De Meillon, Botha

Kokernot, R.H.

Paterson, H.E.

English

AJOL - African Journals Online

546 S.A. MEDICAL JOURNAL 24 May 1958STUDIES 0 THE TRANSMISSIO OF WESSELSBRON VIRUS BY AEDES(OCHLEROTATUS) CABALLUS (THEO.)*R. H. KOKERNOT, D.V.M., M.D., M.P.H., Rockefe/ler FoundationH. E. PATERSO ,RSe. Ho's. (RA ID), Poliomyelitis Research Foundation, South African Institute for Medical ResearchandBOTHA DE MEll_LO ,D.Se. (RA D), PH.D. (S.A.), South African Institute for Medical ResearchTABLE I. COURSE OF WESSE1.S8RON INFEcrJON L'l LAMB AN 2348 WHICH WAS SOURCEOF INFECTIVE FEED FOR LAllORATORY-!lEAR£D Aedes cabal/us, LOTS 35 AND 36Dau Hour Temperature Remarks9.4.57 18.00 ot recorded Exposed I hour to 40 wild-<:aught Aedescabal/us collected 9.4.57 biting man.On the 19th post-feeding day one of the 14 Aedes cabal/usin lot 35 died. A suspension was made of this mosquito andinoculated into infant mice. An identification test confirmedthe presence of Wesselsbron virus in this mosquito (Table lIT,experiment 2). After the lapse of 21 and 22 days, selectedFate a/miceI moribund 9th day-eonfirmedtransmission (Table Ill, experi-ment 3). 6 remained well.I sick 7th day. 6 sick 8th dayConfirmed transmission bymeans of pooled brains or 2mice.7/722 days3536r08.3O 100· 2°F11.4.57 112.00 101·615 50 104·4 14 Aedes cabal/us. lot 35, engorged; lamb22.15 103·6 then bled and Wesselsbron viraemia con-firmed.f8.25102'81 .00 103·812.4.57 16.00 105·3 Confirmed Wesselsbron viraemia.20.00 104'4 {During this 2-hour interval 6 A. cabal/us22.00 104·0 lot 36, engorged.f 08.5O 102'713.4.57 11.50 102·8L16.15 104·3 Anorexic, dyspnoeic and weak.14.4.57 {OS.15 101'3 Critically ill.12.00 Died-Wessel bTOn virus isolated fromJiver, kidney and spleen.TABLE 11. FATE OF ;£\VBQRN MICE BITTEN BY Aedes cabal/us WHICH HAD PREV10USLYENGORGED ON A LAMB (AN 2348) CIRCULATING WESSEl.SBRON VIRUSIntervalbetween umber of ApparentMosquito feeding on mosquitoes trans-JOt no. Jamb and biting missionmice mice rate21 days 6RESULTSWessel bron virus was isolated from the 6 Aedes caba/lus(AR 731) that engorged on lamb A 2348 on 9 April 1957,and from the 34 (AR 732) showing no evidence of engorge-ment.On the 2nd, 3rd and 4th days after exposure to 40 Aedescaba/lus mosquitoes (AR 731 and AR 732), lamb A 2348had a temperature in excess of 104°F. The 4th day it wasanorexic, dyspnoeic and weak. Death occurred on the 5th day.Lots 35 and 36 Aedes caba/lus were allowed to engorge whenthe temperature was in excess of 104°F and during a periodwhen Wesselsbron virus was circulating, as confirmed bysubsequent specificity test. These details are shown in Table I.ob erved to probe or engorge on 3I newborn mice heldfirmly over the orifices of the tubes. Each mouse was thusbitten by more than one mosquito. After exposure, theinfant mice were returned to their respective mothers. Themosquitoes were then pooled in lots of 50, 50 and 34 anddesignated AR 749, AR 750 and AR 751 respectively. Eacblot was killed by being placed at - 20°C and then processedin the usual manner.During the tudy of an epizootic in the eastern Cape Province,Wessel bron viru was isolated from sheep (R. A. A1exanderl ).In the course of subsequent investigations by us on the farmCrowborough lei undertaken principally to identify the vectoror vector of the disease, further i olations were made fromsheep, from 2 species of mosquitoes (Kokernot et a1. 2) andfrom man (Heymann et al. 3). Aedes (Ochlerotatus) caba/lus(Theo.) was the prevalent mo quito and one of the two speciesfrom which we i olated Wes el bron virus. The purpose ofthis report is to present results which demonstrate the abilityof Aedes cabal/us to transmit that agent.METHODS AND MATERIALSWith only the few exceptions mentioned below the methodsand material employed in these experiments were the sameas those previou Iy described (Kokernot et al.4).lamb (A T 2348) obtained on 9 April 1957 from Groot-fontein Agricultural College, Middelburg, Cape Province,was exposed to 40 Aedes caba/lus females caught in theepizootic area the afternoon of the same day. The head andneck of the lamb were placed through the sleeve of a gauzecage measuring 14 x 14 x 14 inches. The legs of the animalwere secured to assure restraint for a period of approximatelyI hour between 18.00 and 19.00 hours. During this interval6 of the mosquitoes engorged and were pooled and designatedAR 731. The remaining 34 mosquitoes showed no visibleevidence of having