/10 of a 24-hours old serum agar culture._ _ _ __;_I -- - - ' - - - -
Rabbit :J died after a \Yeek, but ]Jasteurellae could not be found in media inoculated with heart-blood and no outstanding lesi·ons \\·ere re,·ealecl at autopsy. 'l'n·o more rabbits were inoculated \Yith 1 I 10 of a hYenty-four hours old serum-agar culture; one of these died after four days, but ag-ain heart-blood cultures remained negatiYe f·or pa:;te?ttellae and no lesions were obsen ed. The other rabbits all remained healthv.
(c) For Pi.oeons.
Pig·eons 1 and 2 were inor-ulated respectively \l·ith 1/10 and 1/100 of a twenty-four hours old serum-agar culture. Pigeon 1 cliecl after forty-eight hours, but pigeon 2 surviYed. The inoculations
\\·ere made intramuscularlv.
(d) For Sheep.
Sheep 37285 '"as inoculatell intrapulmonarily \Yith 10 c.c. of a tiYenty-four hours old broth culture of JJasteurella 182 (15.3.34). On
~farch 16th and 17th it was listless with an elevated temperature (107·5° F.) and accelerated respiration. On :Yiarch 18th it dierl and an autopsy reYealed a necrotic fibrinous pleuro-pneumonia "·ith hydrothorax and h:rdropericanlium. Culture media seeded with heart-blood yielded a pure eulture of pasteurella.
Sheep 28078 \Yas injected iutrapulmonarily (19.3.34) with 2 c.e. of a twenty-four hours old broth culture. A.fter t\Yelve hours tlw animal became listless, its temperature shot up to 108° F. and it:;;
hreathiJJg became rapid and d1stressed. The temperatme suddenly clr·opped to 106° F. ·on the next rlay aml then to 104° F. on the day after that. On the f.ourth day the temperature \Yas 102° F. and on the sixth day the animal died. The most outstanrling lesions presented \Yere hyperaemia and -oedema of the lungs, and hydr-operi- carrlium. The earcase was fairl~' clecomposed at the time of antop"y allCl no cultures were made.
Sheep 38880 \Yas inoculated intrapulmonarily (25.3.34) with a :2·5 c.e. of a t\\·enty-four hours ol<'l broth culture. On the eighth clay the temperature rose to 106° F. remained at that level for two rla~-s
anrl then dropped to 103° F. on the tenth da:v a fe,,- hours before the anim:1l died. Symptoms of listlessness and distressed breathing were also manifested during the febrile stage. An autopsy revealed lesions of fibrin·ous pleuritis, slight pericarditis, icterus anrl gastro- enteritis. Pastem·ellae were -obtained from the media seeded with
heart-blood.
117
l'ASTEl:REJ.LOSIS: AK Ol:TllltEAK AMONGST SHEEP .
.Sheep 37744 IYaS inoculated intravenously (15.3.34) ·with 10 c.c. of a twenty-four hours old broth eulture. The temperature shot up to 108° F. within twehe hours and the animal was found dead on the folio"· i ng day, sho1Y ing lesions of pulmonary congestion and oedema, and epicardial haemorrhages. A pure culture of ]Jasi"euJ·ella was obtained from the heart-blo-od.
Sheep 02606 ""ilS iuoeulatetl inh·aYPnously with 2 c.c. of n tiYellty- four hours old broth culture. On the fourth day the tem.perature suddenly shot up to 107° F. while the animal becaine listless and showell rapid distressed breathing. During the f.ollo"ing two clays the temperature dropped, reaching 103 · 2° F. a few hour. before the sheep died on the sixth day. Cultm·es made from the heart-blood yielded paste1t1'ellae and the chief lesions obsen e<l ITere fibrinou,.;
plemo-pneumonia, ateledasis of both lungs and ente1·itis .
.Sheep 33993 "·as inoculated intran nously (22.3.~H) with a saline emulsion of half a twenty-four hours old serum-agar culture. On the f.ollm1·i ng day the temperature was 105 · 6° F. and the breathing clishessecl. After the ti rst <l a.v the telll perature ra pi <ll5· d roppell all d IYas 103° F. a few hours before the animal "·r1s kille<l in extremis on the f011rth <lay. 'l'lte only pathological changes obsen e<l at autopsy wNe atelectasis of the right lung, enteritis and enlargement of the liver.
.SheezJ :36988 was inoculated intraYenou,;ly with 1/10 ·of a twenty- four hours old serum-agar culture. On the following day its temperature had risen to 106·5° F. and its breathing IYas Yery rapi<l;
on the .·econd day the temperature had <lroppecl to 102° F. and after that it :fluctuatecl for several davs between 102° F. and 100° F.
