Cryptosporidium parvum: the many secrets of a small genome

International Journal for Parasitology 30 (2000) 553±565

www.elsevier.nl/locate/ijpara

Cryptosporidium parvum: the many secrets of a small genome Furio Spano a, Andrea Crisanti b, * a

Istituto di Parassitologia, UniversitaÁ di Roma ``La Sapienza'', P. le A. Moro, 5, Box 6 Roma 62, 00185 Rome, Italy Department of Biology, Imperial College, Alexander Fleming Building, Imperial College Road, London SW7 9AX, UK

b

Received 30 July 1999; received in revised form 8 November 1999; accepted 8 November 1999

Abstract The coccidium Cryptosporidium parvum is an obligate intracellular parasite of the phylum Apicomplexa. It infects the gastrointestinal tract of humans and livestock, and represents the third major cause of diarrhoeal disease worldwide. Scarcely considered for decades due to its apparently non-pathogenic nature, C. parvum has been studied very actively over the last 15 years, after its medical relevance as a dangerous opportunistic parasite and widespread water contaminant was fully recognised. Despite the lack of an ecient in vitro culture system and appropriate animal models, signi®cant advances have been made in this relatively short period of time towards understanding C. parvum biology, immunology, genetics and epidemiology. Until recently, very little was known about the genome of C. parvum, with even basic issues, such as the number and size of chromosomes, being the object of a certain controversy. With the advent of pulsed ®eld gradient electrophoresis and the introduction of molecular biology techniques, the overall structure and ®ne organisation of the genome of C. parvum have started to be disclosed. Organised into eight chromosomes distributed in a very narrow range of molecular masses, the genome of C. parvum is one of the smallest so far described among unicellular eukaryotic organisms. Although fewer than 30 C. parvum genes have been cloned so far, information about the overall structure of the parasite genome has increased exponentially over the last 2 years. From the ®rst karyotypic analyses to the recent development of physical maps for individual chromosomes, this review will try to describe the state-of-the-art of our knowledge on the nuclear genome of C. parvum and will discuss the available experimental evidence concerning the presence of extra-chromosomal elements. # 2000 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Apicomplexa; Chromosome structure; Cryptosporidium parvum; Extra-chromosomal DNA; Genetic markers; Genotypes; Microsatellites; Molecular karyotype; Nuclear genome; Telomeres

1. Introduction The phylum Apicomplexa (or Sporozoa) consists of approximately 5000 species of symbiotic protozoans that share a number of morphological traits, in particular a set of highly specialised organelles and structures which form the so-called ``apical complex''. The phylum includes some of the most important human and animal parasites, which fall into two major orders. The Eucoccidida, e.g. Toxoplasma, Eimeria, Isospora,

* Corresponding author. Tel.: +44 171 594 5426; fax: +44 171 594 5439. E-mail address: [email protected] (A. Crisanti).

Sarcocystis, Cryptosporidium and Neospora spp., infect preferentially the gastrointestinal tract of the host, whereas members of the Haemosporida (e.g. Plasmodium, Babesia and Theileria spp.) are haematic parasites transmitted through the bite of arthropod vectors. Representatives of the phylum Apicomplexa have been studied in depth for decades because of their impact on human health (Plasmodium falciparum, Toxoplasma gondii), the economic losses they cause in livestock (Eimeria spp., Babesia spp., Theileria spp.) or their suitability as models for elucidating ultrastructural features (Sarcocystis spp.) or, more recently, for genetic studies (T. gondii). On the other hand, some apicomplexans have attracted the interest of the scienti®c community only recently, as their relevance as

0020-7519/00/$20.00 # 2000 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 0 - 7 5 1 9 ( 9 9 ) 0 0 1 8 8 - 5

554

F. Spano, A. Crisanti / International Journal for Parasitology 30 (2000) 553±565

pathogens of humans and/or animals has become evident. This is the case for Cryptosporidium parvum, an ``emerging'' parasite to which this review is dedicated. After a brief introduction on the history and biology of this fascinating and singular parasite, we will review the studies that have contributed to our currently limited but rapidly expanding knowledge about the C. parvum genome.

viral therapy [24, 25], the parasite still represents a major public health problem, because of its high opportunistic potential and its increasingly frequent involvement in water-borne outbreaks [26, 27], the largest of which a€ected several hundred thousand immunocompetent individuals [28].

1.1. Historical background

The current classi®cation of the genus Cryptosporidium into a distinct family (Cryptosporidiidae) of the suborder Eimeriorina takes into account the evident similarities with other members of the suborder, such as Toxoplasma and Eimeria, as well as morphological, biological and genetic features that render cryptosporidia rather peculiar parasites. The taxonomy of Cryptosporidium is much less de®nitive and less widely accepted at the species level, which on recent ®ndings seems to be comprised of at least eight species with di€ering host ranges [12, 29]: Cryptosporidium parvum, infecting humans and a wide range of mammals, especially ruminants; C. muris, infecting mice and a limited number of other mammals; Cryptosporidium wrairi, isolated from guinea pigs; Cryptosporidium felis, found in cats and very recently in humans [30]; Cryptosporidium baileyi and C. meleagridis in birds; Cryptosporidium nasorum in ®sh; and Cryptosporidium serpentis, associated exclusively with reptiles. The recent application of molecular techniques for typing Cryptosporidium isolates and the discovery of genetically distinct isolates in pigs, mice, koala and other hosts suggests that the composition of the genus may be much more complex [31]. Over the last 4 years several groups, including ours, have investigated the intra-speci®c variability of C. parvum by typing isolates from di€erent geographical and host origins on the basis of polymorphisms detected by comparing their isoenzyme pro®les [32] or by analysing selected genetic loci by PCR-based techniques [33±37] or direct DNA sequencing [38]. Regardless of the typing technique used and the number of polymorphic markers analysed [39], all investigators found that C. parvum isolates could be classi®ed into two genetically distinct, apparently clonal subpopulations. Genotype 1 (or H) is con®ned to a fraction of human infections and is thought to be maintained through an anthroponotic transmission cycle. Genotype 2 (or C) is implicated in zoonotic transmissions and appears responsible for all C. parvum infections in animals and some infections in humans. Some authors have reported the existence of less common genetic variants, in addition to the two major genotypes [38, 40, 41]. The results of these genetic studies present a serious challenge to the concept of C. parvum as a homogeneous species and raise important questions concerning (i) the overall population

The genus Cryptosporidium owes its birth to Ernest Edward Tyzzer who, between 1907 and 1912, reported the identi®cation of novel coccidia-like organisms in the gastric glands [1, 2] or small intestine [3] of laboratory mice and provided a remarkably accurate description of the parasites' life cycle. Based on the di€erent sites of infection and morphology, Tyzzer classi®ed these novel protozoans as distinct species and coined the names Cryptosporidium muris for the gastric and Cryptosporidium parvum for the intestinal parasites. The description of Cryptosporidium meleagridis in turkeys in 1955 [4] and the identi®cation of cryptosporidia in diarrhoeic calves in 1971 [5] provided the ®rst indications that infection with Cryptosporidium spp. might be associated with morbidity and mortality. Their pathogenicity in humans was demonstrated in 1976 with the report of the ®rst two cases of cryptosporidiosis [6, 7], and further in the 1980s by the prevalence and consequence of Cryptosporidium infections in immunocompromised subjects [8±10], especially AIDS patients. Cryptosporidium parvum is now acknowledged as a pathogen of medical and veterinary importance [11, 12]. It is a major cause of diarrhoea in young livestock [13], especially calves [14], being associated with signi®cant morbidity and mortality. In humans, C. parvum is one of the most prevalent enteropathogens worldwide and the aetiological agent of a diarrhoeal disease whose course is dependent largely on the immunological status of the a€ected individual [15, 16]. In immunocompetent subjects, infections are usually self-limiting, lasting 1±2 weeks, and evoke a durable CD4 + T-celldependent immunity; in contrast, immunocompromised individuals frequently experience a severe and unremitting watery diarrhoea that can be fatal [17]. Over the last 15 years, C. parvum has been recognised as one of the most common and dangerous opportunistic parasites [18] a€ecting malnourished children in developing countries [19, 20]. It a‚icts 5±50% of AIDS patients, depending on the geographical area [21±23]. In spite of a recent substantial reduction of C. parvum infections in AIDS patients (Costagliola, 5th Conference on Retroviruses and Opportunistic Infections, Chicago, 1998; abstract no. 182), brought about by the introduction of a combined anti-retro-

