User manual | Heartwater in Sheep.-The W eil-F elix reaction

Ondetstepoo-tt J ou1·nal of V etehnary Sci'ence and Animal
lndust·r y, Vol1tme 3, Number 1, July , 1934.
Heartwater in Sheep.-The W eil-Felix reaction
and an investigation into the bacterial content of the blood with particular reference
to the use of " K " medium.
By J. H. M:ASOK, F.R.C.Y.S., F.RS.E., and R A. ALEXANDER, B.Sc.Agric., B.V.Sc., Empire Marketing Board
Research :Fellows, Onderstepoort.
THE W E ll-FELIX REACTION.
THE existence ·of a common antigenic factor [ Castanida and Zia
(1933), -White (1933) J in R ickettsia. and Bacillus proteus X is known
to be responsible for the specific 0 agglutination of B. proteus· X
by typhus serum, originally described by vVeil and Felix and now
known universally as the W ei l-Felix reaction.
Attention was again focu ssed on this reaction when Felix and
Rhodes (1931) confirmed the observation of Fletcher and Lesslar
( 1925) that there ex isted two serologically distinct types of tropical
typhus which reacted specifically with B. p1·oteus X19 on the one
hand and B. p1·ote:ns XK on t he other. An additional interesting
feature of t his finding was that JJ. p1·oteus XK was merely a nonindologenic variant of X19, :mc1 h a d been obtain ed inadvertently
during t h e course of 1·outine subf'u ltivation.
l"i'urther investigati-on into the serological types of typhus virus
h as l ed Felix (1933) to correlate " the agglutinogenic and immunogenic properties of differ ent types of virus '' . This has led to t he
su ggestion that t here is a variant of B. p1·oteus X that corresponds
serologically with each of t h e Riclcettsiae cau sing the many different
diseases of t h e typhus group.
Cowdry (1926) sh owed that a Rickettsia, R. 1·uminantium, is
the causative agent of hearbYater, and consequ ently the possibility
of demonstr ating a positive \'\Teil-Felix rea ction with one or other
of the p1·ote1ts OX strains was con sidered, sinee the est ablishment
of su ch a r eaction would be -o f t h e greatest value in the further
study ·of t h e disease.
7
HEART"'ATK11 IN SHEEP.
Our colleague, Dr. E . .:\1:. Robinson, infonned us (personal
communication) that previously he had inYestigated this reaction
in heartwater-affected and -recover ed sheep but " ·ith negative results.
N eYertheless we decidecl to take up the problem ane\\· using( a) A larger number .of sera hom sheep in Yarious stageR of
the disease, that is during the reaction, at Yarious periods
after 1·ecovery and after " hyperimmunization "
by
several injections of virulent blood.
(b) Antigens consisting of single cell cultures of OX2, OX19,
and OXK in the hope of the chance isolation -of a specific
variant as was the ease " ·ith OXK.
·
(c) Antigens consist~ng of cultures of organisms isolated from
sheep infected "·i th heart\\·a ter .
(c~) The initial p1'oteus strains. u sed were cultures of OX2,
OX19, and OXK, received through the courtesy of Dr. A. Felix, to
whom "·e IYish to express ·OUr thanks. To serve as c~mtrols to these
cultures we prepared agglutinating sera in ·rabbits by repeated
intravenom; injections of saline suspensions killecl by heating at
60° C. for half an hour, and in goats by inhavenous injections of
saline suspensions of single cell cultures killed " ·ith 0.1 per cent.
formalin. Cross agglutination tests were carried out with liv ing
suspensions in saline of approximately the same density; the V·olume
of fluid in each tube was made up to 2.0 c.c. with saline and readings
were taken after two hours at 45° C. and overnight storage at room
iemperature (22°-26° C. ). The results are recorded in Table I.
I.
TABLE
Agglutinating Serum.
Antigens.
OX2
(s.c.)
I
~
OX2 ·(s.c.) ...
OX19 (s.c.)
OXK (s.c.) ..
OX2 (orig.).
OX19 (orig.)
OXK (orig.)
~ .c.
I
I
I
1,280
20
40
2,560
40
40
(3)
(1)
(1)
(1)
(0)
(1)
OX19
(s.c.)
40
10,240
40
40
10,240
40
I
(2)
(3)
(1)
(2)
(1)
(2)
OXK
(s.c.)
80
40
10,240
80
80
10,240
(3)
(2)
(2)
(1)
(1)
(2)
I
OX2
(orig.)
1,280
20
20
1,280
20
20
(3)
(0)
(0)
(3)
(1)
(0)
I
OX19
(orig.)
80
2,560
20
20
5,120
20
(1)
(3)
(0)
(1)
(2)
(0)
I
I
--OXK
(orig.)
20
20
5,120
20
20
10,240
(1)
(0)
(2)
(1)
(0)
(2)
I
=s ingle cell culture.
orig. = original culture.
(3), (2), etc.= degrees of agglutination, (3) being complete. Double t he
dilution poted was negative or trace, and half was complete (3).
The lowest dilution u sed was l / 20.