Finally the tem11erature remainecf regulnt' and the animal recoYered . .Sheep 34333 was inoculate<l intrawnously with 1/50 of a saline emulsion of a twenty-four hours ol<l Rerum-agar culture. K o obvious r1 i stu rha nee of health w.as noti eecl at any time.
The lungs and spleen of ram 2 IYere passed through a La !"a pie mincer and the emulsion ohtaiue<l 11·as utilised as follows:-
Sheep 27240 IYas inoculated inhaYenously (14.3.3-:1-) ~~·ith 10 e.c.
of the Latapie emulsiou. On the second day the temperature was 105·6° F. an<l then fiuduated between 105·5° F. allll 1040 F. 1mtil the tenth day when it rose to 106 ·4° F. anrl ·on the hwlfth it was lOG· 8° F. After this the temperature gradually chopped to 103° F.
ancl the animal died on the twenty-fifth day. The most outsatHl ing leRions recordecl were purulent pleuro-pneumonia, (there IYere frac- hues of ribs on the right sicle 11·ith laceration of the cone. poncling lung) hyclropericarclium, fatty degeneration of the liYer, ancl kidneys, a1Hl atelectat>is of the right lung .
.Sheep 38914 was injected int.rapulmonarily (1-±.3.0-±) IYith a 10 c.c. of the Latapie emulsion. rrhe temperature l"QSe to 106·0° F.
IYithin twent~·-four hours and then <lroppecl to 102° F. on tlH~ se<·oncl
<la;v; on the thirteenth clay the temperature again rose to 106·8° F.
HIHl dropped -on the following clay as before. The slwep finally recoYered.
ll R
:M. W. IIENNIKG AKJJ M. H. V. llRO\\"K.
On }farch 16th sheep 37698 was inoculated intravenously with 10 c.c. -of a Berkefelcl filtrate of the Latapie emulsion. On the second and fifth days the temperature suddenly rose to 105° F. ancl subsided on the foUowing days. The animal showed no further
reaction and remained healthy.
On }larch 16th sheep 37417 was inoculated into the right lung
\vith 10 c.c. of a Berkefeld filtrate -of the Latapie emubion. Apart from a moderate rise in the temperahue the sheep re1nained healthy.
Sheep 37831, 37818, 37798 were kept as controls in dose coubc:t with the above experimental animals. All remained healthy.
ltmnunis ing Properties.
An atempt \\·as made to immunise laboratory animals with either formoli,;e<l -or live cultures of pasteurella 182. Guinea-pigs \rere inoculated ,,·ith formalised saline emulsions of serum-agar cultures m ''"ith f.onnolisecl broth cultures and rabbits \Yere injected with live emulsions.
Eighteen guinea-pigs were in-oculated subcutaneously at weekly internls with progressively increasing <loses of a formalised fort;y- ei ght hours old broth culture. The first close was 0 · 5 c. c. and the final d-ose \Yas 5 c.c. inoculated on two sides of the body. Six injections in all \Yere given.
Six guinea-pigs were repeatedly inoculated \l·ith progressiYely increasing closes of a formalised saline emulsion of serum-agar cul- tures. The density -of the emulsion corresponded to the -opacity of the nephalometer tube (Burwughs and \iV ell come) iucli eating a concentration of B. coli 3 x 10" per c.c. The close varied from 0:5 c.c. t-o 2 c.c.
Rabbits ''"ere inoculated with increasing doses of live cultures;
1 (1,000, 1/100, and 1/10 of a twenty-four hours ·old serum-agar slant being injected iniTaYenously at weekly interYals, commencing with the smallest close.
A \Yeek after the last inoculation the guinea-pigs were tested for immunity. They ''"ere inoculated intraperit<meally with Yarious dilutions of saline emulsions made from twenty-four hours old serum- agar cultures. Those injected with amounts in excess of 1/200,000 of a culture all died, showing paste1tTellae in the heart-blood, "·hile about 50 per cent. of the guinea-pigs injected "·ith 1/200,000 of a cult1ne sun·i ,·eel.
On account of the poQor immunity produced in the guinea-pigs.
it was decided to ascertain the agglutinin titre of the serum of some -of the animals which had recei vecl the injections. Accordingly, four of them were bled to death and the serum separated. An agglutination test was made with the homologous organisms com-
mencing with a dilution of 1/10. The test was entirely negatiYe. The same test was performed with £-our of the rabbits injected with the live emulsions, but apart from an incomplete agglutination in a dilution -of 1 : 50 in one case the tests were negative.
119
PASTEUHELLOSIS: AK OuTBREAI\: AMONGST SHEEP.
The results of other workers, Schiit:r.e (1929), Cornelius (1929) also seem to indicate that the antigenic properties of pasteurellas are generally very feeble and that they are unreliable for the produc- tion ·of sera of a reasonable titre.
