Facts and trends in advanced systems of Cryptosporidium culture over the last decade

The success of axenic in vitro culture for mass production of the parasite will render the use of experimental animal models unnecessary. Compared with an in vivo model, an in vitro system is less expensive and more convenient for screening anti-cryptosporidial agents and for assessing the efficacy of drugs. Many efforts have been undertaken using cell lines, however, the development rate of the parasite was found to be rather small, although many oocysts were used only a few of them managed to develop into further phases. At that time a cell culture system really did not exist (Arrowood, Reference Arrowood2002). In many reports after a certain period of cultivation with C. parvum or other Cryptosporidium species (with developmental stages from circular trophozoites to the oocyst stage) light micrography of HCT-8 cell monolayer were performed. However, after critically observing the cells, it was not clear whether these stages truly developed intracellularly. Nevertheless, such systems in the past assisted in enabling the release of large amounts of funding over the last 30 years in attempt to develop a functional system for cell culture cultivation of Cryptosporidium or of evaluation of strategies, including reports of the evaluation of disinfection technologies, as well as the studies of drugs and evaluations against Cryptosporidium testing of the cytotoxicity of the drugs in such cells. An easy culture system does not yet exist; nevertheless, in 2017, we look forward to such a functional system. The existence of a ‘suitable cell line’ is not excluded. The complete development could be accomplished inside these cells with many oocysts ultimately produced for practical use, however, this possibility remains to be determined. Several advances and system improvements have also been reported in the last decade (Perez Cordón et al. Reference Perez Cordón, Marin, Romero, Rosales and Sánchez Moreno2007; Alcantara Warren et al. Reference Alcantara Warren, Destura, Sevilleja, Barroso, Carvalho, Barrett, O'Brien and Guerrant2008; Castellanos-Gonzalez et al. Reference Castellanos-Gonzalez, Cabada, Nichols, Gomez and White2013; Varughese et al. Reference Varughese, Bennett-Stamper, Wymer and Yadav2014, see more details below). Perez Cordón et al. (Reference Perez Cordón, Marin, Romero, Rosales and Sánchez Moreno2007) used HCT-8 cell cultures, for which the culture was renewed for seven days, infected with C. parvum sporozoites in RPMI-1640 medium with 10% IFBS, CaCl₂ and MgCl₂ 1 mm at pH 7·2 increased parasitism by 71% at 48 h vs 14·5%. An increase in the percentage of extracellular stages was clearly documented (25·3%). Morada et al. (Reference Morada, Sangun, Gunther-Cummins, Weiss, Widmer, Tzipori and Yarlett2016) adapted hollow fibre technology to provide an environment that mimicked the gut by delivering nutrients and oxygen, and they reported oocyst yields for >6 months, producing approximately 1 × 10⁸ oocysts ml⁻¹ day⁻¹. A recent report from a novel bioengineered three-dimensional (3D) human intestinal model for long-term infection of C. parvum was published by DeCicco RePass et al. (Reference DeCicco RePass, Chen, Lin, Zhou, Kaplan and Ward2017), and it promised a more productive in vitro culture of C. parvum in order to study the different developmental phases in more details. Varughese et al. (Reference Varughese, Bennett-Stamper, Wymer and Yadav2014) proposed a system with culture survival of almost 2 months. However, the successes of the above reports have not yet been proven by other researchers.

The observations of Karanis et al. (Reference Karanis, Kimura, Igarashi, Nagasawa and Suzuki2008) indicated that Cryptosporidium could also develop outside of its host and it also indicated that exclusive endogenous development in the host intestine is not necessary for this parasite to complete its life cycle (Karanis et al. Reference Karanis, Kimura, Igarashi, Nagasawa and Suzuki2008). One major limitation of host cell-free cultivation is the difficulty involved in finding, identifying and recording visual evidence for evaluation by the scientific community of the life cycle stages because they are very small, they are morphologically difficult to identify and they are dispersed throughout the media. Microscopic visualization is, in fact, a limitation of such studies; however, for hundreds of years an unlimited number of previous studies utilized only light microscopic evaluation, yielding invaluable outcomes with various microorganisms, including parasites. For Cryptosporidium, electron microscopy studies have shed light not only on the morphology at the ultra-structural level of the emerging stages, but also on the processes during which they transform into other stages (Aldeyarbi and Karanis, Reference Aldeyarbi and Karanis2016a, Reference Aldeyarbi and Karanisb, Reference Aldeyarbi and Karanisc). The Cryptosporidium species oocysts were first identified by PCR and LAMP and were genetically characterized by subsequent sequencing before the process of culturing. Using thin sections stained with uranyl acetate and lead citrate, EM investigations were performed and the results have been described in detail in three series: (a) the early stages of development in in vitro axenic culture (Aldeyarbi and Karanis, Reference Aldeyarbi and Karanis2016a); (b) the fine structure stage of development and sporogony in the same axenic system (Aldeyarbi and Karanis, Reference Aldeyarbi and Karanis2016b); and (c) the ultrastructural similarities between C. parvum and gregarines (Aldeyarbi and Karanis, Reference Aldeyarbi and Karanis2016c). The cultivation process included direct excystation of sporozoites from the oocysts in a cell-free medium. Excystation was conducted in two steps, and the oocysts were exposed to different supplemented media (SFM, RPMI-1040) (Karanis, unpublished data, Aldeyarbi and Karanis, Reference Aldeyarbi and Karanis2016a, Reference Aldeyarbi and Karanisb, Reference Aldeyarbi and Karanisc) to evaluate their effects on excystation as well as the possibility for Cryptosporidium development. The excystation suspension was centrifuged and examined microscopically. For the cell-free cultivation, the Cryptosporidium oocysts were placed into culture flasks containing cell-free maintenance media. The cultures were incubated at 37 °C, and the developmental media were then fixed in formaldehyde for different lengths of time after incubation for subsequent examination by transmission electron microscopy (TEM). The details of the findings have been reported in the original papers as cited above (Aldeyarbi and Karanis, Reference Aldeyarbi and Karanis2016a, Reference Aldeyarbi and Karanisb, Reference Aldeyarbi and Karanisc).

