Characterisation of a Cryptosporidium parvum-specific cDNA clone and detection of parasite DNA in mucosal scrapings of infected mice1

Molecular and Biochemical Parasitology 95 (1998) 21 – 31

Characterisation of a Cryptosporidium par6um-specific cDNA clone and detection of parasite DNA in mucosal scrapings of infected mice1 Franz Petry a,c,*, Martin W. Shirley b, Michael A. Miles c, Vincent McDonald c a

Institute of Medical Microbiology and Hygiene, Johannes Gutenberg-Uni6ersity Mainz, Augustusplatz/Hochhaus, D-55101 Mainz, Germany b Institute for Animal Health, Compton, Berkshire, RG20 7NN, UK c Departments of Medical Parasitology and Clinical Sciences, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK Received 16 February 1998; received in revised form 23 April 1998; accepted 27 April 1998

Abstract A cDNA library was constructed using total RNA extracted from oocysts and sporozoites of the protozoan parasite Cryptosporidium par6um. The expression library was screened with an anti-C. par6um antiserum and a clone, Cp3.4, with a 2043 bp insert, was extracted. Southern blot analysis demonstrated a single copy gene that was located on a 1.6 Mb chromosome. The gene was found to be C. par6um specific as Cp3.4 did not cross-hybridise with chromosomal DNA from three other apicomplexan parasites. The cDNA encodes a polypeptide with a predicted membrane helix at its C-terminal end which is flanked by stretches of acidic amino acids. Overall, the polypeptide has a low isoelectric point (pI) of 3.94. A total of 21 glycine/proline-rich octapeptides were identified which represented variations of a consensus sequence. The function of this protein is yet unknown. Using Cp3.4-specific PCR primers, this C. par6um gene could be amplified from as little as 0.8 pg of purified parasite DNA in a single polymerase chain reaction. Less than 0.1 ng of DNA from the ileum mucosa of immunosuppressed adult mice that had been infected with C. par6um oocysts was required to detect the parasites. In non-immunosuppressed mice that were infected and which did not shed oocysts in numbers detectable by acid-fast staining, parasite development could be detected in 25 ng of total mucosa DNA. This PCR approach may be a valuable technique for the detection of parasite infections in situations where conventional staining methods fail, such as chronic, low-grade infections or the detection of parasites in potential reservoir hosts. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Cryptosporidium par6um; Gene cloning; Chromosomal localisation; Repetitive peptide motif; Detection; PCR

Abbre6iations: Bp, base pairs; Kb, kilo bases; Mb, megabase pairs; PCR, polymerase chain reaction; PFGE, pulsed field gel electrophoresis; RT-PCR, reverse transcription PCR. * Corresponding author. Tel.: +49 6131 173139; fax: + 49 6131 173439; e-mail: [email protected] 1 Nucleotide sequence data reported in this paper are available in the EMBL, GenBank™ and DDJB data bases under the accession number Y09042. 0166-6851/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved. PII S0166-6851(98)00063-2

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1. Introduction The increasing importance of cryptosporidiosis as a complication of immunocompromised patients such as AIDS sufferers demands an in depth study of the molecular biology of the causative agent, Cryptosporidium par6um. The apicomplexan parasite causes a profuse watery diarrhoea which is self-limiting in the immunocompetent host but can develop into a chronic, severe and life-threatening disease in individuals with an impaired immune system. To date there is no reliable chemotherapy available and alternative ways to control the infection must be pursued. The mechanism of pathogenesis as well as the molecular and cell biology of C. par6um are poorly understood. Therefore, we launched a project to characterise immunodominant molecules of C. par6um which may be involved in the host–parasite interaction by molecular cloning. The limited data presently available on C. par6um genes have shown that there is no extensive homology with genes of other apicomplexan parasites, such as Eimeria, Toxoplasma or Plasmodium. One exception might be the thrombospondin related anonymous protein (TRAP) gene of Plasmodium falciparum [1] of which homologues have been found in other species including C. par6um (accession numbers X77586, X77587). Of the few C. par6um genes that have been isolated, one coding for an oocyst wall protein shows a cysteine-rich repetitive sequence motif [2,3]. Here we report a C. par6um-specific cDNA sequence that encodes a polypeptide which harbours a repetitive octapeptide sequence. A PCR protocol that has been developed using oligonucleotide primers derived from the sequence of this C. par6um gene is presented that allows the detection of parasite DNA in nanogramms of total DNA preparations of infected ileum.