engorged and were pooled and designatedAR 732. Both lots were stored on dry ice overnight and thefollowing day processed and inoculated into litters of newbornand adult mice in Johannesburg.lmmediately after exposure the lamb and mosquitoes weretransported by car in an overnight journey to Johannesburg.Aedes caba/lus that had been reared in the laboratory fromlarvae were allowed to engorge on lamb A 2348 on 2occasions. The first time was 46 hours after the initial expo-Sl:ITe to AR 731, when the lamb's temperature was 104'4°F,and 14 of the laboratory-reared Aedes caba/lus (lot 35)engorged through the gauze covers of glass vials in which theywere confined. On the third day, when its temperature was104°F or over, the lamb was placed in ide a plastic-screenedcage measuring 30 x 24 x 21 inches, and in a 2-hour periodfrom 20.00 to 22.00 hours 6 laboratory-reared Aedes caba/lus(lot 36) engorged.Other Aedes cabal/us caught biting man in the epizooticarea on the afternoon of 9 April were collected in glass tubeslined with strips of saturated absorbent paper and transportedalive to Johannesburg. The next day 134 of these were• The studies and observations on which this paper is basedwere financed jointly by the South African Institute for MedicalResearch, the Poliomyelitis Research Foundation, the SouthAfrican Council for Scientific and Industrial Research, the UnionHealth Department, and the Rockefeller Foundation, and wereconducted with the collaboration of the Veterinary Division ofthe Department of Agriculture.24 Mei 1958 S.A. TYDSKRIF VIR GENEESKUNDE 547mosquitoes of lots 35 and 36 respectively were allowed tobite newborn mice. The results are summarized in Table II.Although 6 mosquitoes of lot 35 were utilized in biting new-born mice many times, only one mouse developed illnessduring the 21-day observation period. The illness in thismouse was confirmed as being due to Wesselsbron irus(Table ill, experiment 3). The 3 mosquitoes of lot 36 bitanother group of 7 newborn mice with 100% apparent trans-mission rate. Two of these mice, sick on the 8th post-bitingday, were sacrificed and their brains pooled and then set upin a neutralization test to confirm Wesselsbron virus trans-mission. Over 4 logs of the agent in the pooled mou e brainswas neutralized by a post-inoculation Wesselsbron guinea-pig serum when compared to the pre-inoculation serum.Thus, laboratory-reared Aedes caballus from 2 lots differen-tiated by the time and method of exposure for engorgementon a lamb circulating Wesselsbron virus were able to transmitthe virus to infant mice. The details confirming Wesselsbronvirus as the agent involved in a successful lamb--Aedescaballlls-mouse transmission are shown in Table ill.In addition to the transmission of Wesselsbron virus bythe technique described above with laboratory-reared Aedescaballus, it was also transmitted by wild-caught mosquitoes(AR 749, AR 750 and AR 751). However, only one of the31 mice bitten 2 to 4 times by a group of 134 such mosquitoessickened. In a neutralization test the aetiology of the illnessas having been due to Wesselsbron-virus infection was con-firmed.The three lots into which the 134 wild-caught Aedescaballlls were divided for processing after having bitten mice(lots nos. 749, 750 and 751) yielded 4 strains of virus. FromAR 749 a mixture of Middelburg (Kokernot et at.") andWesselsbron viruses was isolated. Wesselsbron virus wasisolated from AR 750 and Middelburg virus from AR 751.DISCUSSIONWesselsbron virus was isolated from tbe wild-eaught Aedescaballlls (AR 731) that fed on lamb AN 2348 and this animalwas subsequently proved to be infected with that virus.Tevertheless, this cannot be regarded with certainty as anexperimental transmis ion becau e the lamb wa obtainedfrom a flock of sheep in which morbidity and mortality dueto Wesselsbron virus were occurring.