The establishment of how the culturing of self-renewing liver and pancreas 3D organoids from humans and mice will favour such expectations is still to determined (Broutier et al. Reference Broutier, Andersson-Rolf, Hindley, Boj, Clevers, Koo and Huch2016; Bartfeld and Clevers, Reference Bartfeld and Clevers2017), whereas others have proposed the CRISPR/Cas9 system as a promising player in gene therapy, with experiments resulting in the first gene knockout in C. parvum, in order to address cryptosporidiosis (Striepen, Reference Striepen2013; Vinayak et al. Reference Vinayak, Pawlowic, Sateriale, Brooks, Studstill, Bar-Peled, Cipriano and Striepen2015).

The challenge of the in vitro culture and the recognition of Cryptosporidium as a human and animal pathogen

Why have all attempts in culture remained unsuccessful so far in developing an effective system? Although several approaches for promising results exist, the reproduction of the parasite under cultured conditions has not yet been accomplished. Almost all the scientific reviews about the current most advanced cryptosporidiosis research tasks have come to a clear consensus: the formidable task of cryptosporidiosis disease control will not be accomplished without the development of an effective in vitro culture system.

Although there was the development of parasites in some epithelial cells, the use of different cell lines led to different results. In some cell lines, only the asexual part of the life cycle of Cryptosporidium had been developed successfully. Full development of the parasite and the production of oocysts have been described, however, many details and clear images of the parasite stages during development remain enigmatic. The question remains whether the cell culture system offers an advantage over an animal model with a susceptible host especially for the evaluation, e.g., of disinfection studies, or for oocysts from contaminated environmental samples that are associated with a high degree of biological variability. For this reason, it is necessary to perform additional studies in the future and compare both systems for their ability to detect small numbers of infected oocysts from various samples. It has been reported that mouse models should be the gold standard for the evaluation of the infectivity of Cryptosporidium oocysts (Karanis and Schoenen, Reference Karanis and Schoenen2001) but the establishment of the in vitro culture system would make the use of experimental animals unnecessary.

The development of Cryptosporidium species by axenic in vitro cultivation is applicable but has been limited in its success to produce large numbers of stages, either of the asexual or sexual part of the life cycle. Stages of C. parvum and C. hominis were developed in different media devoid of host cells, and all the further phases of the life cycle were observed (Karanis et al. Reference Karanis, Kimura, Igarashi, Nagasawa and Suzuki2008; Hijjawi et al. Reference Hijjawi, Estcourt, Yang, Monis and Ryan2010). Although the system was functional, the process has not yet been developed for routine use. The cultivation of C. parvum and C. hominis under axenic conditions is possible, however, the system should be developed further by using different strains of the parasite and media manipulation. The definition of an established culture system indicates the survival of sub-cultures and consistent harvest yields from specific inocula within a designated period of incubation.

Equally important in the analysis of Cryptosporidium history and evolution is the understanding of the major Impact of the Cryptosporidium taxonomy and its cellular position within the gut. Furthermore, and specific for the in vitro culture development, it is vital that for such research the researchers themselves have the required knowledge and are familiar with the Cryptosporidium life cycle peculiarities, as well as the basic biology. The researchers will also need to be able to manage the parasite in both the in vitro and in vivo studies.