2. Materials and methods

2.1. Parasites C. par6um parasites were passaged in Jersey

calves infected with 5 × 107 oocysts of a laboratory strain (MD strain) originally isolated from deer. Oocysts were purified as described elsewhere [4]. Any remaining bacterial contamination was removed by incubating the oocysts in 1.5% sodium hypochlorite (10 min on ice) after which they were washed three times in distilled water (15 min, 1600× g, 4°C) and stored in 2.5% potassium dichromate at 4°C until required. Oocysts were excysted for 1 h at 37°C in RPMI medium in the presence of 0.5% bile salts.

2.2. cDNA library construction and antibody screening An excystation mix was spun and resuspended in RNA extraction buffer [5] and subjected to four freeze–thaw cycles (liquid nitrogen/95°C) in order to rupture intact oocysts. An acid phenolchloroform extraction was performed and precipitated with ethanol [5]. The pellet was resuspended once more in RNA extraction buffer and precipitated for a second time. cDNA was synthesised from 20 mg of undegraded total RNA using oligodT priming and standard procedures. EcoRI adaptor-linked cDNA was size fractionated in a 1.2% agarose gel and molecules \400 bp were ligated into lgt11 arms and packaged in vitro. The resulting cDNA library consisted of 2.9× 106 individual phage particles with a frequency of 88% recombinant phages. The cDNA library was plated on E. coli strain Y1090 and screened with a rabbit anti-C. par6um antiserum following the method of Young and Davis [6] with minor modifications. For the screening of the library, a polyvalent rabbit antiserum was used that had been generated with a homogenate of C. par6um oocysts. The antiserum recognised numerous antigens of the parasite on a Western blot of an oocyst lysate. After several rounds of rescreening, lDNA was prepared from phage particles using chromatography on DEAEcellulose, proteinase K digestion and phenol/chloroform extraction (Amersham lgt11 cloning manual).

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2.3. Sequence analysis

2.5. Pulsed field gel electrophoresis (PFGE)

The lCp3.4 EcoRI fragment was subcloned into pBluescript and the entire fragment was sequenced in both orientations using overlapping restriction enzyme fragments and synthetic oligonucleotides as primers to complete the sequence. For analysis of the data, the PC/GENE program package was applied.

Chromosomes were separated by size by PFGE. Pieces cut from chromosomal DNA blocks were placed along an origin of 1 cm of a 21× 21 cm 1.5% agarose gel. A clamped homogeneous electric field (CHEF) DRII electrophoresis cell (BioRad) provided a pulse time of 60–250 s for 40 h. Saccharomyces cere6isiae (Bio-Rad) chromosomes were included in the run as size standards. Electrophoresed gels were stained with ethidium bromide and then irradiated with UV light at 254 nm prior to transfer to nylon membranes. Filters were baked at 80°C for 2 h.