1.2. Cryptosporidium taxonomy

F. Spano, A. Crisanti / International Journal for Parasitology 30 (2000) 553±565

structure of C. parvum, (ii) the reproductive isolation of the various subtypes, (iii) the epidemiological relevance and route(s) of transmission of each subtype to humans and animals, and (iv) the possibility that the clinical course of the infections may be determined by the genotype(s) involved. 1.3. Biology of Cryptosporidium parvum A detailed description of the life cycle of Cryptosporidium is beyond the objectives of this review and can be found in a series of excellent publications [12, 29, 42, 43]. We would rather like to summarise the parasite development by highlighting the morphological and biological features that render Cryptosporidium a unique parasite. The entire life cycle of C. parvum takes place within the gastrointestinal tract of the same host, preferentially in the brush border of the small intestine. This is typical of coccidia of the genera Eimeria and Isospora, and unlike the tissue cyst-forming parasites T. gondii, Sarcocystis spp. and N. caninum, whose sexual and asexual reproductions occur in distinct host species. The key developmental stage of C. parvum is the oocyst, which is shed with the faeces by the infected host and is responsible for the environmental contamination and transmission of the disease via the oral route. Mature oocysts consist of four infective haploid sporozoites enveloped within a double-layered and highly resistant cyst wall [44]. Unlike other coccidia, the sporozoites are free within the oocysts and not surrounded by sporocysts. Another distinctive feature is the presence, in the oocyst wall, of a scissure, a specialised longitudinal structure along which the oocyst wall ruptures during excystation, thus allowing the sporozoites to escape. Oocysts and sporozoites are the most studied stages and the almost exclusive source of parasitic material for molecular and biochemical analyses, due to the lack of an established and ecient in vitro culture system for propagating C. parvum and the diculty of isolating the intracellular forms of the parasite from infected animals. Excystation is induced by physico-chemical stimuli within the gastrointestinal tract, releasing the sporozoites which bind to speci®c ligands on the surface of the host's enterocytes. Morphological and molecular evidence suggests that the invasion process is basically the same in all apicomplexans [45, 46]. However, the penetration of C. parvum sporozoites and merozoites within the host cell presents interesting peculiarities [42, 47]. Upon attachment to an enterocyte and discharge of secretory molecules from the zoite apical organelles, C. parvum does not simply induce the typical invagination of the host plasma membrane (described, for example, in Plasmodium, Toxoplasma and Eimeria), but additionally initiates a profound

555

structural rearrangement of the enterocyte microvilli, which elongate and eventually fuse around the invading zoite. The parasite thereby establishes itself intracellularly within a parasitophorous vacuole that is delimited by a host-derived membrane, but which lies in a singular extra-cytoplasmic position, giving the impression of being attached to the apical surface of the enterocyte. As a consequence of this peripheral location with respect to the host cell cytoplasm, all C. parvum intracellular stages (trophozoites, type I and type II meronts, gametocytes, zygotes and immature oocysts) develop the so-called feeder organelle [48]Ð another peculiarity of the Cryptosporidium genus. This unique organelle has a multilamellar structure and is situated at the base of the parasitophorous vacuole. It is believed to mediate the uptake of nutrients from the host cell. At the end of the schizogonic cycle, merozoites originating from type II meronts di€erentiate into male and female gamonts inside newly invaded host cells, leading to the sexual phase. The fertilised macrogamont (zygote), the only diploid stage in the whole life cycle, develops into an oocyst that will sporulate in the host with formation of four mature sporozoites. Interestingly, it has been observed that two types of oocysts are shed into the intestinal lumen; thickwalled, double-layered oocysts that are shed with the faeces, and thin-walled oocysts [11, 12] that are thought to excyst within the same host (thus leading to an ampli®cation of the infection through a mechanism that has not been described in other coccidia). 1.4. Diculties in the study and control of C. parvum Despite the considerable advances made over the last decade in the biology, immunology, genetics and epidemiology of C. parvum, we have to recognise that little or no progress has been made towards resolving those problems that still make the study and control of C. parvum problematic. The study of the parasite is signi®cantly impeded by our inability to propagate it inde®nitely by in vitro culture. Using di€erent cell lines and culture conditions [49], only the asexual phase of the C. parvum life cycle has been reliably reproduced in vitro, although reports exist which describe the development of sexual stages and oocysts [50±52]. Cryptosporidium parvum-infected cell lines have been useful for the analysis of gene expression in intracellular stages by reverse transcription±PCR [53, 54], but most studies still depend on the availability of oocysts prepared from faecal material by laborious and time-consuming manipulations [55, 56]. In addition, unlike other apicomplexans and despite the number of di€erent protocols tested, the long-term storage of C. parvum remains problematic [57], due to the deterioration of cryopre-

556

F. Spano, A. Crisanti / International Journal for Parasitology 30 (2000) 553±565

served oocysts. This has hampered the establishment of a bank of reference strains. Moreover, the lack of convenient animal models mimicking the course of infection in humans has greatly hindered our understanding of the nature of the immune response elicited by the parasite, the evaluation of the pathogenicity of di€erent strains, the identi®cation of virulence factors and, most importantly, the assessment of drug ecacy. Griths et al. [58] recently described a new model of cryptosporidiosis in interferon-g receptor knockout mice. Although this model o€ers signi®cant advantages over the neonatal, immunode®cient or immunosuppressed animal models [59], an e€ective in vivo model for the propagation of C. parvum isolates of genotype 1, found exclusively in humans or in rhesus monkeys [39], still needs to be developed. Environmental control measures against C. parvum are dicult to implement. Oocysts are resistant to common chlorinated disinfectants [60] and their elimination from drinking water supplies relies mainly on the accuracy of serial treatments of waters (coagulation, ¯occulation and ®ltration), as well as on proper ®lter upkeep [61]. Exposure to ozone was shown to reduce oocyst viability and infectivity [62]. Nevertheless, further e€orts to ®nd highly e€ective chemical agents are required. Unfortunately, the ability to inhibit parasite development in the infected host is extremely limited. Since 1990, over 100 di€erent antibiotic and antiprotozoan agents have been tested against C. parvum with no or limited success [12, 15, 63]. The resistance of C. parvum to such a broad range of chemotherapeutic agents, some of which are e€ective against other apicomplexan parasites, might be related to the peculiar location of the intracellular stages and to their almost complete sequestration from the host cell cytoplasm.

was con®rmed by further molecular studies. Only one gene (encoding b-tubulin) out of approximately 25 sequenced so far has been found to possess intervening sequences, in this case a single intron of 85 bp [66]. In 1996, Char et al. [67] published a study in which the codon usage in C. parvum genes was compared with that determined for other members of the phylum Apicomplexa. Surprisingly, this analysis failed to show any close relationship between C. parvum and two other Eimeriorina species (T. gondii and E. tenella), but rather a higher similarity to P. falciparum, Babesia bovis and Theileria parva (Haemosporida spp.) and to Entamoeba histolytica. The predominantly intronless structure of C. parvum genes and the distinctive codon usage add to the many peculiar traits of this parasite, and strengthen the hypothesis that cryptosporidia have an evolutionary history that is distinct from that of related members of the phylum Apicomplexa [68].

2. First insights into the C. parvum genome

As mentioned previously, the ®rst attempt to delineate a molecular karyotype for C. parvum was carried out in 1988 by Mead et al. [78]. With the main objective of detecting chromosome size polymorphisms among clinical isolates of C. parvum and between C. parvum and the avian-derived species C. baileyi, these authors subjected the chromosome-sized DNA of the parasites to ®eld-inversion gel electrophoresis (a modi®ed PFGE methodology). Of the ®ve C. parvum isolates that were analysed (four from animals and one from a human), all yielded the same ®eld-inversion gel electrophoresis pro®le, consisting of ®ve bands migrating in the 1.4±3.3 Mbp size range. The electrophoretic pro®le of C. baileyi was distinct, and consisted of six chromosomal bands ranging in size between 1.4 and over 3.3 Mbp. The second systematic approach to the establishment of the C. parvum karyotype was published by Hays et al. 7 years later [79]. This followed other

Until 1988, when C. parvum was ®rst analysed at the karyotypic level using pulsed ®eld gradient electrophoresis (PFGE) [64], the information on the nuclear content of the parasite was essentially morphological [42, 65]. Electron microscopic observations of most stages had shown a prominent nucleus with electron-lucent and electron-dense areas, possibly re¯ecting a di€erent transcriptional status of the chromatin. Microgamonts were characterised by a highly condensed nucleus, whereas in trophozoites and macrogamonts the nucleus appeared large, with an evident nucleolus indicating a very active transcription of ribosomal genes. Nucleotide sequence analysis of the ®rst cloned C. parvum genes revealed an elevated A/T content (approx. 65%), and a lack of introns interrupting the protein-encoding regions. The absence of introns as a general feature of the C. parvum genome