Cryptosporidium from its beginning and after the first discovery has stood ‘under bad star conditions’. The pioneer Edward Tyzzer first discovered Cryptosporidium in 1907 in the gastric glands of asymptomatic laboratory mice (Tyzzer, Reference Tyzzer1907). In his descriptions, he clearly emphasized the extracellular position of the parasite (Tyzzer, Reference Tyzzer1910, Reference Tyzzer1912). Many years later, this pathogenic protozoan was reported in fowl with fatal enteritis (Slavind, Reference Slavind1995). Cryptosporidium parasites have travelled a long road towards being recognized as an important human pathogen (Hedstrom, Reference Hedstrom2015). It took seventy more years to establish this notion and recognize that Cryptosporidium infections cause severe diarrhoea, it then took another thirty years before the recent Global Enteric Multicenter Study (GEMS) (Kotloff et al. Reference Kotloff, Nataro, Blackwelder, Nasrin, Farag, Panchalingam, Wu, Sow, Sur, Breiman, Faruque, Zaidi, Saha, Alonso, Tamboura, Sanogo, Onwuchekwa, Manna, Ramamurthy, Kanungo, Ochieng, Omore, Oundo, Hossain, Das, Ahmed, Qureshi, Quadri, Adegbola, Antonio, Hossain, Akinsola, Mandomando, Nhampossa, Acacio, Biswas, O'Reilly, Mintz, Berkeley, Muhsen, Sommerfelt, Robins Browne and Levine2013) revealed that Cryptosporidium is the second leading cause of diarrhoeal mortality in small children, after the Rotavirus (Kotloff et al. Reference Kotloff, Nataro, Blackwelder, Nasrin, Farag, Panchalingam, Wu, Sow, Sur, Breiman, Faruque, Zaidi, Saha, Alonso, Tamboura, Sanogo, Onwuchekwa, Manna, Ramamurthy, Kanungo, Ochieng, Omore, Oundo, Hossain, Das, Ahmed, Qureshi, Quadri, Adegbola, Antonio, Hossain, Akinsola, Mandomando, Nhampossa, Acacio, Biswas, O'Reilly, Mintz, Berkeley, Muhsen, Sommerfelt, Robins Browne and Levine2013; Hedstrom, Reference Hedstrom2015).

Cryptosporidium has served as a vehicle in obtaining funding for water detection and sample analysis, it was a ‘driving force’ for almost all of research related to Cryptosporidium for a long period. Prior to the recognition of Cryptosporidium as an important human pathogen in the early 1980s, interest in research into the parasite was stimulated by the occurrence of community outbreaks of gastroenteritis in the USA in the 1960s and 1970s (Karanis et al. Reference Karanis, Kourenti and Smith2007; Baldursson and Karanis, Reference Baldursson and Karanis2011; Efstratiou et al. Reference Efstratiou, Ongerth and Karanis2017a, Reference Efstratiou, Ongerth and Karanisb). Initial monitoring efforts were conducted to determine the presence and distribution of Cryptosporidium in the water and to develop a risk assessment framework for the water industry to address Cryptosporidium. Most of the activities at that time in both the USA and the UK were directed towards helping to determine the risk posed by the presence of this protozoon in drinking and recreational waters in these countries (Efstratiou et al. Reference Efstratiou, Ongerth and Karanis2017b).

The rudimentary nature of background information, along with the early stage of technological knowledge regarding sampling and analysis resulted in a large proportion of negative results, creating distrust between the related research and governmental authorities. Although in the early 1970s, many research applications related to HIV and opportunistic parasites, e.g., Toxoplasma, were highly appreciated, most issues related to Cryptosporidium were questioned because Cryptosporidium had apparently ‘no clinical significance’. Finding target organisms in only a very small percentage of surface water samples suggested that they were only present intermittently, rather than continuously and that their dispersal through water and food was always perhaps ‘only by accident’; it was a parasite that was mainly prevalent in underdeveloped countries and water authorities in developed countries finally denied to have it in their water. For many decades, Cryptosporidium and cryptosporidiosis were under- and misdiagnosed (Kotloff et al. Reference Kotloff, Nataro, Blackwelder, Nasrin, Farag, Panchalingam, Wu, Sow, Sur, Breiman, Faruque, Zaidi, Saha, Alonso, Tamboura, Sanogo, Onwuchekwa, Manna, Ramamurthy, Kanungo, Ochieng, Omore, Oundo, Hossain, Das, Ahmed, Qureshi, Quadri, Adegbola, Antonio, Hossain, Akinsola, Mandomando, Nhampossa, Acacio, Biswas, O'Reilly, Mintz, Berkeley, Muhsen, Sommerfelt, Robins Browne and Levine2013; Checkley et al. Reference Checkley, White, Jaganath, Arrowood, Chalmers, Chen, Fayer, Griffiths, Guerrant, Hedstrom, Huston, Kotloff, Kang, Mead, Miller, Petri, Priest, Roos, Striepen, Thompson, Ward, Van Voorhis, Xiao, Zhu and Houpt2015; Hedstrom, Reference Hedstrom2015; Caccio and Chalmers, Reference Caccio and Chalmers2016; Lanternier et al. Reference Lanternier, Amazzough, Favennec, Mamzer-Bruneel, Abdoul, Tourret, Decramer, Zuber, Scemla, Legendre, Lortholary and Bougnoux2017). More than 100 years after its first discovery the turning point in Cryptosporidium research was the Global Enteric Multicenter Study (GEMS) highlighting Cryptosporidium as a clinically important pathogen (Kotloff et al. Reference Kotloff, Nataro, Blackwelder, Nasrin, Farag, Panchalingam, Wu, Sow, Sur, Breiman, Faruque, Zaidi, Saha, Alonso, Tamboura, Sanogo, Onwuchekwa, Manna, Ramamurthy, Kanungo, Ochieng, Omore, Oundo, Hossain, Das, Ahmed, Qureshi, Quadri, Adegbola, Antonio, Hossain, Akinsola, Mandomando, Nhampossa, Acacio, Biswas, O'Reilly, Mintz, Berkeley, Muhsen, Sommerfelt, Robins Browne and Levine2013). Funding and research have galvanized new research into Cryptosporidium with its new ‘reputation as an important clinical pathogen’ responsible for nearly a million deaths every year.

