Identification and quantification of rodent malaria ...

21

Identification and quantification of rodent malaria strains and species using gene probes

G. Sl'\OU�Ol_:, T. BOUR N E, W. J A R R A, S. VIR I Y AKOSOL and K.l'\. B ROW N Di·vision of Parasitology, National Institute for 1\1edical Research, The Ridgeway, 1\1ill Hill, London NW7 JAA (Receiud 21 October 1991; ren"sed

5

February 1992; accepted

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February 1992)

SC:\L\IARY

A D:\"A probe PCsv4 and a subclone thereof PCsv4.1, hybridize specifically to rodent malaria DJ\: A. DNA purified from a small volume ( 10 pi) of infected mouse blood was used to determine the composition of the parasite population present. The hybridization signal following PCsv4 probing of slot-blotted DNA correlated directly with parasitaemia. The hybridization pattern and intensity, resulting from probing restriction enzyme digested and Southern-blotted genomic D:\'A, determined the identity of the infecting parasite line(s), and provided a semi-quantitative measure of parasite burden. Fifteen parasite lines representative of all four Plasmodium species infecting rodents can be differentiated in this way. Key words: Plasmodium, rodent malaria, D:\'A probe, RFLP, diagnosis.

1:\TRODCCTION

Studies of the effects of biological and antigenic diversity of parasites on interactions between the host, the parasite and the vector, are of great importance. In malaria, specificity in the protective immune response of the host has been demonstrated in the human and simian host (J ames, Nicol & Shute, 1 932; :\Iulligan & Sinton, 1933 a, b). In rodent models, using a number of cloned parasite lines, species- and strain-specific immune differences have been conclusively shown to be important factors both in initial parasite clearance and in immunity to reinfection (J arra & Brown, 1985, 1989; Snounou et al. 1989). In these studies the presence, in any one experimental group, of more than one parasite line complicates such analyses, since in many cases detailed biochemical analysis is required for differ­ entiation between the lines. Monospecific and di­ agnostic immunological reagents are available, but only for some lines, moreover their effective use depends on the nature of the antigen which they recognize. A variety of Di\'A sequences (both coding and non-coding), have been shown to be specific to particular parasite species and strains. A DNA clone, PCsv4, derived from the AS strain of the rodent malaria parasite Plasmodium chabaudi chabaudi, is specific to rodent malaria parasites and can be used to differentiate between them (Viriyakosol, Snounou & Brown, 1989). A subclone, PCsv4. 1, derived from PCsv4 has also been used to develop an assay in which the parasites present in blood can be semi­ quantitatively detected (Snounou et al. 1989). In this paper we extend our previous observations and describe the use of both PCsv4 and PCsv4. 1 m Parasitology (1992), 105, 21-27

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the identification and quantification of different rodent malaria species and strains in daily blood samples from infected mice.

:\IATERIALS AND :\IETHODS

Parasites Parasites were obtained from cloned lines of the following: P. c. chabaudi (AJ, AS, BS, CB, CR, ER strains, and clone F1 of the IP-PCI strain), P. chabaudi adami (DS and 556 KA strains), P. berghei (ANKA and KSP 1 1 strains), P. vinckei lentum (DS strain), P. vinckei petteri (CR strain) and P. yoelii yoelii (A and YM strains - both derived from the 17X strain). All but two of these parasite lines were originally provided by Professor D. Walliker (WHO Registry of Standard Strains of Malaria Parasites, Edinburgh University) and with three exceptions the origins of these lines have been extensively described elsewhere (Carter, 1978; Walliker, 1983; Jarra & Brown, 1985). P. c. adami DS and P. v. lentum DS were supplied as clones from the ( 408XZ) isolate originally described asP. ·v. lentum, and P. c. chabaudi ER as a clone from the original 56L P. c. chabaudi isolate. P. c. chabaudi, clone Fl of the IP-PCI strain (see Coombs & Gutteridge, 1975) was obtained from Dr L. Pereira de Silva (Institut Pasteur, Paris), and P. berghei, KSP 1 1 strain (Yoeli & Most, 1965) obtained from Professor M. Yoeli and cloned in our laboratory. :vlale CBA/Ca mice (24-26 g) were infected by intraperitoneal (i.p.) injection of parasitized erythro­ cytes (PE) derived from cryopreserved stabilates of the above cloned lines as previously described (Jarra