3. Establishment of the molecular karyotype The lack of chromosome condensation during metaphase in unicellular eukaryotes has hampered the use of classic genetic and cytogenetic approaches, and rendered PFGE techniques as invaluable tools for the karyotypic analysis of diverse protozoan parasites [69]. These include Leishmania [70], Trypanosoma [71] and Giardia [72], as well as numerous apicomplexans (Eimeria [73], Toxoplasma [74], Plasmodium [75], Babesia [76] and Theileria [77]). The technique has recently provided estimates on the number and size of C. parvum chromosomes. 3.1. Pulsed ®eld gradient electrophoresis analysis of intact chromosomes

F. Spano, A. Crisanti / International Journal for Parasitology 30 (2000) 553±565

reports [80±82] which, in the course of characterising newly cloned genes, had found C. parvum to possess ®ve to nine chromosomes smaller than 1.6 Mbp, in sharp contrast with the observations of Mead et al. [78]. Hays et al. [79] subjected the genomic DNA of a single C. parvum isolate to contour-clamped homogeneous electric ®eld (CHEF) electrophoresis, a widely used PFGE technique. Adopting a series of electrophoretic conditions, these authors consistently resolved the chromosomes of C. parvum into seven discrete bands ranging in size from 0.94 to 2.2 Mbp, with the suggested presence of an eighth co-migrating chromosome. At this stage, the results obtained by di€erent authors in the chromosome assignment of speci®c genes were hardly comparable, and in the absence of a reference karyotype there was more confusion than certainty. Indeed, the C. parvum karyotype remained a rather controversial issue until 1997, when Blunt et al. [83] described a new characterisation of the C. parvum chromosomes, combining CHEF electrophoresis with a densitometric scanning of the ethidium bromide-stained chromosomal bands. Blunt et al. reproducibly obtained an electrophoretic pattern comprising ®ve chromosomal bands of 1.54, 1.44, 1.24, 1.08 and 1.04 Mbp. Densitometric analysis showed that the largest (1.54 Mbp) and middle (1.24 Mbp) bands contained two and three distinct chromosomes respectively, and that the genome of C. parvum therefore consisted of eight chromosomes for a total size of approximately 10.4 Mbp. The electrophoretic pro®le described by Blunt et al. was consistent with those reported in some of the previous studies and it was subsequently con®rmed by several groups of investigators [39, 84±86]. 3.2. Contour-clamped homogeneous electric ®eld gel electrophoresis of chromosomes after restriction cleavage The molecular karyotype of C. parvum was more ®rmly elucidated in 1998 by CaccioÁ et al. [87].

557

Chromosomes were cleaved with rarely cutting restriction enzymes (S®I or NotI) and subsequently analysed by CHEF gel electrophoresis and Southern blot hybridisation. Using this integrated approach, the authors showed that the restriction and hybridisation patterns obtained were consistent with the presence of eight chromosomes, some of which had very similar molecular sizes, as suggested by Blunt et al. [83]. The eight chromosomes, individually designated with roman numerals, were attributed the following molecular sizes (in Mbp): I, 0.94; II, 1;04; III, 1.10; IV, 1.13; V, 1.14; VI, 1.36; VII, 1.43; VIII, 1.44. The calculated overall size (9.4 Mbp) showed good agreement with the 10.4 Mbp estimate of Blunt et al. and identi®ed the C. parvum genome as one of the smallest so far characterised among protozoan parasites (Table 1). In the course of their karyotypic analysis, CaccioÁ et al. also assigned 20 genetic markers (17 cloned genes, one anonymous and two repetitive sequences) to individual C. parvum chromosomes. The selected markers included most of the genes mapped previously by di€erent authors using disparate PFGE approaches and the analysis assigned linkage groups consisting of at least two markers to seven out of eight chromosomes.

4. Development of physical maps for C. parvum chromosomes 4.1. A HAPPY map of the whole genome A crucial step towards understanding the organisation of whole genomes is represented by the development of physical maps for individual chromosomes. For C. parvum, a major contribution reported recently by Piper et al. [88] produced a so-called ``HAPPY'' map for each of the eight C. parvum chromosomes of the Moredun strainÐa widely used cervine isolate routinely passaged in sheep. ``HAPPY'' mapping [92] is a relatively easy and fast methodology, particularly suited for the study of small genomes and also success-

Table 1 Main structural features of the nuclear genomes of apicomplexan parasites Organism

Genome size (Mbp)

Number of chromosomes

Chromosomal size range (Mbp)

Cryptosporidium parvum Toxoplasma gondii Eimeria tenella Plasmodium falciparum Plasmodium vivax Plasmodium malariae Plasmodium ovale Theileria parva Babesia bovis

9.6 80 50 27±30 35±40 35±40 35±40 10 9.4

8 10 14 14 14 14 13±14 4 4

0.95±1.45 2±40 1± > 6 0.7±3.5 1.2 ± > 3.5 1.6 ± > 3.5 1.6 ± > 3.5 2.2±3.1 1.4±3.2

References [87, 88] [74] [73] [75, 89] [90] [90] [90] [77, 91] [76]

558

F. Spano, A. Crisanti / International Journal for Parasitology 30 (2000) 553±565

fully applied to human chromosome XIV [93]. It is a PCR-based in vitro technique involving random shearing of intact genomic DNA, subdivision into aliquots of approximately haploid amounts by limiting dilution, followed by the evaluation of the frequency of co-segregation of markers among the aliquots. Closely linked markers have a high frequency of co-segregation due to the low probability of a DNA breakage between them. The random shearing of DNA acts as a physical equivalent of the meiotic recombination between homologous chromosomes on which traditional linkage mapping is based. The ``HAPPY'' mapping approach also o€ers signi®cative advantages over biological mapping methodologies, as it is largely una€ected by intrinsic features of the genome under study, e.g. the AT content or the existence of recombination hotspots. In addition, it requires small amounts of genomic DNA, a characteristic that is particularly desirable when working with C. parvum, for the reasons mentioned earlier. In their study, Piper et al. [94] employed a total of 237 markers, of which 192 were downloaded from the GenBank database, while 38 and seven respectively were derived from characterised genomic fragments identi®ed within M13 and P1 arti®cial chromosome C. parvum libraries constructed by the authors. Two hundred and four markers out of 237 were successfully mapped and 10 linkage groups were identi®ed and assigned to the corresponding chromosomes, leaving one un®lled gap on both chromosomes V and VIII. The resulting map covered all eight C. parvum chromosomes with an average marker spacing of approximately 50 kb. It was veri®ed by the authors in several ways, including chromosome assignment of 45 markers by CHEF gel electrophoresis and Southern blot hybridisation and, at a ®ner level, by comparing the predicted distance between selected markers (calculated from the map) with that deduced by analysis of P1 arti®cial chromosome inserts. No major discrepancies were reported. With respect to the size of individual chromosomes, the values deduced from the ``HAPPY'' map did not match perfectly those based on PFGE analysis. Chromosomes I and III exhibited 78% and 124% of their predicted electrophoretic mobilities, whilst the remaining chromosomes showed a ¯uctuation within 10% of their electrophoretic sizes. These discrepancies are somewhat inherent to the approach used and may re¯ect, especially for the smaller C. parvum chromosomes, the under-representation of near-telomeric markers. 4.2. A long-range restriction map of chromosome VI Another karyotypic study that has generated information about the physical organisation of C. parvum chromosomes was recently carried out by our group using the Moredun strain as reference [95]. Employing