These perceptions contradict any recent data clearly demonstrating that this pathogen is continuously present and exists in almost any animal (domestic or wild) examined, in any surface waters investigated, in many different food groups and finally also in the increase in waterborne outbreaks. This pathogen is prevalent even in industrialized countries, not only in the USA and the UK where considerable efforts were achieved to establish a surveillance system, but also in Central and North European countries that have high standards of water quality levels, such as Germany, Sweden and Norway (see outbreak reviews of Karanis et al. Reference Karanis, Kourenti and Smith2007; Baldursson and Karanis, Reference Baldursson and Karanis2011; Adler et al. Reference Adler, Widerström, Lindh and Lilja2017; Efstratiou et al. Reference Efstratiou, Ongerth and Karanis2017a). Cryptosporidium was one of the four major contributors to moderate-to-severe diarrhoeal disease during the first 5 years of life in the low-to-middle income earning countries (Kotloff et al. Reference Kotloff, Nataro, Blackwelder, Nasrin, Farag, Panchalingam, Wu, Sow, Sur, Breiman, Faruque, Zaidi, Saha, Alonso, Tamboura, Sanogo, Onwuchekwa, Manna, Ramamurthy, Kanungo, Ochieng, Omore, Oundo, Hossain, Das, Ahmed, Qureshi, Quadri, Adegbola, Antonio, Hossain, Akinsola, Mandomando, Nhampossa, Acacio, Biswas, O'Reilly, Mintz, Berkeley, Muhsen, Sommerfelt, Robins Browne and Levine2013); it was only second to the Rotavirus as a cause of moderate-to-severe diarrhoea in children younger than 2 years old and it was also associated with a two to three times higher risk of mortality among children aged 12–23 months with moderate-to-severe diarrhoea compared to controls without diarrhoea (Kotloff et al. Reference Kotloff, Nataro, Blackwelder, Nasrin, Farag, Panchalingam, Wu, Sow, Sur, Breiman, Faruque, Zaidi, Saha, Alonso, Tamboura, Sanogo, Onwuchekwa, Manna, Ramamurthy, Kanungo, Ochieng, Omore, Oundo, Hossain, Das, Ahmed, Qureshi, Quadri, Adegbola, Antonio, Hossain, Akinsola, Mandomando, Nhampossa, Acacio, Biswas, O'Reilly, Mintz, Berkeley, Muhsen, Sommerfelt, Robins Browne and Levine2013). The most recent Global Burden of Disease study listed Cryptosporidium as an important cause of diarrhoea and death in children younger than 5 years of age (especially under the age of two) in sub-Saharan Africa (GBD Diarrhoeal Diseases Collaborators 2017; Platts-Mills et al. Reference Platts-Mills, Babji, Bodhidatta, Gratz, Haque, Havt, McCormick, McGrath, Olortegui, Samie, Shakoor, Mondal, Lima, Hariraju, Rayamajhi, Qureshi, Kabir, Yori, Mufamadi, Amour, Carreon, Richard, Lang, Bessong, Mduma, Ahmed, Lima, Mason, Zaidi, Bhutta, Kosek, Guerrant, Gottlieb, Miller, Kang and Houpt2015).