(b) Agglutination of Single Cell st·rains of Proteus OX.Mention has been made of the chance isolation of a serologically
distinct mutant of OX19, which reacts specifically " ·ith the sera ·of
scrub typhus patients. By the u se of this Yariant OXK and OX 19
scrub typhus may be differentiated from shop typhu8, two types -of
the tropical disease encountered in Malaya and the Dutch
East Indies. Therefore it was considered possible that by isolating
8
.J. II. MASOX .\KIJ lL A. ALEXAXDElL
from the origin~d prote11s culture~:; a large number of single bacilli.
a variant might be obtained which would be agglutinated by heart" ·ater sera, the assumption being made that there '"as present an
ao·glutinable .-ariant , hut in su ch small numbers t hat itt; agglutinati~n '"as marked by the much gTeater number of non-ugglutinable
organisms. It " ·as r ealize<l that if su ch a v~uiant di<l exist its
isolation woulcl be a matter of pure chanGe unless many thousands of
single-cell cultures '"ere examined . A oompr·omise "·~u; made by
obtaining 50 single cell cultmes from OX2, OX19, ancl OXK. The
resulting 150 antigens ,,.e1·e tested against normal sheep sera all<l
heartwater immune sera.
None was agglutinated by any serum at a dilution of 1-40.
(c) Attempt to Isolate a Prot eus X or other Serolo,qi!'ally Similar
Or.r;a11isms from fl earhcater Shnp.-It i:> the routill e practice at
these laboratm·ier:; to maintain the virus of heartwater by passage
through sh eep .
Ample material " ·as a.-ailable therefore f.or all
attempt to isolate n specifit~ proteus or other orga nism '"hich might
have some relation t-o the h earhn1ter .-ini:->. Other nuthorr:; [Anigstein
(1933), Kuczynski (1927), and Martin (1931)], working with diseases
of the typhus gr-oup, have been able to isolate from vari om; patient~>
str ains of p1·ote11s X " ·hich have been shO\Yn to have a clefiuite relation
with the disease in question.
Ken<bll (1931) repmte<l that, hy the use of his medium, nonfilterable bacteria had been rendered filterable, and, further, the
inoculation of this medium with apparently harteriol-ogi t·ally r:;terile
blood from influenza patients had resultecl in the i~-olation of a
coccu s '"hich possessed a definite relation to the virus. We 'vere
further stimulated in our im·estigation by the report of Hadley,
et alia (1931), in which Shiga's clysenter,\- bacillus was sho11·n to
have a filterable stage.
Our main object in undertaking the \Yor.k \Yas in the hope of
isolating a germ which would be agglutinable by heartwater serum,
or which could be u secl as an antigen in the prevention of the disease.
As our result,; " ·ere entirely negatiYe, "·e consi(le1· it nece:;sar:v to
record -onl:v the main points, omitting- details.
METITODS.
In all, material fr-om 51 sheep was investigated-of these 25
\Yere infected 11·ith h eart,Yater, 13 ,,·ith blue tongue, 5 wi th enteritis
[cause unknown ( ?) dietetic J, and 8 were normal animals. All
were M:e1·inos, the majority castr:J tetl males and the ages varied
from 1 to 5 years. The blood of 20 hearbnte1 sheep (87 bleedings),
8 normal sheep (17 bleedings), 13 blue tongue sheep (25 bleedings),
and of 5 " enteritis " sh eep (5 bleedings) was cultured. In 11
heartwater sheep, scrapings from the jugular vein were cultu red,
and in four instances the urine. In some cases the blood, jugular
scrapings, and the urine from the same sh eep were in.-estig-ated.
Collection of Blood.
Prior to commencing the investigation, an attempt was made to
work out a method -of obtaining blood in a sterile manner. The
method u sed is given by Mason (1934), and, in brief, consisted in
9
HEAR'l'\\'A'l'Elt IN SHEEP.
closely clipping the v;·ool from the jugular region, the application
of absolute alcohol and then ether to this area, the insertion, with
one thrust, of the needle into the vein, the allowing of 30.0 c.c . of
blood to escape, and finally the collection of the sample in a sterile
tube.
Jugulm' Sc1'apinys and Urine.
The sheep was pithed and with steril e precautions, the jugular
vein (cervical portion) exposed, and about 20.0 em. dissected out.
The intima was exposed and thoroughly scraped with a knife, the
scrapings being transferred to one or other medium. Urine was
obtained direct from the bladder bv means of a bulb pipette, aseptic
precautions being adopted.
Jll edia.
1. Ordinary beef infusion peptone broth.
2. Clot broth. About 10.0-12.0 c.c. of blood was allowed to clot
in a test tube and the serum discanled. Broth was t hen added to
replace this serum.
3. Horse flesh infusion peptone agar, plus 5 per cent. of a mixture
-of equal parts of sheep serum and heep haemolysed red cells [ Mason
(13) J.
1'his medium was used in the large flat-bottomed tubes
described by Mason (14), and "·as ab·ays incubated for three days
at 37° C. prior to use.
4. Litmus lactose agar in the tubes noted under (3).
5. Kendall (K) medium. Three separat e lots prepared fr-o m
sheep gut according to Kendall, and one sample obtained from
Difco were used.
6. " E.B." medium prepared from sheep brain according- to
Eberson and Mossman (15).
7. Robertson's meat broth (horse flesh) medium.
All media were autoclavecl for half-an-hour at 120° C. prior
to use. [In the case of medium (3) the agar was autoclavecl and
having cooled to 45°-50° C. the serum-haemoly:>ed-cells mixture was
added.]
Gene?'al Scheme .
When blood was investigated, bleedings 'Yere usually made at
the first rise of temperature and then at 1 or 2 days intervals until
t h e death {)f the animal. When broth was used from 0.5 c.c. to
1.0 c.c . of blood was pipetted into 10.0 c.c. -15.0 c.c . of medium, and
this incubated at 37° C. for 14 cla:p>; a sub-culture 'ns then made
into fresh broth, this incubateu for a further 7-14 days, and finally
a further sub-culture made on serum-haemol:vsed-cells-agar or
Haucluroy's (19) serial plating method on either serum-haemolysedcells-ag:u -o1· litmus l actose agar waR carried out.
