On the Question of Priority with Regard to certain ... · portant Recent Researches on Malaria. 1893 and 1895, Sacharoff demonstrated the presence of chromatic substance within the

THE ETIOLOGY OF MALABTAL DISEASES. 429

On the Question of Priority with Regard tocertain Discoveries upon the ./Etiology ofMalarial Diseases.

By

George If. F. Nnttall, M.A., M.D., Ph.D.,University Lecturer in Bacteriology and Preventive Medicine, Cambridge.

THOUGH it has long been a popular belief in certain countriesthat malaria is communicated to man by means of mosquitoes,experimental proof was lacking until a recent date. Thehistory of the mosquito-malaria theory has been amply dis-cussed elsewhere by the writer, to whose papers the readeris also referred for a detailed description of the experimentalwork on the part played by mosquitoes in the propagation ofmalarial diseases.1 It is not the object of this paper todiscuss these matters in detail.

Persons who read the medical literature of but one countrywill naturally become biassed in their judgment. This ac-counts for the fact that at present different investigatorsreceive the credit of having definitely established the partplayed by mosquitoes in malarial diseases. In view of theconfusion which will naturally result from the claims made

i Nuttall, G. H. i\ (1899-1900). I. " On the Role of Insects, Arachnids,and Myriapods as Carriers in the Spread of Bacterial and Parasitic Diseasesof Man and Animals: a critical and historical Study;" 'Johns HopkinsHospital Reports,' vol. viii, pp. 1—154, 3 plates (Bibliography). II. "DieMosquito-Malaria-Theorie," ' Centralbl. f. Bakteriologie,' vol. xxv, pp. 162—170, 209—216, 245—247, 285—296, 337-346 (Bibliography). III." Neuere Forschungen iiber die Rolle der Mosquitos bei der Verbreitung derMalaria: Zusammenlass,endes Referat;" ' Centralbl. f. Bakteriologie,' vol. xxvi,pp. 140—147, and vol. xxvii, pp. 193—196, 218—225, 260—264, 328—340(exhaustive Bibliography).

430 GEORGE H. F. NUTTALL.

in vai'ious quarters, it seems eminently desirable to give abrief impartial summary of the experimental work which hasbeen done, relying solely upon published researches, thesebeing cited in their chronological order. With the factsthus marshalled before him every reader is at liberty to drawhis own conclusions.

The study of the liEemocytozoa begins with the discoveryby Ray Lankester in 1871 of Drepanid ium ranarum.Human malarial parasites were seen, but their significancenot comprehended until Laveran published his investigationsin November, 1880. Following upon, the fundamentnl workof Laveran, the most important discovery was that of Golgi(November, 1885), who demonstrated the relationship exist-ing between the life-cycle of the parasites within the humanbody and the occurrence of the febrile attack. "With regardto these investigations there has never been any dispute onthe question of priority, but this is far froni being the casewith the discoveries which followed. Any further disputesregarding the priority of subsequent discoveries should bedisposed of by such a chronological record as that whichfollows, in which not only the year, but also the month andeven day of publication are given.

Chronology r e l a t i n g to ce r t a in of the more Im-por t an t Recen t Researches on Malaria.

1893 and 1895, Sacharoff demonstrated the presence ofchromat ic subs tance within the " f l a g e l l a " ofcertain avian parasites by means of the Romanowskystain.

December 17th, 1895, Ross observed t he process of" f l age l l a t ion " of crescentic parasites to occur in thestomach of mosqui toes (species not determined) fedon the blood of a malarial patient.

1896, Bignami and Dionisi report the negative results of two experi-ments made in 1893-4 with mosquitoes (species uncertain) collected inmalarious localities, the insects being permitted to bite healthy per-sons. They attribute the failure of the experiment to the dispersion of

THE ETIOLOGY OF MALARIAL DISEASES. 431

the insects in the room where they were liberated, and to the experimentnot having been continued long enough. They cite Calandruccio ashaving observed the degeneration of malarial parasites in the stomachof mosquitoes (species not stated).

November 13th; 1897, MacCallum, in Baltimore, foundthat the "flagella" of Halteridium and ofEestivo^autumnal parasites constitute the maleelement, and serve to impregnate the "pig-mented spheres" or female element. In the caseof Halteridium the impregnated spheres became con-verted into motile " vermicules." This transforma-tion was, however, not observed in the human parasites.