CHEF gel electrophoresis and Southern blot hybridisation, the chromosomal location of 12 C. parvum genes (four of which were cloned in our laboratory) was established. Five of the markers, corresponding to the genes Cryptosporidium parvum oocyst wall protein [96], b-tubulin [66], sporozoite cysteine-rich protein (Spano et al., unpublished), ribonucleotide reductase R1 subunit (Balakrishnan et al., unpublished) and protein disulphide isomerase [97], were found to form a linkage group on chromosome VI. These were exploited to develop a long-range restriction map of this chromosome. Intact C. parvum genomic DNA was incubated with the restriction endonucleases EagI and SmaI, which cleave the AT-rich genome of the parasite at rare sites. The CHEF-separated restriction fragments were tested for hybridisation with each of the ®ve chromosome VI-speci®c markers. This identi®ed three EagI restriction fragments of 0.65 (b-tubulin), 0.51 (sporozoite cysteine-rich protein, ribonucleotide reductase R1 subunit and protein disulphide isomerase) and 0.17 (Cryptosporidium parvum oocyst wall protein) Mbp. Although the sum of the EagI fragments (1.33 Mbp) matched almost perfectly the molecular size of chromosome VI (1.36 Mbp) as estimated by CaccioÁ et al. [87], Southern analysis of C. parvum chromosomes partially digested with EagI suggested the presence of an additional fragment. In order to unequivocally map the chromosomal terminal ends, CHEF-separated chromosome VI (one of the three in C. parvum that migrates as a distinct band) was excised from the agarose gel and incubated with EagI. Following CHEF electrophoresis, the restriction fragments were hybridised with an oligonucleotidic probe consisting of ®ve tandemly repeated copies of the C. parvum telomeric repeat TTTAGG [98]. This con®rmed the presence of a fourth (0.17 Mbp) EagI restriction fragment that was derived from a terminal position. The long-range restriction map produced from these data shows chromosome VI to have a size of 1.5 Mbp. This value is likely to represent a more accurate estimate than those reported previously, due to the better resolution by CHEF gel electrophoresis of DNA molecules in the 1.0±0.1 Mbp range. 5. Genome sequencing projects The growing interest of the scienti®c community in C. parvum is clearly re¯ected by the number of DNA sequences deposited in the databases over the last decade (about 40 in 1995, rising to 5200 currently). This dramatic increase is due to recently initiated genome projects whose aim is to sequence the entire parasite genome. Three main sequencing projects are in progress, two in the US and one in the UK. An Expressed Sequence Tag (EST) project (web site http://mercury.e-

F. Spano, A. Crisanti / International Journal for Parasitology 30 (2000) 553±565

bi.ac.uk/parasites/cparvEST.html) is being carried out at the University of California, with the objective of determining 300- to 600-bp-long single-pass sequences from about 1000 genes expressed in the sporozoite stage. The project involves sequencing the 5 0 ends of cDNA inserts randomly selected from three directionally cloned cDNA libraries, which were constructed in the phage vector Uni-ZAP XR using the mRNA of C. parvum sporozoites (Iowa isolate, genotype 2). Almost 600 ESTs have been analysed so far, of which 68% represent unique sequences corresponding to about 265 kb of novel coding genetic information. Thirtyseven percent of the non redundant ESTs shared signi®cant homology with GenBank sequences. A collaborative Gene Sequence Tag (GST) project (web site http://mercury.ebi.ac.uk/parasites/cparvGST.html) is being conducted by researchers at the Universities of California and Minnesota. Unlike the EST approach, the GST project consists in the generation of short DNA sequences from random genomic inserts of the Iowa isolate, thus enabling identi®cation of genes that are expressed in all developmental stages of the parasite, as well as genomic sequences devoid of coding function. Over 2000 GSTs have been characterised so far by sequencing about 500 bp from both ends of randomly sheared genomic DNA inserts derived from a pBlueScript II (SKÿ) plasmid library. Overall, the

559

sequenced GSTs account for approximately 10% of the C. parvum genome, with about 20% of unique GSTs showing signi®cant homology with sequences present in GenBank. A slightly di€erent approach to the characterisation of random C. parvum genomic sequences, a Sequence Tagged Sites project (web site http://mercury.ebi.ac.uk/parasites/cparvSTS.html), has been undertaken by scientists at the MRC in Cambridge, UK Cryptosporidium parvum genomic DNA (Moredun isolate, genotype 2) was cut to completion with either AluI or RsaI and the fragments were cloned into the M13mp18 phage vector to obtain two distinct libraries. Randomly selected clones were analysed by single-pass sequencing, yielding 161 Sequence Tagged Sites >100 bp for a total of 43 kb of unique sequence information. The same group has recently commenced a new project aimed at determining the nucleotide sequence of the whole genome of the Iowa C. parvum isolate. Although started with some delay with respect to other parasites, the ongoing C. parvum genome projects are likely to achieve the complete sequencing of the small parasite genome within the next 2 or 3 years. This will not only provide important insights into the genome structure and dynamics, but also expedite gene discovery in C. parvum. This will o€er new opportunities to identify and study parasite-encoded molecules

Table 2 Completely sequenced Cryptosporidium parvum genes Gene

De®nition

Cloning strategya Originb GenBank accession number Reference

a-Tubulin b-Tubulin Actin eIF-4A EF-1a EF-2 Hsp70 Hsp90 pHEM2 CppA-E1 CpABC HemA DHFR-TS RNR-R1 PDI Acetyl-CoA synthase rDNA unit (Type A) rDNA unit (Type B) COWP TRAP-C1 GP900 CP15/60 p23 CP15 SCRP

Cytoskeletal component Cytoskeletal component Cytoskeletal component Translation initiation factor Elongation factor Elongation factor Heat shock protein Heat shock protein Zinc ®nger protein Cation transporter Transporter protein Haemolysin Enzyme Enzyme Enzyme Enzyme 18S, 5.8S, 28S ribosomal RNAs 18S, 5.8S, 28S ribosomal RNAs Oocyst inner wall component Sporozoite micronemal protein Sporozoite micronemal protein Sporozoite surface antigen Sporozoite surface antigen Sporozoite surface antigen Sporozoite adhesive protein

H P H P/H IS P/H IS/H n.a. n.a. P/H P/H A P/H n.a. IS/H IS/H P/H P/H IS P/H IS IS IS IS P/H

a

gDNA gDNA gDNA mRNA mRNA gDNA mRNA gDNA gDNA gDNA gDNA gDNA gDNA gDNA gDNA gDNA gDNA gDNA gDNA mRNA gDNA mRNA mRNA mRNA mRNA

AF082877 Y12615 M86241 AF001378 U69697 U21667 U69698 AF038559 U48717 U65981 AF110147 U18120 U41366 AF043243 U48261 U24082 AF015773 AF015774 Z22537 AF017267 AF068065 U22892 U34390 L34568 AF061328

A, activity; H, hybridisation; IS, immunological screening; n.a., not available; P, PCR ampli®cation. gDNA, genomic DNA; mRNA, messenger RNA (cDNA).

b

[80] [66] [81] Spano et al., unpublished [99] [100] [101] Woods et al., unpublished Steele et al., unpublished [102] [103] [84] [85] Balakrishnan et al., unpublished [97] [104] [86] [86] [96] [105] [106] [107] [108] [109] Spano et al., unpublished

560

F. Spano, A. Crisanti / International Journal for Parasitology 30 (2000) 553±565

that may serve as metabolic or antigenic targets of novel control measures. Despite the fact that the number of C. parvum sequences deposited in the databases has increased by two orders of magnitude in the last 4 years, less than 30 C. parvum genes/proteins have been extensively characterised so far (Table 2). This highlights the need for whole-genome approaches to be adequately paralleled by basic molecular biology and biochemical studies. 6. Repetitive sequences In addition to the discovery of new protein-encoding sequences, an important outcome of genome projects is the identi®cation of repeated sequences having a functional or structural relevance within the non-coding fraction of genomic DNA. Telomeric and microsatellite sequences, two important elements of the repetitive component of eukaryotic genomes, are currently being characterised in C. parvum based on genetic information made available by the genome projects. 6.1. Telomeric repeats The ends of eukaryotic chromosomes possess specialised structures, called telomeres [110, 111], that consist of simple tandem DNA repeats. These protect the chromosomes from loss of genetic information during DNA replication and minimise the fusion between chromosomal termini. For those lower and higher eukaryotic organisms whose telomeres have been studied, all show the presence of conserved repeated motifs of 5±8 bp. In 1998, Liu et al. [98] reported the characterisation of four distinct C. parvum genomic clones, each containing multiple copies of the hexanucleotide TTTAGG, which resembles the telomeric repeat units of Plasmodium spp. [112] and humans [113]. Using one of the four putative telomeric clones (CpGR254, spanning the TTTAGG-rich region and a non-repetitive ¯anking sequence) as a probe, the authors obtained multiple-band hybridisation patterns on restricted C. parvum genomic DNA and strong hybridisation signals corresponding to all ®ve CHEF-separated chromosomal bands constituting the electrophoretic karyotype. Though consistent with the TTTAGG sequence representing the telomeric repeat of C. parvum, these results did not rule out the possibility that the repeat units were located internally within the chromosomes. Direct evidence that the TTTAGG motif is indeed part of the telomeric region in C. parvum was obtained in another study [95], in which an oligonucleotidic probe (Tel5) consisting of ®ve tandem copies of the putative telomeric repeat was shown to hybridise selectively with the chromosome