The current stage of the achievements in Cryptosporidium in vitro axenic culture

Based on novel observations (Karanis et al. Reference Karanis, Kimura, Igarashi, Nagasawa and Suzuki2008; Hijjawi, Reference Hijjawi2010; Hijjawi et al. Reference Hijjawi, Estcourt, Yang, Monis and Ryan2010; Aldeyarbi and Karanis, Reference Aldeyarbi and Karanis2016a, Reference Aldeyarbi and Karanisb, Reference Aldeyarbi and Karanisc) regarding the cultivation of human and animal pathogenic species (C. parvum and C. hominis) under axenic conditions, the results have confirmed the possibility of axenic propagation of C. parvum in different media. The findings have shown a natural pressure on C. parvum for further development outside of hosts; perhaps endogenous development in the host intestine is not necessary for this parasite to complete its life cycle. Hijjawi et al. (Reference Hijjawi, Estcourt, Yang, Monis and Ryan2010) confirmed findings of Karanis et al. (Reference Karanis, Kimura, Igarashi, Nagasawa and Suzuki2008) for C. parvum, yielding similar findings for C. hominis. For cell-free cultivation, Cryptosporidium oocysts were placed into culture flasks containing cell-free maintenance media RPMI-1640 (Hijjawi et al. Reference Hijjawi, Estcourt, Yang, Monis and Ryan2010) or RPMI-1640 and/or Express Five Serum Free Medium (Karanis et al. Reference Karanis, Kimura, Igarashi, Nagasawa and Suzuki2008; Aldeyarbi and Karanis, Reference Aldeyarbi and Karanis2016a, Reference Aldeyarbi and Karanisb, Reference Aldeyarbi and Karanisc; Karanis, unpublished data). Express Five Serum-Free Medium (SFM) is a serum-free insect cell medium that contains vitamins, which allow for long-term cell growth and recombinant protein expression by using the Baculovirus Expression System (BEVS) in the Tricholplusia ni BTI-5B1-4 (High Five) cell line. Cultures were incubated at 37 °C and were fixed in formaldehyde at different times after incubation for subsequent preparation for EM. The development in vitro has been observed by a light microscope (LM) (Karanis et al. Reference Karanis, Kimura, Igarashi, Nagasawa and Suzuki2008, Karanis, unpublished) and after ultra-structural studies by electron microscope (Aldeyarbi and Karanis, Reference Aldeyarbi and Karanis2016a, Reference Aldeyarbi and Karanisb, Reference Aldeyarbi and Karanisc). Both methods (LM and EM) have provided images of different cycle stages of C. parvum growth in vitro and have confirmed the in vitro development of C. parvum and/or C. hominis (Hijjawi et al. Reference Hijjawi, Estcourt, Yang, Monis and Ryan2010), indicating that the in vitro axenic culture system is possible. In other trials, it has been noted that the development conducted with pure Express Five SFM medium without supplements had the same yield of Cryptosporidium life stages as trials with supplements added (Karanis, unpublished).

There are three main tasks of such research: (a) identification of strain suitability for the in vitro growth of C. hominis and C. parvum; (b) the screening of different cultivation media and substances to accelerate the development of Cryptosporidium life cycle stages; and (c) the screening of other Cryptosporidium species for their ability to develop in an in vitro axenic system. Based on the above findings from all the research tasks, it is possible to find solutions in the future. Media manipulation will enable the development, propagation and establishment of several C. hominis and C. parvum isolates/genotypes/strains or other Cryptosporidium species in in vitro axenic cultures. For this reason, it is necessary to isolate as many strains as possible of C. hominis and C. parvum from humans and animals. These strains should be first genetically characterized and then subjected to cultivation trials in vitro using many different culture media until it is determined which strain and which medium are the most compatible with each other. The possibility of the exogenous in vitro development independent of ‘oocyst stage maturation’ has been confirmed (Karanis et al. Reference Karanis, Kimura, Igarashi, Nagasawa and Suzuki2008) and it has also been recorded for certain developmental stages (immature or mature merozoites) in the asexual part of the Cryptosporidium life cycle (Hijjawi et al. Reference Hijjawi, Estcourt, Yang, Monis and Ryan2010).

A strain's suitability to grow in vitro or not is still a common discussion regarding parasitic pathogens, it has, however, been proven many times in the past. ‘Around the same time that Tu was investigating Chinese herbs against Malaria parasites, Omura at the Kitasato Institute was looking for solutions in the soil. ‘Equipped with extraordinary skills in developing unique methods for large-scale culturing … Omura isolated and characterised new strains of Streptomyces from soil samples and successfully cultured the strains in the laboratory’, affirmed the Nobel Assembly. Under a partnership agreement with the pharmaceutical company Merck, Omura shipped batches of bacteria cultures of interest to the USA, where a team led by Merck scientists tested their potential use in treating parasitic diseases. In the batch of 54 such cultures there was one included that was taken from the soil of a golf course outside Tokyo, a culture that would be known as avermectin’ (Honouring pioneers of treatments for malaria and nematode infections/http://www.thelancet.com/infection; Vol. 15, December 2015).

Pioneers in the field of Cryptosporidium such as Ernest Edward Tyzzer (1875–1965) and Huw Smith (1947–2010) and most likely – other less prominent investigators in the field of Cryptosporidium diagnosis will not be able to see the current achievements in Cryptosporidium research or be honoured for their invaluable observations, however, the scientific community will always be thankful because most of the knowledge we have today and the achievements we have accomplished is mainly because of their significant observations and their invaluable research work in the past.