'l'he use of precipitating sera, as described by Yuse£ (1935), for tl:c reoognition of pasteurellas has not been tried.
Results.
Pasteu1'ella 182 has been found to be highly pathogenic for guinea-pigs and sheep, but ra bhi ts and pigeons "·ere extremely resistant t.o infection and withstood doses of virulent culture that proved to be lethal for sheeJl. 'l'he organism .apparentl~- has a pre- dilection for pulmonary tissue and serous membrane~ heca use the lungs and pleurae \Yere almost inYaria bly affected extensiYel)· even when the infection was made h)· the intraYeuous route. Affede(l lungs passecl through the Lata pie miuce1· produced a definite reaction in sheep ,,·hen the injection ''"ail made either intraYenou:dy or intnqml- monarily. The animal inocula ted by the former 1·outP d i eel hom fibrinous pleuro-pneumonia, "·hile the sheep that I·ec·pi...-ed the emul- sion by the latter route IecoYerNl. :Uerkefielcl filtrates of the Latapie emulsions ''"ere harmless f.or sheep.
The immunity produced in guinea-pigs by repeated injections of formalised cultures \YaS negligible, and in both guinea-pigs and rabbits inoculatecl with dead and live cultures 1·espedively, appreciable amounts of agglutinin could not be demonstrntell.
(2) pASTEURELLA 181.
In morphology, and staining charaderistics this ·organism could not be differentiated from pasteuTella 182. Cultures of zJasteu1·ella 181 on serum-ag·nr retained the moist, translucent appearance longer than the other organism, they became less adherent to the underlying medium anrl did not develop the dullness s·o early. 'l'he primary freshly isolated coloni8s ''"ere smaller than the single eolonies of pasteurella 182, but on subculturing the colonies becnme larger.
Biochemical Reactions.
Fermentation of sugars "·as found to be irr8gnlar; generally acid but no gas was formed in glucose, saccharose, maltose, mannite, and inosite. Indol "·as not f.onnecl, nitrates were not reduced and the methyl-red and Y oges-Proskaues tests \Yere neg a ti Ye; but ammoma and hyclrogen sulphide \Yere formed. The methylene blue reductase and catalase tests were both positive, but litmus milk remamecl unaltered.
V£1'1.tlence Tests.
(a) For Rabbits.- Rabbit 1 was inoculated intraYenouslv with 1 c.c. of a twenty-four hours old broth culture and rabbit '2 \Yas inoculated with a 2 c.c .. of the same culture.
120
l\I. " ·· IIEK:NIKG AND l\I. H. V. DRo"·~.
(b) For Guinea-pigs.-Guinea-pigs 1 t·o 5 were inoculated as follows:-
Guinea-pigs. Method. :Material.
l.. . . Intraperitoneal. ..... . 0 · 05 c.c. of a 24-hours old broth culture.
0 ·l c.c. of a 24-hours old broth culture.
0 · 25 c.c. of a 24-hours old broth cultnre.
0 · 5 c.c. of a 24-hours old broth culture.
l c.c. of a 24-hours old broth culture.
2. 0 •• 0 • • ••• 0 0 • • • • 3. 0 •• 0 • • • • • 0 0 • • • •
4 .... 0 • • • • • 0. 0 0 0.
5 .... 0 • • • • • 0 0 • • 0 .
After fo1-ty-eight hours guinea-pigs 4 and 5 "·ere dead, ]Jasteurellae beiug recovered from cultures made from the heart-blood. Both the rabbits and the other three guinea-pigs remained healthy. The test was repeated on guinea-pigs ''"ith saline emulsions of serum-agar cultures, but death resulted only in those animals that 1·eceived Yery la rg·e doses-1/5 and 1
/1
0 of an agar slant.Res1dts.
Pasteurella 181 "·as only very slightly pathogenic £.or guinea- pig:-;, anil rabbits remained unaffecte<l eYen by employing compara- tiYely large doses of virulent cultme. It differed from pasteV?·ella 182 culturally, biochemically and in virulence.
(3) PASTECRELLA 247.
In morphology, cultural and stainiug characteristics this org-amsm \Yas found to be identical with ]Jasteurella 182.
Biochwmical Reactions.
Like strain 182 this organism formed acid hut no gas in glucose, saccharose, mannite and maltose. It did not change litmus milk or liquify gelatin. It gaYe negative methyl red and Voges-Proskauer tests, but positiYe methylene blue reductase and catalase reactions.
It reduced nitrates, formed ammonia, a small amount of hydrogen s;.1lphide, and indol.
Virulence.
In Yirulence also this organism closely resembled pasteurella 182. Virulence Tests.
(a) For Guinea-pigs.
