Rift valley fever or enzootic hepatitis

229 Proceedings of an Ordinary Mooting of the Soeioty, held at the L o n d o n School of Hygiene and Tropical Medicine, K e p p e l Street, L o n d o n , W.C.1, at 8.15, on T h u r s d a y , 10th D e c e m b e r , 1931. Dr. G. CARMICHAEL LOW, F.R.C.P., President, in the Chair.

PAPER. RIFT

VALLEY

FEVER

OR ENZOOTIC

HEPATITIS.

BY

G. M. FINDLAY.*

WeUcome Bureau o/ Scientific Research, London. INTRODUCTION. SUSCEPTIBLE SPECIES. CLINICAL SYMPTOMS. METHODS OF INFECTION. MINIMAL INFECTIVE DOSE. POSTMORTEM APPEARANCES. HISTOLOGICAL APPEARANCES. " INTRANUCLEAR INCLUSIONS."

DISTRIBUTION OF THE VIRUS IN THE TISSUES. DURATION OF INFECTIVITY. IMMUNITY. PHYSICAL PROPERTIES OF THE VIRUS. RELATION TO OTHER DISEASES. DISCUSSION. CONCLUSIONS. INTRODUCTION.

Rift Valley fever, or enzootic hepatitis, was first described by DAUBNEY, HUDSON and GARNHAM (1931) in K e n y a Colony, British' East Africa. T h e same disease is almost certainly described b y MONTGOMERY (1913). I n 1912 a mortality of 90 per cent. was recorded a m o n g the l a m b s at G o v e r n m e n t F a r m , Naivasha. O t h e r f a r m s in the Rift Valley also suffered considerable loss. I n some cases the s y m p t o m s were very acute and death occurred within a few hours. I n others the disease ran a m o r e subacute or chronic course. I n the acute f o r m the only s y m p t o m s shown were dullness, rapid respirations, collapse and death within a few hours. At p o s t m o r t e m the liver was soft and friable and the kidneys congested. N o h u m a n cases were recorded and the causal agent was believed to be a coliform bacillus. I n July, 1930, on a f a r m in the Rift Valley, on the shores of Lake Naivasha, there occurred a heavy * I wish to express my gratitude to Mr. R. DAUBNEYfor his generosity in placing at my disposal the virus of Rift Valley fever and for his advice and assistance. My thanks are, also due to Dr. H. J. PAaISH and Major T. DALLING, of the Wellcome Physiological Research Laboratories, for the information and material they have so willingly tendered. Finally, I should like to place on record my indebtedness to my laboratory assistant, Mr. A. E. Green, whose constant help, rendered with an enthusiasm which not even illness could damp, has been of the greatest value.

280

RIFT VALLEY FEVER OR ENZOOTIC HEPATITIS.

mortality among ewes and newly-born lambs. In the course of some seven weeks approximately 3,500 lambs and upwards of 1,200 ewes died from the disease, the death rate in very young lambs being over 95 per cent., while in ewes the mortality, though less, was associated with a high rate of abortion. The symptoms in lambs were somewhat indefinite. On the day before death they were listless, disinclined to feed, and through weakness, unable to stand. Diarrhoea was not a feature of the disease which usually ended in death within twenty-four hours of the first signs of illness. The ewes were either found dead without having shown any previous symptoms or were observed to be ill for but a few hours before death. In some cases they vomited in the evening and were found dead the next morning. When ill the sheep showed a thick mucopurulent nasal discharge and, on occasions, the stools consisted of almost pure blood. They did not feed, and were disinclined to rise, though at this stage the temperature was normal. Ewes that aborted were not visibly ill at the time, with the exception of a few animals that developed septic metritis or peritonitis. At necropsy the most striking change was a focal necrosis of the liver. Attempts to transmit the disease to sheep and lambs demonstrated the fact that the ~etiological agent was a filterable virus. During the course of the investigation, the four Europeans engaged all developed a dengue-like fever characterized by rigors, pains in the back and a fever temperature persisting for from twelve to thirtvsix hours. Upon further enquiry it was found that almost every native engaged in herding sheep during the original outbreak had been ill for some four days and had complained of fever accompanied by severe pains. A native volunteer was subsequently inoculated with filtered virus and reacted with an acute attack of fever on the third day after inoculation, his blood being infective to sheep for nine days after inoculation. In addition to sheep, goats and cattle were also found to be susceptible while a natural outbreak was observed in a dairy herd on a farm in Solai, also in the Rift Valley, though some fifty or sixty miles distant from the site of the outbreak in sheep. The disease, it was found, was not contagious, but there was evidence to suggest that in nature it may be transmitted by a mosquito, probably Tceniorhynchus brevipalpis. The virus in infected blood, preserved in oxalate-carbol-glycerine was brought to this country by Mr. R. DAUBNEY,and in May of this year was inoculated into two lambs by Major DALLING, M.R.C.V.S., of the Wellcome Physiological Research Laboratories, Beckenham. Both the lambs died within thirty-six hours of their inoculation with typical pathological changes, and five and a half days after performing the postmortem examinations Major DALLING became acutely ill with rigors, fever up to 103 ° F., headache and pains in the back. Twelve hours later his laboratory assistant, Mr. C. E. Hart, who had helped at the necropsies, also developed similar symptoms. Subsequent evidence has shown that both these illnesses were due to the virus of Rift Valley fever. Blood was removed from the second case by Dr. H. J. PARISH on the second day of the illness. This blood, together with that from lambs infected in Kenya and those

G. M. FINDLAY.

~)3r

dying at Beckenham constituted three possible sources of the virus which were then tested for their infectivity to laboratory animals. The human blood and the lamb bloods were inoculated intraperitoneally into monkeys (Macacus rhesus), rabbits, guineapigs, rats, mice, chickens and canaries. The blood from the lambs had had 0.5 per cent. carbolic acid added to it, and had been preserved in the ice chest at a temperature of 4 ° C. for eighteen days. It was found to be extremely virulent to mice, six mice inoculated intraperitoneally with 0.2 c.cm. dying in from thirty-six to forty-eight hours after inoculation, mrobic and anmrobic cultures from their blood and organs remaining sterile. Four rats similarly inoculated with 0.3 c.cm. of lamb blood died in from two to four days, while two monkeys, 3/lacacus rhesus, reacted with fever twentyfour and thirty-six hours after inoculation. The rabbits, guineapigs, chickens and canaries showed no signs of illness. The human blood acted in a precisely similar manner. The blood from lambs infected in Kenya, however, was rather less virulent. It had been preserved in oxalate-carbolic-glycerine for nearly four months, and owing to the exigencies of travel had not always remained at a low temperature. Two out of six mice were, however, killed in from four to six days, and two out of four rats. The monkeys, however, did not show any significant rise in temperature and subsequent investigation showed that they were not immune to the virus of Rift Valley fever. The blood, liver and spleen of the mice and rats thus killed by the inoculation of blood containing the virus of Rift Valley fever were found to be infective for other mice and rats both when inoculated directly or after suspension in saline and filtration through Berkefeld N, V or W filter candles. Blood from the monkeys removed during the febrile attack was also found to be infective for mice and rats, while blood from mice and rats dying of the disease was infective for monkeys. The virus is now at its sixty-second passage through mice and rats. As a routine mice are killed when moribund ; the livers and spleens are removed under aseptic conditions, finely minced, ground up in ordinary broth to make a dilution of roughly one in twenty and centrifuged for five minutes at 2,000 revolutions per minute to throw down gross particles ; the supernatant liquid is pipetted off and filtered through a British Berkefeld candle. The filtrate is then inoculated into fresh mice, each mouse receiving 0.5 c.cm. intraperitoneally. Death invariably occurs in from thirty-six to ninety-six hours. The heart blood is cultured ~erobically and anaerobically in broth and, except in very few instances, has proved sterile. After five passages through mice and two through rats, a young goat about one month old, was inoculated with rat blood, as owing to the season of the year it was not possible to obtain young lambs. It reacted with a rise of temperature twenty-four hours after inoculation, while its blood was infective for mice and rats. After thirty passages through mice a second goat, about a year old, was also inoculated with the filtrate of a mouse liver emulsion. It too reacted with fever, although possibly owing to its age, the symptoms of illness were not as severe as in the first goat. After fifty-six passages through mice, a Dorset horned

~8~

RIFT VALLEY FEVER OR ENZOOTIC HEPATITIS.

e w e a n d h e r f o r t n i g h t o l d l a m b w e r e i n o c u l a t e d w i t h t h e filtrate o f a m o u s e l i v e r e m u l s i o n . T h e ewe h a d a s e v e r e a t t a c k o f fever, b u t r e c o v e r e d . T h e l a m b was k i l l e d t w e n t y - f o u r h o u r s a f t e r i n o c u l a t i o n , w h e n o b v i o u s l y e x t r e m e l y ill. T h e b l o o d s b o t h o f t h e e w e a n d t h e l a m b w e r e i n f e c t i v e to m i c e . F i n a l l y six d a y s a f t e r p e r f o r m i n g t h e n e c r o p s y on t h e l a m b , m y l a b o r a t o r y a s s i s t a n t , M r . A. E . G r e e n b e c a m e a c u t e l y ill w i t h fever. H i s b l o o d was also i n f e c t i v e f o r m i c e . T h e v i r u s o f Rift V a l l e y f e v e r has t h u s r e t a i n e d its v i r u l e n c e b o t h f o r s h e e p , goat a n d m a n a f t e r p r o l o n g e d p a s s a g e t h r o u g h r a t s a n d m i c e . T h e p a t h o g e n i c i t y o f t h e v i r u s has also b e e n t e s t e d o n a n u m b e r o f o t h e r s p e c i e s o f a n i m a l s a n d b i r d s [FINDLAY a n d DAUBNEY ( 1 9 3 1 ) ] , as s h o w n in T a b l e I. TABLE I. A N I M A L SPECIES INOCULATED ~ ' I T H THE VIRUS OF

Species

Susceptibility ..

Man

Monkey ( Macacus rhesus) Cat . . . . Cow

.

.

.. •. ..

. . . .

Sheep .. Lamb .. Goat .. •. Horse .. Pig . . . . .. Mouse Wood Mouse'( Apodemus sylvaticus) . . . . Field Vole (Microtus agrestis) Dormouse (Muscardinus avellanarius) .. + + + + + + -" + + + ±

++ ++ ++ t+÷ +++~

++++ '~ ~ . " ~ + + + + + + +

Species

RIFT

VALLEY FEVER.

Susceptibility

Golden Hamster (Cricetus (Mesocricetus) auratus) + + + + Rat + + + Grey squirrel (Sciurus carolinensis) .... + + Rabbit . . . . . . zE Guineapig . . . . . . -Mongoose ( Herpestes ichneumon) Hedgehog .. . -Tortoise (Emys orbic'ularisi Frog . . . . . . -Hen . . . . . . . . -Pigeon . . . . . . -Canary . . . . . . -Parakeet . . . . . . --

= = = = =

nearly always fatal. about 50 per cent. mortality. severe, but non-fatal reaction. slight reaction. virus may survive for a time in the blood, but without causing any reaction. - - = non-susceptible.