2.4. Southern blot analysis For preparation of chromosomal DNA from C. par6um and C. muris, oocysts were resuspended in DNA extraction buffer (100 mM EDTA, 20 mM Tris/Cl, pH 8.0, 1% SDS, 200 mg ml − 1 Proteinase K) and subjected to four freeze–thaw cycles. The suspension was incubated at 50°C for 24 h. DNA was extracted with phenol/chloroform and precipitated with ethanol. Residual RNA was digested with RNase and DNA was precipitated again. The DNA concentration was estimated spectrophotometrically at 260/280 nm. Each microgram of chromosomal DNA was digested with 4 U of restriction endonuclease overnight at 37°C in reaction buffer supplied by the manufacturers (Amersham, Pharmacia). Restriction fragments were separated on a 0.8% agarose gel and processed according to Maniatis et al. [7]. After blotting onto nylon membranes, DNA was UV-crosslinked. Insert DNA fragment of clone Cp3.4 was labelled with [a 32P]dCTP. Prehybridisation was done at 42°C for 4 h in 6 ×SSC (150 mM NaCl, 15 mM Na3Citrate, pH 7.0), 1% SDS, 50% deionized formamide and 100 mg ml − 1 heat denatured sonicated salmon sperm DNA. Hybridisation was performed overnight in the same solution plus labelled DNA probe. After hybridisation, the filters were washed twice for 30 min in 2 × SSC plus 0.5% SDS at room temperature and once in the same buffer for 30 min at 65°C (low stringency) or, additionally, 30 min in 0.2 × SSC plus 0.5% SDS at 65°C (high stringency). The filters were air dried and exposed to Kodak X-omat AR film at − 70°C with intensifying screens.

2.6. DNA and RNA preparation from mouse ileum mucosa Infected and non-infected C57BL/6 mice [8] and C57BL/6 × 129 F1 hybrids were killed by cervical dislocation. An :10 cm fragment of the terminal ileum was cleared from faecal debris, slit open and flushed with sterile PBS. The mucosa was scraped off using microscopic glass slides. For preparation of chromosomal DNA the mucosa was resuspended in 6 M guanidinium HCl and processed according to [9]. Total RNA was isolated from the mucosa as described elsewhere [5]. In some experiments, total RNA was treated with DNase I, extracted with phenol/chloroform and precipitated with ethanol.

2.7. Preparation of cDNA from total RNA for RT-PCR Approximately 5 mg of total RNA in a vol. of 16 ml were heated to 70°C for 1 min and cooled on ice for 1 min. To the denatured RNA, 24 ml of a reaction mix was added which included 5 mM DTT, 1 mM of each dNTP, 120 U of RNase inhibitor (Promega), 800 ng oligo-dT-primer, 2 mg actinomycin (Boehringer Mannheim) and 400 U of Moloney–Murine leukemia virus reverse transcriptase (M-MLV-RT, Gibco) in reaction buffer (Gibco). The reaction was incubated at 37°C for 60 min, followed by 5 min at 95°C and then chilled on ice. Then 1 ml of this reaction, the equivalent of 125 ng RNA, was used in the PCR.

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2.8. Amplification of the Cp3.4 gene and mRNA by PCR and RT-PCR Two oligonucleotide primers were synthesised for PCR amplification of the Cp3.4 sequence. The forward primer (5%-GATGGATTAATTGTTCCACC-3%) spans the sequence at positions 7 – 26 (see database accession number Y09042), the reverse primer (5%-CCTCCATTTTCACCTTGTGG-3%) is complementary to positions 637 – 656. The PCR was carried out as followed: Various amounts of DNA and cDNA in a volume of 1 ml, were mixed with 20 pmol of each primer, 5 ml of 10 ×standard PCR reaction buffer (Eurobio, Raunheim, Germany), 0.2 mmol dNTP in a final volume of 50 ml. After a 3 min hot start, 2.5 U of Taq DNA polymerase were added. A total of 35 thermocycles were performed with 45 s denaturing at 94°C, 45 s annealing at 57°C and 1 min at 72°C. After the last cycle a 5 min elongation at 72°C was done. Of the total reaction, 20 ml were analysed on a 1% agarose gel in the presence of ethidium bromide.

