Para;sitol Res (1991) 77:266-268 004432559100030J

Parasitnlngy Research 9 Springer-Verlag1991

Short communications Restriction-fragment-length polymorphisms among Toxoplasma gondii strains N. Cristina, B. Oury, P. Ambroise-Thomas, and F. Santoro D6partement de Parasitologie-MycologieM~dicaleet Molbculaire(DP3M), CNRS URA 1344, Facult~de M~decine, Universit6 Joseph Fourier Grenoble I, 38706 La Tronche, France Accepted October 19, 1990

Molecular biology techniques are currently being used for the characterization of strains in a range of clinically important parasites. For example, restriction-fragmentlength polymorphisms (RFLPs), which are obtained by restriction enzyme analysis with or without Southern blot hybridization and DNA cloning, have been reported for different protozoa, including Trypanosoma (Paindavoine et al. 1989), Plasmodium (Oquendo et al. 1986), Leishmania (Barker 1989), Naegleria and Acanthamoeba (McLaughlin et al. 1988), and helminths such as Schistosoma (Rollinson et al. 1986), Echinococcus (Rishi and McManus 1987), Brugia (Cameron etal. 1988), and Taenia (Rishi and McManus 1988). RFLPs provide a great potential for investigating differences among parasites o f phylogenic proximity or among strains of the same species that present a particular origin or pathogenicity. In the pathogenic protozoan Toxoplasma gondii, differences between several strains have been established by isoenzymic (Barnert et al. 1988; Darde et al. 1988) or immunologic (Ware and Kasper 1987) analysis. Different zymodemes associated with strain pathogenicity in mice have been described (Darde et al. 1988). Other authors have used chromosome analysis by pulsed-field electrophoresis to study T. gondii strains (Candolfi et al. 1989). This heterogeneity in the biological behaviour of the strains suggested the existence of DNA polymorphisms.

The present investigation demonstrated the presence of RFLPS among six strains of T. gondii using a specific repetitive DNA sequence with 353 bp, TGR1E (patent submitted for nucleic sequence), which was cloned from a genomic DNA library of the T. gondii RH strain. A brief summary of the T. gondii strains used in this study appears in Table 1. The strains were maintained by murine passage, and all but the RH strain were cultured on human embryonic cell line MRC5 (Biomerieux; Lyon, France). RH, Prugniaud and M77,1 were cloned strains. Parasites of each strain were lysed with proteinase K, the genomic DNA was extracted and precipitated with ethanol (Johnson et al. 1986). Following standard techniques of restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting and hybridization, genomic DNA that had been hybridized with the [32p]_ labelled TGR1 E sequence generated four different RFLP patterns (Fig. 1). The smallest band appearing in the six profiles resulted from homologous hybridization of the TGR1E sequence. Major differences between strains were demonstrated in bands showing >1.6 kb pairs. These are schematically represented in Fig. 2. The current classification of T. gondii strains according to their their pathogenicity in mice does not explain the RFLP pattern distribution observed in the present study. The closely related B and C patterns seem to correspond to strains of intermediate pathogenicity, which were isolated from chicken (C56) and sheep

Table 1. Characteristicsof the Toxoplasrnagondii strains studied Strains

Pathogenicity

Source

Year isolated

Place isolated

References

RH C C56 Prugniaud M7741 Haran

Virulent Chronic Intermediate Chronic Intermediate Chronic

Human acute encephalitis Human placenta Chickenovary and oviduct Organs from a newborn human Ovine diaphragm Blood from an aborted human foetus

1939 1981 1961 1964 1958 1987

USA Paris, France USA Limoges, France USA Grenoble, France

Sabin 1941 Beauvais et al. 1982 Araujo et al. 1976 Martrouet al. 1965 Jaeobs et al. 1960 -