A p a r t f r o m t h e s u s c e p t i b i l i t y of s h e e p , l a m b s a n d goats, t h e p a t h o g e n i c i t y of t h e v i r u s for m a n a n d m o n k e y s a n d f o r m a n y s m a l l r o d e n t s is e s p e c i a l l y noticeable. CLINICAL

SYMPTOMS.

Man.--Three h u m a n cases of Rift Valley f e v e r h a v e o c c u r r e d in t h i s c o u n t r y as t h e r e s u l t o f l a b o r a t o r y i n f e c t i o n s . I n t h e first t w o , T . D . a n d C . E . H . , t h e disease was c a u g h t as t h e r e s u l t o f p e r f o r m i n g n e c r o p s i e s o n t h e t w o l a m b s i n f e c t e d w i t h t h e K e n y a v i r u s . N o s p e c i a l p r e c a u t i o n s h a d b e e n t a k e n to g u a r d

288

G. M . F I N D L A Y .

against infection as the high infectivity of the virus for man was not at the time recognised. The third case, that of A.E.G., presents certain features of interest. Before commencing laboratory work on Rift Valley fever, A.E.G. had been injected with 10 c.cm. of serum from C.E.H., this serum having been found to contain protective antibodies against Rift Valley fever when tested in mice. For the next five months though constantly working with the virus in mice and other rodents there was no evidence of infection, but on testing A.E.G.'s serum it was found to be entirely lacking in protective antibodies against Rift Valley fever. One week after this test a postmortem examination was carried out on the lamb which had been infected by passage virus. Rubber gloves and surgical operating gowns, but no masks, were worn when carrying out the necropsy. Six days later, A.E.G. developed an acute attack of fever. Although the evidence is not sufficient to warrant any general conclusion it is of interest that all three cases were infected from lambs. The incubation period of the three cases varied from five and a half to six days. The clinical symptoms were also very similar. A.E.G. on the evening of the sixth day after infection experienced a feeling of nausea and a sensation of fulness over the region of the liver. His temperature was 98 ° F. Next morning he woke with severe headache and pain in the back. When seen about 10 a.m., his temperature was 102.6 ° F., pulse 120. He complained of violent headache, shivering and general malaise. The face was flushed but the conjunctiwe were not injected. There was marked photophobia. The tongue was covered with a whitish fur, and the breath was foul. Pains in the shoulders, back and legs continued throughout the day, the temperature in the evening being 102.8 ° F. A restless night was spent and in the morning the temperature was again 102.6 ° F., although the patient felt better. During the morning there was nose-bleeding. An injection of 10 c.cm. of immune human serum was given in the afternoon, the temperature falling in the evening to 101.4 ° F. In the morning, the tenth day after infection and the fourth day of illness, the temperature had fallen to

A.E.G.

1o4" o

tos" 102' f-

< ,o~' oe '" O- i00' z taJ I- qq' qs'

~ 0~ ";,ooc

~00c >- ~0~ o ,.,-~ I0~ -J I

~AYS

I

i

2.

I ,,

i

i

3

i

4, s 6 7 TOTAL L E U C O C Y T E S P O L Y M O P J ' H O N U C L E A & S --HONONUCLEAP, S ....

Text Fig. I.

9,84

RIFT VALLEYFEVER OR ENZOOTIC HEPATITIS.

97.8 ° F., accompanied by profuse sweating. Thereafter the temperature remained normal and the patient made an uninterrupted recovery, though complaining for some days of weakness and a tendency to sweat on the least exertion. The bowels were constipated throughout the illness. The urine was deep yellow in colour, but contained neither albumin, bile nor casts. Bile was also absent from the blood serum. At no time was there any rash or naso-pharyngeal catarrh, nor could enlargement of the spleen or superficial lymph glands be detected. Blood removed during the height of the fever was injected into four mice, each mouse receiving 0.5 c.cm. Two of the mice died after thirty-six hours, the others after forty-eight hours, all with typical pathological changes in the liver, while the blood and livers when cultured failed to show the presence of bacteria. An emulsion of one of the mouse livers in broth was filtered through a Berkefeld filter and passaged into four mice all of which died within seventy-two hours of the injection. Urine, filtered through a Berkefeld filter and inoculated in amounts of 1 c.cm. into four mice failed to infect, thus confirming the observation of DAUBNEY, HUDSON and GARNHAM(1931) that human urine is not infective, tErobic and anmrobic cultures of the patient's blood taken on the second day of fever remained sterile. Total and differential leucocyte counts were made throughout the course of the illness. A leucocytosis, affecting chiefly the polymorphonuclear leucocytes, was followed by a leucopenia which persisted into convalescence. The leucocyte changes and temperature chart are shown inText Fig. 1,together with the duration of infectivity of the blood for mice. Occasional myelocytes and one or two polymorphonuclear leucocytes with vacuolated nuclei were noted in the blood films. There was no change in the number of eosinophils or large mononuclears. Protective antibodies were detected in the blood serum nine days after the onset of the illness. T.D. was taken ill five and a half days after carrying out a postmortem on infected lambs. He had rigors, a temperature of 103 ° F., headache, and pains in the shoulders and back of such severity that they were at first thought to be due to acute rheumatic fever. Fever continued for three days, then fell to normal, but rose again on the sixth day to 103 ° F. with some return of the muscular pains. This febrile relapse, which lasted for a further twenty-four hours, is similar to that recorded in one of the Kenya cases and suggests the saddle-back temperature so characteristic of dengue. Convalescence was somewhat prolonged in this case, headache being of frequent occurrence. For the above details, I am indebted to the patient himself. Blood was not removed during the acute stage of the disease, but the serum from blood removed fifteen days after the onset of fever was found to have protective antibodies against the virus of Rift Valley fever when tested in mice. C.E.H. was taken ill twelve hours after T.D., with very similar symptoms. Rigors, headache, pains in the shoulders, back and legs were all present and the temperature was 103 ° F. This case differs somewhat from the other two

Go M. VI~V,DLA.Y.

~:~

I

~

~1~ I I

~

~

~,-: ~ ~

, -~ I u

n~ o

U

s

"

.~ u

°

Q,~5

~

~--

~

°

~

"

..

~

u

-~

p.,~

~

~

~

~'

"~

"~ " - . ~ . ~

u.oO

O0

u"~ b r~

~

.~

~

o

I ~..,~

I

I

o

o

~

~

o u u u o

.~ .~

u u

I

,~I

~o]~

I

'-,~.

°

~

co

I ~

~

o,1

I

I o,)c',l

I

cqo,)

~ ~1~'~ ~ ~I~

,~

JI~

-u

.

z l

¢~

oo

-~.~

~

6..

~,

I ~ IR=~o ~

oo

~l °

I

~I

o

~°--

I~o~_~

0 ~' ~ ",Ul ~

~ co

'

°

~ co.

oo~.~.._ ~o

~-~ co

"0u ~0" u~

~'-~ '~'-~ ~co

~moo~

~ co

~ co

0

o~oo~o® ~o~oooooo

""~ '~'~ ~'~ '~'~ ~'-~ O~ '~'-~ cooococoooc:;co

~ oo

"~ co

O~ c::;

¢0~ ,~,-:

~86

RIFT VALLEY FEVER OR ENZOOTIC HEPATITIS.

as the febrile period, in which the temperature ranged from 101 ° to 103 ° F., lasted for ten days and convalescence was as a result somewhat prolonged. For the above particulars I am indebted to Dr. H. J. PARISH who kindly removed blood on the second day of the illness, when the fever was at its height. This blood, as detailed above, killed mice and rats and produced a febrile reaction in monkeys. Serum obtained on the seventeenth day after the onset of illness and one week after the return of the temperature to normal was found to contain protective antibodies against the virus of Rift Valley fever when tested in mice. Protective antibodies were still present five months after the attack. The virus of Rift Valley fever thus produces in man a disease resembling in many respects dengue and sandfly fever. Blood removed during the first few days of the illness is pathogenic for mice, rats and monkeys and by means of a filtered emulsion of the organs or blood of these animals, the disease can be carried on by passage. In addition to these definite infections with the virus it is possible to acquire immunity, as in many other virus diseases, without manifesting any definite symptoms of ill health. At the commencement of the experiments the writer's serum was tested for protective antibodies and was found not to contain them. He then received an injection of 10 c.cm. of human serum from C.E.H. at the same time as A.E.G. Five months later his serum was found to protect mice against 100 M.I.D. of the virus. Active immunity had therefore been acquired as the result of working with the virus, though there had been no attack of fever nor any symptoms pointing to an infection with Rift Valley fever.

Monkeys.--Fourteen monkeys, 3¢acacus rhesus, have been inoculated with the virus of Rift Valley fever. Details of the inoculum, the method of inoculation, t h e onset and duration of fever are shown in Table II. Of eleven monkeys inoculated intraperitoneally or subcutaneously only one, Monkey No. 4, failed to give any evidence of reacting to the infection and on subsequent inoculation was not immune. When inoculated with lamb, human or mouse blood fever appeared in from twenty-four to ninety-six hours, but when the infection was passaged from monkey to monkey a rise of temperature was not invariable. No fever occurred in Monkeys 7 and 10, though the virus was present in the blood stream, as is shown by the infectivity of the blood for mice and also for Monkey 12. In order to determine the infectivity for mice, blood was removed from the ear vein of the injected monkeys shortly after inoculation and on each succeeding day; 0.5 c.cm. was then injected into each of four mice. With the exception of Monkeys 4, 13 and 14 the virus was invariably present in the peripheral blood stream twenty-four hours after inoculation, and remained in the blood stream for from five to thirteen days after inoculation. The infectivity of the blood was therefore present both before and after the attack of fever. When fever occurred the interval after inoculation of the virus varied from 24 to 96 hours, while the actual duration of the fever varied from

G. M. F|NDLAY.

~37

;\

~

<+

(6 >.. hl V

) :+i: ,~ 1

Z o

.¢-..~ E~

+,8 l

i

~. --

~

nlVm3dm3.L

++

.

,

& o

,+~-

8

°

,m

"lAl,,I "0 +++ +.71.LL00Of'PJ7 A

~-wtJ t..,} t,.j c,,p ,c'-. O ::) : ) "JZZ

<

)W

.~

.D : : ) 0 0

Z

I--J

O

Z

E~

3~nlV~3dlA31

WW :) ~3+ 5"]1190DC137

+i +,

L

,~:T

)-L~J

i

8

Z c) E

o

i"

r-o

~

~

I I/

, P,~°!"~o~----? I.... _o

~_ ? o_ 3~n1Y~3~ ~.i31

o

,,,P

",+,-~

,

•

• ,4,

~

"

.

o

"IAIAI3 '.3+

~31A303fl]7

~c

o

°

238

RIFT VALLEY FEVER OR ENZOOTIC HEPATITIS.