Regarding the current stages of in vitro cultivation –, (either in cell lines or in axenic culture), – more information on Cryptosporidium's pathogenesis, parasite-cell reactions and developmental biology can be achieved by focusing on the ability of Cryptosporidium to grow under different conditions in the laboratory, with an understanding of its fascinatingly close position to gregarine species (see more details below). Accumulated evidence suggests the important results of examining the potential linkage between Cryptosporidium and the gregarines (Aldeyarbi and Karanis, Reference Aldeyarbi and Karanis2016c). This work could hold the key to a better understanding of the Cryptosporidium as forgotten gregarine (see further details below). Research in this direction could provide some productive clues to successful axenic cultivation, which in turn could lead to the investigation of factors contributing to the limitation of parasite development in vitro for the sake of developing an adequate model for the propagation of Cryptosporidium isolates, aimed at overcoming restrictions to developing new anti-cryptosporidial drugs and vaccines in the future.

Developmental stages and cell-free culture: different points of view

Hijjawi et al. (Reference Hijjawi, Meloni, Ng'anzoc, Ryan, Olson, Cox, Monis and Thompson2004) reported the complete development of Cryptosporidium in an axenic in vitro cultivation system. This report was the first of its kind and it provoked the idea of the possibility of in vitro axenic cultivation of Cryptosporidium. It was a surprise for the scientific community and it provided us with hope that we are able to cultivate Cryptosporidium in an in vitro axenic system; however, it was preliminary in nature and still not reproduced by other research groups (Girouard et al. Reference Girouard, Gallant, Akiyoshi, Nunnari and Tzipori2006) not even by the original publishers, as no further papers have since been published. It was a surprise because no one believed at that time that Cryptosporidium could develop without a cell culture system, as the scientific community strongly believed that the Cryptosporidium parasite was and is an intracellular parasite, using the term of ‘intracellular-extra-cytoplasmatic’, even though the original and very first description of Tyzzer (Reference Tyzzer1907, Reference Tyzzer1910) clearly stated the ‘extracellular’ position of Cryptosporidium. Looking carefully at the paper by Flanigan et al. (Reference Flanigan, Aji, Marshall, Soave, Aikawa and Kaetzel1991), infection of the differentiated HT29·74 cell line was easily and reproducibly quantifiable by counting schizonts with light microscopy, the authors at that time described the schizonts as intracellular and the merozoites within the schizonts stained avidly with haematoxylin, distinguishing the parasite from the host cell, but this position was ‘epi-cellular’. In the meantime, several researchers hypothesized and clearly argued for the ‘epi-cellular’ position of the parasite (Barta and Thompson, Reference Barta and Thompson2006; Valigurová et al. Reference Valigurová, Hofmannová, Koudela and Vávra2007; Karanis and Aldeyarbi, Reference Karanis and Aldeyarbi2011; Clode et al. Reference Clode, Koh and Thompson2015; Aldeyarbi and Karanis, Reference Aldeyarbi and Karanis2016c), and several other researchers reported the extracellular developmental stages of the parasite (Rosales et al. Reference Rosales, Peréz Cordón, Sánchez Moreno, Marín Sánchez and Mascaró2005; Borowski et al. Reference Borowski, Clode and Thompson2008, Reference Borowski, Thompson RC, Armstrong and Clode2010; Karanis et al. Reference Karanis, Kimura, Igarashi, Nagasawa and Suzuki2008; Hijjawi et al. Reference Hijjawi, Estcourt, Yang, Monis and Ryan2010), including the development and propagation of Cryptosporidium life cycle stages (sporozoites, trophozoites, type I and II meronts) in aquatic biofilms (Koh et al. Reference Koh, Clode, Monis and Thompson2013, Reference Koh, Thompson, Edwards, Monis and Clode2014).