Guinea-pigs 1 to 6 were inoculated as f.ollmYs:-
Guinea-pigs. Method.
l. . . . Tntraperitoncal. ..... . 2 ... 0 0 •• • • 0 0 • • •
3 ...... 0 • • •• 0 0 • • • 4 ...... 0 . 0 0 . 0 0 0 0.
5. 0 •• • 0 0 0 0 0 0 0 0 0 ••
6 ... 0 • • 0 •• • •••• ••
Material.
0 · 5 c.c. of a 24-hours old broth culture.
0 ·1 c.c. of a 24-hours old broth culture.
1/20,000 of a 24-hours old serum agar slant. l/20,000 of a 24-hours old serum agar slant.
1/200,000 of a 24-hours old serum agar slant.
1j200,000 of a 24-hours old SC'l'um agar slant.
121
PASTE"CHELLOSIS: AK OU'l'BREAK AMONGST SHEEP.
After forty-eight guinea-pigs 1, 2, 3, 4, 5, \Yere deatl, showing lesions of hydrothorax, hydropericardium and intense fibrinous peritonitis; ]Jasteun llae \Yere obtained in pure culture from the heart- blood of the dead animals. Guinea-pig 6 survived. Later four more guinea-pigs were inoculated each with 1/200,000 of a twenty-fuor hours .old serum-agar slant; of these three died.
(/1) For Rabbits.
Rabbit 1 was inoculated intravenously with 1/10, rabbit 2 with 1/100, and rabbit 3 with 1/1,000 of a twenty-four hours old serum- agar slant. After forty-eight hours rabbit 1 "·as dea<l, but rw pasteurellae were found in media inoculaterl with heart-blood and no outstanding lesions were detected in the carcase. Two more rabbits \Yere inoculated each "·ith 1/10 of a twenty-fours hours old serum-agar slant and the result was the same as before. Ra hbits 2 and 3 survived.
(e) For Pigeons.
Pigeon 1 ''"as inoeulate<l intramuscularly "·ith 1/10 and pigeon 2 with 1/100 of a twenty-four hours old serum-agar slant. After forty-eight hours pigeon 1 was dead, but pigeon 2 survived.
(d) For Shee]J.
,Sheep 40531 was inoculated intravenously (8.10.34) 'vith half the emulsion from a twenty-f·our hours old serum-agar slant of J!a.stew ella. 24 7. On the following day the temperature was very high (10G·8° F.) and the animal sho,Yed <listressed and. rapid breathing. The sympt{))US persi steel for :fi. ve days when the animal
<lied. An autopsy revealed intense fibrinous pleuritis, marked oedema and eongestion of the lungs, hydrothorax, hydropericardium, fatty degeneration an<l bile pigmentation of the liver and enteritis.
Cultures were made from the heart-blood, liver, pleuritic fluid and lungs, fr01n all of 'vhi ch paste1trellae were obtained.
SJ,eerJ 40444 was inoeubtecl intravenously (8.10.34) with 1/10 of a t"·enty-four hours old serum-agar slant. Within t'venty-four hours the temperature rose to 107 · 7° F. but dropped to normal on the following rlay; on Kovember 2nd and 9th the temperature again rose to 10[J° F. After this the temperature dropped and the animal recovered.
Sheep 40994 was injected intratracheally (17.10.34) with 1/10 of a serum-agar slant; the same close was repeated after a "·eek. 'fhe sheep Temained apparently healthy.
Sheep 41034 was injected intratracheally (17.10.34) 'vith one- half of a t'venty-four hours old serum-agar slant, the same close being repeate<l after a week. A certain amount of the first dose was expelled through the nose while the animal was being flrenchecl. 'fhe day follo1Ying the second injection the temperat1.ue rose to 106° F. anrl then gradually subsided for a week until it reached 102° F. After this the temperature rose perimlically to 105° F. but the animal reeovered.
122
M. W. IIEX~I:XG AND M. H. V. BRO,YX.
Sheep 40488 was drenched (15.10.3-!) with 20 c.c. and sheep 40538 ''"ith 450 c.e. of a forty-eight hours old broth culture of
£Hlsteurella 247. Both nnimals remained apparently \Yell.
Sheep -!0414 was <henched (12.10.34) with about 100 c.c. of an emulsion made from the lungs of sheep 40531 (which died from an intravenous injection of pastmu'ella. 247). On the tenth day its temperature 1·ose to 1070 F. and then suddely dropped to 102° li'. on the follmYing day; on the twenty-second day the temperature rose to 105 ·4° F. and again dropped to 102° F. within twenty-four hours;
on the thirty-first day there \\as another rise to 105° F., after which the temperature gradually dr-opped to 101·80 F. and no further renction oeeured.
