1 Department of Veterinary Pathobiology, College of Veterinary Pathobiology, Texas A&M University, 4467 TAMU, College Station, TX 77843-4467, USA
2 Faculty of Genetics Program, Texas A&M University, 4467 TAMU, College Station, TX 77843-4467, USA
3 Department of Veterinary Pathobiology, University of Minnesota, St Paul, MN 55108, USA
Correspondence
Guan Zhu
gzhu{at}cvm.tamu.edu
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ABSTRACT |
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INTRODUCTION |
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Although C. parvum can be cultured in vitro by infecting various primary or transformed human or animal cell lines, the development of this parasite in vitro has been mostly limited to the asexual development (merogony) (Arrowood, 2002; Upton, 1997
; Upton et al., 1995
). Well-developed C. parvum can typically be observed in vitro after infecting host cells for 24 and 48 h, in which most parasites are in the stages of Type I and II meronts (with a few developing gametes), respectively. However, the growth of parasites apparently starts to decline after cultivation in vitro for 72 h or longer, in which many of them have typically developed into macrogametes or microgametocytes. Although limited numbers of C. parvum oocysts produced in vitro have been reported, most of them are not biologically viable (Hijjawi et al., 2001
; Yang et al., 1996
). As yet, it is unclear why most C. parvum can not complete sexual development under in vitro conditions.
C. parvum possesses a compact genome with short intergenic regions, and only a limited number of introns have been reported in the C. parvum genome (Bankier et al., 2003; Caccio et al., 1997
; Deng et al., 2002
). Since introns have to be removed by splicing mature RNA, primers spanning the intron regions have been frequently used in RT-PCRs to distinguish RNA-originating amplicons from those amplified from DNA. Using this approach, however, the presence of intron-containing transcripts was unexpectedly observed in late developmental stages of C. parvum cultured in vitro, but not in those obtained from infected calves. These observations imply a possible abnormality in intron-processing by C. parvum during some in vitro life cycle stages, and these can be correlated with the dramatic arresting of growth and sexual development of parasites cultured in vitro.
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METHODS |
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For cultivation of C. parvum in vitro, HCT-8 or Caco-2 cells (106 per well) were seeded in six-well plates and allowed to grow overnight or until they reached confluence. Free sporozoites were added to the monolayers at parasite-to-host cell ratios of either 1 : 5 (for infections lasting up to 24 h) or 1 : 10 (for infections lasting longer than 24 h), respectively. Free sporozoites were removed after 3 h infection by a medium replacement. After monolayers were infected with sporozoites at various time-points (e.g. 3, 6, 12, 24, 36, 48, 60 or 72 h), cells were washed three times with PBS and treated immediately with RNAlater (Ambion) to preserve RNA integrity. Total RNA was then isolated from RNAlater-treated monolayers using an RNeasy isolation kit (Qiagen). For the isolation of total RNA, uninfected host cells cultured for 24 h were similarly treated and used as one of the negative controls. In addition, total RNA was also isolated from the small intestinal epithelial cells from four calves (in vivo samples): two were uninfected and the other two were infected with C. parvum for 72 h. All RNA samples were further treated with RNase-free DNase and purified again. In some cases, the DNase treatment procedure was repeated up to five times to ensure no contamination of genomic DNA occurred in any samples.
RT-PCR.
Since total RNA isolated from infected host cells or intestines might contain different ratios of parasite 18S RNA, the concentration of parasite total RNA in all samples was normalized using a semi-quantitative RT-PCR method described previously (Abrahamsen & Schroeder, 1999). Briefly, using an AccessQuick RT-PCR kit (Promega) and a pair of 18S RNA-specific primers (995F, 5'-TAG AGA TTG GAG GTT GTT CCT-3'; 1206R, 5'-CTC CAC CAA CTA AGA ACG GCC-3'), the amplifications were performed for 22 thermal cycles to ensure the amounts of RT-PCR products were within the linear range. The amplicons (10 µl each) were separated in 2 % agarose gels, intensities were measured and then were used as a guide for normalizing parasite total RNA in the various diluted samples. These adjusted total RNA samples were subject to a second or third round semi-quantitative RT-PCR and the concentrations were further adjusted until the intensities of the amplicons were comparable. Therefore, although the concentrations of total RNA (a mixture of host and parasite RNA) might vary among different samples, those of parasite RNA in different samples were relatively similar (if not identical) and suitable to serve as templates for comparative amplification of parasite transcripts.