C. parvum oocysts were developed in the RPMI-1640 medium (Hijjawi et al. Reference Hijjawi, Meloni, Ng'anzoc, Ryan, Olson, Cox, Monis and Thompson2004) or in Express Serum-Free Five Medium (Karanis et al. Reference Karanis, Kimura, Igarashi, Nagasawa and Suzuki2008; Aldeyarbi and Karanis, Reference Aldeyarbi and Karanis2016a, Reference Aldeyarbi and Karanisb, Reference Aldeyarbi and Karanisc; Karanis, unpublished) devoid of host cells and all the phases of the life cycle were reported. Most likely, all the researchers that had previously worked with Cryptosporidium culture and had attempted Cryptosporidium culture (either in cell monolayers or axenic) propagated the same stages in their tubes and culture wells, however, they were unable to observe them because the supernatant of the intended cultures was always discarded, including the ‘free swimming’ stages, during the technical work of subcultures. After the fresh medium was added to the monolayer, the observations focused on the putative and intracellular culture developments because the expectation was that Cryptosporidium developed only intra-cellularly. Therefore, free stages developed in the medium of the cells and were not visible under low magnification, they were also never seen by researchers under the microscope because the expectation was ‘intracellular-extra-cytoplasmatic’. This hypothesis has in fact been confirmed by the observations of Perez Cordón et al. (Reference Perez Cordón, Marin, Romero, Rosales and Sánchez Moreno2007), in which HCT-8 cell cultures, for which the medium had not been renewed in 7 days, had a large percent of extracellular stages augmented Perez Cordón et al. (Reference Perez Cordón, Marin, Romero, Rosales and Sánchez Moreno2007). These extracellular stages were minimal in short-term cultures in all the reports; thus, it was considered necessary to review the culture techniques in order to improve the conditions for studying (Perez Cordón et al. Reference Perez Cordón, Marin, Romero, Rosales and Sánchez Moreno2007). Researchers in the working group under the leadership of Professor Andrew R. C. Thompson at Murdoch and Rosales et al. (Reference Rosales, Peréz Cordón, Sánchez Moreno, Marín Sánchez and Mascaró2005) could make this observation; thus, it was considered necessary to review the culture technique for better study and they could develop the possible axenic propagation of C. parvum (Hijjawi et al. Reference Hijjawi, Meloni, Ryan, Olson and Thompson2002, Reference Hijjawi, Meloni, Ng'anzoc, Ryan, Olson, Cox, Monis and Thompson2004). Rosales et al. (Reference Rosales, Peréz Cordón, Sánchez Moreno, Marín Sánchez and Mascaró2005) reported similar observations of extracellular gregarine-like stages. It appears that as, per the methodology used in the in vitro culture and the Cryptosporidium species/genotype used, some extracellular phases or intracellular/epicellular were developed (Rosales et al. Reference Rosales, Peréz Cordón, Sánchez Moreno, Marín Sánchez and Mascaró2005; Perez Cordón et al. Reference Perez Cordón, Marin, Romero, Rosales and Sánchez Moreno2007; Karanis et al. Reference Karanis, Kimura, Igarashi, Nagasawa and Suzuki2008).

Although the system appeared to be functional, it has not yet been reproduced by other researchers, for example, by Girouard et al. (Reference Girouard, Gallant, Akiyoshi, Nunnari and Tzipori2006), who had used a similar but not identical, serum-free cultivation system. Other researchers suggested the possibility of misinterpretations of the original photomicrographs (Woods and Upton, Reference Woods and Upton2007). Their evaluation of the report of Hijjawi et al. (Reference Hijjawi, Meloni, Ng'anzoc, Ryan, Olson, Cox, Monis and Thompson2004) suggested that the photomicrographs were misinterpreted, suggesting that the reported forms could alternatively be budding yeast, host cells, contaminating debris or fungal conidia resembling Bipolaris australiensis and/or Colletotrichum acutatum. Further uncertain reports of C. parvum proliferation in a cell-free culture were raised by Zhang et al. (Reference Zhang, Sheoran and Widmer2009), who attempted the multiplication of C. parvum under cell-free conditions and identified the developmental stages via immuno-fluorescence and qPCR. They reported observations indicating modest proliferation of C. parvum in the cell-free culture. Perhaps the only ‘oversight’ made by Hijjawi et al. (Reference Hijjawi, Meloni, Ng'anzoc, Ryan, Olson, Cox, Monis and Thompson2004) at that time was that they presented their findings in a schematic diagram as the ‘new typical protozoan life cycle’, giving the scientific community the impression that the developmental stages generated in a certain time frame, one after another and overloaded expectations. We know that this type of development does not adhere to the same ‘rules’ of subsequent development in the stages one after another, under the reported conditions, as they are in other protozoan and/or coccidia species, such as Eimeria and/or Giardia, or any other protozoan, because the culture conditions were not optimal for growth. The generation time has never been recorded. The developed stages (trophozoites, merozoites I and II, sexual stages) are observed at the same time in culture (or subcultures) and after a short period of culture, indicating that the release or generation of different stages is a matter of every single oocyst's maturation and culture conditions. The generation of such stages has ‘genetic drivers’, which must be clarified in the future.

In other protozoan parasites, such as Giardia lamblia, the in vitro axenic culture was established by Meyer (Reference Meyer1976), and although its culture system seems to be simpler and easier than that of Cryptosporidium. It was only possible to establish successful ‘first cultures’ in vitro after hundreds of attempts. Approximately 10% of the tried isolates or strains of animal or human origin were successfully established in vitro (Karanis and Ey, Reference Karanis and Ey1998). Several isolates could later be routinely propagated sub-culturally in vitro because the isolates already adapted to the cultivation conditions which will not change the results obtained for that the ‘first isolation and cultivation’ of G. lamblia isolates remains a challenge, and many cultures have been established ‘accidentally’ or with ‘luck’, whereas others have shown visible growth only weeks after the first initiation of cultivation experiments in the tube. However, it was always possible to reproduce this system and generation times were reported, some isolates displayed different generation times and were categorized as ‘slow’, ‘medium’ or ‘rapid’ growers (Karanis and Ey, Reference Karanis and Ey1998). More attention should be focused on above facts regarding the Cryptosporidium in the future.