Bloocl clots were incubated at 37° C. for one month (reduction
in volum e due to evaporation being made up with sterile distilled
water), " ·hen a smear was made on serum-haemolysed-cells-agar, and
this incubaied for 48 hours.
10
,J. H. MASON AND lL A. ALEXANDER.
K medium (30.0 c.c.-50.0 c.c. with 0.5 c.c.-1.0 c.c. of blood)
was incubated at 30° C. or 37° C. for 14-28 days. Two further subcultures (0 .5 c.c. -1.0 c.c. into 10.0 c.c.-15.0 c.c. medium) were made
at 10-14 days interval. If no growth was· demonstrable (either by
naked eye ·o r by stained smear) Hauduroy's serial plate procedure
on serum-haemolysed-cells medium or litmus lactose agar was
adopted, fr-om 3-8 su ch platings being conducted.
Throughout, smears stained by Giemsa's and Gram' s methods
were examined even in apparently negative tubes.
Special attention was given to any tube, which, by stained
smear examination, showed what could be developmental forms of
bacteria. Serial platings of such material were carried out as many
as 25 times before a negative result was accepted.
Every micro-organism which grew, with the exception of
obvious contaminants from the sheep's skin -or fr-om the air was
investigated. This consisted in (1) carrymg out with every germ
agglutination tests with the heartwater sera previously mentioned,
(2) in some cases injecting a K culture or a saline suspension of the
germ intracerebrally and intraperitoneally and sometimes intratesticularly into guinea pigs and mice and subinoculating the brains
of such animals intracerebrally into fresh guinea pigs and mice,
and (3) injecting living K culture and saline suspensions of the
microbes subcutaneously and intravenously into sheep and later
testing with virulent blood for immunity against heartwater.
Organisms which developed quickly, within 1-3 days, in the
original culture tube, and which on further examination proved to
be aer·obic Gram positive spore-forming bacilli, staphylococci, large
copiously growing aerobic Gram positive bacilli (usually pigment
producers) were c-onsidered to be c-ontaminants and were discarded.
All diphtheroids (·of which a considerable number was isolated) and
Gram negative bacilli were retained and examined in some detail;
in addition to the tests already mentioned, the Gram negative bacilli
were investigated biochemically.
Results.-These may be briefly summarised
headings:(1) No ]J1'oteus X-like bacillus was isolated.
under
fiye
(2) No germ, which was isolated, was agglutinated by heartwater or proteus OX sera at a titre significantly higher than that
obtained with normal serum.
(3) It was not p-ossible to produce a transmissable disease,
symptom or reaction in guinea pigs or mice by the intracerebral.
intraperitoneal or intratesticular inj ection of living K culture o1·
saline suspension of 8 organisms obtained from 6 sheep.
(4) K cultures and saline suspensions of 14 germs from 12 sheep
injected intravenously and subcutaneously (simultaneously) into
28 sheep, apart from a transitory rise in temperature, pr-oduced no
reaction. Sheep, treated with two such inoculations at from 2-3
weeks interval, were not immune to heartwater, when tested 10-20
days later with intravenous injections of virulent blood.
11
HEARnVATEH. IN SHEEP.
(5) Formol-killed saline suspeusious of p1·oteus OX2, OX19 anrl
OX2 (two subcutaneous injections at 14 days interval) dirl 11ot
immunise sheep against heartiYater (as tested by the intravenou s
injection of virulent blood).
DISCUSSION.
In Appendix 1 a n~sume is given of the t reatmeut a dopted >Yith
each sh eep and of the germs isolated therefrom. It will be noted
that from t he heartwater sheep the following germs were isolated :
22 diphtheroids (small Gram positiYe rather sparsely growing
bacilli), 11 staphylococci (1Thite or yello"·), 7 Gram negative bacilli
(B . cloa.crn, B. JYI.JOcycLneus, a member of the a lkaligenes group, and
three unidentified bacilli, not members -o f the colontyphoicl or
proteus groups), b1·o tetrads, bvo streptococci, ancl 10 Gram positi.-e
copiou sly g-ro" ·ing aerobic bacilli (8 sporers), and one Gram negati..-e
coccus. From the 26 r emaining :sh eep (normal , blue tongue aJH1
" enteritis ") four dipht h e1·oids, one staphylococcu s and one G-nnn
positiYe aerobic sporer \Yere ohta iuec1 .
Although t h e number of
bleedings ic; not large en oug-h to l end itself to accurate statistic<ll
analysis, it is inte1·esting to noie that basing 1·esuit:; on the munbt-r
of bleec1ingc;, G of 37 (16.2 per ct-nt.) of the non-h eartwaie1· bleedings
gave positive growth results, and 55 ·of 87 (63 .2 per cent.) of the
h eart\Ya ter bleedings and jugular scrapings \Tere positiYe. Reckoning only diphtheroids, the figmes become 4 of 37 (ll per cent.) and
22 of 87 (25 per cent.).
\V e a1·e of th e opinion that po~sibly in non-heart\Yater sheep the
figure 11 per cent. for diphtheroids is actually too h igh. This is
hased on results obtained in similar but not identical investigations
(unpubli sh ed) on t h e sterility -of the blood of normal sheep. 'l'h ese
r esults showed that blood taken c1 irect from the heart of a killed
lamb or sheep \vas sterile; the occasional gr-owth obtained was
obviously of aerobic origin.