December 18th, 1897, Ross fed mosquitoes upon humanblood containing crescentic parasites. The ex-periments were made at Secunderabad, and werereported upon at the time as follows :

After examining hundreds of mosquitoes fed onmalarial blood, always with negative results, he obtained afew which belonged to a species with spotted wings, whichhe had hitherto not used. As Eoss distinctly describesthe egg of this species, there is no doubt whateverbut that he was dealing with a species of Ano-pheles. The insects were bred from larvae, and fed•with blood containing crescentic parasites. Pour to fivedays later peculiar pigmented cells were obsei-ved lyiugwithin the walls of their stomachs. These cells wereround or oval; they measured 12—16 /x on the fourth, and20 fx on the fifth day after feeding, and the pigment theycontained was similar to that within the malarial para-sites in the blood upon which the insects had been fed.Such bodies could not be found in control mosquitoes.Eoss concluded that he had found the mosquitowhich served as a host for the parasite.

February 26th, 1898, Eoss refers again to his experimentswith crescentic parasites. After examining somescores of " dapple-winged" mosquitoes unfed or fedwith healthy blood, all the results were negative until

432 GKOfiOK K. F. NTTTTALT,.

" at last two of this species were, persuaded to feed on apatient with crescents. One of them was killed nextday; no pigmented cells could be found. The secondwas killed forty-eight hours after feeding ; numerouspigmented cells were pi'esent. They were a l l small ,much smaller than epithelial cells, ovoid, about7 n in the major axis, and each contained abouttwenty granules of typical pigment, which wereoften arranged circmnferentially, just as in the malarialparasite." Though it is not stated in this publicationthat he raised these mosquitoes from larvae, reference toBoss's previous paper (p. 1786) will show this to havebeen a part of the method he employed.

E x p e r i m e n t s wi th T e r t i a n P a r a s i t e s . — " A hundred or more grey or'barred-back' mosquitoes, unfed or fed on healthy or crescent blood,have been dissected without finding the pigment cells. At last one wasobserved feeding on a patient whose blood that morning had been seento contain numerous mild tertian parasites." Killed on t he t h i r dday, t h e i n s e c t c o n t a i n e d m a n y p i g m e n t e d ce l l s m e a s u r i n g8—25 p, (Ross subsequently discarded this experiment, as it waspossible that the insect which was not raised from the larva had becomeinfected with some other parasite.)

May 21st, 1898, Exper imen t s on Proteosoma.—Work-ing in Calcutta, Ross observed the development ofProteosoma in a species of Culex (subsequentlydetermined as C. fat igans, Wied.), the insects beingfed on the blood of infected crows, larks, and sparrows.The parasites found in the external coat of the insects'stomachs measured 6 fi after thirty hours, 60 (i after sixdays. "Successive feeds by the same mosquito on thesame bird are followed by fresh crops of young coccidia.. . . Similar pigmented cells" had been previouslyobserved in mosquitoes fed on human parasites. Ninety-four per cent, of the mosquitoes fed on blood containingmature Pro teosoma became iufected.

September 24th, 1898.—Manson reported to the British

THK ÊTIOLOGY 01' MALAHIAL DISEASES. 433

Medical Association Meeting at Edinburgh (July) onbehalf of Boss regarding further experiments withProteosoma. These observations showed that theencapsulated parasites, on reaching a certain size, rup-tured and emptied their contents into the coelom of theinsect. The contents of the ruptured capsules consistedof minute spindle-shaped bodies, and these bodies sub-sequently accumulated in the salivary gland of theinsect. When this had occurred the insects werecapable of communicating the proteosomal infection tohealthy birds. Of twenty-four sparrows exposed to thebites of insects fed on matnre parasites, twenty-twobecame infected.

October 1st, 1898, Grrassi reported that he had reason forsuspecting three species of Culicidae as being carriers ofmalarial infection, claiming that they were confined intheir geographical distribution to those regions wheremalaria was prevnlent in Italy. The three species wereCulex penic i l la r i s , Anopheles c laviger (syn.A. maculipennis), and a purported new species,Culex malariae.1 It has since been proved that only

1 In his paper in the 'Policlinico' (October 1st, 1898), Grassi writes: "Inconclusione, io sono d'avviso che it Culex penicillaris e 1' Anophelesclaviger o per Io meno il Culex penicillaris, fors' anche il Culexmalaria, nella malaria si comportano come le zeeca nella febbre del Texas."Grassi therefore makes a misstatement in a later paper (December 1st, 1900)when he writes, " Proclamai come iudiziati due specie di culex, ma sopratutto1'Anoplieles olaviger." It is curious that Grassi should subsequently havecontinued to lay stress upon the geographical coincidence having led him tothe discovery of Anopheles claviger being a host of malarial parasites,for two out of three species which he for this reason supposed must be hostswere afterwards proved not to be such. He certainly considered A. clavigerat first to be of quite secondary importance; we have his own words for it:" Certi casi di malaria sviluppatisi in Settembre a Locate Triulzi, nei qualigli Anopheles di certo o non puusero o soltanto rarissime volte, den uncianodecisamente come trasmissore il Culex penicillaris, enorma-mente comune in tut t i i luoghi malarici." (The italics are Grassi's.)It is but fair to Ross to state here that Grassi in his paper of the 1st ofOctober refers to the experiments made by Smith and Kilborne upon Texas .fever; and by Ross upon avian malaria as having been a " forte argumento "