24 hours to 120 hours in Monkey 9 (Text Figs. 2--5) where, however, the temperature curve was of the saddle-back variety. Apart from some decrease in activity during the height of the fever, there were no definite symptoms of ill health. There was no diarrhoea and repeated examinations of the urine failed to disclose the presence of either albumin, bile or blood. Total and differential leucocyte counts were made from all monkeys at intervals of twenty-four hours. With the exception of Monkey 4 at its first inoculation, and also Monkey 12, there was at first a leucocytosis, involving chiefly the polymorphonuclear leucocytes, followed by a leucopenia also affecting the polymorphonuclear leucocytes. Leucocytes curves of Monkeys 6, 8, 9 and 13 are shown in Text Figs. 2 to 5. After the temperature had returned to normal occasional myelocytes were seen in the blood films, while the proportion of young polymorphonuclear leucocytes appeared to be increased. There was no evidence of nuclear degeneration. Monkey 5, inoculated with human blood had a rise of temperature from 101.4 ° F. to 105-8 ° F. in forty-eight hours. Monkey 11 which was inoculated intracerebrally showed a rise of temperature to 103 ° F. twenty-four hours after inoculation, while virus was present in the liver and blood. Monkey 13 (Text Fig. 5), which was inoculated intranasally showed a rise of temperature to 104 ° C., sixty hours after inoculation ; the blood at the same time became infective to mice. Monkey 14, similarly inoculated, h~.d a rise of temperature seventy-two hours after inoculation. It is thus possible to produce in monkeys a non-fatal disease closely resembling Rift Valley fever as seen in man. The infection can be transmitted by subcutaneous, intraperitoneal or intracerebral inoculation and by intranasal instillation as in the case of the viruses of yellow fever and vaccinia. The infection can be passaged from monkey to monkey though there is some evidence that with monkey passage the virulence of the virus for monkeys may become attenuated. Even after three passages in monkeys, however, there was no loss of virulence for mice.

Sheep and Lambs.--The symptoms observed in sheep and Iambs inoculated in this country were entirely similar to those already fully described by DAUBNEY, HUDSON and GARNHAM(1931) in Kenya. The two lambs, I and lI, inoculated as already mentioned by Major DALLINGwith the virus from Kenya collapsed and died within thirty-six hours. On 26th October, 1931, a Dorset horned lamb (Lamb III), about a fortnight old, was inoculated intraperitoneally with 2-0 c.cm. of blood from Lambs I and II. This blood had been kept at 4 ° C. for five months. Seven hours after inoculation the rectal temperature had risen from 103.4 ° F. to 106.8 ° F., and the lamb was less active than usual. The next morning the temperature had fallen to normal and the animal appeared more sprightly. The blood was infective to mice for five days after inoculation. Protective antibodies appeared in the serum ten days after inoculation. The total number of leucocytes showed at first a moderate increase followed by a fall. The temperature and leucocyte

G.

LAMB

3.

M.

FINDLAY.

989

changes are recorded in Text Fig. 6. The virus had thus survived for five months in the ice chest, but its pathogenicity for lambs had been decreased. Four mice and two rats inoculated with this same preserved blood died, however, in from two to four days.

108" 10 7"

i06" 105°

Lamb IV, the twin of Lamb III, was inoculated intraperitoneally on 27th October, 1931, with 1 c.c. of blood from Mice 823 and 824 (fifty-sixth mouse passage). In seventeen hours the rectal temperature rose 4.4 ° F. while the leucocytes also increased in numbers :--

~L 104.°

E t ~ 105o I0~ I01"

20,000

Z ~,ooo

d to o. tf)

i

i

i

,

i

I

i

i

27th October, 1931, 5 p.m. Temperature 103.0 ° F. Total leucocytes~ 8,750per c.mm. Differential leucocyte count =Polymorphonuclears 27 per cent., small lymphocytes 70 per cent., large mononuclears 1 per cent., basophils 2 per cent.

28th October, 1931, 10 a.m. Temperature 107.4 ° F. Total leucocytesz J I J i i r i i 16,280 per c.mm. Differential leucocyte I 2 3 4 ~ 6 7 8 DAY5 count~Polymorphonuclears 74 per cent., TOTAL LEUCOCYTES small lymphocytes 24 per cent., large POLYrlORPHONUCLEARS...... mononuclears 1 per cent. basophils 1 per rlONONUC LEAR3 cent. The lamb was extremely weak and T e x t F i g . 6. could barely stand, while its bleat was reduced to a plaintive squeak. It was therefore killed and blood was inoculated into four mice all of which died within forty-eight hours of inoculation. /Erobic and anmrobic cultures from the heart blood were sterile. The urine contained neither blood, bile nor albumin. The urine did not infect mice. A Dorset horned ewe, the mother of the two Iambs described above, was inoculated intraperitoneally with 5 c.cm. of a Berkefeld filtrate from the livers of Mice 848 and 850 emulsified in sterile broth. Tile rectal temperature rose from 103.6 ° F. to 106.8 ° F., thirty-six hours after inoculation and remained high for forty-eight hours. The temperature curve and total leucocyte changes are shown in Text Fig. 7. Blood was infective for mice for seven days after the inoculation of the mouse livers. With the exception of a disinclination to rise there were no symptoms of illness. No nasal discharge was noticed. tJ

O

0

Goats.--Two goats were inoculated with the virus of Rift Valley fever. Goat l, a kid of about one month old, was inoculated intraperitoneally with

9,,4:0

RIFT VALLEY FEVER O R ENZOOTIC HEPATITIS.

C,OAT I

EWE.

.

&OAT

7_.

108o

lOT

105" 107 =

UJ

LIJ

-5

e~

I06°

/

-n

I-< i05 o

rv" L~ O_

ny- 104."

103° k~

,,, 103° I--

F-

K

to£

102" I01'

lot" V~Yus I

,

I

,n blood. i,

9..soo(

~

i

I

~

i

I

i

,

I

,

E

y-

F j ,~ooo

~5 a~

o. I 1 ~ u'~

Io,o0o F-

0 0 ~

.J

DAYS

s,/ % // %% ......• %%

o

0

_

i

|

2 TOTAL

,

3

.

4

,

5

.

6

LEUCOCYTES

P OL¥1"IORPHONUCLEAR5 MONONUCLEAIRS

Text

F i g . 7.

t~J .J

¢

7

8

DAYS

t

Z

,5

4

t

z

.~

4

- -- ......... ,

TOTAL LEUCOCYTE5 , POLYMORPHONUCLEARS MONONUCLEAES ............... .

.

.

.

T e x t F i g . 8.

2 c.cm. derived from Rats 9 and 10, the virus having been passaged five times through mice and twice through rats. Twenty-four hours after inoculation its temperature rose from 102.4 ° F. to 106.8 ° F., it lay with its head down, shivering at intervals and taking no notice of its food. Next day the temperature had fallen to 104-0 ° 17. and the kid appeared much more active. It was killed and the blood inoculated into four mice. These mice were all dead within three days, with typical pathological lesions, the heart bloods, cultured aerobically and anaerobically remaining sterile. The temperature chart and total leucocyte curves are shown in Text Fig. 8. A moderate leucocytosis was followed by a leucopenia, 66 per cent. of the polymorphonuclear leucocytes present being immature. The urine was free from albumin and bile.

G. M. FINDLAY.

")41

Goat II, an animal about a year old, was inoculated with 2.5 c.cm. of the bloods of Mice 420 and 421, the thirtieth passage through mice. Twelve hours after inoculation the temperature rose from 102-8 ° F. to 104.2 ° F. reaching 105-2 ° F. in the evening of the same day. Next morning the temperature fell to 101.8 ° F. and remained within normal limits till the animal was killed seventytwo hours after inoculation (Text Fig. 8.) Blood from this goat only killed one out of four mice inoculated, probably owing to the fact that the virus was being rapidly removed from the blood stream in an animal which was becoming immune. The histological changes in the liver, however, were similar to those found in Goat I.

Cats.--In view of the fact that MONTEIRO (1930) has recently shown that cats are susceptible to the virus of yellow fever it appeared to be of interest to determine whether cats are also susceptible to Rift Valley fever. Kitten I was inoculated intraperitoneally with 0.25 c.cm. of blood from Rat 10. After forty-eight hours its temperature rose from 102.4 ° F. to 103"2 ° F., reaching 104.4 ° F. twelve hours later. The cat at this time was very quiet. Blood removed from the femoral vein was inoculated into four mice (0.2 c.cm. per mouse) all of which died in from thirty-six to ninety-six hours with the lesions typical of Rift Valley fever. The blood, cultured in broth, ~erobically and an~robically, remained sterile; a total leucocyte count gave a total of 17,500 per c.mm., with polymorphonuclear leucocytes 85 per cent., lymphocytes 15 per cent. Seventy-two hours after inoculation the temperature had fallen to 103.2 ° F. Blood taken for inoculation into mice on the sixth day after injection of the virus failed to kill the mice while the total leucocyte count had now fallen to 12,200 per c.mm., with polymorphonuclear leucocytes 73 per cent. (42 per cent. of immature forms) lymphocytes 24 per cent., large mononuclears 3 per cent. Three weeks after the first inoculation the cat was again injected with a Berkefeld filtrate of the liver of Mouse 123. There was no temperature reaction, no change in the leucocyte count and no evidence that the blood was pathogenic for mice. Kitten 2 was inoculated intraperitoneally with 0-25 c.cm. of blood of Mouse 142. Sixty hours after inoculation its temperature rose from 101-6 ° F. to 103.0 ° F., reaching 105-8 ° F. eighteen hours later. The cat was now very quiet and had not eaten its food. It was killed and the blood inoculated into four mice (0.2 c.cm. per mouse) all of which died within three days. Kitten 3 was inoculated at the same time and with the same blood as Kitten 2. It failed to show any rise in temperature or any definite change in the leucocyte count. Blood removed from the femoral vein forty-eight hours after inoculation killed two out of four mice, but blood removed ninety-six hours after inoculation was not pathogenic to mice. The virus of Rift Valley fever may therefore survive for from forty-eight to seventy-eight hours in the blood of kittens and may give rise to fever associated with changes in the total leucocyte count.

249,

RIFT VALLEY FEVER OR ENZOOTIC HEPATITIS.