\Ve are 1mable to state if the c1iphthe1·oid s (·or other germs)
obtainerl hom the blood -of h eal-t\TH ter sheep ha.-e actually any
c-onnection wit h the virus of the c1i sea. e. There is no result \\-hich
would indicate any relation, bu t the pos ibility does exist that one or
more might h ave been a developmental stage of the virus, but not at
any time obtained by us in a phase capable of being specifically
agglutinated or of setting up disease symptoms in laboratory animals
-o r sheep. Ho"·ever, \Ye consider it more like]~, t h at they had th eir
origin in the intestin e a nd \Ye1·e able to escape easily into the blood
stream t hrough the inflamed gut. 'l'his opinion is .strength ened by
t h e faci. that the hig h est percentage of positive blood cult ures was
obtained when the disease was well advanced, bleedings taken prior
t-o t h e rise in temperature usually being sterile.
One g-erm only desen-es a somewhat detailed description; its
g-rowth ch:uacteristics, the apparen t developmental phase throu gh
which it went, and its pleomorphism gave us cau se for hope t hat
we h ad obtained a bacillary stage of the causative rickettsia.
Sheep 34443 .- Inj ected l7 / 10 / 32 with 10.0 c.c. of virul ent
blood from sh eep 34910. This latter animal died fr-om and showed
t h e typical lesions of h ear twater on 28 / 10 / 32. On 24th October,
12
J. H. MASON AND R. A . ALEXANDER.
1932, 1.0 c.c. ·of blood \Yas so1Yn in 30.0 c.c. of K medium, and
incubated at 37° C.
After four days the medium was slightly
cloudy, but a Giemsa smear showed nothing to arouse suspicion.
Sub-cultures on serum-haemolysed-cells-agar, incubated aerobically
and anaerobically remained apparently sterile.
A Giemsa smear
fr·o m the original K tube made on 3rd November, 1933, revealed a
large number ·of tiny reel-blue bacilli, which when t h ey occurred
in clumps, closely resembled rickettsiae .
Aerobic and anaerobic
sub-cultures on agar remained without visible growth. Hauduroy's
technique on litmus lactose agar was now adopted and on the t hird
passage (24 hours incubation between each) what appeared to be
Hadley's G colonies \Yere obtained. These \Yere so tiny as to be
visible only by the aid of a lens when the t ube was held in a certain
position in relation to the source of light. Giemsa smears showed
a very pleomorphic organism; every gradation between a r ickettsialike microbe and a medium-sized bacillus was seen , bu t the predominant form was what might b e termed " knobbly ", i.e. cocci
showing protuberances, t apering bacilli ending in swellings <Or
nodular-curved rods.
.
At this stage t h e germ would not grow on " dry " serum agar or
m ordinary brot h, hut did so on t h e surface of " flooded " agar.
The serial passages were c·ontinued 13 times, at whi ch stage the
germ gre\Y reasonably well-sub-cultures on " dry " agar or in broth
gave, after 2-4 days' incubation at 37° C., a scanty, rath er dry
grey growth or a faint turbidity. Smears made at this time revealed
a bent <Or curly Gram positive bacillus, still " knobbly ", and only
a very occasional coccus, or rickettsia-like organism. Cultures in K
medium, broth, serum-haemolysecl-cells-agar and litmu s l actose agar
m a de during a periorl of six months did not r esult in any furth er
ch ange in morphology or type of growth. The bacillus was not
agg lutinated by h eart,mter or p1'oteus OX ser a at a dilution of 1 I 40.
K cultures (2 and 10 days at 30° C. and 37° C.) and saline suspensions hom " :flooded " agar injected intraperitoneally, intracranially
and intesticularly into g uinea pigs cau sed no disease; th e brains
·of the intracranially injected animals passaged into fresh guineapigs (and t hese later again passaged) gave n egative results . The
same technique in mice (omitting the int r atesticular route) gave the
sam e n egative results. As appendix shows, sh eep were not demonstrably affected by the intravenous and I or subcutan eous injection
of K culture or saline suspensions and were not later shown to he
immune to heartwater.
CoNCLUSIONs.
(1) The H and 0 Yariants of p1'oteus X2, X 19 and XK are not
a gglutinated by serum from sheep , affected ''"ith, recovered from or
" hyperimmune " to h eartwater .
(2) None of 50 single-cell cultures (150 in all) from the 0
variants was agglutinated by these sera.
(3) Blood cultures from sheep infected with h eart"·ater gave
more positive g'rowth results than did blood from normal sh eep ·Or
from sheep affected with blue tongue or an enteritis of unknown
13
HBAKl'"'ATELt IN SHEEP.
ongm. A considerable percentage of the organisms isolated pmbably had their origin in the intestine an d escaped via the inflamed
gut into the blood stream.
(4) 0£ the microbes investigated, none wa s specifically agglutinated by heartwater or p1'oteus OX sera. In addition none set up
disease symptoms in guinea pigs, mice or sheep, and sheep injected
twice with living cultures 'vere not protected against heartwater.
REFERENCES.
ANIGSTEIN, L., 1933. Observations concernant la variabilite des souches
cultives du typhus tropical. O.R . Soc. Biol., Vol. 112, p. 1449.
CASTANIDA, M . R. , AND ZIA, S . (1933). The Antigenic Relationship between
P1·oteu X19 and Typhus R-icke:ttsiae. A study of the vVeil-Felix
Reaction. J. of E:cp. Jl ed., Vol. 158, No. 1, pp. 55-62.