VI-speci®c EagI restriction fragments corresponding to the chromosomal ends. The identi®cation of telomerespeci®c sequences in C. parvum represents an important step in the elucidation of chromosome structure and the development of accurate genetic maps. 6.2. Microsatellites Microsatellites are genomic DNA sequences which consist of up to 50 tandem repeats of a short nucleotidic motif, usually a dinucleotide or a trinucleotide [114]. Two basic features make microsatellites powerful markers for both genome mapping and intra-speci®c population studies. First, they are usually highly polymorphic, the number of repeats at a particular locus varying among individuals of the same species as a consequence of uneven crossover or slippage of the DNA polymerase during replication. Second, the study of microsatellites in humans, plants, insects and several protozoan parasites (P. falciparum [115], Leishmania infantum [116], Trypanosoma cruzi [117]) has shown that they are densely distributed throughout eukaryotic genomes, making them useful markers for high-resolution genetic mapping. Microsatellites have been identi®ed in C. parvum and partly characterised only very recently, as a result of sequence data made available through the genome projects. In a survey of the available GSTs, more than 200 dinucleotidic and trinucleotidic microsatellites have been identi®ed in C. parvum DNA (Abrahamsen et al., Nelson et al., CaccioÁ et al., 6th International Workshop on Opportunistic Protists, Raleigh, 1999; abstract nos W4, W5 and W32). Most of these involve an AT-rich sequence, as might be expected on the basis of the overall nucleotidic composition of the C. parvum genome. A limited number of the C. parvum microsatellite loci are suciently polymorphic that they allow discrimination of isolates belonging to genotypes 1 or 2 (Spano et al., unpublished; CaccioÁ et al., Aiello et al., 6th International Workshop on Opportunistic Protists, Raleigh, 1999; abstract nos W32 and P1). The availability of microsatellite markers is likely to open a new phase in the study of the population structure of this parasite, in addition to expediting the mapping of the whole C. parvum genome. 6.3. Ribosomal genes Concerning the coding component of the C. parvum genome, the available gene cloning data indicate that organisation of genes into multicopy clusters is not common in C. parvum. This is also true for the ribosomal genes, which in most organisms have a typical repeated structure, being organised into clusters consisting of approximately 100 (T. gondii) [118] or even

F. Spano, A. Crisanti / International Journal for Parasitology 30 (2000) 553±565

thousands (mammals) [119] of gene units located on one or a few chromosomes. Le Blancq et al. [86] have reported that C. parvum possesses only ®ve copies of the ribosomal unit per haploid genome. These are dispersed among at least three chromosomes and belong to two sequence classes, designated Types A and B. This arrangement of the rDNA is more similar to that of Plasmodium [120], Babesia [121] and Theileria [122] (possessing four to eight, three and two ribosomal units respectively) than T. gondii. This con¯icts with the current taxonomic classi®cation of C. parvum. 7. Extra-nuclear genomes While our knowledge of the structural organisation of the C. parvum genome is rapidly evolving, very little is known on the presence in this organism of extranuclear DNA elements. Unlike other members of the phylum Apicomplexa, for which morphological, molecular and biochemical data concur in demonstrating the existence of extra-chromosomal genomes in the mitochondrion and in the plastid-like organelle (apicoplast) [91, 123±129], there is controversy about whether these specialised compartments occur in C. parvum. E€orts to detect a mitochondrion-like organelle in the various stages of C. parvum have so far proved unsuccessful. A structure resembling the mitochondrion of other apicomplexans has been observed in the merozoites of C. muris [130]. This result requires independent con®rmation. On the other hand, EM observations seem to support the presence, in C. parvum sporozoites, of a membrane-bound organelle [131] whose perinuclear location and morphology are compatible with it being the plastid-like organelle found in Toxoplasma and Plasmodium. Speci®c in situ labelling experiments are needed to corroborate these morphological indications. At the karyotypic level, none of the aforementioned PFGE studies provided clear evidence for the presence of linear or circular extra-chromosomal DNA elements in C. parvum. Non-chromosomal bands have occasionally been observed in ethidium bromide-stained pulsed ®eld gels, most frequently above the larger C. parvum chromosome [79, 86, 95]. In one instance [86], the DNA comprising this high-molecular-weight band was shown to hybridise weakly with a probe speci®c for the nuclear ssrRNA gene of C. parvum. It was suggested that this might re¯ect a partial complementarity between the nuclear ssrRNA probe and ribosomal genes in a putative organellar genome that migrates anomalously during CHEF electrophoresis. In another study [83], a DNA element distinct from C. parvum chromosomes was reported to migrate in CHEF gels with a mobility similar to that of circular plasmids, in the 28±32 kb range. The authors envi-

561

saged the possibility that this relatively small DNA molecule might be related to the 35-kb circular DNA which constitutes the genome of the plastid organelle of Toxoplasma and Plasmodium. This hypothesis deserves further investigation, but it should be noted that most of the PFGE studies carried out so far on C. parvum have failed to reveal the presence of a 35kb-like element. At the molecular level, PCR-based approaches aimed at the cloning of putative C. parvum apicoplastencoded genes (using degenerate oligonucleotides for the genes rpoB and ORF470 of the 35-kb element of Toxoplasma and Plasmodium) have been unsuccessful (Spano, unpublished). Similarly, attempts to detect the 35-kb circle in C. parvum using DNA sequences from the apicoplast genome of other apicomplexans as probes showed no signi®cant cross-reactivity (Keithly et al., 6th International Workshop on Opportunistic Protists, Raleigh, 1999; abstract no. W59). On the other hand, three putative mitochondrial markers, the proteins adenylate kinase 2, Cpn60 and valyl tRNA synthase, have recently been found to be encoded by C. parvum nuclear genes (Keithly et al., 6th International Workshop on Opportunistic Protists, Raleigh, 1999; abstract no. W59). In spite of biochemical data suggesting that the energy metabolism of C. parvum does not strictly depend on oxygen [132] and the fact that a recent detailed ultrastructural study of the sporozoite supported the lack of a mitochondrion [131], the cloning of chromosomeencoded mitochondrial proteins represents a strong, though indirect, indication for the existence of mitochondria (or mitochondrial remnants) in C. parvum. 8. Concluding remarks After almost a century since the discovery of C. parvum, important questions concerning many aspects of the parasite biology, immunology and epidemiology remain unanswered. This re¯ects our inability to develop adequate models for the study of this novel parasite. To some extent, C. parvum had to be discovered twice before its importance could be fully recognised: a ®rst time at the beginning of this century, when Tyzzer came across new coccidia-like organisms, unusually ``attached'' to the host cell surface and apparently ``non-pathogenic'', and a second time (between 1970 and 1990), when C. parvum was de®ned as the agent of a novel ``zoonosis''. We now know that this peculiar intracellular parasite can also follow an anthroponotic cycle, that water represents one of the main transmission routes and that what we used to call a ``species'' is indeed something more complex. Years of study have taught us that often, when dealing with C. parvum, things are just not as they appear.

562

F. Spano, A. Crisanti / International Journal for Parasitology 30 (2000) 553±565

Most of what we know about C. parvum is the result of studies carried out in the last decade, with an important contribution provided by molecular biology. With more than 5000 DNA sequences deposited in public databases, C. parvum is now an apicomplexan parasite subject to intense and systematic genetic characterisation, second only to P. falciparum and T. gondii. This e€ort has produced a series of important results, including: (i) the cloning of about 30 C. parvum genes, some of which are involved in key biological processes; (ii) the identi®cation of potential immunological or metabolic targets of future anticryptosporidial measures; (iii) the characterisation of polymorphic loci, allowing dissection of the C. parvum populations at the genetic level; (iv) elucidation of the overall structure of the parasite genome; and (v) the development of the ®rst physical chromosomal maps. Given the wealth of genetic information being generated on C. parvum, the complete sequencing of its small genome within the next 2 or 3 years seems a realistic goal. However, in order to convert such a huge amount of sequence data into an increased ability to control the parasite, it will be essential to considerably enhance our ability to obtain functional and developmental information on the many parasite-encoded molecules. Moreover, while investigating the epidemiological and clinical relevance of genetically distinct C. parvum subpopulations, whole-genome approaches should be extended to strains of the parasite that are found in humans. Acknowledgements We would like to thank Lorenza Putignani for the invaluable contribution to the molecular work carried out in our laboratory on C. parvum, and Manlio Di Cristina and Federico Gata for helpful and stimulating discussions. We also thank the AIDS Research Program, Ministero della SanitaÁ, Istituto Superiore di SanitaÁ (Italy) and the BBSRC (UK), who provided support for our studies on C. parvum. References [1] Tyzzer EE. A sporozooÈn found in the peptic glands of the common mouse. Proc Soc Exp Biol Med 1907;5:12±13. [2] Tyzzer EE. An extracellular coccidium, Cryptosporidium muris (gen. et sp.nov.), of the gastric glands of the common mouse. J Med Res 1910;18:487±509. [3] Tyzzer EE. Cryptosporidium parvum (sp.nov), a coccidium found in the small intestine of the common mouse. Arch Protistenk 1912;26:394±412. [4] Slavin D. Cryptosporidium meleagridis (sp.nov). J Comp Pathol 1955;65:262±6. [5] Panciera R, Thomassen RW, Garner FM. Cryptosporidial infection in a calf. Vet Pathol 1971;8:479±84.