G. Snounou and others 2

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M, (kb)

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23 9·5 6·3 4·6

2·3 2·0

PCsv4

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PCsv4.1

Fig. 1. Southern blots of Eco RI-digested genomic DNA (ea. 1·5 f1g) from: (1) and (2) Plasmodium chabaudi chabaudi AJ and AS lines respectively and (3) and (4) P. yoelii yoelii A and YM lines respectively. Duplicate nitrocellulose filters were probed with the two different probes as indicated. vVashing was performed at Tm 22 oc (0·4 x SSC, 0·1 "o SDS, 50 °C). Exposure was performed for 6 h (PCsv4 probed filters) and 60 h (PCsv4. 1 probed filters) at -70 °C. Fragment sizes in kilobase pairs (kb) of the lambda Hind Ill markers are indicated. =-

& Brown, 1985). In the experimental group, the infection was initiated in 5 mice by i.p. injection of 5 x 104 P. c. chabaudi AS PE/mouse. Parasitaemias were assessed on daily Giemsa­ stained, thin tail-blood smears from each mouse and expressed as the log10 of the number of parasites/ 105 erythrocytes. In contrast to previous studies (Jarra, 1982) the log10 of the arithmetic mean, rather than the geometric mean, has been used in order to allow for direct comparison between the parasitaemia data from microscopy and from scin­ tillation counting (see below). Numerical differences between the arithmetic and geometric means are of significance in statistical manipulation of the data, but have no consequence for the conclusions drawn in this paper.

Purification of parasite DNA Genomic DNA from the various lines was isolated as described previously (Viriyakosol et al. 1989). Blood samples from the experimental P. c. chabaudi (AS) infected group were obtained as follows. Sampling was performed daily between days 4 and 14 inclusive, post-infection. All samples were taken between 09.00 and 10.00 h, which corresponds to the ring stage of the parasite which, in this

particular parasite�host combination, IS highly synchronous. Ten ,ul tail blood samples from each mouse in the group were immediately mixed with 450 ,ul of Kreb's saline (KGS �see J arra, 1982) containing 0·2 °0 glucose and 25 u/ml heparin. The erythrocyte concentration was determined by flow cytometry (Coulter counter) on three 1 ,ul aliquots. Two ,ul of 10 °0 saponin (final concentration 0·04 °0) were added to the remainder of the sample in order to lyse the erythrocytes. The parasites were then collected by centrifugation (5000 x g, 5 min at room temperature), stored at -70 °C and the individual daily samples then processed simultaneously to purify the DNA. Each parasite pellet was resuspended with 80 ,ul of lysis buffer ( 10 m:vt Tris, pH 8·0, 20 m:vt EDTA, 0·5 mgjml pronase and 0·5 °0 SDS) and incubated at 37 °C for 1 h. The volume was then made up to 400 ,ul with 375 m:vt Na acetate, pH 5·5 (final con­ centration 0·3 :VI) and the resulting solution thoroughly mixed before further incubation at 37 °C for 1 h. Contaminating proteins were removed by extraction with an equal volume of Tris (0·1 :VI, pH 7·0) -equilibrated phenol / chloroform/ iso- amyl alcohol (50:48:2 v/v/v). The aqueous phase was recovered by centrifugation ( 13 000 x g, 5 min at room temperature). DNA was precipitated at -20 °C overnight with 2 volumes of cold absolute ethanol and recovered by centrifugation ( 13 000 x g, 10 min at room temperature). The D0;A pellet was then washed with cold 70 ethanol, briefly vacuum­ dried and resuspended in 50 ,ul of TE ( 10 m:vi Tris, 1 m:vt EDT A, pH 8·0). Probe isolation and primer extension labelling D.l\.'A from an M 13 mp11 clone harbouring the plasmodial DNA fragment to be used as a probe was purified following the usual procedure (Maniatis, Fritsch & Sambrook, 1982). The PCsv4 and PCsv4. 1 insert DNAs were purified, following restriction endonuclease diges­ tion, from agarose and acrylamide gels respectively as described previously (Viriyakosol et al. 1989; Snounou et al. 1 989). Insert DNA was labelled with 32P a-dATP following the method of Feinberg & Vogelstein ( 1984) and purified on GSO Sephadex columns (Maniatis et al. 1982). The probes obtained had specific activities of> 108 cpm/,ug of DNA. Southern blotting The plasmodial DNA isolated as above was digested with the appropriate restriction enzyme and electro­ phoresed on agarose gels in E buffer (40 m:VI Tris, 20 m:vt Na acetate, 2 m:VI EDTA, pH adjusted to 7·7 with glacial acetic acid). After staining in ethidium bromide to visualize the DNA, the DNA was

