Annals of Tropical Medicine & Parasitology

ISSN: 0003-4983 (Print) 1364-8594 (Online) Journal homepage: http://www.tandfonline.com/loi/ypgh19

The chemotherapy of rodent malaria, XXX. The enigmas of the ‘NS lines’ of P. berghei W. Peters, M. L. Chance, R. Lissner, H. Momen & D.C. Warhurst To cite this article: W. Peters, M. L. Chance, R. Lissner, H. Momen & D.C. Warhurst (1978) The chemotherapy of rodent malaria, XXX. The enigmas of the ‘NS lines’ of P. berghei, Annals of Tropical Medicine & Parasitology, 72:1, 23-36, DOI: 10.1080/00034983.1978.11719276 To link to this article: http://dx.doi.org/10.1080/00034983.1978.11719276

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Date: 30 June 2017, At: 06:15

Ann als of Tropi ca l l\1 cdi cinc a nd Paras it ology, Vol. 72 , No. I ( 1978 )

The chem other apy of roden t m alaria, XXX T he enigm as of the 'NS lines' of P. bergltei* Bv W. PETER S, M . L. C li ANCE , R . LT SSNE Rt, H . MOME t AND D . C . WAR H URSTt

Departm ent of J>arasitology, Liverpool School rif T ropical ,\/edicine, Pembmke Place, l.iverpool 1~3 5QA Received 16 Februa1y 1977 Vin ckc a nd Lips ( 1948) iso la ted a number of strain s of Plasmodium berghei a t Lub11mb as hi near E li sabe th vill e, but th e on ly iso la te th a t survived was th a t ident ified as Kcybcrg 173 . Thi s was passaged from rodent to rode nt , first in Vin c kc's a nd la ter in ma ny other la bora tor ies by th e sy ringe tra nsfer of infec ted bloo ::l , sin ce it was o nl y w ith th e g reates t diffi cu lt y th a t mosqu it o transmis sion cou ld be ac hi eved in th e laborato ry. l nd cec.l it was severa l years a ft er its isolat ion before Yoeli a nd Wall ( 195 1) achi eved cycli ca l la boratory tra nsmi ss ion of P. berghei, a nd that was with a different isola te la bell ed ' S.P. 28S K cibcrg'. Tn th e co urse of innum erable syringe passages th e Keyberg 173 stra in lost th e ab ilit y to produ ce ga rnctocy tcs, and th e spec ies carn e to be cycli cally tra nsmiucd on a rcg11la r b as is on ly a ft er Yocli, Most a nd Bone ( 1964) d emonstr at ed (in a strain iso la ted th e prev ious yea r) th a t th e op timum tempera t ure for sporogo ny wa s a low 18- 2 1°C . Nl ca nw hil c th e K cy bcrg 173 stra in had bee n don a ted to research la bora tories throug hout the world , a nd th is was th e paras ite on wh ic h was based th e vas t majority of a ll papers on P. berghei from 1948 to a bo ut 1964, when th e cycli call y tra nsmi ss ib le N K65 stra in was wid ely di str ibut ed by Yocli . I n th e m eantim e P. yoelii h ad been describe d by Landau a nd Killi ck-Kcnd ri ck ( 1966 ) a nd thi s was to be followed by the subspec ies P. y. killicki Landau , Mi chel a nd Adam 1968 a nd P.y. nigeriensis K illi ck-Kend r ick 1973. The las t p a rasit e, und er the la bel 67 , was sent to a few selec ted laborato ries by Killi ck-Kcn cl rick soon a fter its d iscovery, a nd o n I D ece mber 1967 it reac hed L ive rpool w here it was m a inta ined in reg ul ar cyclica l tra nsm iss ion th roug h Anopheles steplzensi, and used for numero us ex pe riments, such as those report ed by P ete rs ( 1968a, b), Di ggens a nd Gregory ( 1969) a nd Gregory a nd P e ters ( 1970). From F e bru a ry 1967 we a lso ma int a in ed Yoeli 's NK65 stra in cycl icall y. Othet· strains o f P. berghei a nd P. yoelii were introd uced to Liverpo ol from 1966 onwa rd s. The importa nce of thi s chrono log ica l accou nt w ill be ev id ent from th e fo llowing pages in which we d escribe the apparen t selec tion from P. berghei K eyberg 173 of severa l lin es of pa rasite that we labe l th e NS and BS se ri es whi ch appea r, so far, to be a lmost indi sti ng ui sha b le from P. y. nigeriensis. Sin ce th e develop m ent of th e orig in a l NS a nd BS series has a lread y bee n fu ll y d escribed *The work di sc ussed in this paper was suppor ted by gra nts to various indi vidu als. In particula r we a rc ind eb ted to th e Direc tor, the Di vision of M ala ri a a nd O ther Paras iti c Diseases, WHO ; M edi ca l R csen rch Council ; C l l3A-GE IGY AG , Basic, Swi tzerland ; U .S. Arm y M ed ical R esearc h a nd Developm ent Co mmand, through th e European R esea rch Office und er Contrac t DAji\3769-C-0 7!.12. In relation to U.S. i\rm y su ppor t, thi s paper is co ntributi on no. 14·62 from th e Army R esea rch Program. t Prese nt addresses : Miss R. Liss ner, Ca rlett Pa rk College of Further Education , Eas th a m , M crsc ysid c, L62 OA Y. Dr. H. M om en, l nstituto Mi crobiolog ia, ni vcrsid ade Federal do Ri o de J a neiro, Bra zil. Dr. D . C. War hurst, D epa rtment of Protozoology, Lond on School of Hygiene and Trop ical M edi cin e, K eppel Street Lo ndon WC IE 7HT.