Sheep 40777 was drenched on (12.10.34) with about 100 c.c.
pleural fiuid from sheep 40531 (which died from an intraYenous injection of pasteurella 2-H). On the thirteenth day the temperature sml<lenly rose to 107° F. and then as sml<lenly dropperl to 103° F. on the f.ollrming rlay. After this the :mimal remained healthy.
Ues·ults.
Paotc?trella 247 was shown to be pathogenic for guinea-pigs and sheep, hoth ra bhi ts aml pigeons being remarkably resistant to infection. In guinea-pigs infection was very readily set up by intraperitoneal inoculations, while in sheep death was produced by the intraYenous route. Although a fatal infection did not result from an intratrache:.l injection of virulent material, sheep so injected shOIYe<l a severe thermal reaetion. \Vhen virulent cultures were given per os no reaction was observed, but when organ material from an experimentally infected sheep was given to t'IYO sheep by the same route a definite thermal reaction was set up in Goth eases.
The org.anism apparently has a preclilection for pulmonary tissue and serous membranes as the most outstanding lesions rleYeloped in in the lungs and pleurae, even when Yirulent material 1nts given
intraY~:;nously.
(4) PASTELTRELLA 247 (a).
In morphology and staining charaeteri sti cs pasteurella 24 7 (a)
\Yas indistinguishable from pasteuTella 24 7 but differed from it culturally, b1ochemieally, and in virulence. In these respects it resembled pasteurella 181. Primary colonies of this organism on serum-agar 11·ere much smaller than those of pasteurella 247. After subculturing single colonies became much larg-er.
Biochemical React·ions.
Pasteurella 247 (a) formed acid hut no gas in glucose, saccharose, mannite, maltose, and inosite. It did not change litmus milk, the methyl-reel and Yoges-Proskauer tests \Yere negative; indol '"as not formed and nitrates \Yere not reduced; ammonia and hydrogen sulpbirle 11·ere formed in small quantities; the methylene bh1e reductase and catalase reactions were positive.
123
Pol.s'fE~llELLOSIS: AX OUTBREAK A:MOJ'\GST SHEEP.
V i1·ulence Tests.
Guinea-pig 1 was inoculated with 0 ·1 c. c. of a twenty-four hours old hroth culture, guinea-pig 2 was iujected with 0·:!5 c.c. anu guinea-pig 0 with 0·5 c.c. Guinea-pig 3 died after f.orty-eight hours, but Nos. 1 and 2 remained healthy. The test "·as repeateil with three more guinea-pigs with similar results. Pastettrellae were recovered from the heart-blood of the dead animals.
These results indicated that paste1t1·ella 24 7 (a) resembled pasteurella 181 not only in morphology, cultural aucl stam mg- characteristics, but aho in biDchemical reactions and Yirulence. It cannot be regard eel .as i<lenhcal "·it h ]Jastem·ellc~ 24 7 anrl 182.
(5) PASTE~RELLA 247 (c).
ln morphology and staining characteristics pasteurella 247 ( c
1
was also very much like pasteurella 247, but differed from it culturally, biochemically and in Yiruience.
Although this organism also grew best on serum-agar it grew only moderately well in broth and poorly on nutrient agar; but the colonies were very much smaller than those of pasteurella 24 7 and a much weaker growth "·as obtained on laboratory media, including· serum-agar.
H iochemical Reactious.
Ko change occured in litmus milk, indol "·as not formed, the Y oges-Proskaues and met h~·l-recl tests \\·ere negahYe, nih a tes '"ere very slightly reduced, ::mel ammonia was formed in Yery small quantities. the methylene blue reductase test '"as weakly positive anrl tlHl catalase test was negative. Sugars were not fermented at all. The gTo\l·th on sugars and on the Yarious media used for bio- chemical tests was very p-am·, a fact "·hich possibly explained the a bsPnce or mildness of reactions.
Virulence Tests.
Guinea-pig 1 was injected intraperitoneally \Y.ith 0·6 c.c. of a hYllnty-four hours old broth culture and guinea-pig 2 with 1·0 e.c. of the same culture. Both animals remained heaHhy.
These results show that a third and entirely difteren t type of pastettrella was obtained fr-om the cultures made from the lungs of ram 3. It differed from strains 247, 247(a), 182, and181, and it was the least pathogenic of all the strains isolated and stucliell.
CuNICAL OBSERVATIONs.