Mice.--The death rate in mice inoculated with the virus of Rift Valley fever is from 98 to 100 per cent. Mice thus constitute an extremely sensitive test for the presence of the virus. Death usually occurs in from thirty-six to seventy-two hours, though very occasionally mice may survive for as long as seven to ten days. The first noticeable symptom is a slight roughening of the coat, associated with decreased activity. Very rapidly the mouse becomes more lethargic, lying with its back legs wide apart. At first, if the skin is pinched or the animal is rolled upon its side it regains its equilibrium with convulsive movements. Sometimes at this stage, also there is noticeable a fine tremor. Soon, however, there is complete unconsciousness and death. Quite often an animal will be found dead in its cage within an hour of the first symptoms of lethargy. Examination of the urine for albumin and bile has always proved negative. There is no rise in the rectal temperature which varies from 101 ° F. to 102 ° F. until shortly before death. In the blood there is usually a considerable increase in the percentage number of polymorphonuclear leucocytes, many of which show vacuolization of the chromatin. Myelocytes are often present and there is marked vacuolization of the cytoplasm of the mononuclear cells. House mice are as susceptible to the virus as are tame mice. Wood Mice and Field Voles.--Through the kindness of Mr. C. ELTON, of the Department of Zoology and Comparative Anatomy, Oxford, to whom my sincere thanks are due, it became possible to test the pathogenicity of the virus of Rift Valley fever for wood mice and field voles. Ten wood mice, caught in a wood near Oxford, and six field voles from Roxburgshire were available. They were inoculated subcutaneously with 0.2 c.cm. of a Berkefeld candle filtrate of the livers of Mice 127 and 143. Death, preceded by lethargy merging into unconsciousness, took place in all the animals in from thirty to sixty hours. Blood removed from the heart failed to give any growth of bacteria when cultured ~erobically and anaerobically but was pathogenic to mice in the usual manner. Dormice.--The pathogenicity of the virus of Rift Valley fever was tested on three dormice, which were each inoculated intraperitoneally with 0.2 c.cm. of a Berkefeld filtrate of an emulsion of the liver of Mouse 742 in sterile broth. One dormouse was killed when moribund thirty hours later, the others died seventy-two hours later. Cultures from the heart blood remained sterile, but mice inoculated with the blood succumbed. Golden Hamster.--Through the kindness of Dr. HINDLE, who for the first time has succeeded in breeding this animal in this country, two specimens of this rare species were available. The golden hamster inhabits a small area of country in the neighbourhood of Aleppo. The animals were injected intraperitoneally with 0-5 c.cm. of a Berkefeld filtrate of a liver emulsion of Mouse 904. Thirty hours later both hamsters were unconscious and were killed when moribund. Cultures, made from the heart blood, remained sterile, but the blood injected into four mice killed them in forty-eight hours.

O. M. FINDLAY.

248

Rats.--Of forty-three rats injected, either intraperitoneally or subcutaneously, with virus containing material twenty-two or 51 per cent. died in from two to five days, the average time of death being four days after inoculation. As in the case of mice the symptoms of illness came on very rapidly, roughening of the coat, a disinclination to feed or move about the cage and a staggering gait were quickly succeeded by RATS. complete unconsciousness and death. The deep golden colour of the urine, as it stained the white fur, was very noticeable, while in some animals r the margins of the eyelids were covered with a sticky exudate. As in the case ~- gS'! I of mice the temperature remained between 101 ° F. and 103 ° F. until just T e x t Fig. 9. before death, when it became subnormal (Text Fig. 9). In rats that survived e.g., Rat 43, the temperature fell to 98.4 ° F. but as the animal recovered rose again to its normal level. Attempts to determine total leucocyte counts met with somewhat discordant results, in some cases a leucocytosis was present at death, in others a leucopenia. More characteristic especially in animals that recovered was the number of polymorphonuclear leucocytes in which the nuclei showed vacuolization of the chromatin and inability to take up the basophilic stain, while in the mononuclears vacuolization both of cytoplasm and nuclei was not uncommon. These changes are similar to those noted in man by JOANNIDES (1929) during the course of dengue fever. Phagocytosis of red cells by polymorphonuclear leucocytes was also occasionally seen. Intracerebral inoculation of the virus in rats is invariably fatal. Four intracerebral passages of the virus were made using an emulsion of rat brain filtered through a Berkefeld candle and injecting 0-i15 c.cm. Death took place in from twenty-four to seventy-two hours, virus being present in the heart blood and liver at death. Intratesticular inoculation is also usually fatal in forty-eight hours. Of ten rats inoculated intratesticularly there has been but one survivor. Grey Squirrel.--A grey squirrel inoculated intraperitoneally with 0"3 c.cm. of a Berkefeld filtrate of Mouse 221 liver showed no signs of ill health and no characteristic rise in temperature. Sixty hours after inoculation it was killed and the blood inoculated intraperitoneally into four mice, two of which died within three days while the others survived. Another squirrel inoculated with the blood of Mouse 972 died in thirty-six hours, its blood being infective for mice.

244

RIFT VALLEY FEVER OR ENZOOTIC HEPATITIS.

Rabbits.--Attempts to reproduce the disease in rabbits have so far failed either by intravenous, subcutaneous, intraperitoneal, intradermal or intracerebral inoculation of the virus. No rise in temperature, no blood changes and no evidence of the presence of the virus in the blood have been obtained except in one rabbit out of twelve. In this animal from which blood was removed daily for inoculation into mice the virus survived in the blood stream for twenty-four hours after injection by the intravenous route. Guineapigs.--Twenty-five guineapigs have been injected either intradermally, subcutaneously, intraperitoneally or intracerebrally but without producing any signs of illness or even obtaining evidence of survival of the virus in the blood stream. The mongoose and hedgehog both proved insusceptible to the virus. For the loan of the former animal, 1 am indebted to Major DUNKIN, M.R.C.V.S. Reptiles, amphibians and birds, so far as they have been tested, have proved refractory to ini:ection. ~-V[ETHODS OF INFECTION. Apart from subcutaneous, intraperitoneal, intracerebral and intratesticular methods of injection, it is possible to infect susceptible animals by percutaneous methods, by intranasal instillation and by infecting the conjunctival sac. Four mice were epilated on the abdomen by means of sodium sulphide. The skin was lightly scarified and the blood of Rat 45 was placed on the skin. Three of the mice died in four days. A similar experiment was carried out with four rats but in this case only one of the rats died in three days. Two rats had their conjunctival sacs flooded with the blood of Mouse 129. One rat died after five days with the typical lesions of Rift Valley fever ; its blood was infective for mice. The effect of intranasal instillation in Monkeys 13 and 14 has already been described. Numerous attempts have been made to infect both mice and rats by feeding them on infected livers and spleens. In no case has infection resulted. This Confirms the experiments of DAUBNEY,HUDSON and GARNHAM (1931) who were unable to infect lambs by drenching them with blood containing the virus. No evidence has yet been obtained that the virus can be transmitted from infected to healthy animals by direct contact. It seems probable that in the laboratory infections which have occurred both in this country and in Kenya the virus has passed either through an abrasion on the skin, through the conjunctival sac or by the mucous membrane of the nose. MINIMAL INFECTIVE DOSE. In order to test the minimal infective dose for mice, the blood of Rat 45 was passed into heparin and dilutions of this blood in phosphate buffer at

G. M.

FINDLAY.

245

pH 7.2 were prepared ranging from 1 in 100 to 1 in 100,000,000. Three mice were inoculated intraperitoneally with 0.1 c.cm. of each dilution. All the mice inoculated with dilutions up to and including 1 in 1,000,000 died within four days, as did two of those inoculated with the dilution 1 in 10,000,000. A similar experiment was carried out with epilated and scarified mice. In this case the mice inoculated with dilutions of 1 in 100 and 1 in 1,000 all died, but only one of the mice inoculated with a dilution of 1 in 10,000. POSTMORTEM

CHANGES.

The lesions encountered in laboratory animals at postmortem examination are very constant and bear a close resemblance to those found in sheep and lambs whether naturally or experimentally infected.

Monkeys. In monkeys there are few naked eye changes. In Monkey 5, killed after twelve hours' fever with a rectal temperature of 105.8 ° F. the liver was light yellow in colour with some faint reddish mottling, more pronounced near the free border. There was no enlargement of the spleen and no congestion of the intestine. The mesenteric vessels were congested and in the mesentery were one or two small scattered hmmorrhagic spots. The mesenteric glands were also congested and on section appeared to have small hmmorrhages in the cortex. The kidneys, lungs and heart were normal. The appearances met with in Monkeys 2, 4 and 11 were similar.

Sheep. No adult sheep have been killed during the course of the present investigations. The postmortem appearances in the three lambs, however, were similar in every way to those described by DAUBNEY and his colleagues. Liver.--The organ was not enlarged but was paler in colour, in one case a yellow chamois leather colour. On the surface of the liver were pale whitish areas of necrosis about the size of a pin's head, associated with tiny sub-capsular h~emorrhages. In other parts the surface of the organ appeared relatively unchanged. In Lamb IV, which was killed seventeen hours after inoculation, the lesions were more akin to those described as being present in adult sheep. The surface of the liver was pale but scattered throughout were reddish-brown areas varying in Size from a pin point to a threepenny bit. On section the cut surface was somewhat pale but no necrotic areas were visible.

Spleen.--This organ also was not enlarged in any of the cases but beneath the capsule and more especially near the free border of the organ there were constantly present tiny little h~emorrhages, often associated with capillary arborescence. Kidneys.--The capsule stripped readily, showing congested stellate veins. C

246

RIFT VALLEY FEVER OR ENZOOTIC H E P A T I T I S .

On section the organ was pale in colour with congested vessels more especially in the boundary zone between cortex and medulla. Lungs.--There was no congestion of the larynx, trachea or bronchi and the lungs themselves were normal in appearance except that at the bases in Lamb IV there was some slight congestion. Circulatory System.--Subpericardial ha~morrhages were seen in the region of the coronary grooves on the ventricles of the heart while in the left ventricles there were present the small sub-endocardial extravasations which appear to be very characteristic. The mesenteric and omental vessels were deeply engorged--another very characteristic feature of the disease. Lymphatic System.--In all three cases the mesenteric lymph glands were enlarged, and moist and in two there were h~morrhages into the cortical portion of the glands. Alimentary Tract.--The mouth, pharynx and three compartments of the stomach showed nothing abnormal, but in the mucous membrane of the abomasum there were a few small pin point h~emorrhages near the cardiac end. The intestines in all three cases were free from catarrhal changes ; there were no impacted pultaceous masses. The pancreas, suprarenals and thyroid showed no naked eye lesions. Nervous System.--The brain was only examined in one case; beyond congestion of the meningeal vessels there were no macroscopic lesions.

Goats. The changes found in the two goats at the" postmortem examinations were similar, though most marked in Goat 1. The livers were pale and on the surfaces both anterior and posterior there were small areas studded with reddish mottlings about the size of a pin's head which stood out in contrast to the paler ground colour of the organ. The kidneys were pale, with tiny sub-capsular h~emorrhages, while on section the cortical striation appeared to be more pronounced than usual. The spleens were not enlarged but pin point ecchymoses were present beneath the capsule in the region of the free border. In both goats there were a few scattered petechi~e in the mesentery, while the mesenteric vessels were engorged. In Goat I a single lymph gland in the small intestine mesentery was greatly enlarged and h~emorrhagic. A portion of the ileum also of this goat was congested for a distance of about 10 inches, the mucous membrane thickened and dusted with pin point h~emorrhages. This change was associated with a heavy infection of Eimeria faurei. The testicles of Goat I on section appeared very congested. There were no hremorrhages in the heart.