434 GEORGE H. F. NUTTAIJ,.

the second of the three species named can serve as ahost for human malarial parasites. The coincidence inthe geographical distribution of ague and malaria-bear-ing mosquitoes in Italy, as claimed repeatedly by Grassi,has been disproved by Celli. The claim that this geo-graphical agreement would probably be found to holdin other parts of the world has been disproved by Nut-tall, Cobbett, and StrangewaysPigg (1901) in England.We cannot, therefore, accept Grasses statement that hediscovered the malarial mosquito because of its geo-graphical distribution, pretty and ingenious as thehypothesis seemed in the beginning. It seems certainthat Grassi was after all entirely guided by Ross's pub-lication of December 18th, 1897, in which he describesan insect with spotted wings and eggs like those whichcharacterise Anopheles .

November 6th, 1898, Infec t ion Exper iment on Man.—Grassi mentions that Bignami had made an infectionexperiment by means of inosquitoes (the three speciesabove named were employed) collected at Maccarese,a malarious locality. The result was positive in thiscase, the person acquiring sest ivo-autumnal fever.(Several infection experiments were subsequently car-ried out by Bignami, Bastianelli, and Grassi in colla-boration, these being reported in various papers oflater date. The first experiment did not prove whichspecies harboured the parasites, and of itself was insuf-ficient to establish the theory on a firm basis.)

December 4th, 1898, Bast ianel l i , Bignami, and Grrassiobserved the development of c rescent ic pa ras i t e sin Anopheles claviger, the appearances correspond-ing to those described by Ross for Proteosoma on the

in favour of the mosquito-malaria hypothesis. In the paper read on the nextday at the Accademia dei Lincei, under the same title as that which appearedin the ' Policlinico," Grassi omits to mention Ross, though he refers to whatwas known regarding Texas fever. The paper, published in the ' Transac-tions ' of the Accademia, differs in several respects from that which appearedjn the 'Policlinico.'

THK ETIOLOGY OP MALARIAL DISEASES. 435

fourth day in Culex. Referring to his experiments with,human parasites, they write, " Verisimilmente i duemosquitos coli ali macchiate nei quali il Ross in Indiatrovo stadi di sviluppo simili a quelli del proteosoma (3°giorno circa) appartenevano pure alia specie An ophel esclaviger, Fabr." (This statement is of interest in viewof Grassi's subsequent claim that Ross might very wellhave been working with insects belonging to the genusCulex, and not with Anopheles at all.) They, more-over, consider that Ross had not certainly determinedthe development of the crescents in his mosquitoes, forhis observations had been broken off at too early a date;besides which the insects might have infected themselveswith hsematozoa from some other animal. We have seenthat the latter supposition is unwarranted, because Ross'sAnopheles were raised from larvse. Moreover theythemselves neglect to state that they raised their Ano-pheles from larvae, so we must presume that they didnot.

Infection Experiment on Man.—In a foot-note to theabove publication it is reported that the authors hadsuccessfully infected a person with tertian fever bymeans of infected A. claviger, collected at Maccarese.

December 22nd, 1898, Grassi, Bignami, and Bastianellifollow the development of crescentic parasitesin Anopheles claviger to the formation of "sporo-zoites," the escape of the latter into the coelom of theinsect, and their accumulation in the salivary gland.The development was found to be slower at 20° to 22°than at 30° C. The fully developed capsules measured70 /u, the sporozoites measured 14 ju. The process ofdevelopment, the size of the fully developed capsules,and of the sporozoites, were the same as Ross hadobserved in Proteosoma.

The development of tertian parasites was observed totake place in A. claviger up to the fifth day.

February 2nd, 1899, Koch published a preliminary note

436 GEORGE H. F. NUTTALL.

upon the results of the investigations conducted by theGerman Malaria Commission, consisting of himself, E.Pfeiffer, and H. Kossel. Further details will be foundin a publication which appeared September 8th, 1899.The Commission observed the development of Pro-teosoma in Culex nemorosus, from the formation ofthe " vermiculi" described by MacCallum for H a l t e r i -dium to their appearance in the salivary gland of theinsect. The pi'ocess of fertilisation was found to occurin Proteosoma, as MacCallum had found for Ha l t e r i -dium and human crescentic parasites. Healthy birdswere successfully infected by means of infected insects.The later publication, which is illustrated by excellentmicrophotographs, completely confirms the observationsof Ross and others.