'rhese are confined to the th1·ee natural cases seen in tlw Rvelancl rams and the cases of pasteurellosis produced in sheep by the in.]eetion of strains 182 and 247 of the organism. Of the three natural cases seen one was noticed to be sick for only hYenty-four hotus before death occured. The other t"·o showed symptoms of pneumonia for periods of t\YO to three weeks before succumbing. In eli vidual experimental sheep varied clinically t·o a large extent apparently
124
).l. \\". liE::\ .\TXG ,\::\D ~\1. 11. V. JJ RO\\' .X.
depen1ling upon tlw amount of material and the method used. Fur example, ;-;heep 07744 1·ecei,·ecl 10 e.t·. of a broth culture on the aftenwon of 10.J.J4 and clie1l on lhe next day, the dit>eac;e assuming a septicaemic form. On the other hand, sheep :27240 reeei.-ed 10 c.c.
of a lung emubion frolll one of the e;n-ly r·ases on J4.3.:l4 and died hom pleuro-pneumonia on 9.4.35 nearly a month after ihe injection. In the animals that died from the septicaemic or acute form of the dic;ease t>J"lli]Jtom,; -ohser.-ed consi,;ted of seYere respiralm·y distres:;.
11·ith acceleratetl breathing- and pulse rates ancllll<lrke!l hyperthermi<t.
\\'ithin twentv-four hours the animals ,,-el-e clencl. A n1unber of the experimental "sheep, ho,,-e,·er, a~ 11·ell as t 11·o of the natur;d C:<bes sh·mYcd consi!h•rnble si1nilarit~· in clinic:1l s~·mptoms. The hig·b ft>hrilt• rea!'!ion ouuring- a fe11· da_,·:; after the inje!'!iou was !'ilal'il!'- t<'ristif· Ul}(l it 11·as ahYays assof'interl ''"ith <l!'!'elernted respiration <1!1!1 pulse mte; the brenthing- in <Hldit·ion beconti11g· !]i,;h·e,;,;ed. After JPaching- its hig-hest point u;,unlly h!•t1n•en 107° an1l 101)0 JC. the tPmpPrahll·r-• al11·ays rlropperl to alHnd I 04° F. shortl,,- h~:·fore de;d h.
Sheep 32()0(; ""hid1 1·ece i ,-ed 2 e.('. of a ln·ot h C1ilt urP i u t ra- Yenousl_,-on V).T:l-t ma:.- he Liken <1s wPll rPJHPsPnt.d iYe -of th1· usu<ll f'ase seeu. 'l'hP tPmperahue renwinrd lJ('l\,·een 102° nn1l 104° F.
up till thE' morning of :n_:l_:J-t, ll"ill'll it !'OJnmell!·ed to rise and by the a Hen10ou of the :2:lnl it ha1l reach ell 101° F. Then it tlrop1wrl almost as suddenly as it had risen <111!1 "·a~ 104° F. on the :1ftPrnoon of 2-±.:L:)4 odtorih· hPfOl"E' the animal's death on the 2:}th. ( Jn the 2:lnl ihe animal l'elll;1i.ne1l tl(JI\"11 11·ith a greatly acf'elerated pulsP <11111 respiration rntes, and had n slight bilateral muco-purulent nnsal rliseharg-e. In the ani lll als 11· hidt reeoYerecl an i.n term i tten t £eyer was mainL1ined for periods of up to three -or fmu· iYPeks. The temperature fluduatecl hetlnen 103° F. <lllcl 108° F. eYentuall_,.
reaching- a normal leYel. During the perio!l of feYel· the pulse nnrl J·espir<dion rate,; \H'l'e increased but tlr·oppecl ;~gnin :1s the £eyer
cleL:re;~sed.
\YhiLl it would he mtwise to make a rliagno,;i.s of pasteurellosis upon clinical gTounds alone the symptonJ::; seE'n cnn he usefull~
conelaterl 11·ith post-morltm appearanf'e:· ;mrl laboratory ex<lmina.- tions.
PATHOLOGICAL ANATOMICAL CHANGES.
'l'h('se <Jn• !'Cmfine1l t·o t lte Jlost-llun·felll appear<mCE's seen in sheep;
both in tit!• n;~tural !'ases fnnn thP "C"niYersity farm <illcl in the cases artifi1:i:lll_v produce1l "·ith sh;~ins .2-n and 1S2 of the organii'JU an!l en1ling L!Ldl\. 'fhe:-· ma~- he roughl.)· cla.-sed 11·ith those f'ren in sheep that rlie1l from the septicaemic or anite form i.e. 11·ithin one or hYo rlnYs <lll!l those seen in nnimab "·hich ''"ere sick for a week or more.
Changes in the animals 11·hich died from the acute or scptic- aPmif' fonll 11·ere li mi. ted to se1·ere hy perapm i.a and often oe<h•ma of the lungs with enlnrg-ement of the spleen. Tl1e latter change wns J•Pver Yery seYerf', tlw larg-est spleen sePn mrnsuring l0x7·5x2 ems. J-1_,-drothorax anrl h~·dropericard \l·erf' usually seen, and in some instan1·e:-; there was a h~·pernemi.a of the mucou::; membrane of the
125
l'ASTE"CHELLOSIS : AK O"LTDREAI~ AMONGST SHEEP.
ga1>tro-intestinal tract. Slight fatty degeneration of the liver and kidneys was noticed in these cases. K o emaciation was seen except m the case of the third Ryeland ram "·hich was losing oondit~on
for some months befme it developed acute pneumonia and d1ed
~:~ucldenl:y.