Cats. The liver was rather pale, with a few tiny reddish mottlings near the free border. The spleen was not enlarged ; there was one small h~emorrhage

G. M. FINDLAY.

247

near the free margin. The mesenteric vessels were congested and the mesenteric glands rather enlarged, two of them showing small h~emorrhages in the cortical regions. There was no enteritis. Mice The postmortem appearances have now been studied in between nine hundred and a thousand mice. The most noteworthy changes are seen in the liver. It is rarely enlarged but may vary in colour. The commonest appearance, found in nearly 90 per cent. of cases, is of small dark purplish red areas about the size of a pin's head uniformly spread on a lighter red background; in some 5 per cent. of cases the spots are brick red in colour scattered on a background of a light salmon pink and in the other 5 per cent. of cases the spots are of salmon pink hue, the background of a light buff colour. The appearances are shown in Plate I. When exposed to the air for a short time the'contrast between the purple spots and the red background becomes dulled. On section the mottled appearance is not so well seen but the organ is often softer than normal. Postmortem changes come on very rapidly. The spleen is never enlarged and there is no obvious lesion except an increase in the intensity of the surface mottling. The mesenteric vessels are generally congested and occasionally there are tiny h~emorrhagic loci in the mesentery and omentum while the mesenteric lymph glands often exhibit small h~emorrhages. As a general rule there is no enteritis but in a few cases there may be patches of inflammatory change involving more especially the transverse colon. This inflammatory change is probably associated with an invasion of bacteria from the lumen of the bowel. The lungs, heart, kidneys, suprarenals, pancreas, and brain do not show any naked eye lesions. Rats. The postmortem appearances have been studied in fifty-two rats. As in the case of mice the colour of the liver shows considerable variation. The commonest appearance is of purplish red spots uniformly distributed on a rather paler red background, but in a few cases, as is also the case in sheep, the liver is of a yellowish buff colour, with tiny reddish areas about the size of a pin's head scattered over the surface. These colour changes may possibly be correlated with the amount of fatty infiltration which has occurred in the liver, the red areas which might at first be taken for petechi~e being in reality areas of more normal liver tissue. The spleen is not enlarged but the surface mottling is very distinct. The mesenteric vessels are engorged and there are usually present one or more tiny petechi~e in the substance of the mesentery. The mesenteric lymph glands are generally enlarged and may exhibit h~emorrhages. The tendency to h~emorrhage is greater in the rat than in the mouse. Thus, though in some rats there may be no h~emorrhages, in others, h~emorrhages have been found in the wall of the stomach at the cardiac end, in the lungs, the pericardium, over the left ventricle, a not uncommon site, in the testicle,

9~48

RIFT VALLEY FEVER OR ENZOOTIC HEPATITIS.

ureters and bladder. There is as a rule no true enteritis but in two cases there have been found pultaceous masses impacted in the lumen of the descending colon with a h~emorrhagic inflammatory condition of the gut wall in their neighbourhood. In two cases the subcutaneous tissues have exhibited a definite lemon yellow tint, though examination of the urine and blood for bile proved negative.

Other Rodents. In wood mice, field voles, dormice and hamsters infected with Rift Valley fever there have been noted reddish-purple mottling of the liver, absence of splenic turnout, congestion of mesenteric and omental vessels and petechial h~emorrhages in the mesentery and mesenteric glands. The postmortem appearances are therefore very similar in all the small rodents susceptible to the virus and are closely akin to those found in sheep and Iambs dying from this virus infection. HISTOLOGICAL

CHANGES.

Technique. Tissues were fixed for histological examination in Zenker's fluid with and without the addition of acetic acid, in Bouin's fluid and in neutral formol-saline. Schridde's method was used for the study of mitochondria, while Feulgen's technique was used for the study of chromatin. Frozen sections were stained for fat by heematoxylin and Scharlach R or Sudan III. Paraffin sections were stained by ha~matoxylin and eosin, ha~matoxylin and van Gieson, Mallory's methylene blue and eosin, methylene blue and phloxine, Giemsa's stain, Mann's stain and Goodpasture's aniline-fuchsine.

Liver. Sheep.--The changes seen in the liver of the three lambs though varying somewhat in degree are essentially similar in type. In the two lambs, infected by Major DALLING at Beckenham, there is so complete a necrosis that sections are scarcely recognizable as liver tissue. With the exception of a few poorly stained parenchymatous cells in the neighbourhood of the portal space or round the central vein the liver lobule is represented by faintly stained and irregular shaped masses of broken down cells separated from one another by infiltrating polymorphonuclear leucocytes and histiocytes. The typical architecture of the liver is almost entirely lost. Here and there may be seen the remains of a nucleus of one of the liver cells, represented by a thin hair-like ring of chromatin. Scattered throughout the Iobule both intra- and extra-cellularly, are masses of chromatin of irregular size and shape, derived in part from the remains of the nuclei of the liver parenchyma, in part from the nuclei of infiltrating cells, polymorphonuclear ceils and histiocytes which have also undergone karyorrhexis. The Kupffer ceils can no longer be distinguished and the majority of the parenchyma cells are mere amorphous masses with little affinity for eosin or other acidophilic stains. Occasionally, however, a few definitely oval or

PLATE

I.

Livers of M i c e w i t h Rift Valley fever. T h e dark m o t t l e d appearance is seen in about 90 per cent. of cases.

G.

M.

FINDLAY.

249

rounded cytoplasmic masses can be distinguished--the last stages of the Councilman lesions which are characteristic of a less advanced stage of the hepatic necrosis. The cells of the larger bile ducts are relatively unaffected. In Lamb IV, killed during the height of the fever the changes are somewhat less advanced. The liver is studded with necrotic loci, situated irregularly throughout the whole lobule, sometimes close to the portal space or central vein, more often in the mid zone of the lobule. These necrotic loci show in many places a tendency to coalesce and had the animal been allowed to live it is probable that in a few hours the whole of the hepatic tissue would have been involved. The size of the foci varies considerably ; in some cases only a few cells are involved ; in others large masses of liver cells are undergoing degeneration, in the centre of each necrotic focus there is a mass of cellular debris, the faint outlines of broken down cytoplasm and chromatin granules of varying shapes and sizes. Around this are numerous polymorphonuclear leucocytes and a few histiocytes, the former especially showing pyknotic nuclei on the point of breaking down. Here also liver parenchyma cells can still be distinguished. The nucleus is represented by a faint hair-like ring of chromatin, while in the central space may sometimes be seen a small irregular acidophilic mass. Many of the cells at the edge of the foci exhibit a cytoplasmic degeneration which is very characteristic. A portion of the cytoplasm stains intensely with eosin; it soon becomes rounded in shape and though at first contained within the cytoplasm the hyaline globular mass, as it now appears, is eventually separated off from the portion of cytoplasm which contains the degenerating nucleus. In larger loci, instead of a portion of the cell only undergoing the hyaline change, the whole of the cytoplasm is involved and the cell is represented by a rounded or oval hyaline body which stains intensely with acid stains. Occasionally two or more hyaline bodies may appear in the cytoplasm of the same cell. These hyaline bodies are identical with the lesions originally described by COUNCILMAN (1890) and more recently by KLOTZ and BELT (1930) as occurring in the liver in yellow fever in man. The earliest lesion would seem therefore to be a focal degeneration of the liver parenchyma, which is next invaded by polymorphonuclear leucocytes and histiocytes. These invading cells are in their turn attacked and destroyed by toxins, so that in the fully formed focus there is a mass both of degenerated leucocytes and liver cells. That the earliest lesion is a degenerative change of the liver cells is borne out by a study of Schridde preparations in which the rounding up of the small rod shaped mitochondria in the liver cells is well seen. These changes will be described in detail in a further publication. In portions of the liver not actually the site of necrotic loci the Kupffer cells were swollen, with deeply stained nuclei. In the livers of the three lambs there was no excessive congestion of the capillaries. In sections of Lamb IV stained with Scharlach R there was little or no fat in the actual foci of degeneration but a fine fatty degeneration in the intact parenchyma cells. (Plate II. Fig. 1--6.)

250

RIFT VALLEY FEVER OR ENZOOTIC HEPATITIS.

Goats.--The changes present in the livers of the two goats were similar to, but less marked than those found in Lamb IV. Foci of necrosis were present in both livers but were more numerous and more extensive in the liver of Goat I. Councilman lesions were noticeable in both cases though they were less frequent than in the liver of Lamb IV. Otherwise the histology of the loci was identical. There were no fatty changes in the livers of the goats. The absence of intense fatty change in the liver of animals suffering from Rift Valley fever constitutes one of the most striking differences from the hepatic lesions found in yellow fever. (Plate III. Fig. 1-3.) Monkeys.--In these animals discreet foci of necrosis were present, irregularly distributed throughout the liver lobule and varying considerably in numbers in different sections. Hyaline degeneration of the cytoplasm was not as well marked as in the sheep and goats. (Plate III. Fig. 4-6.) Cats.--In the liver of the cat there were seen here and there very small foci of degeneration containing a few polymorphonuclear leucocytes. (Plate IV. Fig. 1.) Mice.--The lesions in the livers of mice were very similar in character to those seen in the most acute stage in lambs. In many animals it was impossible to recognize the section as being one of liver tissue, except for the occurrence of occasional portal spaces. The liver cells were for the most part feebly stained ; some however were deeply stained with eosin and had become separated off and converted into round or oval masses ; in some cells the nucleus consisted of a thin ring of chromatin ; in others it was either hyperchromatic or had entirely disappeared, the contents of the rounded cell being reduced to a thin shadow still contained within a delicate cell membrane. Nuclear debris was scattered throughout the tissue while occasional polymorphonuclear leucocytes were seen, many of them undergoing karyorrhexis. Many of the endothelial cells were swollen, and their nuclei deeply stained. In many livers also there was much engorgement for not only were the capillaries and central veins packed with red cells, but in many areas there were definite loci of haemorrhage. (Plate VI. Fig. 2 and 3). Rats.--In these animals also the lesions were of a diffuse rather than a focal character. The liver cells were, however, somewhat better preserved than in the mouse; in many the cytoplasm was granular and acidophilic in character, while in others it had become rounded off into a circular or oval non-nucleated mass. Many of the nuclei were intensely pyknotic, while in others there was but a thin layer of chromatin round the nuclear membrane. Polymorphonuclear leucocytes were diffusely scattered through the tissue. The central veins were usually congested. In some cases the rats had died before the necrosis had become general. Foci of feebly stained degenerated liver cells were being invaded by polymorphonuclear leucocytes which were also present in large numbers in the central veins and sinusoids. (Plate IV. Figs 3-6.)

G. M. FINDLAY.

0'51

Wood Mice, Field Voles, Dormice and Hamsters.--In all these animals the liver changes were similar in character to those described in mice. In Apodemus and Microtus a noticeable feature was the intense congestion of the capillaries. (Plate VI. Figs. 5 and 6.)

Sp/een. The subcapsular h~emorrhages present in the Iambs, goats and occasionally in the rats, mice and other small rodents were not extensive. The sinusoids though somewhat swollen with red ceils did not contain an excessive number of leucocytes in the sheep and goats but in some of the rats' spleens there were many degenerated polymorphonuclear leucocytes with some nuclear debris both free and phagocytosed. (Plate V. Fig. 1).