3. Results and discussion Initial screening of 1.2× 105 recombinant phage plaques resulted in the identification of several positive signals. One particularly strong reacting phage plaque was purified and lDNA was prepared. Restriction of the recombinant clone, designated lCp3.4, with EcoRI and BamHI liberated a single fragment. The EcoRI fragment was subcloned into pBluescript for sequence analysis. The total length of the clone was 2043 bp (excluding the EcoRI adapters from the library construction). One long open reading frame of 1695 bp was found which is in frame with the lgt11 b-galactosidase gene and is followed by a 325 bp 3% non-coding sequence leading into a poly(A)tail. This open reading frame was classified ‘coding’ with a probability of 98% [10]. The G+ C content is relatively low with overall 37.1 and 40.2% in the coding region. Analysis of the deduced peptide sequence revealed no cysteine residues but a high glycine (11.3%) and proline (11.1%) content. However, no similarity to colla-

gen or any other reported sequences has been found. Applying the method of Klein et al. [11] and Rao and Argos [12] to predict membrane spanning segments and helices, one stretch of hydrophobic residues has been found (Fig. 1). The protein was classified as an integral protein. The transmembrane spanning segment was flanked by stretches of acidic amino acid residues; overall the polypeptide was richly acidic with a calculated pI of 3.94. There were five putative N-glycosylation signals at amino acid positions 25, 105, 135, 156 and 549. Further analysis of the deduced peptide sequence identified 21 octapeptide repeats which consisted of variations of a consensus glycine/proline-rich sequence. The anchoring positions of this sequence motif consisted of a threonine or serine in 76% of the repeats at position 1, glycine in all repeats at position 2, a hydrophobic residue in 81% of the repeats at position 5 and an aspartic acid in 71% of the repeats at position 6 (Fig. 1B). Interestingly, there were no two identical repeats in this sequence. Short peptide repeats have been identified in major surface proteins of a number of protozoan parasites, well known examples being the tetrapeptide repeat within the circumsporozoite protein of Plasmodium falciparum [13] or the p30 protein of Toxoplasma gondii [14]. These proteins are major antigens and may distract the host immune system from parasite receptor proteins essential to the invasion process. An oocyst wall protein of C. par6um, which has been cloned independently by two groups, consisted of two different types of cysteine-rich repeats [2,3]. In this case, it was postulated that the repeats are important to the structure and/or function of the polypeptide rather than representing relevant antigens which are involved in an immune escape mechanism. Southern analysis of restricted chromosomal DNA revealed that Cp3.4 appeared to be encoded by a single copy gene as judged by the unique hybridisation signals after EcoRI, DraI and HindIII digestions. Hybridisation of PstI digested DNA revealed two bands, as predicted from the cDNA sequence where a PstI site was located at position 837 (Fig. 2A). The Cp3.4 probe hybridised specifically with C. par6um DNA and did not react with DNA from the closely related

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Fig. 1. (A) Graphical representation of the peptide sequence analysis of Cp3.4. The coding region is represented as a box; the 3%-non-coding region is shown as a bar. The locations of the glycine/proline-rich repeats (arrows), the putative N-glycosilation sites (diamonds), the hydrophobic helix (grey) and the acidic regions (black) have been highlighted. The scale below the graph is given in amino acid residues. (B) Alignment of 21 glycine/proline-rich octapeptides and the consensus sequence. Numbers indicate positions within the peptide sequence deduced from the nucleotide sequence deposited at the sequence data bases (accession number Y09042). (C) Carboxy-terminus of the deduced peptide sequence showing the acidic regions (bold) flanking the putative membrane spanning region (underlined and italic).

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species C. muris, or any of the tested species including Eimeria acer6ulina, Toxoplasma gondii or human DNA, even under low stringency. The Southern blot in Fig. 2A was overexposed in order to detect even weak hybridisation signals. A sequence data base search did not reveal any known homologous sequences. This fact underlines the findings of the Southern blot which suggest that Cp3.4 represents a C. par6um-specific sequence. Separation of C. par6um chromosomes by PFGE revealed five bands ranging from 1.0 to 1.6 Mb (Fig. 2B). A C. par6um chromosomal band of 1.3 Mb probably consisted of two co-migrating chromosomes with similar sizes as judged by the intensity of the ethidium bromide staining. These results were comparable to findings of Kim et al.