In the animals which died after a more lengthy course of the
!hsease, much more significant and severe lesions were noted at autopsy. 'l'hey usually showed some degree of emaciation; those that had been ill f.or some time were often very poor in condition. ln one sheep (27240) two ribs on the right side were fractured but this ''"as apparently the result of a traumatic injury. 'l'he changes which may be classed as ·occurring almost invariably were those in the respiratory tract. '!'here \vas frequently a muco-puruleJ?-t discharge from the nostrils and in many cases a severe hyperaenna of the pharynx, larynx and trachea. The lungs and pleural mem-
brane~ were the seat of severe damage in .almost every animal, either fihriuous pleuro-pneumonia or purulent broncho-pneumonia l)eing present. usually the lung \Yas found partially deflated and heavier than normal. It was firm to the touch and of a mottled bluish-re(l or light reel colour. The pleural membranes, both costal and pulmonary were frequently covered with a fibrinous yello''"
deposit up to 2 ems. in thickness and the lung was often attached by fibrinous material to the c-ostal wall over Yarying areas. A large amount of turbid yellow or red :fluid was usually present in the
thoracic and, to a lesser extent, the pericardial cavity.
On sectioning the lung, which cut like a solid organ such as liver, the cut surface varied in colour from a light pink to a clark reel, often with lighter coloured yellow areas. A reddish turbid fluid could he expressed and was present in the bronchioli. ]<'requently light c·olourecl areas of soft necrotic tissue were seen. The whole of the lung tissue was never hepatised- the parts usually affected being the anterior ancl inferior parts of hoth lungs- and the junction behYeen the normal and healthy tissue was ahYays clearly demar- cated. In some of the eases parts of the lungs were atelectatic, being completely collapsed an(l clark red in colour.
Changes in the other organs \Yere limited to fatt~· (~egenerahon
of Yar~·ing severity in the kidneys and IiYer and occasionally a slight enlargement of the spleen. The gastr.o-intestinal tract sometimes showed hyperaemia in the mucosa.
HISTO-PATHOLOCICAL CHANCES.
In eYery case that terminated fatally specimens of internal organs \Yere collected and fixed in forma lin; sechons were cut by either the freezing or the paraffin embedding method. Organ smears were also made and stained with Giesma. The stains used in the section,; were Haematoxylineosin, van Giesen, Sudan III, and Giemsa.
'J'he sheep that died fr.om the acute form of the disease showed very fe11· histological changes: the blood content of the spleen was increasecl ancl the lymphoicl follicles enlarged. BirJOlar organism were demonstrated without cliffieulty in spleen smears from these cases.
126
M. W. I-IEKNJ~G AND M. H. V. mw"·N.
In the lungs hyperaemia and oedema "·as observed. 'rhe aheoli were :fillerl with clear serous material and the capillaries were distended with blood.
In animals that lived a week or more after the infection characteristic changes developed in the respiratory organs. In the lungs a constant feature was the severe hyperaemia present. Every capillary blood vessel "·as packed with blood cells. In almost every case areas of c-onsoli<lation were present. 'l'he alveoli in these areas were :filled with a mass ·of fibrinous material, neutrophiles and red blood corpuscles. The fibrin "·as in the form of a network of Yery fine strands in which the infiltrating cells and red corpuscles were enmeshed. Large phagorytic cells (macrophages) \Yere also present.
'l'he :tlbrin, rather difficult to identify with the Haewatcxylii1-eosin stain ,,·as very dearly shown up by vViegert's methotl (Fuchsin, M:ethylviolet and Lugol's iodine). In these cases of fibrinous pneumonia in sections cut by the paraffin method and staine<l ''"ith Giemsa numerous bacteria were present amongst which bipolar stain- ing organisms could be dearly identified. Necrotic areas were seen in which all cell structure had <lisappeared and which sho1Yed up as light pink staining (Haematoxylin-eosin) homogeneous structure- less foci surrounrled by a zoue of neutrophiles.
Cases were seen in 11·hich as \Yell as a fibrinous pneumonia, a rem:
of purulent brondw-pueumonia \Yere also present. Here thP alveoli contained no fibrin but were blocked "·ith a. mass of neutrophiles and large phagocytic cells. In some of the sheep this type of pneumonia only was present, and no bipolar mganisms were seen, although numer<JUS cocr,i and short bacillary organisms could be demoiiStrated.