February 5th, 1899, Grassi, Bignami, and Bast ianel l iobserve the development of qua r t an paras i tes inA. c laviger . Eoss (September 2nd, 1899) observedthe development of quartan parasites in a species ofAnopheles in Sierra Leone.

January 23rd, 1899, Danie ls reported to the Royal Societythat he had been able to confirm Ross's observationswith Proteosoma. He followed their development ina species of Culex, and successfully infected healthybirds by means of infected iusects. He added nothingto what Ross had already found,

April 19th,1899,Bastianelli and Bignami reported furtherstudies upon the development of tertian parasites inAnopheles claviger , and describe three successfulinfection experiments on man by means of A. c lavigerpreviously fed on t e r t i an parasites.

May 7th, 1899, Grassi, Bignami, and B a s t i a n e l l i reportto the Accademia dei Lincei that they had observed thedevelopment of tertian and crescentic parasites inAnopheles bifurcatus.

June 18th, 1899, Grassi observed the development oftertian and crescentic parasites in Anopheles

fHE JfiTIOLOGt OF MALAEIAL DISEASES. 437

pseudopic tus , but not in va r ious species ofCulex. The latter result again obtained later (October4th, 1899).

June 28th, 1899, Ross stated that P ro t eosoma scarcelydeveloped in Culex at 21°, and that the growth of theparasites was already slowed at 27° C. in Calcutta. Thedevelopment of tertian parasites in spotted-wingedmosquitoes raised from larvae was also observed (letterdated February 22nd, 1899, to Nuttall; see 'Centralbl.f. Bakteriologie,' vol. xxv, p. 908).

September, 1899, Bas t i ane l l i and Bignami give a de-tailed description of tertian and crescentic parasites, thepublication being accompanied by the best colouredplates hithei'to published, illustrating their development.They prove that a single infected Anophe les c lavigermay communicate malaria (tertian) to man.

May 4th, 1900, Ziemaun, working in Cameroon, observes thedevelopment of the p a r a s i t e s of t rop ica l malar ia intwo species of Anopheles , as also the development oft e r t i an parasites in one species of Anopheles . Hefollowed the development to the appearance of sporozo-ites in the salivary glands of the insects. He subse-quently (November 22nd, 1900) found that the parasiteswould not develop in Cimex l ec tu la r ius nor in sand-flies.

September, 1900, van der Scheer and van Berlekom, inHolland, observe the development of t e r t i a n parasitesin A. claviger .

September 29th, 1900,Manson reported apositive infect ionexper imen t with tertian-infected Anopheles (spec. ?)imported' from Rome, the insects being permitted tobite his son in London.

October 6th, 1900, Rees reports a similar experiment tothe former.

After perusing the above chronology, and rememberingthe question most disputed—the discovery of the develop-

438 GEORGE H. 1?. NDTTALL.

ment of human parasites in Anopheles, we must concludethat the pigmented encapsulated bodies observed by Eoss in" spotted-winged mosquitoes" at Secunderabad were cres-centic parasites in early stages of development. In his firstpaper Ross definitely states that he raised the imagos fromlarvae kept in bottles; that the parasites which subsequentlydeveloped within them contained a pigment similar to thatof the parasites iu man; and his description of the insects'eggs leaves no room for doubt but that they were Auo-phe les . (In their paper of December 4th, 1898, Bastianelli,Bignami, and G-rassi even made the statement that it isextremely likely that Ross's spotted-winged mosquito wasA. c lav iger ! ) The work done subsequently on P r o t e o -sotna quite rightly confirmed Ross in his belief. We are,hpwever, indebted to the Italian investigators named forcompleting the study of the further development of humanparasites in various species of Anophe les , these studiesbeing subsequently pursued by still other investigators inother countries.1 Ross is perfectly justified in laying stressupon the fundamental importance of his discoveries in thedevelopment of P r o t e o s o m a , and there can be no doubtwhatever about his work having served as a guide to sub-sequent investigators. There is no denying that both thehuman and avian parasites referred to offer great points ofsimilarity throughout. The assumption was, therefore, per-fectly justified that the further stages in the development ofcrescentic parasites such as Ross had observed at Secunder-abad would be identical with what he saw in the case ofP ro t eosoma afterwards in Calcutta.

In conclusion we must not forget to mention the name ofPatrick Manson, who until recently took no part in theexperimental solution of the problem, but who throughoutRoss's investigations, which he stimulated, did much tofurther the studies which iu one direction at least havereached such a satisfactory conclusion.

1 It lias not been deemed necessary to refer to all of these.

THE AETIOLOGY OF MALARIAL DISEASES. 439

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VOL. 4 4 , PAKT 3 . NEW SERIES,