Fig. 2. Southern blot analysis of chromosomal DNA from various species (A) and pulsed field gel electrophoresis of C. par6um chromosomes (B) hybridised with Cp3.4. (A) Lanes 1–4, C. par6um DNA (5 mg) restricted with the endonucleases PstI, HindIII, DraI and EcoRI, respectively; lane 5, C. muris DNA (5 mg); lane 6, Eimeria acer6ulina DNA (1 mg); lane 7 Toxoplasma gondii DNA (5 mg); lane 8, human DNA (10 mg). DNA in lanes 5 – 8 were restricted with EcoRI. Positions of l HindIII DNA size markers (in bp) are indicated on the left. The blot was washed at low stringency (2× SSC, 65°C) and exposed for 24 h. (B) Lane 1 represents an ethidium bromide staining of five bands of chromosomal DNA from C. par6um (highlighted by bars on the right) separated by pulsed field gel electrophoresis; lane 2, hybridisation signal of the corresponding blot after probing with Cp3.4. The positions of S. cere6isiae chromosome size markers (Mb) are given on the left.

[15] and Khramtsov et al. [16] who have both demonstrated five discrete chromosomal DNAs by orthogonal field alteration gel electrophoresis ranging in size from 900 to 1400 kb [15] and by clamped homogeneous electric field electrophoresis in the 1.05–1.65 Mb range [16]. Mead et al. [17] have also found five DNA bands of C. par6um by field inversion gel electrophoresis; however, the sizes of the chromosomes were considerably larger (1400–3300 kb). A recent paper by Blunt et al. [18] identified eight chromosomes by densitometry of a clamped homogeneous electric field electrophoresis gel in the range of 1.04 and 1.54 Mb. This analysis suggests the highest band being a doublet and the middle (1.24 Mb) being a triplet. The hybridisation of the pulsed field gel in this study could localise the Cp3.4 gene on the largest chromosome with a size of : 1.6 Mb. Northern blot analysis of total sporozoite RNA hybridised with the Cp3.4 probe and washed at high stringency showed a mRNA species of : 6 kb in size in addition to hybridisation signals of high molecular masses. This 6 kb band was extremely weak in comparison to the strong signal obtained from the pulsed field gel which was hybridised in parallel, although 10 mg of total RNA were used (data not shown). In order to confirm the presence of the Cp3.4 transcript in RNA preparations of C. par6um, sporozoite RNA was reverse transcribed into cDNA and amplified in a RT-PCR using Cp3.4 specific primers (Fig. 3). Amplification of 125 ng of total RNA gave a strong product suggesting that the RNA preparation was contaminated with chromosomal DNA. After DNase treatment of the same amount of RNA, no PCR product was found and the cDNA from this preparation gave only a weak band (Fig. 3, lane 4). These results suggest that the Cp3.4 mRNA is not abundant in the RNA preparation from sporozoites used in this experiment. However, specific PCR products could be seen in cDNA generated from 125 ng of DNase-treated total RNA from the intestine of mice infected with C. par6um. The mRNA was easily detectable in cDNA from the ileum (lane 8) and to a lesser extent in caecum cDNA (lane 12). It appears that Cp3.4 gene expression takes place in intracellular stages of the parasite and that the isolation of the

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Fig. 3. PCR amplification of Cp3.4 from total RNA and cDNA preparations. In order to compare the quantities of PCR products, 125 ng of RNA or cDNA generated from 125 ng of mRNA were used in the reactions. PCR results from cDNA generated from DNase I-treated total RNA (lanes 4, 8 and 12) are indicated by arrows. Lane 1, total RNA from purified C. par6um oocysts/sporozoites; lane 2, cDNA from RNA of lane 1; lane 3, DNase I-treated total RNA from purified C. par6um oocysts/sporozoites; lane 4, cDNA from DNase I-treated RNA of lane 3; lane 5, total RNA from the ileum of a mouse infected with C. par6um6cDNA from RNA of lane 5; lane 7, DNase I-treated total RNA from the ileum of a mouse infected with C. par6um; lane 8, cDNA from DNase I-treated RNA of lane 7; lane 9, total RNA from the caecum of a mouse infected with C. par6um; lane 10, cDNA from RNA of lane 9; lane 11, DNase I-treated total RNA from the caecum of a mouse infected with C. par6um; lane 12, cDNA from DNase I-treated RNA of lane 11; lane 13, C. par6um genomic DNA; lane 14, Cp3.4 plasmid DNA; lane 15, negative control (no DNA). M, 100 bp DNA marker (GIBCO-BRL).