In some sheep the pneunwn ic changes \Yere aecompanierl by a fibrinous pleuritis. In sedions the pleural membrane of both the lnng and the cor;tal "·all "·as roughened and thickened anrl c-o\·ererl with a dense la)·er of fibrin which again appeared on a network of fibrils enclosing here and there rerl blood corpuseles and a fe"· neutro- philes. In the Rections stain eel w·i t h Giemsa hi polar organisms, as well as other bactera, coul(l he rlemoJistratecl fairly easily. In smem·s made from the consolidate<! parts of the lungs numerou,.; bacteria were present including frequent bipolan;.
'rhe histological changes in the lung can then be summarised as including acute and !';uh-ac11te fibrin·ous anrl purulent hroncho- -pneurnonia, frequently with areas of 11ecrosis and fibrinous pletn·itis.
In other org-ans such as liver and spleen a hypentemia w·as oecasionall_v noted, whilst in the liver and kidneys fatty (lPg-enera- tion to a greate1· or lesser extent was usually present.
DISCUSSION.
A ,·indent form Qf pasteurellosis iu Ryeland sheep at the experimental farm of the University of P1·etoria was inwstig-atecl.
Of the three natural cases stuclie<l, hYo harl been suffering- fr.om a chronic pulmonar_y infection for several weeh before the~· died while the thirrl (ram 3) was not noticed sick for more than twent~·-four
hours before death. Ryeland ewes kept under identical con<lihons
127
l'ASTE"CRELLOSIS: AX O"C"Tl:lHEAI~ AMOK<.7ST SHEEP.
and in dose contac:t with the rams did not suffer in the same waY. lt is true that at one time these eiYes were unthrifty and that th~y impro1·ed in condition after they had been inoculated with a forma- lised emulsion of pasteurella 182, but there is no proof that they suffered from pasteurellosis or that the imp1·ovement could be attributed to the inoculation; ram 3 whieh received the same treat- ment finally succumbe<l to a pulmonary disease from which an organism (zJasteurella 24 7) identical with pasteurella 182 was obtained.
:Primary cultures made fr.om the lungs of ram 1 procluce<l a gr·owth containing se1·eral colonies of rlifferent sizes; ·one of the largest of these "·as picked anrl subculture<l for a detailed study. It yielcled
~ulture 182 "·hich was composed o£ small 0\·oicl (short bacillary), bipolar-staining, Gram-negative mganisms correspom1ing to the descripti-on of paste1uellae giYen by Topley and ·wilson (1929). This
·Organism proved to be highly pathogenic for guinea-pigs and sheep, but appnently non-pathogenic for rabbits and pigeons; it 11·aB found to have a prerlileetion for pulmonary tissue and serous membranes;
when a liYe culture was injec:ted inhaYenously into sheep patho- logical changes developed in the lungs, pleurae and pericardium in vreferenee to other parts of the 11ody. In the tluee natural cases stn<1ied the lungs, pleurae an<l pericardium we1·e afff'r-ted most extensiYely. ~fmeover, the lesions presented by the experimentally I}l'·oduced cases closely resembled those observed in the natural cases.
He pea te<l inoculations of formalised cultures of ]Jastew·ella 182 did not appreciably increase the resistance of guinea-pigs to infection
>Yith Yirnlent live cultures and the serum of the " immunised "
guinea-pigs was devoid of any agglutinin content. Like"·ise no agglutinins could be demonstrated in the serum of rabbits in·oculate<l with liYe cultures.
On serum-agar media see<lerl 11·ith material from the diseased lungs of the Reconcl ram several colonies of different. sizes tl eYelopecl ; one of the smaller colonies was picked, subculturecl anrl stmlie(l.
The organisms of this culture (pasteurella 181) were also oYoi<l, bipolar-staining and Gram-negative; but although morphologically indistinguishable from pa:;teutella 182, it differed fr·om it culturally, in Yirulence and in biochemical reactions, the most shiking of which was its inability to form ind-ol ano to reduce nitrates. For guinea- pigs it IYas barely pathogenic, Yery larg·e doses being necessar.Y for the procluchon disease.
On account of the similarity of the pathologi!'al ehanges vresentecl by these two rams and the apparent similarity ·of the primary gro"·ths ·On serum-agar seeded with pulmonary material from ram::; 1 and 2 it is suggeste<l that each of the primary e 1ltures con- tained at least bY·O d i:fferen t types of colonies, one of them yiel<ling virulent organisms corresponding to ]Jasteurella 182 IYhile the other g'ave rise to n-on-virulent bacteria like pasteurella 181. It is further sugge,;tecl that the small colonies of the p1·imarr growths contained the non-Yirulent organisms while the large ones yielded Yirulent cultmes. 'rhe bacteriology of the cultures -obtainecl by seeding
}J~llmonary material from ram 3 on serum-agar slants r:;upports this VIe\\·.
128