[6] Nime FA, Burek JD, Page DL, Holscher MA, Yardley JH. Acute enterocolitis in a human being infected with the protozoan Cryptosporidium. Gastroenterology 1976;70:592±8. [7] Meisel JL, Perera DR, Meligro C, Rubin CE. Overwhelming watery diarrhea associated with a Cryptosporidium in an immunosuppressed patient. Gastroenterology 1976;70:1156±60. [8] Anonymous. Human cryptosporidiosis. Morbid Mortal Weekly Rep 1982;31:252±4. [9] Current WL, Reese NC, Ernst JV, Bailey WS, Heyman MB, Weinstein WM. Human cryptosporidiosis in immunocompetent and immunode®cient persons. Study of an outbreak and experimental transmission. N Engl J Med 1983;308:1252±7. [10] Forgacs P, Tarshis A, Ma P et al. Intestinal and bronchial cryptosporidiosis in an immunode®cient homosexual man. Ann Intern Med 1983;99:793±4. [11] Tzipori S. Cryptosporidiosis in animals and humans. Microbiol Rev 1983;47:84±96. [12] O'Donoghue PJ. Cryptosporidium and cryptosporidiosis in man and animals. Int J Parasitol 1995;25:139±95. [13] de Graaf DC, Vanopdenbosch E, Ortega-Mora LM, Abbassi H, Peeters JE. A review of the importance of cryptosporidiosis in farm animals. Int J Parasitol 1999;29:1267±87. [14] Harp JA, Woodmansee DB, Moon HV. Resistance of calves to Cryptosporidium parvum: e€ects of age and previous exposure. Infect Immun 1990;58:2237±40. [15] Current WL, Garcia LS. Cryptosporidiosis. Clin Microbiol Rev 1991;4:325±58. [16] Flannigan T, Whalen C, Turner J et al. Cryptosporidium infection and CD4 counts. Ann Intern Med 1992;116:840±2. [17] Petersen C. Cryptosporidiosis in patients infected with the human immunode®ciency virus. Clin Infect Dis 1992;15:903±9. [18] Guerrant RL. Cryptosporidiosis: an emerging, highly infectious threat. Emerg Infect Dis 1997;3:1±10. [19] Sallon S, Deckelbaum RJ, Schmid II, Harlap S, Baras M, Spira DT. Cryptosporidium, malnutrition and chronic diarrhea in children. Am J Dis Child 1988;142:312±5. [20] Agnew DG, Lima AA, Newman RD et al. Cryptosporidiosis in northeastern Brazilian children: association with increased diarrhea morbidity. J Infec Dis 1998;177:754±60. [21] Casemore DP. Epidemiological aspects of human cryptosporidiosis. Epidemiol Infect 1990;104:1±28. [22] Adal KA, Sterling CR, Guerrant RL. Cryptosporidium and related species. In: Blaser MJ, Smith PD, Ravdin JI, Greenberg HB, Guerrant RL, editors. Infections of the gastrointestinal tract. New York: Raven Press, 1995; 1107±1128. [23] Pedersen C, Danner S, Lazzarin A et al. Epidemiology of cryptosporidiosis among European AIDS patients. Genitourin Med 1996;72:128±31. [24] Carr A, Marriott D, Field A, Vasak E, Cooper DA. Treatment of HIV-1-associated microsporidiosis and cryptosporidiosis with combination antiretroviral therapy. Lancet 1998;351:256±61. [25] Foudraine NA, Weverling GJ, van Gool T et al. Improvement of chronic diarrhoea in patients with advanced HIV-1 infection during potent antiretroviral therapy. AIDS 1998;12:35±41. [26] Fricker CR, Crabb JH. Water-borne cryptosporidiosis: detection methods and treatment options. Adv Parasitol 1998;40:241±78. [27] Smith HV, Rose JB. Waterborne cryptosporidiosis: current status. Parasitol Today 1998;14:14±22. [28] McKenzie WR, Hoxie NJ, Proctor ME et al. A massive outbreak in Milwaukee of Cryptosporidium infection transmitted through the public water supply. N Engl J Med 1994;331:161± 7. [29] Fayer R, Speer CA, Dubey JP. The general biology of Cryptosporidium. In: Fayer R, editor. Cryptosporidium and cryptosporidiosis. Boca Raton, FL: CRC Press, 1997; 65±92.

F. Spano, A. Crisanti / International Journal for Parasitology 30 (2000) 553±565 [30] Pieniazek NJ, Bornay-Llinares FJ, Slemenda SB et al. New Cryptosporidium genotypes in HIV-infected persons. Emerg Infect Dis 1999;5:444±9. [31] Morgan UM, Xiao L, Fayer R, Lal AA, Thompson RCA. Variation in Cryptosporidium: towards a taxonomic revision of the genus. Int J Parasitol 1999;29:1733±51. [32] Awad-El-Kariem FM, Robinson HA, Dyson DA et al. Di€erentiation between human and animal strains of Cryptosporidium parvum using isoenzyme typing. Parasitology 1995;110:129±32. [33] Morgan UM, Constantine CC, O'Donoghue P, Meloni BP, O'Brien PA, Thompson RCA. Molecular characterization of Cryptosporidium isolates from human and other animals using random ampli®ed polymorphic DNA analysis. Am J Trop Med Hyg 1995;52:559±64. [34] Bonnin A, Fourmaux MN, Dubremetz JF et al. Genotyping human and bovine isolates of Cryptosporidium parvum by polymerase chain reaction±restriction fragment length polymorphism analysis of a repetitive DNA sequence. FEMS Microbiol Lett 1996;137:207±11. [35] Carraway M, Tzipori S, Widmer G. Identi®cation of genetic heterogeneity in the Cryptosporidium parvum ribosomal repeat. Appl Environ Microbiol 1996;62:712±6. [36] Spano F, Putignani L, McLauchlin J, Casemore D, Crisanti A. PCR±RFLP analysis of the Cryptosporidium oocyst wall protein (COWP) gene discriminates between C. wrairi and C. parvum, and between C. parvum isolates of human and animal origin. FEMS Microbiol Lett 1997;150:209±17. [37] CaccioÁ S, Homan W, van Dijk K, Pozio E. Genetic polymorphism at the b-tubulin locus among human and animal isolates of Cryptosporidium parvum. FEMS Microbiol Lett 1999;170:173±9. [38] Peng MM, Xiao L, Freeman AR et al. Genetic polymorphism among Cryptosporidium parvum isolates: evidence for two distinct human transmission cycles. Emerg Infect Dis 1997;3:567± 73. [39] Spano F, Putignani L, Crisanti A et al. Multilocus genotypic analysis of Cryptosporidium parvum isolates from di€erent hosts and geographical origin. J Clin Microbiol 1998;36:3255± 9. [40] Patel S, Pedraza-Diaz S, McLauchlin J, Casemore D. Molecular characterisation of Cryptosporidium parvum from two large suspected waterborne outbreaks. Comm Dis Pub Health 1998;1:231±3. [41] Widmer G, Tchack L, Chappell CL, Tzipori S. Sequence polymorphism in the b-tubulin gene reveals heterogeneous and variable population structures in Cryptosporidium parvum. Appl Environ Microbiol 1998;64:4477±81. [42] Vetterling JM, Takeuchi A, Madden PA. Ultrastructure of Cryptosporidium wrairi from the guinea pig. J Protozool 1971;18:248±60. [43] Tzipori S. Cryptosporidiosis in perspective. Adv Parasitol 1988;27:63±129. [44] Petry F, Harris JR. Ultrastructure, fractionation and biochemical analysis of Cryptosporidium parvum sporozoites. Int J Parasitol 1999;29:1249±60. [45] Dubremetz JF, Garcia-ReÂguet N, Conseil V, Fourmaux MN. Apical organelles and host-cell invasion by Apicomplexa. Int J Parasitol 1998;28:1007±13. [46] Naitza S, Spano F, Robson KJH, Crisanti A. The thrombospondin-related protein family of apicomplexan parasites: the gears of the cell invasion machinery. Parasitol Today 1998;14:479±84. [47] Marcial MA, Madara JL. Cryptosporidium: cellular localization, structural analysis of absorptive cell±parasite membrane±membrane interactions in guinea-pigs, and suggestion

[48] [49]

[50] [51] [52] [53]

[54]

[55] [56] [57] [58]

[59] [60] [61] [62]

[63] [64] [65] [66] [67] [68]