The intron-containing -tubulin gene from C. parvum has been characterized previously (Caccio et al., 1997
). In this study, the following two primers flanking the 85 bp intron were used for detecting C. parvum
-tubulin transcripts by RT-PCR: CpBTUB-F137 (5'-ATG TTC AAG GAG GAC AAT GTG-3') and CpBTUB-R611 (5'-GAG TGA GTG ATT TGG AAA CCC-3'). (Note: the two underlined nucleotides may represent sites of variations between isolates based on the recent deposit of alternative sequences in GenBank, e.g. Y12615 vs BX538353). Each 25 µl reaction contained 50 ng each of the primers, 2·5 U reverse transcriptase (RTase), 2·5 U DNA polymerase, 1 µl normalized total RNA and other reagents as specified by manufacturer's instructions (Promega). The synthesis of first strand cDNA was performed at 48 °C for 45 min, followed by a heat-inactivation of RTase at 95 °C for 2 min and subsequent 2832 thermal cycles of amplification (95 °Cx30 s, 50 °Cx30 s and 68 °Cx60 s for each cycle). All amplicons were analysed by 2 % agarose gel electrophoresis.
Fluorescence in situ hybridization (FISH).
The following three probes were synthesized by Integrated DNA Technologies (IDT, Coralville, IA) to confirm the presence of intron-containing -tubulin transcripts in C. parvum. (i) Intron-antisense (5'-GTT CAA ATT ATT TCA ATT ATT TTC AAA ACT-3') corresponding to a nucleotide sequence within the C. parvum
-tubulin intron region for detecting intron-containing mRNA. This probe was labelled with TAMRA (carboxytetramethylrhodamine) at both ends. (ii) Intron-sense oligonucleotide (5'-AGT TTT GAA AAT AAT TGA AAT AAT TTG AAC-3') complementary to the Antisense-intron probe as a negative control, TAMRA-labelled at both ends. (iii) Exon-antisense probe (5'-TCT AAC TGA ATC CAT TGT TCC TGG CTC AAG-3') corresponding to a C. parvum
-tubulin exon as a positive control. This oligonucleotide was linked with FAM (carboxyfluorescein) at both ends. Since our observation by RT-PCR indicated that intron-containing
-tubulin transcripts started to appear after C. parvum was cultured for 48 h or longer, only HCT-8 monolayers infected with C. parvum for 36 and 60 h, respectively, were selected for comparative FISH analysis. Briefly, C. parvum-infected monolayers growing on poly-L-lysine-coated glass cover-slips were fixed in PBS-buffered formaldehyde (4 %) and acetic acid (10 %) for 10 min at room temperature. After two washes in RNase-free PBS, the monolayers were permeabilized in 70 % ethanol overnight or longer at 4 °C, hydrated for 5 min at room temperature in 2xSSC/50 % formamide and then hybridized overnight at 37 °C with 30 ng each individual or dual probes in a buffer containing 2xSSC/50 % formamide/10 % dextra sulfate/2 mM vanadylribonucleoside complex/0·02 % RNase-free BSA/40 µg Escherichia coli tRNA. After three washes at 37 °C for 30 min with 2xSSC/50 % formamide, cells were incubated with a monoclonal anti-fluorescein/Oregon Green and/or a rabbit anti-tetramethylrhodamine isothiocyanate (TRITC) antibody (Molecular Probes) in PBS containing 0·5 % BSA for 2 h at 37 °C, followed by three washes with PBS/BSA (0·5 %). Samples were then labelled with FITC-conjugated goat anti-mouse IgG (H+L) and/or TRITC-conjugated goat anti-rabbit IgG antibodies. After three washes to remove free conjugates, samples were mounted onto clean glass slides using a SlowFade Light Antifade mounting medium (Molecular Probes) and examined using an Olympus BX51 Epi-Fluorescence microscope system equipped with FITC/TRITC filters.
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RESULTS AND DISCUSSION |
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The study of C. parvum biology is greatly limited by the inability of the parasite to complete its life cycle in vitro to produce vital oocysts. This parasite can grow after infecting host cells, but optimal growth is mostly limited to the asexual development (merogony) that occurs within the first 48 h (Upton, 1997). The appearance of an unprocessed
-tubulin intron in C. parvum correlates well with the decline or arrest of growth of this parasite under in vitro conditions, implying that an abnormality in intron-splicing might be associated with difficulties in culturing this parasite. If this is true, investigations on the factors contributing to the intron-splicing in cultured C. parvum may provide important information for overcoming the limitations in culturing this parasite. Furthermore, the co-existence of processed and unprocessed introns, at least in
-tubulin transcripts in vitro, may also be utilized as a potential model to study the intron-splicing factors in this parasite.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 10 November 2003;
revised 3 January 2004;
accepted 4 February 2004.
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