Cp3.4 cDNA clone from a sporozoite expression library was due to the presence of only a few or even one mRNA molecule during cDNA library production. The function of the gene product remains unknown but preliminary results from IFAT of an oocyst/sporozoite preparation suggest that Cp3.4 antigen is a constituent of the oocyst wall (data not shown). The same Cp3.4 primers were used to detect C. par6um in chromosomal DNA preparations of the small intestine of mice infected with the parasite. Fig. 4 shows the results of Cp3.4 PCR in non-infected mice (A), non-infected mice treated with dexamethasone (B) and dexamethasone-treated mice infected with 106 oocysts 3 days prior to the DNA preparation from mucosal scrapings. Whereas no PCR products were amplified from the mucosa of non-infected mice even at 1.6 mg of template DNA (A and B), strong bands of the expected size (649 bp) were seen in mucosa DNA preparations of infected mice. In order to determine the sensitivity of this PCR and to calculate the proportion of parasite DNA in total DNA of infected mucosa, purified C. par6um oocysts/ sporozoite DNA was titrated against mucosa DNA from infected mice (Fig. 5). Amplification of the Cp3.4 gene from as little as 0.8 pg of purified C. par6um DNA yielded a detectable band, which is equivalent to : 140 oocysts as

calculated from the genome size of C. par6um of 10.4 Mb [18]. This value is comparable to the results of Laxer et al. [19] who detected a 400 bp PCR product from 300 fg of purified C. par6um DNA by ethidium bromide staining of a gel and who could increase the detection by a factor of ten by hybridisation of the PCR fragments with an internal oligonucleotide probe. From mucosa of infected mice, amplification of B 0.1 ng of total DNA from mice at days 3 and 8 post infection gave a PCR product. These results indicate that the amount of parasite DNA contributes to B 1% of the total chromosomal DNA of ileal mucosa of mice infected with C. par6um. The mice used in these experiments shed C. par6um oocysts at a high level. More than ten oocysts were seen per × 1000 oil immersion field of a Ziehl–Neelsen stained faecal smear at day 3 post infection. But even at day 8 post infection when the oocyst number drops : 10-fold, the amount of PCR product was nearly unchanged. We therefore screened for C. par6um DNA in the mucosa of non-dexamethasone-treated C57BL/ 6 ×129 F1 mice that were given 106 oocysts 3 or 8 days before DNA preparations. In these mice, oocysts cannot be detected by acid fast staining of faecal smears (unpublished observation). However, Cp3.4 DNA can be amplified from as little as 25 ng of total mucosal DNA at day 3 and from 800 ng of DNA at day 8 (Fig. 6).

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Fig. 4. PCR amplification of the Cp3.4 gene from total chromosomal DNA extracted from the ileal mucosa of mice. Minimal template amounts giving a PCR product are indicated. (A) DNA from non-infected C57BL/6 mice. (B) DNA from non-infected mice treated with dexamethasone. (C) DNA from dexamethasone-treated mice infected with 106 C. par6um oocysts 3 days prior to DNA preparation. Lanes 1–6, 2-fold serial dilutions of 1.6 mg template DNA; lane 7, Cp3.4 plasmid DNA; lane 8, negative control (no DNA).

Several investigators have concentrated on the detection of C. par6um oocysts by PCR approaches in order to increase the sensitivity of detection levels over conventional staining and immunodetection of oocysts from faeces and wa-

ter samples. Current detection limits reach one to ten oocysts using either nested PCR [20] or hybridisation of PCR product with labelled internal oligonucleotide probes [21–25]. This is equivalent to 5.7–57 fg of chromosomal DNA. Besides direct

Fig. 5. Amplification of Cp3.4 in DNA preparations from purified C. par6um oocysts using the method described in Section 2 and in DNA preparations from ileum mucosa of C. par6um infected C57BL/6 mice day 3 (B) and day 8 (C) post infection using the guanidinium proteinase K method [9]. Minimal template amounts giving a PCR product are indicated. A. In lanes 1 – 16, 2-fold serial dilutions of C. par6um DNA were used for PCR amplification starting with 1.6 ng DNA in lane 1; lane 17, negative control. Parts B and C. In lanes 1–10, 2-fold serial dilutions of ileum mucosa DNA were used starting with 100 ng DNA.