· When obtained, thelamb appeared healthy, but it is po sible that Wessel bronvirus infection, acquired from a different ource than mos-quitoes of lot AR 731 or AR 732, was in an incubation phase.Regardless of the source of the lamb's infection, the We els-bron virus \ hich was present in its blood served a theinfecting feed for 2 lots of laboratory-reared Aedes caballlls(lots 35 and 36).A succes rul transmission by means of mo quitoes collectedfrom nature within such a short period as 24 hours mightinclude specimens capable only of mechanical transmission.However, the results presented include successful transmissionby laboratory-reared Aedes caballlls after a 21- and 22-dayspost-infective feeding period. There is a significant differencebetween the transmission rate to mice by bite of mosquitoesin lots 35 and 36. Lot 35 mosquitoes were exposed to A 2348a day earlier in the course of the illness, and it is probablethat at that time the viraemia level was at a low infectivethreshold. The two lots of mosquitoes were expo ed to thelamb by different methods. Lot 35 mosquitoes were main-tained individually in glass vials. They were allowed toprobe and feed upon the lamb through the gauze-coveredorifice. Lot 36 mosquitoes became engorged with blood byfeeding upon the lamb restrained in the cage with the mos-quitoes.The significance of Aedes caballus as a vector of Rift Valleyfever virus (Gear et al. 5 ) and Middelburg virus (Kokernotet a1.4) has previously been reported. It is apparent from thecurrent results that it is also a vector of Wesselsbron virus.The bionomics of this little-studied species merits intensiveinvestigation with the hope that some practical method ofcontrol will be revealed.Aedes caballus is the second member belonging to this• The assistance of Dr. C. W. A. Belonje, Senior State Veteri-narian, in the collection of field specimens from sheep is gratefullyacknowledged.TABLE ill. RESULTS OF 3 NEUTRALlZATlON EXPERIMENTS THAT CONFIRM \VESSELSBRON VIRUS AS BEl G THE AGE"'T IN A SUCCESSFUL LAMB-Aedes Cabal/us(LOT 35)·INFANT MOUSE TRANSMlSSIONPo t-3. Identily of ageUl cau ing illness in I of 7 Mouse moribund on lhe 9th poSI-mo quiloinfanl mice billen by Aedes cabal/us on biring daylheir 21st post-feeding day Pre·2. Identity of agent pre ent in 1 Aedes caba/lus Mouse inoculated with t.he mosquito suspensionon lhe 19th posl-feeding dayResults(Fate of mice)Died Lived Titre3 In5 3-8600 12J0 n 0-5t012 n12I 4'6t0 6J0 12} 0'5t0 106n126 5·11200 12} 0'5tU 10r10-'10-'110-'10-'llO-'{'IO-'10-'-{IO-'10-'10-'10-'{1O-110-'Dilution ofinfectedbrainsuspensionadded toequal volumeofn'rumPre·Pre-POSI-POSI-Serum pre-or post-Wesselsbronvirus·Source of infected infant mouse brainExperiment No.1. Identity of agent circuJating in blood of Mice inoculated with serum taken from Alamb A 2348 which was lhe source of 2348 immedialely after feeding 14 Aedesinfective feed for lot 35 A edes cabal/us cabal/us, 101 35• Sera in experiments I and 2 were from guinea-pigs and serum in eX'Perimen 3 was from a sheep.t Approx.imate titre since no dear end-point could be caJculated.: UnsalisfaclOry litter548 S.A. MEDICAL JOURNAL 24 May 1958genus hown in this laboratory to be capable of transmittingWessel bron viru. Mu pratt et af.6 demonstrated the abilityof Aedes (Banksinella) circumluteolus to tran mit the viru .Furthermore, the virus has been isolated in nature from thelatter pecies (Smithburn et af.1).S MMARY1. Laboratory-reared Aedes caballus were allowed toengorge on a lamb circulating We sel bron virus.2. On the 21st and 22nd po t-engorgement days thesemo quitoes tran mitted the virus by bite to new-born mice.3. 134 wild-caught Aedes caballus were allowed to bite31 new-born mice. From this a ucce sful transmissionresulted and Wes el bron virus was confirmed in the singlemouse showing illness.REFERE CESt. Alexander. R. .: Personal communication.2. KokemOl, R. H.• de Meillon. B. and Pate~n. H. E. In preparation.3. Heymann. C. S., Kokernot, R. H. and de Me.Hon, B. (195): . Afr. Mf'<!. J.,4. il~~::~ot, R. H., de Meillon, B.. Paterson, H. E., Heymann, C. S. andSmithbum. K. C. (1957): S. Afr. l. Med. Sei., 22, 145.5. Gear. l., de Meillon, B.. Le Rou~, A. F.. Kofsky, R .. Rose [nnes, R.. Steyn,J. J.. Oliff, W. D. and Sehulz, K. H. (1955): . Afr. Med. J., 29, 514.6. Muspran. l.. mithbum, K. C., Paterson, H. E. and Kokernot. R. H. (1957):S. fr. J. Med. Sei.. 22. 1217. Smithbum. K. c., Kokernot, R. H., Weinbren. M. P. and de Meillon, B.