23

Genotyping of rodent plasmodia with DNA probes

denatured and neutralized before blotting on to nitrocellulose filters using 10 x SSC (1 X SSC is 1 50 m:vi NaCI, 1 5 mM Xa citrate, pH 7·0) (Maniatis et al. 1 982). After blotting, the filters were baked for 2 h at 80 °C under vacuum. The baked filters were pre-hybridized at 37 oc in 5 x SSPE (1 x SSPE is 1 50 m:v1 NaCI, 1 0 m:-.1 ::\aH2P04, 1 m:vr EDTA, pH 7-4), 0·1 °0 Na pyrophosphate, 0·2% SDS and 5 ,ug/ml heparin for 4 h and hybridized overnight at 37 °C in 5 x SSPE, 0·1 °0 Na pyrophosphate, 0·2% SDS, 50 ,ugjmi heparin, 10 ° 0 dextran sulphate, 30 °0 formamide and the radio-isotope labelled probe. Washing was performed at the appropriate SSC and temperature conditions (see legends to Figs 1-4) and in the presence of 0·1 °0 SD S. The melting temperature (Tm) was determined as a guide for the stringency of washing. The formula used is the following: Tm 1 6·6 log10 [Na+]+0·4 1 (% G+C)+ 81 ·5. The % G+ C of the two probes is calculated as being approximately 25 °0. Hybridizations were performed at Tm 35 °C. Autoradiography was performed using an intensifying screen and on pre-flashed Fuji RX X-ray film. =

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Slot blotting was performed on a Schleicher and Schi.ill apparatus according to the manufacturer's recommendations. Briefly, a small volume of DNA (5-15 pi) was denatured in 200 ,ul of 0·3 M NaOH by a 1 0 min heating step at 80 °C, followed by rapid chilling on ice. Then 300 ,ui of neutralizing buffer (1 2·5 x SSC, 250 mM Tris and 250 m:-.1 HCl) were added and the samples immediately applied onto the nitrocellulose membrane by vacuum suction. The nitrocellulose membrane was then baked for 2 h at 80 °C under vacuum. Hybridization to the 32P­ labelled probe was assessed either by auto­ radiography (as for Southern blots) or by scintillation counting. In the latter case the portion of the filter corresponding to each daily sample was cut out, and the amount of radioactivity determined by Cerenkov counting. The resulting counts were corrected by subtracting the counts obtained from a portion of the same filter that was free from DNA. RESULTS

Choice of probe In order to determine the efficacy with which DNA

\)tobes could be used to monitor the course of the parasitaemia, two experiments were performed using DNA samples obtained throughout an experimental P. c. chabaudi AS infection. The PCsv4 DNA

fragment generates a complex hybridization pattern

upon probing of a Southern blot of restriction enzyme digested rodent malaria genomic DNA (Viriyakosol et a l. 1989). A fragment of PCsv4, PCsv4.1 (Snounou et al. 1 989) hybridizes only to a

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