COWDRY, E . V . (1926) . Cytological Stadies on Heartwater, 1. The Observation of R. ntminantit~7n in t he Tissues of Affected Animals. 11th and
12th R epoTt s of the Director of Vet. Ed. and B es., Union Dept. of
AgTic., pp . 161-180.
COWDRY, E. V. (1926). Cytological Studies on H.W. 2. R . ntnvinatiwm
in t he Tissues of Affected Anima ls. 11th and 12th R eports of lhT. of
Vet. Rd. and Res. , Union Dept. of Ag1ic., pp. 181-198.
EBERSON, F ., AND MOSSMAN, W . G . (1932). Proeedures in the I solation
and Cultivation of an Organism from Berkefeld N . Filtrates of Poliomyelitis virus. PTocs . Soc. Exper. Biol. and Med., Vol. 30, No. 1,
pp. 92-97.
FELIX...' A., AND RHODES , M. (1931). Serological Varieties of Typhus Fever.
J . of Hyg'., Vol. 31, No. 2, pp. 225-246.
J!'ELIX, A. (1933). Serological Types of Typ hus Virus and Corresponding
Types of P roteus. 'l'Tans. of Boycd Soc. of TTop. jJ[ed. and Hyg. ,
Vol. 27, No. 2, pp. 147-172.
FLETCHER , W. , AND LESSLAR, J. E. (1925).
Malay States, No. 2.
Bttll. lnst. Med. Res. Fed.
HADLEY, P. ; DELVES, E.; A~D KL1~NEK, J. (1931). The Filterable
Forms of Bacteria : A Filter able Stage in the Life Histor y of the
Shiga Dysentery Bacillus. J . lnf. Dis., Vol. 48, pp. 1-159.
HA UDUROY, P. (1929). Les nltravirus et les formes filtrantes des
Masson et Cie, Paris.
microbe~.
KENDALL, A. I. (1931). ObserYations upon the Filterability of Bacteria,
including a Filterable Organism obtained from Cases of Influenza.
Science N.S., Vol. 74, No. 1910, pp . 129-139.
KUCZYNSKI. M. H . (1927) .
Springer, Berlin .
MARTIN, P. H. (1931).
S t ates, p. 48.
Die Erreger des Fleck und Felsenfiebers.
J.
Annual Repo1·f , lnst. Ji ed. Res. Fedemted Malay
MASON, J. H. (1934). A Note on the Cultin1tion of Anaerobes. This J ouTnal.
MASON, J. H . (1933). A New Culture Tu be.
Vol. 4, No. 2, pp. 89-90.
.T. S.A. Vet . Jied. Assoc.,
WHITE, P. BRUCE (1933). The 0-Receptor Complex of .B. pTote1ts
BTit . .J. of E xp. Path ., Vol. 14, No. 3, pp. 145-148.
14
X
19.
.T. H. MASON AND lL A. ALEXANDER.
APPENDIX 1.
(" Not agglutinated" means that the organisms were not agglutinated
by heartwater o·r JYrotetLs OX serum at a d ilution of ·1 -50.)
Sheep 31281.-Bled fi,·e times, at daily intervals, during fever. B lood
allowed to clot, serum decanted, and clots incubated aerobically and anaerobically. Sub-cultures on serum ag.ar and serum broth put up daily from depth
of clot and these incubated aerobically and anaerobically. The first two bleedings sterile; from remainder a streptococcus, large Gram negative bacillus,
small Gram negati,·e bacillus, and a diphtheroid isolated. The bacilli not
agglutinated.
Sheep 31763.-Bled t hree times during fever, as clot broths. A staphylococcus, a Gram positive aerobic sparer and a pleomorphic diphtheroid isolated.
Last germ not agglutinated .
She ep 31098.-Bled six times during fever, as clot broths and into meat
broth . First two and fifth bleedings sterile. The third, fourth, and sixth
bleedings positive in 1-3 days.
B . pyacyanetLS B. cloacae, member of alkal igenes group, Gram negative bacillus (not colon-typhoid or protetis group)
staphylococcus and a dipththeroicl. None agglutinated.
Sheep 32120.-Bled seven times during fever as clot broths. The third,
fifth , and seventh bleedings sterile. Bleeding 1 positive 9 clays Gram positive
sparer; bleeding 4 stap hylococcus 14 days; bleeding 6 Gram positive sparer
l'i' days; bleeding 2 by Hauduro~·'s technique diphtheroid-not agglut inated
and non-pathogenic.
Sheez1 32118.-Bled nine times prior to and during fever as clot broths.
First, second, fourth, and n inth bleedings sterile . From remainder a staphyloccccus and Gram positive sparer. Pithed at height of fever. Jugular scrapings
into its own clot broth, clot Tyr ocle and into clot Tyrode and clot broth from
a nor mal sheep. One (its own) clot broth positive 10 days, Gram positive
sparer. Remainder sterile.
Sheep 31720.-Pithed at height of fever. Jugular scrapings into normal
sheep clot broth and its own clot broth, and in addition a clot broth put up .
All tubes sterile after 14 days. With Hauduroy' s technique a fine diphtheroid
isolated from one tube. After one month's sub-culturing this grew as a profuse,
yellow paint-lifle growth. Non-agglutinated.
Sheep 32049.-Bled e ight t imes prior to and during fever. The first
three bleedi n gs sterile. F r om remain der after 3-8 days, a Gram positive sporer,
a streptococcus, a staphylococcus a nd a fine diphtheroid obtained. The dipht heroid not agglutinated.