Mesenteric Glands. H~emorrhagic loci were found in relation to the cortical lymph nodes in lambs, goats, monkeys, cats, rats, mice and hamsters. Sometimes there was also an associated infiltration of polymorphonuclear leucocytes, many of which were undergoing karyorrhexis. (Plate IV, Fig. 2 and Plate VI, Fig. 4.)

Kidneys. In the lambs dying in the acute stage there was noticeable merely a cloudy swelling of the cells of the convoluted tubules. In the goats which had survived somewhat longer, the lesions in the kidneys were more extensive than in the lambs. The nuclei of many of the cells in the convoluted tubules stained poorly, the cytoplasm was granular and the edges of the cells somewhat ragged. At the same time there was congestion of the vessels both of the cortex and medulla, with occasional small h~emorrhages beneath the capsule. There was no increase in the number of polymorphonuclear leucocytes present nor was there any evidence of the formation of necrotic loci in the kidneys. In mice and rats the changes in the kidney were as a rule slight, owing to the extreme rapidity of the toxic process. Cloudy swelling and in some cases a loss of nuclear staining were the most noticeable features together with congestion of the cortical and medullary vessels. The same changes were present in monkeys. In Monkeys 2 and 4 and in certain of the rats the cells of the convoluted tubules showed evidence of a fine fatty degeneration when stained with Scharlach R.

Alimentary Tract. Apart from the occurrence of h~emorrhages in the submucosa at the cardiac end of the abomason of one of the goats and the occasional presence of ha~morrhages in the cardiac end of the stomach in rats there was present enteritis in Goat I and a ha~morrhagic enteritis in some of the rats and mice. The enteritis was associated with extreme congestion of the vessels

~'5 9'

RIFT VALLEY FEVER OR ENZOOTIC HEPATITIS.

in the villi and with some desquamation of the lining epithelium. The interglandular tissue contained numerous polymorphonuclear leucocytes, many of them undergoing karyorrhexis. (Plate V, Fig. 2.)

Genito-Urinary System. In rats there were occasional petechial h~emorrhages in the wall of the bladder and small h~emorrhages in the intertubular tissue of the testicles. After intratesticular inoculation there was very marked degeneration of the cells lining the tubules in the inoculated testicle. The tubular cells had in many cases lost their nuclei and stained intensely with eosin. Curious giant cells were also encountered. Congestion of the blood vessels and an infiltration with red cells and polymorphonuclear leucocytes were also seen. (Plate V, Fig. 3-5.) Heart. The small subpericardial and subendocardial h~emorrhages met with in lambs, goats and rats do not require any special description. Lungs. In Lamb IV there was some congestion of the interalveolar capillaries which contained an excess of polymorphonuclear leucocytes. Occasionally in rats there were small h~emorrhages due to rupture of the interalveolar capillaries. Central Nervous System. Beyond congestion of the meningeal vessels and occasionally of the small cerebral vessels, there were no histological lesions in the brain and cord. Bone Marrow. H~ematopoietic bone marrow has been examined in rats and mice dying from Rift Valley fever. The capillaries were congested and, as a rule, there was a definite activity of the leucopoietic tissues, together with much congestion of the blood capillaries. In some animals, though by no means in all, a considerable amount of nuclear debris and karyorrhexis involving especially the granular series of leucocytes was noted. Placenta. In view of the frequency with which ewes suffering from Rift Valley fever abort, the placentas of female mice and rats dying while pregnant were examined. The only abnormal change was an invasion of the muscular wall of the uterus and the deeper layers of the decidua by large numbers of polymorphonuclear leucocytes, many of which were breaking down and undergoing karyorrhexis. (Plate V, Fig. 6.) Foetal Liver. Examination of the livers from rat and mouse f~etuses whose mothers had

G. M. FINDLAY.

~5,~

died while pregnant did not show any actual breaking down of the liver cells or neerotic loci. There was, however, a considerable number of polymorphonuclear leucocytes present. (Plate VI, Fig. 1.) " INTRANUCLEAR

INCLUSIONS."

In a disease having certain resemblances to yellow fever search has naturally been made for the presence of what have been perhaps unfortunately named " intranuclear inclusions." These " intranuclear inclusions," which have now been found in association with herpes, virus III infections in rabbits and salivary gland infections in guineapigs as well as in yellow fever cannot be regarded as " inclusion bodies " in the sense that the Bollinger bodies of fowl pox or the Guarnieri bodies of vaccinia are specific although their presence may indicate that the cell has reacted to the presence of a virus. In certain cases these acidophilic " intranuclear bodies " would seem to be the acidophilic nucleoli or plasmosomes which have been rendered more prominent by the disappearance of the thin film of chromatin which normally coats the plasmosome nucleolus. This may be the explanation of the acidophilic bodies which have been described by DAUBNEY, HUDSON and GARNHAM (1931) as occurring in the livers of sheep suffering from Rift Valley fever. In the present study search has been made for these intranuclear bodies and they have been found most plentifully in the liver of Lamb IV, but also in the livers of the goats and in certain of the rat livers. They stain intensely with eosin and other acidophilic dyes but are not stained by the Feulgen technique. (Plate II. Fig. 6.) Very careful search was also made in the livers of mice for similar intranuclear bodies and in the hope of detecting them mice were killed before they had developed any symptoms of illness. In the liver cells of certain mice there were found definite acidophilic bodies, not unlike the " intranuclear inclusions " associated with virus III infections and herpes. These bodies were round or oval in shape and had a finely granular appearance. They were surrounded by a very definite membrane in contradistinction to the small acidophilic masses in the livers of sheep and goats infected with Rift Valley fever. When small the whole of the space within the membrane was occupied by finely granular material, but as the bodies increased in size the granular material failed to occupy the whole of the space. Not infrequently there were two or more of the acidophilic bodies within the same nucleus. In sections stained by the Feulgen technique the granular material failed to stain thus showing that it is not composed of chromatin, the membrane however took on the stain and is apparently composed of chromatin. When very large, these bodies may take up almost the whole of the nucleus, the chromatin of which is reduced to a narrow ring in contact with the nuclear membrane. The fact that the " intranuclear bodies " with a definite limiting membrane

~4

RIFT VALLEY FEVER OR ENZOOTIC HEPATITIS.

found in the mouse differed morphologically from the small acidophilic masses found in the liver cells of the lamb and goats, raised doubts as to whether the mouse bodies had any relation to Rift Valley fever. T h e y were not constantly present in all mice infected with Rift Valley fever and they were not found in the liver cells of other small rodents dying of this disease. Eventually the same " intranuclear bodies " were found in the livers of mice which had not been brought into this laboratory and therefore could not have been exposed to infection with the virus of Rift Valley fever. After examining the livers from mice of various strains it was found that the " intranuclear bodies " were limited to one strain of mice supplied by a certain L o n d o n dealer. So far these intranuclear bodies have not been found in other organs of the mouse except the liver. F r o m their morphological appearances these bodies are most probably hypertrophic nucleoli. T h e y will form the subject of a further communication. DISTRIBUTION

OF THE V I R U S IN THE TISSUES.

T h e presence of the virus in whole blood has already been described. In order to determine whether the virus was wholly attached to the corpuscles or was free in the plasma, blood from Rat 20 was passed into heparin, centrifuged and the plasma pipetted off. T h e cells were twice washed in physiological saline and resuspended. F o u r mice were then inoculated with plasma, four with cells. All mice died within four days. This confirms the experiment of DAUBNEY, HUDSON and GARNHAM (1931) who found that in the sheep virus was present both in the plasma and attached to the blood cells. In order to determine the presence of virus in other tissues, mice were, as far as possible, exsanguinated, the various organs were removed, finely minced and twice washed with saline. F o u r mice were then inoculated with 0.1 c.cm. of a suspension of each organ. T h e following organs were found to contain virus : - Liver Spleen Lung Testicle Adrenal

.. .. .. .. ..

+ + + + +

+ + + + +

+ + + + +

+ + + + +

Kidney .. Brain .. Sciatic nerve Skin ..

+ + + + + + + + + + + + + -f-

T h e suspension of skin alone failed to kill all the mice inoculated, two out of the four mice thus inoculated failing to succumb to the infection. In the case of yellow fever virus inoculated into mice it has been found by THEILER (1930) that the virus is definitely neurotropie and is located in the nervous system and medulla of the suprarenals. In mice inoculated intracerebrally with the virus of Rift Valley fever it has been found that the blood, liver, spleen and other organs all contain the virus at death. T h e virus was not present in the urine either of the human case (A.E.G.) or of L a m b 1V.

C. M. FINDLAY.

~55

The virus of Rift Valley fever may, however, pass through the placenta of pregnant animals and infect the foetal tissues, as is shown by the following experiment. Two foetuses from Mouse 959 dying of Rift Valley fever were removed from the membranes and washed four times in physiological saline. The abdomens were then opened and the foetal livers and spleens removed, minced and ground up in broth, filtered through a British Berkefeld candle and 0.5 c.cm. of the filtrate injected into four mice. All four mice died within four and a half days. Similar results have been obtained in other experiments. DURATION OF INFECTIVITY.

In order to determine how soon the blood becomes infective after intraperitoneal injection, blood was removed from Monkey 9 twenty minutes after inoculation and injected into four mice, all of which died with Rift Valley fever. In this monkey the blood remained infective for seven days after inoculation. Monkey 6 remained infective for thirteen days after inoculation, though it at no time showed any definite fever; Monkey 8 was infective for eleven days after inoculation, long after the cessation of fever. Monkey 13, infected intranasally, did not show the presence of virus in the blood until thirty hours after inoculation. It then remained infective for five days. In the case of the human volunteer infected by DAUBNEY, HUDSON and GARNHAM (1931) the blood was not tested for infectivity during the incubation period but virus could be demonstrated in the blood for six days after the first rise of temperature or nine days after the inoculation. This agrees very closely with the findings in the case of A.E.G. who became ill on the sixth day after being exposed to infection and was found to have virus present in the blood on the seventh and eighth days after infection. The ewe infected intraperitoneally with a sterile filtrate of the livers of Mice 848 and 850, was found to have virus in the blood twelve hours after inoculation. The blood remained infective for six days, that is to say for two days after the temperature had become normal. Lamb III inoculated with virus-containing blood kept for five months in the ice chest at a temperature of 4 ° C. also remained infective for seven days. The blood of mice and rats inoculated intraperitoneally has been found to contain virus thirty minutes after injection. Rat's blood and liver contained live virus eleven and fifteen days after inoculation but was non-infective twenty days after injection. As in the case of yellow fever, it is probable that the apparent disappearance of virus from the blood and tissues is due not necessarily to the death of the virus but merely to its neutralization in the presence of immune bodies. Experiments are at present being undertaken to establish this hypothesis. The fact that in the rat the formation of immune bodies is rather deficient may possibly be correlated with the somewhat longer period, as compared with larger animals, during which the presence of living virus may be demonstrated in the tissues.