24

CHEMOTHERAPY OF RODENT MALARIA, XXX

we will limit the me thod ologica l d esc riptions in thi s pa per to tec hniqu es a nd expe rim ents so far undesc ribed. A stud y of th e DNA rela tionships within th e rodent ma la ri as including th e NS lin e (C hance et al., 1978) produ ced results relevant to th e a na lys is o f th e id entity of th e NS lin e a nd should be consu lted .

MATERIALS AND METHODS Parasite Strains

P. BERGHE! N STRAIN (Pe ters, 1965a). Origin a te d from K eybe rg 173, sent by Professor Vin cke to London , then by Professo r Garnham to Basle in Apr il 1962. Mainta ined th ere in th e CIBA-GEIGY research laborator ie3 by the senior a uthor (W. P .) throug h 2 16 sy ringe passages before being brou g ht to Liverpool in 1964. No other rod ent pa rasite ha d been maintained in Basle up to that time (but the old ha mster-a dapted Adl er stra in of P. vinckei vinckei was held in Liverpool ). RC AND

B

LINES. Drug-res ista nt lin es derived from N stra in by Peters ( 1965a, b) in Bas le.

NK65. Drug sensitive, maintain ed by syringe passage in Basic from Aug ust 1966 wh en it was broug ht from New York by Pe ters ( 1968a), th en cycli call y tra nsmitt ed in Liverpool from Febru a ry 1967. ANKA STRAIN. Broug ht to Liverpool by Bafort from Antwerp wh ere it was cyclicall y passaged 28 tim es from isolatio n in Janu a ry 1969, a nd maintained here a lso by cycli cal transmiss ion. SP 11

LINE (Resistant to pyrime thamine) . Hi story d escribed b y Killi ck-Kendri ck ( 1974), broug ht from Antwerp to Liv~ rpool by Bafort in D ecembe r 1968 and mainta ined by cycli ca l transmiss ion . Source of the RLL line.

HILL). A line of N strain maintained at th e Nat iona l Institute for M edi cal R esearch , Mill Hill , where no P. y . nigeriensis was held , a nd tra nsferred to Liverpoo l by Warhurst in November 1971.

MH ( MILL

P.r. YOELII 17u STRAIN. R eceived from Dr. I. L a ndau in November 1966 and m a intained fro m then by cyclical transmi ss ion.

P.

r. N !GERIENSJS

N67 STRAIN. Mainta ined by cycl ical transmission since its receip t in Liverpool in D ecember 1967 from Dr. R. Killick-Kendrick.

P. VINCKE! VINCKE! 67 STRAIN. Maintained in cyclical passage (Bafort, 197 1) until rece ipt 111 Liverpoo l in April 1968, th en fro zen. All these strains and lin es were preserved in liquid nitrogen with g lycero l a nd Al sever' s solution as protec tive agent when not in regul ar use. Gl ass or plasti c 1-ml a mpoules, cl early and ind elibly labell ed were used for thi s purpose, and every preca ution was taken

PETERS F.T AL.