563

of protozoan transport by M cells. Gastroenterology 1986;90:583±94. Lumb R, Smith K, O'Donoghue PJ, Lanser JA. Ultrastructure of the attachment of Cryptosporidium sporozoites to tissue culture cells. Parasitol Res 1988;74:531±6. Eggleston MT, Tilley M, Upton SJ. Enhanced development of Cryptosporidium parvum in vitro by removal of oocyst toxins from infected cell monolayers. J Helmintol Soc Wash 1994;61:122±5. Buraud M, Forget E, Favennec L, Bizet J, Gobert J, Deluol AM. Sexual stage development of cryptosporidia in the Caco2 cell line. Infect Immun 1991;59:4610±3. Villacorta I, De Graaf D, Charleir G, Peeters JE. Complete development of Cryptosporidium parvum in MDBK cells. FEMS Microbiol Lett 1996;142:129±32. Yang S, Healey MC, Du C, Zhang J. Complete development of Cryptosporidium parvum in bovine fallopian tube epithelial cells. Infect Immun 1996;64:349±54. Schroeder AA, Brown AM, Abrahamsen MS. Identi®cation and cloning of a developmentally regulated Cryptosporidium parvum gene by di€erential mRNA display PCR. Gene 1998;216:327±34. Schroeder AA, Lawrence CE, Abrahamsen MS. Di€erential mRNA display cloning and characterization of a Cryptosporidium parvum gene expressed during intracellular development. J Parasitol 1999;85:213±20. Arrowood MJ, Sterling CR. Isolation of Cryptosporidium oocysts and sporozoites using discontinuous sucrose and isopycnic Percoll gradients. J Parasitol 1987;73:314±9. Petry F, Robinson HA, McDonald V. Murine infection model for mainteinance and ampli®cation of Cryptosporidium parvum oocysts. J Clin Microbiol 1995;33:1922±4. Fayer R, Nerad T. E€ects of low temperatures on viability of Cryptosporidium parvum oocysts. Appl Environ Microbiol 1996;62:1431±3. Griths JK, Theodos C, Paris M, Tzipori S. The gamma interferon gene knockout mouse: a highly sensitive model for evaluation of therapeutic agents against Cryptosporidium parvum. J Clin Microbiol 1998;36:2503±8. Theodos CM. Innate and cell-mediated immune responses to Cryptosporidium parvum. Adv Parasitol 1998;40:87±119. Fayer R. E€ect of sodium hypochlorite exposure on infectivity of Cryptosporidium parvum oocysts for neonatal Balb/c mice. Appl Environ Microbiol 1995;61:844±6. Rose JB. Environmental ecology of Cryptosporidium and public health implications. Annu Rev Public Health 1997;18:135± 61. Finch GR, Black EK, Gyurek L, Belosevic M. Ozone inactivation of Cryptosporidium parvum in demand-free phosphate bu€er determined by in vitro excystation and animal infectivity. Appl Environ Microbiol 1993;59:4203±10. Griths JK. Human cryptosporidiosis: epidemiology, transmission, clinical disease, treatment and diagnosis. Adv Parasitol 1998;40:37±85. Lai E, Birren BW, Clark SM, Simon MI, Hood L. Pulsed ®eld gel electrophoresis. Biotechniques 1989;7:34±42. Goebel E, Braendler U. Ultrastructure of microgametogenesis, microgametes and gametogamy of Cryptosporidium sp. in the small intestine of mice. Protistologica 1982;3:331±44. CaccioÁ S, La Rosa G, Pozio E. The beta-tubulin gene of Cryptosporidium parvum. Mol Biochem Parasitol 1997;89:307± 11. Char S, Kelly P, Naeem A, Farthing MJ. Codon usage in Cryptosporidium parvum di€ers from that in other Eimeriorina. Parasitology 1996;112:357±62. Johnson AM, Fielke R, Lumb R, Baverstock PR. Phylogenetic relationships of Cryptosporidium determined by

564

[69]

[70]

[71]

[72]

[73] [74]

[75]

[76]

[77]

[78]

[79]

[80]

[81]

[82]

[83]

[84]

[85]

[86]

F. Spano, A. Crisanti / International Journal for Parasitology 30 (2000) 553±565 ribosomal RNA sequence comparison. Int J Parasitol 1990;20:141±7. Van der Ploeg LHT, Gottesdiener KM, Korman SH, Weiden M, Le Blancq S. Protozoan genomes. Meth Mol Biol 1992;12:203±23. Giannini SH, Schittini M, Keithly JS, Warburton PW, Cantor CR, Van der Ploeg LH. Karyotype analysis of Leishmania species and its use in classi®cation and clinical diagnosis. Science 1986;232:762±5. Van der Ploeg LHT, Schwartz DC, Cantor CR, Borst P. Antigenic variation in Trypanosoma brucei analyzed by electrophoretic separation of chromosome-sized DNA molecules. Cell 1984;37:77±84. Adam RD, Nash TK, Wellems TE. Giardia lamblia trophozoite contains set of closely related chromosomes. Nucleic Acids Res 1988;16:4555±67. Shirley MW. The genome of Eimeria tenella: further studies on its molecular organisation. Parasitol Res 1994;80:366±73. Sibley LD, Boothroyd JC. Construction of a molecular karyotype for Toxoplasma gondii. Mol Biochem Parasitol 1992;51:291±300. Kemp DJ, Thompson JK, Walliker D, Corcoran LM. Molecular karyotype of Plasmodium falciparum: conserved linkage groups and expendable histidine-rich protein genes. Proc Natl Acad Sci USA 1987;84:7672±6. Jones SH, Lew AE, Jorgensen WK, Barker SC. Babesia bovis: genome size, number of chromosomes and telomeric probe hybridisation. Int J Parasitol 1997;27:1569±73. Morzaria SP, Young JR. Restriction mapping of the genome of the protozoan parasite Theileria parva. Proc Natl Acad Sci USA 1992;89:5241±5. Mead JR, Arrowood MJ, Current WL, Sterling CR. Field inversion gel electrophoretic separation of Cryptosporidium spp. chromosome-sized DNA. J Parasitol 1988;74:366±9. Hays MP, Mosier DA, Oberst RD. Enhanced karyotype resolution of Cryptosporidium parvum by contour-clamped homogeneous electric ®elds. Vet Parasitol 1995;58:273±80. Nelson RG, Kim K, Gooze L, Petersen C, Gut J. Identi®cation and isolation of Cryptosporidium parvum genes encoding microtubule and micro®lament proteins. J Protozool 1991;38:52S±55S. Kim K, Gooze L, Petersen C, Gut J, Nelson RG. Isolation, sequence and molecular karyotype analysis of the actin gene of Cryptosporidium parvum. Mol Biochem Parasitol 1992;50:105±14. Petersen C, Gut J, Doyle PS, Crabb JH, Nelson RG, Leech JH. Characterization of a >900,000-M(r) Cryptosporidium parvum sporozoite glycoprotein recognized by protective hyperimmune bovine colostral immunoglobulin. Infect Immun 1992;60:5132±8. Blunt DS, Khramtsov NV, Upton SJ, Montelone BA. Molecular karyotype analysis of Cryptosporidium parvum: evidence for eight chromosomes and a low-molecular-size molecule. Clin Diagn Lab Immunol 1997;4:11±13. Steele MI, Kuhls TL, Nida K et al. A Cryptosporidium parvum genomic region encoding hemolytic activity. Infect Immun 1995;63:3840±5. Vasquez JR, Gooze L, Kim K, Gut J, Petersen C, Nelson RG. Potential antifolate resistance determinants and genotypic variation in the bifunctional dihydrofolate reductase±thymidylate synthase gene from human and bovine isolates of Cryptosporidium parvum. Mol Biochem Parasitol 1996;79:153± 65. Le Blancq SM, Khramtsov NV, Zamani F, Upton SJ, Wu TW. Ribosomal RNA gene organization in Cryptosporidium parvum. Mol Biochem Parasitol 1997;90:463±78.