256

RIFT VALLEY FEVER OR ENZOOTIC HEPATITIS. IMMUNITY.

Immunity has been studied by attempting to reinfect animals which have previously suffered from an attack of Rift Valley fever and by demonstrating in the serum of recovered animals the presence of antibodies capable of neutralizing the pathogenic action of the virus. As these experiments are still in progress only a very brief summary of the results so far obtained will be given here. Monkeys which have suffered from Rift Valley fever cannot be reinfected with massive doses of the virus for at least six months (the longest period for which it has so far been possible to test). Failure to react here implies the absence of any temperature reaction or change in the leucocyte count and the absence of infective power of the blood for mice. A ewe and a lamb reinfected with a massive dose of virus four weeks after an attack of Rift Valley fever also failed to react in any way. Rats which have recovered from one injection of the virus are immune to further infection for from four to six weeks, but after this period may succumb to large doses of the virus. Comparatively few mice have survived a first injection with the virus and of those which have survived the majority have succumbed to a second injection given at an interval of four weeks. Some evidence has, however, been obtained that a slight degree of immunity may exist in mice for from two to three weeks after an infection. The presence of protective antibodies in the serum has been demonstrated by injecting a series of mice with 0.1 c.cm. of the serum to be tested, and with increasing dilutions of blood containing virus in phosphate buffer at p H 7.2, a control series of mice being injected with the same dilutions of virus and with 0.1 c.cm. of normal serum. At least three mice were injected witheach dilution of virus. By this means it has been possible to determine that (i) in man protective antibodies are present in the blood of recovered cases, (three), for at least five months after infection ; (ii) such antibodies are present in the blood fourteen days after the onset of clinical symptoms (A.E.G.) ; (iii) the antibody titre increases after about three weeks; (iv) protective antibodies capable of neutralizing 100 M.I.D. may appear in the serum of those exposed to infection in the absence of any clinical symptoms due to infection with the virus of Rift Valley fever (G.M.F.). In monkeys recovered from the disease protective antibodies, capable of neutralizing from 100 to 1,000 minimal infective doses have been detected twelve days after injection with the virus. Such protective antibodies have been found to be still present six months after the injection of virus. In the Dorset horned ewe and her lamb (Lamb III) protective antibodies appeared after twelve and sixteen days respectively. In rats the presence of the same antibodies has been detected but only in very low titres, usually a capacity for neutralizing not more than ten M.I.D.

G. M. FINDLAY.

257

PHYSICAL PROPERTIES OF THE VIRUS.

Comparatively few observations have so far been made on the physical properties of the virus. The presence or absence of the virus has in each of the following experiments been determined by the results obtained with injected mice.

Filterability.--The virus in plasma suspended in physiological saline or phosphate buffer at pH 7.2 passes through Berkefeld N, V and W candles; it also passes through Chamberland L2 and Ls candles at a negative pressure of 630 mm. Hg. without any loss in virulence• L.~ and L7 candles, however, caused some loss of virulence but when the plasma was mixed with sterile broth there was no diminution in potency• Survival in vitro.--Blood preserved in oxalate-carbolic-glycerine has retained slight virulence for mice when preserved in the ice chest at 4 ° C. for eight months. Two out of ten mice died in forty-eight hours when inoculated with 0-1 c.cm. of the blood. Defibrinated blood to which had been added 0.5 per cent• carbolic acid retained its virulence for mice unimpaired for six months when similarly kept in the ice chest at ~- 4 ° C. Its pathogenicity for lambs (Lamb III) however had decreased• Blood in phosphate buffer at pH 7.2 was still active when kept at 56 ° C. for twenty minutes but had lost its virulence when heated at the same temperature for forty minutes. As is the case with yellow fever virus, the virus of Rift Valley fever is sensitive to the hydrogen ion concentration of the fluid in which it is kept• Blood from Mouse 388 was withdrawn into heparin ; equal amounts of blood were then added to test tubes each containing 1 c.cm. of phosphate buffers ranging from pH 6-6 to pH 8.0. One series of tubes was kept at room temperature, the other at 37 ° C. ; after twenty hours two mice were injected intraperitoneally with 0-1 e.cm. of each suspension. The results are shown in the following Table III : SURVIVAL OF VIRUS IN RELATION TO TEMPERATURE AND pH.

Virus kept for 20 hours at room temperature, pH 6.6 pH 6.9

Died after 5 days Survived Died after 2 days 3

3 3

pH 7.3 pH 8"0

Survived

Virus kept for 20 hour., at 37° C Survived Died' after 3 days Survived Died after 2 days 3 Su~civecl'

9'~8 •

RIFT VALLEY FEVER OR ENZOOTIC HEPATITIS.

At the temperature of the incubator the virus was inactivated at pH 6.6, while at room temperature there appears to have been partial inactivation. At pH 8.0 the virus was destroyed both at incubator temperature and at ordinary room temperature. Infected mouse blood dried in vacuo over phosphorus pentoxide and kept at a temperature of 4 ° C. has survived for at least six weeks. Infected mouse liver similarly dried retained its activity for four weeks but had become much attenuated in virulence after six weeks. RELATION TO OTHER DISEASES.

Both from its clinical features in man and from the pathological changes which it produces in animals, Rift Valley fever, as DAUBNEY, HUDSON and GARNHAM (1931) point out bears certain resemblances to yellow fever, dengue and sandfly fever. The fact that mice may be protected against infection with Rift Valley fever by the injection, at the same time as the virus, of serum containing immune bodies against Rift Valley fever suggested a method of determining whether yellow fever immune serum has any protective action against Rift Valley fever. The serum of I.H. who suffered from yellow fever twelve months previously was available for this test. Twelve mice were injected each with 0-2 c.cm. of yellow fever immune serum and twelve mice with the same amount of normal human serum. Two mice from each series received0.1 c.cm. of a dilution of infective blood at pH 7.2, the dilutions ranging from 1 in 100 to 1 in 10,000,000. All the mice receiving dilutions up to 1 in 1,000,000 died, whether injected with yellow fever immune serum or with normal human serum. One mouse, injected with the yellow fever immune serum and receiving a dilution of blood 1 in 10,000,000 also died. DINGER and SNIJDERS (1931) have recently made the interesting observation that monkeys previously infected with dengue are subsequently immune to yellow fever. An experiment was therefore carried out to determine whether yellow fever had any power to immunize against Rift Valley fever. Through the kindness of Dr. E. HINDLE, three monkeys, which had suffered from yellow fever eighteen months previously were available, in addition to one monkey which had received a yellow fever vaccine. All these monkeys (Nos. 8, 9, 13 and 14) became infected with Rift Valley fever. There is therefore no evidence to suggest that yellow fever and Rift Valley fever are in any way directly related. Unfortunately it has not yet been possible to determine whether monkeys previously infected with dengue are subsequently immune to Rift Valley fever inoculation. Serum, however, was kindly provided by Dr. M. P. O ' C O N N O R , of the Federated Malay States Medical Service who had suffered from a typical attack of dengue fever three years previously.

G. M. FINDLAY.

259

A protective experiment was therefore carried out on exactly similar lines to that already described for yellow fever. The dengue immune serum had no protective power against the virus in mice. Similar experiments are being carried out with sandfly immune serum. Though there is some uncertainty as to how long immunity to sandfly fever endures, it may be noted that the writer suffered from sandfly fever in 1918, and, as previously described, his serum, when first tested, was found to be entirely devoid of protective antibodies against Rift Valley fever. Owing to the fact that psittacosis is also pathogenic for mice, monkeys and man it appeared to be of interest to determine whether an anti-psittacosis serum has any protective action against Rift Valley fever in mice. Dr. BEDSON of the London Hospital very kindly supplied me with an anti-psittacosis serum which was known to be capable of neutralizing 100 minimal infective doses in mice. A protective experiment, as for yellow fever and dengue, was therefore carried out but no evidence that the anti-psittacosis serum exerted any protective action was obtained. DISCUSSION. The observations of DAUBNEY,HUDSON and GARNHAM (1931) have thus been confirmed and extended by the experiments here described. In addition to the pathogenicity of the virus for men and sheep it has been possible to show that mice and other small rodents are extremely susceptible to Rift Valley fever. The injection of mice with blood from suspected cases of Rift Valley fever thus provides a ready means of diagnosis and serves to differentiate Rift Valley fever from yellow fever, dengue and sandfly fever since adult mice can only be infected with yellow fever from man or monkey by intracerebral injection while dengue and sandfly fever cannot be transmitted to mice. The extreme susceptibility of small rodents to the virus of Rift Valley fever raises the important question as to whether outbreaks of Rift Valley fever in sheep and cattle may not be related to certain epidemic outbreaks in small rodents. There is in fact some evidence to suggest that the rodent " plagues," which, as ELTON (1931) has shown, are always associated with the periodic increase in numbers of small rodents, may coincide with outbreaks of Rift Valley fever. The disease has undoubtedly existed in the Rift Valley in Kenya for some years, and MONTGOMERY (1913) almost certainly encountered the same condition in sheep in 1912 in the neighbourhood of Lake Naivasha. In 1926, as DAUBNEY, HUDSON and GARNHAM (1931) point out, at least three stockowners suffered a heavy mortality among ewes, with a high abortion rate. There is also a history in this year of numerous abortions among cattle and the occurrence of a dengue-like fever both among natives and Europeans. The incidence of malaria was also the highest for some years. Mr. C. ELTON, of the Department of Zoology and Comparative Anatomy, Oxford, informs me that from information furnished by Dr. W. FLETCHER,there are records in 1926 of a large increase in the number of small rodents at a point some fifty miles north of Lake Naivasha.

260

RIFT VALLEY FEVER OR ENZOOTIC HEPATITIS.

Unfortunately when, in July, 1930, the original outbreak was being investigated no observations were made on any other diseases which might have been occurring at the same time in wild animals. Mr. DAUBNEY, however, has recently informed me that an increased mortality among small rodents did occur during the summer months in the Rift Valley, though at the time it was thought that the deaths were due to infection with Bacillus pestis. Field observations are at present being undertaken to determine whether outbreaks of Rift Valley fever in sheep are in any way correlated with similar outbreaks in wild rodents. The investigations of DAUBNEY, HUDSON and GARNHAM (1931),together with the observations here recorded, prove conclusively that certain cases of pyrexia of unknown origin occurring in the tropics are due to infection with a specific virus, the virus of Rift Valley fever, the presence of which can be readily detected by the inoculation of mice with infected material. It is possible that further research will show that this specific virus infection occurs not only in Kenya, but in many other parts of the tropics.