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Fig. 6. Detection of C. par6um by amplification of the Cp3.4 gene. Minimal template amounts giving a PCR product are indicated. A and C, ileum DNA from dexamethasone-treated C57BL/6 mice which have been infected with 106 oocysts. B and D, ileum DNA from non-treated C57BL/6× 129 F1 mice infected with 106 oocysts. In the F1 hybrids, no oocyst shedding can be seen by acid fast staining of faecal smears. A and B, DNA preparations of mice infected 3 days before PCR analysis. C and D, DNA preparations of mice infected 8 days before PCR analysis. Lanes 1–8, 2-fold serial dilutions starting with 1.6 mg of template DNA; lanes 9, negative control (no DNA).

rect amplification of oocyst DNA derived from faecal and water samples, a detection system has been developed that combines in vitro cultivation with RT-PCR [26]. With this method, infectivity of oocysts from water samples can be determined. The detection limit lies between one and ten oocysts from 65 to 100 l of water. Only a few papers have been published that address the detection of C. par6um in infected tissues by PCR. In one report, as a source of DNA specimen fixed and embedded in paraffin were used for amplification [27]. In comparison to our results, the detection of parasite DNA from 0.1 mg of total DNA extracted from a human ileum biopsies appears to be much lower. This 3-log difference may be due to the degradation of target DNA by the fixative, a lower grade of infection of the host tissue or qualitative differences between the PCR amplifications used. Two recent reports describe the de-

tection of parasite DNA in the intestine of infected neonatal mice by a semi-quantitative PCR technique [28,29]. The authors used 100 ng of either total intestine or ileum DNA and found that specific PCR products can be detected in DNA preparations of mice infected with as few as 100 oocysts. The use of terminal ileum for DNA preparation resulted in a 10-fold increase of the PCR signal over DNA extracted from whole intestine. These findings support our observation that ileum DNA is superior to caecum DNA in detecting intracellular parasite development (Fig. 3 and data not shown here). A direct comparison of the two PCR protocols is not possible as Jenkins et al. [28] have not tested the minimal amount of DNA that produces a positive PCR signal but rather determined the minimal number of oocysts required for the detection of parasite infection. Furthermore, we used DNA extracted

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from mucosa which has a higher parasite DNA to host DNA ratio than whole ileum tissue as parasite stages are restricted to the epithelium of the gut. From these results we conclude that in situations where conventional methods fail to detect C. par6um, parasite DNA can still be demonstrated in the intestine by PCR analysis. This method might be useful for the detection of C. par6um in intestine or gall bladder biopsies of individuals that do not shed sufficient numbers of oocysts to be detectable by conventional methods or in broncho-alveolar lavages from patients with suspected pulmonary cryptosporidiosis. Furthermore, this method might be a valuable, sensitive and simple tool for the screening of potential reservoir hosts that could contribute to the spread of C. par6um in the environment.

[4]

[5]

[6]

[7]

[8]

[9] [10]

Acknowledgements We thank Dr Fiona Tomley (Institute for Animal Health, Compton, UK) for Eimeria DNA, Dr Tim McHugh (Royal Free Hospital, London, UK) for Toxoplasma DNA and Sabine Pauls and Inka Kneib for technical assistance. F.P. is supported by grants from the German Ministry of Research and Technology (BMFT)-Infection Programme and the German Research Foundation (DFG). V.M. was supported by grants from the Medical Research Council and the Biotechnology and Biological Sciences Research Council.

[11]

[12]

[13]

[14]

[15]

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