[87] CaccioÁ S, Camilli R, La Rosa G, Pozio E. Establishing the Cryptosporidium parvum karyotype by NotI and S®I restriction analysis and Southern hybridization. Gene 1998;219:73±9. [88] Piper MB, Bankier AT, Dear PH. A HAPPY map of Cryptosporidium parvum. Genome Res 1998;8:1299±307. [89] Triglia T, Wellems TE, Kemp DJ. Towards a high-resolution map of the Plasmodium falciparum genome. Parasitol Today 1992;7:225±9. [90] Carlton JM, Galinski MR, Barnwell JW, Dame JB. Karyotype and synteny among the chromosomes of all four species of human malaria parasite. Mol Biochem Parasitol 1999;101:23± 32. [91] Nene V, Morzaria S, Bishop R. Organisation and informational content of the Theileria parva genome. Mol Biochem Parasitol 1998;95:1±8. [92] Dear PH, Cook PR. Happy mapping: linkage mapping using a physical analogue of meiosis. Nucleic Acids Res 1993;21:13± 20. [93] Dear PH, Bankier AT, Piper MB. A high-resolution metric HAPPY map of human chromosome 14. Genomics 1998;48:232±41. [94] Piper MB, Bankier AT, Dear PH. Construction and characterisation of a genomic PAC library of the intestinal parasite Cryptosporidium parvum. Mol Biochem Parasitol 1998;95:147± 51. [95] Putignani L, Sallicandro P, Alano P, Abrahamsen MS, Crisanti A, Spano F. Chromosome mapping in Cryptosporidium parvum and establishment of a long-range restriction map for chromosome VI. FEMS Microbiol Lett 1999;175:231±8. [96] Spano F, Puri C, Ranucci L, Putignani L, Crisanti A. Cloning of the entire COWP gene of Cryptosporidium parvum and ultrastructural localization of the protein during sexual parasite development. Parasitology 1997;114:427±37. [97] Blunt DS, Montelone BA, Upton SJ, Khramtsov NV. Sequence of the parasitic protozoan, Cryptosporidium parvum, putative protein disul®de isomerase-encoding DNA. Gene 1996;181:221±3. [98] Liu C, Schroeder AA, Kapur V, Abrahamsen MS. Telomeric sequences of Cryptosporidium parvum. Mol Biochem Parasitol 1998;94:291±6. [99] Bonafonte MT, Priest JW, Garmon D, Arrowood MJ, Mead JR. Isolation of the gene coding for elongation factor-1alpha in Cryptosporidium parvum. Biochim Biophys Acta 1997;1351:256±60. [100] Jones DE, Tu TD, Mathur S, Sweeney RW, Clark DP. Molecular cloning and characterization of a Cryptosporidium parvum elongation factor-2 gene. Mol Biochem Parasitol 1995;71:143±7. [101] Khramtsov NV, Tilley M, Blunt DS, Montelone BA, Upton SJ. Cloning and analysis of a Cryptosporidium parvum gene encoding a protein with homology to cytoplasmic form Hsp70. J Eukaryot Microbiol 1995;42:416±22. [102] Zhu G, Keithly JS. Molecular analysis of a P-type ATPase from Cryptosporidium parvum. Mol Biochem Parasitol 1997;90:307±16. [103] Perkins ME, Riojas YA, Wu TW, Le Blancq SM. CpABC, a Cryptosporidium parvum ATP-binding cassette protein at the host±parasite boundary in intracellular stages. Proc Natl Acad Sci USA 1999;96:5734±9. [104] Khramtsov NV, Blunt DS, Montelone BA, Upton SJ. The putative acetyl-CoA synthetase gene of Cryptosporidium parvum and a new conserved protein motif in acetyl-CoA synthetases. J Parasitol 1996;82:423±7. [105] Spano F, Putignani L, Naitza S, Puri C, Wright S, Crisanti A. Molecular cloning and expression analysis of a Cryptosporidium parvum gene encoding a new member of the

F. Spano, A. Crisanti / International Journal for Parasitology 30 (2000) 553±565

[106] [107]

[108]

[109] [110] [111] [112] [113] [114] [115] [116]

[117]

[118]

thrombospondin family. Mol Biochem Parasitol 1998;92:147± 62. Barnes DA, Bonnin A, Huang J-X et al. A novel multidomain mucin-like glycoprotein of Cryptosporidium parvum mediates invasion. Mol Biochem Parasitol 1998;96:93±110. Khramtsov NV, Oppert B, Monteleone BA, Upton SJ. Sequencing, analysis and expression in Escherichia coli of a gene encoding a 15 kDa Cryptosporidium parvum protein. Biochem Biophys Res Commun 1997;230:164±6. Perryman LE, Jamser DP, Riggs MW, Bohnet SG, McGuire TC, Arrowood MJ. A cloned gene of Cryptosporidium parvum encodes neutralization-sensitive epitopes. Mol Biochem Parasitol 1996;80:137±47. Jenkins MC, Fayer R. Cloning and expression of a cDNA encoding an antigenic Cryptosporidium parvum protein. Mol Biochem Parasitol 1995;71:149±52. Henderson E. Telomere DNA structure. In: Blackburn EH, Greider CW, editors. Telomeres. Plainview: Cold Spring Harbour Laboratory Press, 1995; 11±34. Pardue ML, DeBaryshe PG. Telomeres and telomerase: more than the end of the line. Chromosoma 1999;108:73±82. Ponzi M, Pace T, Dore E, Frontali C. Identi®cation of telomeric DNA sequence in Plasmodium berghei. EMBO J 1985;4:2991±5. Allshire RC, Dempster M, Hastie ND. Human telomeres contain at least three types of G-rich repeat distributed non-randomly. Nucleic Acids Res 1989;17:4611±27. Tautz D. Hypervariability of simple sequences as a general source for polymorphic DNA markers. Nucleic Acids Res 1989;17:6463±71. Su XZ, Wellems TE. Plasmodium falciparum: a rapid DNA ®ngerprinting method using microsatellite sequences within var clusters. Exp Parasitol 1997;86:235±6. Rossi V, Wincker P, Ravel C, Blaineau C, Pages M, Bastien P. Structural organization of microsatellite families in the Leishmania genome and polymorphism at two (CA)n loci. Mol Biochem Parasitol 1994;65:271±82. Oliveira RP, Broude NE, Macedo AM, Cantor CR, Smith CL, Pena SD. Probing the genetic population structure of Trypanosoma cruzi with polymorphic microsatellites. Proc Natl Acad Sci USA 1998;95:3776±80. Guay J-M, Huot A, Gagnon S, Tremblay A, Levesque RC. Physical map and genetic mapping of cloned ribosomal DNA

[119] [120] [121]

[122] [123]

[124] [125] [126] [127] [128] [129] [130] [131] [132]

565

from Toxoplasma gondii: primary and secondary structure of the 5S gene. Gene 1992;114:165±71. Cory S, Adams JM. A very large repeating unit of mouse DNA containing the 18S, 28S and 5.8S rRNA genes. Cell 1977;11:795±805. McCutchan TF, Li J, McConkey GA, Rogers MJ, Waters AP. The cytoplasmic ribosomal RNAs of Plasmodium spp. Parasitol Today 1995;11:134±8. Dalrymple BP. Cloning and characterization of the rRNA genes and ¯anking regions from Babesia bovis: use of the genes as strain discriminating probes. Mol Biochem Parasitol 1990;43:117±24. Kibe MK, ole-MoiYoi OK, Nene V et al. Evidence for two single copy units in Theileria parva ribosomal RNA genes. Mol Biochem Parasitol 1994;66:249±59. Gozar MM, Bagnara AS. An organelle-like small subunit ribosomal RNA gene from Babesia bovis: nucleotide sequence, secondary structure of the transcript and preliminary phylogenetic analysis. Int J Parasitol 1995;25:929±38. Ji Y, Mericle BL, Rehkopf DH, Anderson JD, Feagin JE. The Plasmodium falciparum 6 kb element is polycistronically transcribed. Mol Biochem Parasitol 1996;81:211±23. Fichera ME, Roos DS. A plastid organelle as a drug target in apicomplexan parasites. Nature 1997;390:407±9. KoÈhler S, Delwiche CF, Denny PW et al. A plastid of probable green algal origin in apicomplexan parasites. Science 1997;275:1485±9. Wilson RJ, Williamson DH. Extrachromosomal DNA in the Apicomplexa. Microbiol Mol Biol Rev 1997;61:1±16. Dunn PP, Stephens PJ, Shirley MW. Eimeria tenella: two species of extrachromosomal DNA revealed by pulsed-®eld gel electrophoresis. Parasitol Res 1998;84:272±5. Lang-Unnasch N, Reith ME, Munholland J, Barta JR. Plastids are widespread and ancient in parasites of the phylum Apicomplexa. Int J Parasitol 1998;28:1743±54. Uni S, Iseki M, Maekawa T, Moriya K, Takada S. Ultrastructure of Cryptosporidium muris (strain RN 66) parasitising the murine stomach. Parasitol Res 1987;74:123±32. Tetley L, Brown SMA, McDonald V, Coombs GH. Ultrastructural analysis of the sporozoite of Cryptosporidium parvum. Microbiology 1998;144:3249±55. Coombs GH, Denton H, Brown SMA, Thong K-W. Biochemistry of the coccidia. Adv Parasitol 1997;39:141±226.