CONCLUSIONS. (1) The observations of DAUBNEY, HUDSON and GARNHAM (1931) on the occurrence of a specific infection--Rift Valley fever--in sheep, lambs and goats due to a filterable virus have been confirmed. (2) The virus produces a dengue-like disease in man, characterized clinically by fever, rigors, headache, and muscular pains. In the blood there is a primary polymorphonuclear leucocytosis followed by a leucopenia. Three cases of laboratory infection have occurred in this country. (3) Protective antibodies appear in the serum of those who have suffered from Rift Valley fever. (4) Protective antibodies may also appear in the blood of persons who have been exposed to subinfective doses of the virus but have shown no clinical symptoms of illness. (5) The virus of Rift Valley fever produces in monkeys (Macacus rhesus) fever associated with blood changes similar to those recorded in man. The virus can be passaged in monkey but the clinical symptoms are of decreased severity. (6) No fatal cases have occurred in monkeys. (7) Monkeys which have suffered from one attack of Rift Valley fever are immune for at least six months to further inoculations of the virus. (8) Protective antibodies make their appearance in the serum of monkeys recovered from Rift Valley fever.

G. M. FINDLAY.

261

(9) Cats exhibit a transitory infection, associated with slight fever. (10) The virus of Rift Valley fever is highly pathogenic for mice, field voles (Microtus agrestis), wood mice (Apodemus svlvaticus), dormice (Muscardinus avellanarius), and golden hamsters [Gricetus (Mesocricetus) auratus]. The death rate is from 98 to 100 per cent. and occurs in from thirty-six to ninety-six hours after inoculation. (11) Rats are also very susceptible to the virus, the death rate being about 50 per cent. after subcutaneous or intraperitoneal injection, nearly 100 per cent. after intracerebral or intratesticular inoculation. (12) Rabbits are non-susceptible but may possibly on occasions allow the virus to survive for a short time in the blood. (13) The guineapig, mongoose, hedgehog, tortoise, frog, hen, pigeon, canary and parakeet are insusceptible. (14) In susceptible animals the infection can be transmitted by subcutaneous, intraperitoneal, intratesticular or intracerebral inoculation, by application to the scarified skin, and by instillation into the nares or conjunctival sac. (15) After repeated passage through mice and rats the virus has retained its pathogenicity for sheep, Iambs, goats and man. (16) The pathological changes consist of a focal necrosis of the liver. The loci may be discreet, as in adult sheep, goats and monkeys or may coalesce so as to involve the whole of the liver as in rats, mice and other small rodents. (17) Degenerated polymorphonuclear leucocytes and a hyaline degeneration of the cytoplasm are characteristic of the necrosis. (18) Acidophilic material is present in a few nuclei of the liver ceils of the sheep, goat and of some rats and wood mice. (19) Acidophilic bodies were found in the nuclei of the liver cells of certain mice infected with Rift Valley fever, and also in the livers of control mice. (20) Hzemorrhages and karyorrhexis of polymorphonuclear leucocytes were found in other organs--mesenteric lymph glands, spleen, heart, testicle, and cardiac end of stomach. (21) The virus is widely distributed in the body and can pass through the placenta to the foetal organs. (22) Certain observations on the physical properties of the virus are described. (23) Monkeys immune to yellow fever are susceptible to Rift Valley fever. Human immune yellow fever serum does not protect mice against the virus of Rift Valley fever. D

262

R I F T V A L L E Y FEVER O R E N Z O O T I C n E P A T I T I S .

(24) H u m a n d e n g u e i m m u n e s e r u m d o e s n o t p r o t e c t m i c e a g a i n s t i n f e c t i o n nor does psittacosis immune serum. (25) T h e p o s s i b i l i t y t h a t t h e v i r u s m a y p r o d u c e e p i d e m i c s a m o n g r o d e n t s u n d e r n a t u r a l c o n d i t i o n s is d i s c u s s e d .

REFERENCES.

COUNCILMAN,W. T . (1890). Report on etiology and prevention of yellow- fever. Marine Hosp. Service. 151. DAUBNEY, R., HUDSON, J. R. and GARNHAM, P. C. Valley fever. J/. Path. & Bact., xxxi¢, 543. DINGER, J. E., and SNIJDERS, E . P . Trop.-Hyg., xxxv, 497.

(1931).

U.S.

Enzootic hepatitis or Rift o.

(1931). Dengue und Gelbfieber. Arch. f. Schiffs. u.

ELTON, C. (1931). T h e study of epidemic diseases among wild animals. Jl. ofHyg., xxxi, 435.

FINDLAY, G. M., and DAUBNEY,R. (1931). T h e virus of Rift Valley fever or Enzootic hepatitis. Lancet, ii, 1350. JOANNIDES, G. (1929). L'examen morphologique du sang dans la fi~vre dengue. Med. (January.)

Athens

KLOTZ, O., and BELT, T. H. (1930). T h e pathology of the liver in yellow fever. Amer.Jl. Path., vi, 663. MONTEIRO, J. L. (1930). Sobrevivencia do virus amarillico no organismo de certos animaes domesticos. Brazil Med., xliv, 1087.

MONTGOMERY, R. E. (1913). Ann. Rep. Dep. Agriculture, Kenya Colony. Report of the Veterinary Department (R. J. STORDY, Chief Vet. Officer) for the year 1912-13. THEILER, M. (1930). Studies on the action of yellow fever virus in mice. Ann. Trop. Med. & Parasit., xxiv. 249.

PLATE

II.

FIG, 1.--Liver of Lamb I, died thirty-six hours after inoculation. Complete necrosis of liver tissue. × 125. H~em. and eosin. C.B. FIG. 2 . - - L i v e r of Lamb I. Last stages of a necrotic focus. The polymorphonuclea. leucocytes and histiocytes have undergone karyorrhexis. × 250. H~em and eosin. C.Br. Fro. 3 . - - L i v e r of Lamb II, died thirty-six hours after inoculation. Necrosis of the liver with separation and rounding up of the liver cells, h~emorrhage and nuclear karyorrhexis × 250. H~em. and eosin. C.B. FIG. 4 . - - L i v e r of Lamb IV, killed seventeen hours after inoculation. Necrotic loci in the liver tending to coalesce. × 200. Ham1. and eosin. C.B. FIG. 5 . - - L i v e r of Lamb IV. Edge of necrotic focus : hyaline degeneration--" Councihnan " lesion--in the cytoplasm. × 400. Hmm. and eosin. C.B. F l a . 6 . - - L i v e r of Lamb IV. Edge of necrotic focus. T h e chromatin in nuclei of the liver cells forms a ring round the nuclear membrane, leaving acidophilic material in the centre of the nucleus. × 800. Hmm and eosin. C.B.

PLATE II

Figs. 1-6,

PLATE

FIG. 1 . - - L i v e r of G o a t I. FIG. 2 . - - L i v e r FIG. 3 . - - L i v e r Fie,. 4 . - - L i v e r Fro. 5 . - - L i v e r

FIG. 6 . - - L i v e r

III.

N e c r o t i c focus a ~ a c e n t to the central vein. × 125. H m m . a n d eosin. C.B. of G o a t I. N e c r o t i c focus. • 250. H ~ m . a n d v a n G i e s o n . C.B. of G o a t I I . L a r g e discrete focus i n v o l v i n g a portal tract. × 125. Haem. a n d van Gieson. C.lq. of M o n k e y II inoculated w i t h blood of L a m b I. N e c r o t i c focus, × 125. F a t t y d e g e n e r a t i o n of liver cells. H m m . a n d eosin. C.B. of M o n k e y V inoculated w i t h blood f r o m h u m a n case of Rift Valley fever t C . E . H . ) . N e c r o t i c focus. × 2 5 0 . H~em. a n d eosin. C.B. of M o n k e y V small necrotic focu,s, >~ 250. Hoem, and v a n Gie,son, C.B.

PLATE I I I .

Figs. I-6.

PLATE FIG. F~c. Fro. FiG. FiG.

IV.

l . - - L i v e r of Cat 3. Small necrotic loci. :< 25~}. lt~em, a n d cosin. C.B. 2 . - - M e s e n t e r i c gland of Cat 3. H m m o r r h a g e . /. 125. H m m . a n d eosin. C.B. 3 . - - L i v e r of R a t 5. Early stage of necrosis, x 125. Haem. and eosin. C.B. 4 . - - L i v e r of Rat 12. A d v a n c e d stage of necrosis. :~ 125. Hama. a n d eosin. C.B. 5 . - - L i v e r of R a t 12. L i v e r cells separated a n d r o u n d e d : Nuclei necrotic. × 250. Haem. and eosin. C.B. FIC. 6 . - - L i v e r of Rat 67. C o m p l e t e necrosis, x 25ti. H m m . and eosin. C.B.

PLATE IV.

Figs. 1-6.

PI~ATE

V.

l q u . I.---Spleen of P,at 67 : sinuses packed with red blood corpuscles and p o l y m o r p h o nuclear leucocytes, m a n y of w h i c h are u n d e r g o i n g karyorrhexis. " 2S0. lt~em, and eosin. C.B. t~'I(;. 2 . - - S t o m a c h of Rat 2 : haemorrhage into the s u b m u c o u s coat at the cardiac end of the stomach. 125. t-Iaem, and eosin. C.B. Fro. ; L - - T e s t i c l e of Rat 6 inoculated s u b c u t a n e o u s l y with the virus of Rift Valley fever : hmmorrhage, x 125. Hmm. and eosin. C.B. Fro. 4 . - - T e s t i c ! e of Rat 68 inoculated intratesticularly : at the site of injection red blood corpuscles and p o l y m o r p h o n u c l e a r cells u n d e r g o i n g karyorrhexis : degeneration of secreting cells. ,< 125. H m m . and eosin. C.B. I~q<:. 5 . - - T e s t i c l e of Rat 68 : degeneration and formation of giant cells in the secretin~ tubules, x 250. Ha:m. and eosin. C.B. lq'm. 6 . - - P l a c e n t a of Rat 5 : infiltration of d e e p e r layers of decidua with p o l y m o r p h o n u c l e a r leucocytes. :, 25(I. Haem. and eosin. C.B.

PLATE V,

FiRs, 1-6,

PLATE

VI.

FIG, 1 . - - L i v e r of foetus f r o m Rat 5 : m a n y p o l y m o r p h o n u c l e a r leucocytes are p r e s e n t . × 125. H~em, and eosin. C.B. FIG. 2 . - - L i v e r of M o u s e 286 w i t h Rift Valley f e v e r : acute necrosis of liver. × 125. H~em. and eosin. C.B. FIG. 3 . - - L i v e r of M o u s e 780 : separation a n d r o u n d i n g of liver cells : infiltration w i t h p o l y m o r p h o n u c l e a r leucocytes. × 25(i), Harm. and eosin. C,B. Fie,. 4 . - - M e s e n t e r i c gland of M o u s e 33 : h~emorrhage. × 125. H~em. a n d eosin. C.B. Fro. 5 . - - L i v e r of Field Vole : acute necrosis. × 250. H m m . and eosin. C.B. FIG, (:i.--Liver of G o l d e n H a m s t e r : acute necrosis a n d infiltration w i t h p o l y m o r p h o n u c l e a r leucocytes, × 250, Haem. a n d eosin, C.B,

~', 4,

N

PLATE VI

Figs. 1-6.