S h eep 33518.-Bled out at height of reaction. Clot broths made, clot
broths inocu lated with jugular scrapings and clot broths from normal sheep
dot broth i noculated w ith serapings. A Gram negative bacillus (not colontyphoid or pToteus group) , a Gram negative coccus and a Gram positive
cocco--bacillus isolated. T he first germ not agglutinated.
Sheep 34103.-Pithecl at height of reaction. Clot broths made; jugular
serapings into normal sheep clot broth, urine into meat broth and normal
sheep clot bmth. The normal sheep clot broth plus scrapings positive eight
days with diphtheroid. Not agglutin(\t,e!f. Remainder sterile.
Sheep 34607.-Pithed at height of reaction. Jugular scrapings into its
own clot br oth, urine into its own clot broth, meat broth and broth. Urinebroth, positive three days with staphylococcus, scrapings-clot broth positive
four days Gram positive sporer.
Sheep 34103.- Pithed at height of reaction. Jugular scrapings into normal
sheep clot broth and u rine into broth, meat broth and nor mal sheep clot
broth. A clot broth made from heart blood. Scrapings and urine sterile.
Clot broth gave a fine Gr am negative bacillus and a fine diphtheroid. Neither
agglutinated.
Sheep 34095.-Pithed at height of reaction. Jugular scrapings into meat
b r oth-positive seven days Gram positive sporer, blood into meat brothpositive four days staphylococcus, urine into broth-positiYe four days
staphylococcus.
15
IIEAln'\L\TER IK SHEEP .
Sheep 34910.- Bled twice during fever into K medium and into broth.
Ks at 30° C., broths at 37° C. First T\: bleedi ng sterile (serially passaged
on litmu s lactose agar n ine t imes; second K bleechng apparent!;~· steril e on.
sixth serial passage. The fifth and sixth passage tubes sea led off and set
aside fo r s ix "·eeks at r oom temperat ure . A diphtheroid (good growing)
growing on fifth tube and a tetrnd on t he s ixth. 1\either agglutinated.
The br oths treated as the K s. Both sterile.
Sheep 34054.- Blecl six times during fever a& 34.910.
L\.s: First, fourt h , fi fth, and s ixth bleedings sterile . F rom t h e second a nd
third a t iny diphtheroid isolated. Not agg lutinated.
B1·oths: The first fi1·e bleedings incubated at 370 C. for from 25 to 28
days. Subcul t ures in K incubated at 370 C. for one month and smears ma de
0n agar- all sterile. 'L'h e six t h bleeding pos itiYe t hree dn.ys-staphylococcus.
Sheep 3443 .- Blecl six t imes dur ing feve r .as 3491.0.
J{s : By mistake, all but one bleeding 214.10.32 (the t hird) cliscanled. For
descripti<Jn of germ obtained see text.
lJmths : From t he first t hree bleed ings a staphylococcus, and from the fift h
a Gram positiYe sparer obtained; the fourth and sixth sterile after one
month. The positi,·e resul ts were got in t he original t ubes after 7-14 day::;
incubation.
Sheep 35024.- Bied five t .imes during; feYer as 34910.
Ks: The second, t hird , fourth, and s ixth bleedings sub-cultured at 14 day~
interval, four times through K and t hen serially plated three times; all
negative. First bleeding, good gro11·ing diphtheroid iw lated . from original
t.obe after 17 days . Not agglutinated . Fifth bleeding-poorly growing dip hther oid from origi nal tube after 12 days. Not agglutinated.
Broths: Original t ubes negati1·e after one month ; s ub-cul t n es on :tgar
negative afte r one 11·eek.
Sheep 35029.-(Injectecl i. r. 11·ith 35.0 c.c. of a 24 hours K culture of
d1phthero id isolated from the third K tube of 34054. No reaction. After
nine clays bled into K and broth.)
K: By serial platings a Gram positive , fine g r anular bacillus isolated.
On smear, t he or gani sm often appeared to be made up on t iny granules.
Not agglutinated .
Broth: Sterile after one month.
Sheep 35007.-Bled t hirteen times prior to a nd during ferer as 34910.
Ks: T he first to se1·enth , Hinth, eleventh, t welfth , and t hirteenth bleedings
sub-cultured th rough K thr ee times at 14-clay-intetTals . '!'h en serially plated
t hree t imes-all negative. From t he e ighth a n d tenth fine diphtheroids
isolated-neither agglu t inated.
BToths: All incubated on e m<Jnth and then s ub-cult ured on agar for three
days . All nega tiYe, except the seeond-a large, good g r owing Gram positiYe
bacillus.
Sheep 35533: Bled se1·en times durin g fever as 34910.
Ks: The first, thir d, fifth, sixth, and seventh hleed in g incubated for one
month , then serially plated three t imes-all negative. From the fifth a
copiously growing Gram positive bacillus isolated f rom original tube after
four clays incubation. From t he fourth a fine diphtheroid obtained . Not
agglutinated.
Broths: All inc ubated 2.5 clays- then into fresh broth for 10 c1a~·s-then on
agar-all negati,·e.
SheeJJ 34894.-(Rece ived 90·0 <:.c. , int rave nously, of a 24 h ours K cultur e
r•f Gram pos it ire bacillus isolated fro m 34443, 24 I 10 I 32. No rear.t ion.)
Nineteen dn;vs later bled into K . Sub-cultured into fresh K in 7 clays,
ii' to EB in a f\;u·ther 16 da~·s, and 2 clays later seri al plat ing commenced.
By t his technique a rn t be r stout. vacuollatecl "knobhl~· " bacill us isolafed.