(1957): Ibid., 22, 113.PARALYTIC MUSSEL POISONI G IN NATAL. M. M " M.D., M.R.C.P.E., D.C.H. and W. S. WINSHIP, M.B., CH.B. (CAPE TOWN)Department ofPaediatrics, University of atal, and King Edward VIII Hospital, DurbanParalytic mu el poisoning has been reported from manyparts of the world.1 Mo t of the literature refers to thePacific Coast of orth America, where extensive investiga-tions have been conducted.2 American workers3 haveidentified the neurotoxin as an alkaloid occurring in adinoflagellate plankton, Gonyaulax catenella, on which themu el feed. Sapeika4 fir t de cribed it occurrence inSouth Africa and al 0 isolated the alkaloid. A recent leadingarticle5 noted that ca es had not been reported for sometime. We report here the poi oning of a Zulu family bymus el eaten on the coast of atal, 35 miles north of Durban.DESCRlPTIO ' OF CASESOn the morning of 9 December 1957 a young Zulu motherof Tongaat collected mussels from rocks exposed by thelow tide. She killed the mussels by heating them withoutadded water, extracted them from their shells and wa hedthem in rain water to remove sand. She then cooked them ina well-known vegetable oil, adding onion and curry powderfor flavouring. In the evening, the mussels were eaten withdry maize porridge by her elf, her husband, their 2 onsaged g and 3 year, and their daughter aged 5 year.Onset of SymptomsLess than 1 hour after the meal, the mother felt a peculiarlightness of her body, with a tingling around her mouth andin her finger and toes. nperturbed by these symptoms shewent to bed with the rest of the family. About 6 hoursafter the meal she was awakened by her daughter callingout that she wanted to pass urine but could not move. Themother got up to find herself weak. In lighting the paraffinlamp her hand wavered. She said he had 'to pull herselftogether' before she could apply the match to the wick.She managed to drag her daughter from the bed and hadto support her whil t she passed urine. She then noticed thatthe child's breathing wa laboured and that her attempts topeak resulted only in feeble inarticulate noise .She awakened her husband and sent him to summon help.He did not feel ill but noticed that his body felt light and ashe walked he had to concentrate hard to keep on the road.The mother also awakened both her on and found thatthe eldest boy was hardly able to walk and his brother couldneither sit up nor stand. She returned to her daughter, onlyto find her dead in bed.Before leaving for hospital they noticed that a cat whichhad eaten the remnants of their meal was dragging itselfabout on its belly, owing to apparent loss of power in itslower front legs and hind legs.Clinical ObservationsApproximately 16 hours after the meal, the family wasadmitted to hospital. Both husband and wife complained ofa feeling of lightness and tingling in their extremities andaround their mouths. Clinical examination was, however,negative and both were symptom-free within 24 hours ofeating the mussels.The elder boy had a temperature of 99· gOF and weaknessof the erector spinae mu c1es only. He had recovered com-pletely 42 hours after eating the meal.The younger boy's temperature was lOO°F. He had aflaccid pare is of all four limbs and the erector spinae muscleswere paralysed. The action of the intercostal muscles appearedweak. The deep reflexes were absent. He reacted normally topinprick. The cerebrospinal fluid was normal. No treatmentwas given but a respirator was held in readiness. Within24 hours he had improved. His temperature was normal andhe could roll over in his cot and move all four limbs againstgravity. However, it was not until the 5th day that he wasable to sit up unaided. Weakness of the quadriceps femorawas a marked feature, and to stand he had to climb up hislegs like a child with pseudo hypertrophic myopathy. It was14 days before he had recovered completely.We sa the cat 4 days after it had eaten the mussels andit was then moving normally.Investigation ofthe MusselsThe site of the poisoning was visited 4 days afterwards.There was no evidence of the red discolouration of the seacaused by the plankton floating on the surface which hasbeen described by other worker ,1,2,4 nor had tills ever beennoticed by the local inhabitants. Mussels were collectedfrom the same rocks and submitted for examination toMr. W. Pople, of the Zoology Department, University ofatal, to whom we are indebted for this report:'The mu sels were the South African Black Mussel (Mytilusmeridionalis). Attempts to recover a dinoflagellate fromthe crop contents and faeces were unsuccessful, but minutedinoflageUate were discovered in the rejection currentsfrom the gills of all the mussels. These flagellates measured

Studies on the transmission of wesselsbron virus by Aedes (Ochlerotatus) Caballus (Theo.)

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Studies on the transmission of wesselsbron virus by Aedes (Ochlerotatus) Caballus (Theo.)

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