to ensure th a t mate ri a l was clearly id entified both when fro ze n a nd wh en being re tri eved . Good recoveri es a re being made from th e li quid nitrogen a ft er ten years' cryoprc sc rva ti on. Rodent s and Their Handli ng F or a ll routin e syringe passages a nd mos t cyclica l tra nsmiss ion ra nd ombred a lbin o mi ce (Tu ck's TFW , ma le, a pprox im a tely 20 g wei ght ) were used . Occas iona ll y cycli cal tra nsmi ss ion was ca rri ed out in labora tory-bre d Crammomys surdaster in ord er to in c rease ga me tocy te product ion a nd hence improve th e infec ti ons in th e mosquit oes. M a le \1\iistar rat s, ftvc to eight weeks old were used for cross-im mun ity exper iment s sin ce a ll th e pa rasite stra ins prod uced less severe a nd self- limiting infec ti ons in thi s host. The infec ti ons in mi ce wc t·c in va ri a bl y fulmin a ting a nd leth a l, making th e assess ment o f a ny protecti on imposs ibl e. All rod ents were free o f contam in at ing infec ti ons with EfJery throzoon or Haemobartonella. Mi ce a nd ra ts rece ived a sta nd a rd pcll cted di et (Di xo ns 4 1B) a nd wa ter ad Lib . Th e Crammomys rece ived fres h ap ples instead o f wat er, in a dditi on to th e sta nd a rd pell ets. P a ras itaemi a was gauged by counts on thin film s mad e fmm ta il blood and sta ined with G iem sa sta in . Syringe and Cyclica l Passag es Th e techniqu es employe d a rc th ose described by Pe te r ( 1965a, 1968c, 1970a), Grego ry a nd P eters ( 1970) a nd Ba for t ( 197 1). Cloning A diluti on tec hniqu e was used to clon e th e P. berghei MH line. Ten-fold dilut ions o f heparini zed bl ood were carri ed out in icc-co ld 50% calf serum in ph ys iolog ical sa lin e so th a t each 0·2 ml of inoculum fina ll y conta in ed a n es tima ted 0·3 pa ra it es. Further diluti ons redu ced thi s number to 0·2 a n d 0· 1. Each diluti on was inocul a ted via th e ta il ve in into 20 mi ce whi ch were th en examine d dai ly, th e cut tips of th e ta il s being sealed with ferri c a lum to prevent acc id enta l co nta min a ti on o f cage pa rtners. Mice show ing no pat ent paras it aemi a by th e twelfth d ay a ft er inoculat i on (D + 12) were consid ered to be uninfect ed. Ei g ht o f 20 mi ce receivin g 0· 3, three of 20 rece iving 0·2, a nd no ne of 20 rece iving 0· 1 pa ras ites deve loped patent p a rasitae mi a by D 12. The cloning proced ure was repea ted ta king one mouse th a t had been infected from the 0·2 paras ite in ocu lum. Three mi ce o f th e 20 becam e infected a nd blood from one of these was preserve d as a clone.

+

Produc tion of Chloro quine Resista nce by the Relaps e Techni que This procedu re whi ch involves the ad mini stra ti on of a single la t·gc dose o f chl oroq uin e to rec ipi ent mi ce a t th e time o f passage, thi s being ma de wh en th e pa ras itaem ia in th e d onor mi ce first exceed s 2 % of the hos t ct·y throcytes, was describe d in detai l b y Pe ters ( 1968c, 1970b ) a nd P eters et aL. ( 1970) . Isoenzy me Determ ination s Isoenzy me va ri a nt types of paras it e glu co ephosph atc isomera se, 6-ph osph og lu co na te dehydro genase a nd gluta m a te deh ydrogen ase were d ete rmin ed by th e procedu res d esc ribed by Carter ( 1970, 1973, 1974) . Cross- immun ity Studies R a ts were inocul a ted intraperitoneall y with infec ted blood from d onor mi ce cont a mm g a pprox im a tely 10 6 parasiti zed erythroc ytes. D a il y pa ras ite counts were made on thin film s of ta il bl ood, the infecti on level s b eing expresse d as the number of paras itized erythrocy tes in a tota l of I 0 000 red cell s. The infec tions were all owed to run th eir na tura l course, i.e. th e

26

CHEMOTHERA PY OF RODENT MA I.AR IA, XXX TABLE I Summary of attempts to produce NS-tyjJe Lines

Starling maleriat mul passage number

Derived lines

P. BERGHEI N K65 L3185

185 line

N K65 L/9

L 151/213

N L/20

NS, NS /7, NSf i5A

N / L3

NS/ L3

ll L/327 BS Mi ll Hill (uncloned) MH/NS I Mill Hill Nil* Mill Hill (uncloned) MH /NS2 ANKA RLL

Procednre used

b crg he i R~feren ces

Originalor(.r )

and

notes

60 mg/kg chloroquine Peters Peters ( 1968c) in relapse tec hnique Mi ldly chloroq uine Di gg~ n s & Gregory Hooley (197 1) resista nt , a rose IJ iggcns & spon tanco usly Grego ry ( 1969) Ramka ran & Peters ( 1969) 60 mg/kg chloroquine Peters el al. ( 1969) in rel apse tec hniqu e Peters & Port us ( 1970) + freezing Peters ( 1970b ) Peters el a/. ( I970) + freez ing " 20 and 40 mg/kg Lissner Liss ner ( 1974) chloroquin e in rel apse technique

C""

" 60 "mg/kg chloroq ui ne in rel apse technique

Nil"f Nil t

P. VINCKEI VINCKEI 67 strain Nil § 67 strai n 'berghei-like P. vinckei'

rif P .

" spontaneously " Arose

L;o,oo r om