G rew ver.1· poorly. 1\ot agglutinated.
16
J. H. )JASON AXD Jt. A. ALEXANDER.
8/t~•ep
34761.- Bled three times during fe1·er a s 34910.
Inc ubated 20 days, then into fres h K for 7 days-serial platings
three t imes. Th e seco n d , th ird , and fourth bleedings steri le. From the first
a ver_,. profusely gro11·i ng Gram pos it i,-e bacillus.
JJroths: Iueu hated one mon t h-the n in to f resh brot h for 10 days-all
negat ive .
.Sheep 34177 : Pith ed at he ight of r ea ct ion and jugul ar scrapings cultured
ll1 K and broth.
E: Pos itive in 8 days- staphylococcu s .
BToth: Incubated fo r 14 days-th e n into fresh broth-negative 14 days .
Sh eep 35829.- Pith ed at h eight of reaction and jugular scrapings cultured
ido K a nd brot h.
Ii..: Incubate d 10 days; t he n in t o fres h l\: for 10 days; sown on agarstaphylococcus .
lll'flth : l ncuiJated 10 clays : t hen in fresh br oth. Negative 14 days.
811 eep 35035.- Treated a s 35829.
Ii..: ), " knobbly" diph t heroid isolated. Not agglut in ated .
BToth: :\Tegative .
.'·!It ee11 3.~871. -'t'reated a s 35829 .
]{: .-\. profuse[_,. growing Gram positi,·e ba cillus isolated. Not a gglutinated .
.limth: .-\ abph~· lococcu s isolated .
Ks:
Non ::liA I. SHEEr.
S e ,·en normal sheep (:35803, 35808, 34806, 34998, 34093, 35819 and
bled into K and broth a s :!4910.
X)
K s : Incubated for 14 days; t he n sub-c ultured into f resh K and incubat ed
for a furtl1er 14 day~ . T hen c ultured in " flooded " agar for seven days
(evaporated fl uid replaced a s requir ed with sterile distilled water). All n egative.
B ·roth s : Inc ubnted for 14 days;
negative .
th en into fresh broth for 14 clays.
All
Shee1J T: Te n c-lot broths made at one bl eeding. Incubated for one month.
Two tubes posit ive after fou r days i ncu hation , with apparen tly the same
fine growing d i phtheroid.
BLuE To:-<our-: SHEEP.
.Sheep 34681, 34713, 347!51.-Blecl at h e ight of temperature r eaction int o
meat broth a n d a s clot broths . Th e first positi ve in four days-staphylococcus.
R emainder ste ril·e afte r 20 clays' in cubat ion.
Sh eep 34562, 34633, 34750, 34581, 34522, and 34626.- Blecl as clot broths at
h e ight of t e mperature r eact ion. Th e fi rst pos it i,·e in 10 clays- fine dipht heroi d.
R emainde r n egat ive after 20 cla~·s .
Sheep 35007 , :Jsoo:J, 350a:l, 35006. - Bled during fe,'er on each of three
success ive clays into m eat broth and broth . All incu bated 20 days. The first
positive (in meat broth only) "· ith a fi ne growing diphtheroid .
Sheep 35001, :35().'3,3, 350"1 ·1.-B!ed clu rin g fe,·er into br oth and meat broth.
,\11 negative after one month ' s incu bat ion.
" EKTEIUTIS "
SHF:EP.
Sheep 34356, 34547 , .34717. 34653, and 33961.- Bled as clot broths and into
broth. The first posit ive with a Gram posit i,·e sparer nfter four clays.
R emainder negatirt> in 20 clnys.
APPENDIX 2.
Summary of t he inoc ulations of l i1· ing cultures and s us pe ns ions into sh eep
with t h e result of the immuni ty test w it h v irule nt h eart water blood : -
17
HEARTWATEll IK SHEEP .
Sheep.
32957
22 / 3 / 33
32952
5 / 4 / 33
22 / 3/33
32958
5 / 4 / 33
22 /3/ 33
32949
5 /4/ 33
22 / 3 / 33
33117
5 I 4 / 33
22 /3/ 33
:l3118
5 /4/ 33
22 /3/ 33
32948
5 /4/ 33
22 / 3 / 33
32507,
33119
33104'
35803
5 / 4 / ;33
22 / 3 / 33
5 / 4/33
9 / 1 / 33
27 / 8 / 33
35808
9 / 1 / 33
~-l5819
23 / l / 33
9/1 / 33
23/1 / 33
:J4550
9 / 1 / 33
34480
23 / l / 33
1 / 2 /33
3 / 1 / 33
23 / 1 / 33
34576
34891
32109
34390
'Tr-eatment.
Immunity 'l'est.
10.0 c.c. s.c. saline suspension of
fourth bleeding diph. of 35533 ...
18 / 4 / 33 died H .W.
25.0 c.c., ditto.
10.0 c.c:. s.c. saline s uspension of d i.p h .
of 34443
... ... ... .. . ....... .
Do.
20.0 c.c., ditto.
10.0 c.c. s.c. salin e s usp. of Gr am pos.
bacillus of 34871 . . . . . .
. ...... . .
Do.
20.0 c.c. , ditto.
10.0 c.c. s .c. saline su sp. of d iph. of
:15035 ...
Do.
20.0 c.c., ditto.
10.0 c.c. s.c. saline susp. of staphylococcus from 34177
Do.
20.0 c .c., ditto.
10.0 c.c. s.c. saline susp. of staphylococcus of 35829 ..
.. . . .. .. . .. .
Do.
20.0 c.c., d itto.