Offprint request to: N. Cristina

N. Cristina et al. : Identification of Toxoplasma gondii strains

267

12.2 12.2 i

6.1 ira. 6.1 4 . 0 p.-

4.0

2.0 ~

2,0

1.6 m,--

1.6

1.0

1.0

0.5 ~

0.5

0 . 3 ~,-

0.3

a

b

b

A

c

d

e

B

(M77,,1), respectively. Pattern D includes two chronic strains o f h u m a n origin (Prugniaud and Haran). The virulent f o r m of pathogenicity was linked to yet another group ( R H strain; pattern A); surprisingly, the chronic C strain of h u m a n origin presents the same pattern. The possibility that the chronicity of the C strain

m m m

m m m

INto

m

m

m

m

m m

m

m

mmmm

mmmm~

mmmm

m m m

m

n

m

m

A

B

m

C

m

D

Fig. 2. Schematic presentation of RFLP patterns exhibited by the six Toxoplasmagondii strains. A, RH and C strains; B, C56 strain; C, M7741 strain; D, Prugniaud and Haran strains

f

g

h

Fig. 1A, B. Southern blots showing the hybridization of SalI restricted genomic DNA from six Toxoplasma gondii strains with the [32P]-labelled TGR/E sequence. Electrophoresis was performed in 0.8% agarose gel. A a, RH strain; b, C strain. B b, C strain; c C56 strain; d, M7741 strain; e, Prugniaud strain; f, Haran strain; g, human DNA; h, murine DNA. For hybridization experiments, 200 ng parasite DNA was used and l-gg samples of human and routine DNA were deposited on the filters. The latter were hybridized at 42~ C in 50% formamide buffer, washed three times for 5 rain in 3 x SSC and 0.1% sodium dodecyl sulfate (SDS) at room temperature and then autoradiographed for 4 days at - 7 0 ~ C. The molecularweight markers were from BRL (1 kb ladder)

in mice was lost during its passage in culture was ruled out since it remains cystogenic in these animals (Derouin, personal communication). Therefore, at least two different R F L P patterns were associated with the chronic f o r m o f murine toxoplasmosis. Moreover, the four strain groups distinguished in the present study can be c o m p a r e d with the three strain groups previously determined by isoenzyme analysis (Darde et al. 1988). The first group, called zymodeme I, includes three virulent strains (RH, P, and E N T ) ; zym o d e m e II includes three chronic strains ($1, $3 and BOU), to which the Prugniaud strain was recently added (Darde, personal communication); and z y m o d e m e I I I consists of a single strain o f intermediate pathogenicity (M7741). Therefore, for at least three strains, of which two are cloned (Prugniaud and M774.1), each of the R F L P patterns corresponds to a particular zymodeme: the R H strain corresponds to pattern A and zymodeme I; the Prugniaud strain, to pattern D and zymodeme II; and the M7741 strain, to pattern C and zymodeme III. The use of pathogenicity in mice as a p a r a m e t e r for strain differentiation seems to be much too restrictive to explain the differences observed a m o n g the four R F L P patterns identified in the present study. It would be interesting to consider other strain characteristics such as the eventual production of oocysts in cat intestinal epithelium or of pseudocysts in cellular culture or to examine the geographic origin o f hosts or s y m p t o m a -

268 t o l o g y in p a t i e n t s . I n c o n c l u s i o n , the t e c h n i q u e p r e s e n t e d h e r e i n offers a v a l u a b l e m e a n s o f i d e n t i f y i n g d i f f e r e n t T. gondii strains. H o w e v e r , o t h e r a s p e c t s o f the b i o l o g i cal b e h a v i o u r o f these strains s h o u l d be t a k e n i n t o acc o u n t , since t h e y c o u l d p r o v i d e a d d i t i o n a l i n f o r m a t i o n a b o u t the o b s e r v e d R F L P s .

Acknowledgements. This work was supported by ANVAR (Association Nationale pour la Valorisation et l'Aide A la Recherche). The authors would like to thank G. Tavenlier, J. Burnod, B. Mertiny and M.M. Lombard for their technical assistance and O. Brasier for valuable secretarial help. We also thank Drs. F. Derouin and M . L Darde for providing the T. gondii strains and Dr. F. Darcy for critically reading the manuscript.

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