10.0 c.c. s.c. sa line s usp. of g rain p os.
hacill us of 35029
Do.
20.0 c.c., ditto .
10.0 c.c. s.c. of formo l-k illed sa line
sus p. of OX2, OX19. and OXK
r esp ectively
All died H .W.
20.0 c.c., ditto.
2.0 c.c. LV. 15.0 c.c. s.c. saline susp.
of bacillus of 34894
15 / 2 / 33 died H.W.
5.0 c. c. L V. 15.0 c. c. s .c. saline susp.
of bacillus of 34894.
2.0 c.c. LY. 10.0 c.c. s .c. saline su sp .
of first bleeding diph . of 35024
15 / 2 / 33 died H.W.
5.0 C.C. LV. 20.0 C. C., ditto.
2.0 c.c. i.v. 95.0 c.c. s .c. saline susp.
diph. of 34443 ...
..
Do.
5.0 c.c. LV. 17.0 c.c. s.c. saline s usp.
diph. of 34443.
2.0 c.c. L V. 20.0 c.c. s .c. of :).0 days
(S7° C.) K. culture of bacillus of
34894
... ... .. .
Do.
5.0 c.c. l . L 25.0 c.c. s.c., ditto.
5.0 C.C. I.Y. 35.0 C.C. S . C . , d itto.
10.0 c.c. s.c. saline s usp. of diph. of
second bleeding of 34054
15 / 2 / 33 died H.W.
25.0 c.f·. i.v., di tto.
2.0 c.c. LV. 45.0 c.c . of JO d ays K
culture (37° C.) of diph. of ;34443 ...
23 / 1 / 33 5.0 c.c. i. v . 27.0 c.c. s.c. of 10 days
K culture (37° C.) of d ip h . of 34443.
9/1 / 33 2.0 C . C . LV. 20.0 C . C . S . C . 10 days (37°
C.) K culture of diph. of fi r st bleeding of 35014
. .. .. . . ..
23 / 1 / 33 5.0 C . C. LV . 25.0 C.C . S . C., ditto.
9 / 1 / 33 2.0 c.c. i.v. 15.0 c.c. s .c. of saline susp.
of diph. of fifth bleeding of 35024 .. .
23/1 / 33 5.0 C.C. LV. 20.0 C.C. S . C., di tto.
9 / 1 / 33 2.0 C.C. LV. 25.0 C.C. S.C. 10 days (37°
C .) K cultur e of diph. of fifth bleedin g of 35024 . .. . .. . .. .. . .. .
23/ 1 / 33 5.0 C.C. i . V . 20 C.C. S.C., ditto.
9 / 1 / 33
18
Do.
Do.
Do.
Do.
J. H. MASON AND R. A. ALEXANDER.
Sheep.
34420
3(•001
33577
'l'reatment.
9/1 / 33 2.0 c.c. i. v . 15.0 c.c. s.c. saline susp .
o.f eli ph. of <l4910
.. .
23 / 1 / 33 5.0 C.C . i. \·. 15.0 C.C. S. C., ditto.
34281
30031
34894
Do.
Do.
10.0 c.c. i.v. 125.0 c.c. s.c. of 10 days
(37° C.) K cult ure of diph. of 34054
5.0 c.c. i.v . 15.0 c.c. s.c., ditto.
Do.
33.0 c.c. i.v. 4 days (37° C.) ICB.
culture of diph. of 34894 ... ...
26 / 1 / 33 died H.W.
9/1/33
28/12 / 32
12/12/32 25.0 c.c. i.r. 6 days (30° C.) K culture
of diph. of 34910 ... ... ...
29/11 / 32 17.0 c.c. i.r. 4 days (370 C.) K culture
diph. of 34054
9 / 11 / 32 90 c.c. i. v. 24 hours K
dipth. <l4443
34290
16 / 11 / 32
35029
16/11 / 32
12 /12 / 32
4 / 1 / 33 died H.W.
20/12 / 32 died H.W.
culture of
3<l.O <: .c. i. v. 2-! hours K culture of
diph. of 34054 ...
... ... ... ...
<l5 .0 c.c. i.Y. of 24 hours K culture diph.
... ... ...
of 34054
35006 26 / 11 / 32 20.0 c.c. i.r. saline susp. of tetrad of
34910 .......................... . ...
35034 5/12/32 25.0 c.c. i.v. 4 days (30° C.) K culture
of dip h. of <l4443 .. . .. . . .. .. . .. . . . .
85026
Test.
3 / 1/33 10.0 c.c. s. c. saline susp. of diph. of
34054 ... ... ... ... ...
23/1/33 20.0 C.C. S.C., ditto.
23/1/33
34871
Immt~nity
25.0 c.c. i. v. 6 days (30° C.) K culture
of tetrad of 34910
.. . . . . . ..
1.v. = intravenous injection.
s.c. =subcutaneous injection.
susp. = suspension.
dip h.= diphtheroid.
pos. =positive.
29 / 11/32 reacted.
H.W. recovered .
20/12 / 32 died H.W.
20/12 / 32 died H.W.
29/12/32 died H.W.
29/12/32 died H. W.
4/1/33 died H. W.
NoTE.-Where repeated injections were given, fresh saline suspensions or
fresh K cultures were injected. The saline suspensions were prepared by
washing off the growths from the surface of agar (or " flooded " agar) after
24 to 48 hours' incubation. The immunity tests were arranged so that the
routine virus-passage sheep served as controls. All of these died of or reacted
with heartwater, but owing to the negative nature of the experiment have
not been included in the appendix.
19

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