The system of Hijjawi et al. (Reference Hijjawi, Meloni, Ng'anzoc, Ryan, Olson, Cox, Monis and Thompson2004) has been used by others of the same group (Boxell et al. Reference Boxell, Hijjawi, Monis and Ryan2008), but it has not yet been accepted as a routine axenic culture system, however, there were further observations by others (Karanis, unpublished data) regarding the development of C. parvum in vitro and they also confirmed the observations made by Hijjawi et al. (Reference Hijjawi, Meloni, Ng'anzoc, Ryan, Olson, Cox, Monis and Thompson2004).

Hijjawi et al. (Reference Hijjawi, Estcourt, Yang, Monis and Ryan2010) reported the completion of the life cycle of C. hominis in a cell-free culture and they found that efforts to establish development in cultures inoculated with purified sporozoites lagged behind the cultures inoculated with excysted oocysts. This finding was not a new discovery but it was in its basic principle, in concordance with the observations described by Karanis et al. (Reference Karanis, Kimura, Igarashi, Nagasawa and Suzuki2008) for the C. parvum – Japanese HNJ-1 strain with advanced and extended aspects of staining procedures with the Cryptosporidium – specific polyclonal antibody Sporo-Glo and the Cry1 FISH probe. Such observations are worth being evaluated, however, as previously stated by Hijjawi (Reference Hijjawi2010), they drew little attention subsequently to the scientific community at that time.

Although the completion of the parasite's full life cycle or parts of it could be possible, in previous efforts to achieve this cycle the cultures became weak and degraded in a relatively short time of approximately 4 weeks (Karanis et al. Reference Karanis, Kimura, Igarashi, Nagasawa and Suzuki2008; Karanis, unpublished data). These results were found in part to be useful for further in vitro observations on stage developments. Matsubayashi et al. (Reference Matsubayashi, Ando, Kimata, Nakagawa, Furuya, Tani and Sasai2010) reported that the sporozoites excysted from oocysts changed morphologically from banana-shaped to rod-shaped and than finally, to round-shaped. They also presented sporozoite-like stages in the medium after they were examined up to 24 h later by TEM (Harris et al. Reference Harris, Adrian and Petry2003; Petry et al. Reference Petry, Kneib and Harris2009). Karanis et al. (Reference Karanis, Kimura, Igarashi, Nagasawa and Suzuki2008) used the C. parvum Japanese HNJ-1 strain, genotype 2 and reported that the merozoites were released from oocysts directly during incubation and during excystation without previous bleach treatment. The parasites survived for a short time in an axenic in vitro culture system but could not be established in subsequent cultivation in RPMI-1640 medium although they survived for approximately 4 weeks in Express Five Serum Free Medium (SFM). These stages were plentiful, active, polymorphic and mostly spindle-shaped; others were bean-shaped, actively motile and underwent division. This process could be a protective mechanism by which Cryptosporidium can increase its proliferation rate while it is still protected inside the oocyst because the Cryptosporidium stages (trophozoites and meronts) can develop inside the oocysts without excystation (Karanis et al. Reference Karanis, Kimura, Igarashi, Nagasawa and Suzuki2008; Hijjawi et al. Reference Hijjawi, Estcourt, Yang, Monis and Ryan2010). The merozoites, described by Karanis et al. (Reference Karanis, Kimura, Igarashi, Nagasawa and Suzuki2008) were observed to have a central nucleus and clear outer membranes, in contrast to the rounded swollen or distorted zoites, as reported by Matsubayashi et al. (Reference Matsubayashi, Ando, Kimata, Nakagawa, Furuya, Tani and Sasai2010). Furthermore, the observation of Karanis et al. (Reference Karanis, Kimura, Igarashi, Nagasawa and Suzuki2008) was consistent with the shapes of merozoites described in the study by Current and Reese (Reference Current and Reese1986), where they were banana-shaped with a slight curvature. They also presented with a spherical to sub-spherical nucleus located in the central third of the parasite and they displayed gliding and flexing movements. The living sporozoites were comma-shaped they also had a rounded posterior end that tapered to a pointed anterior end and they also presented with a compact nucleus located in the posterior third of the parasite. Petry et al. (Reference Petry, Kneib and Harris2009) could have possibly misinterpreted the oval- and bean-shaped cells described by Hijjawi et al. (Reference Hijjawi, Meloni, Ng'anzoc, Ryan, Olson, Cox, Monis and Thompson2004) and Karanis et al. (Reference Karanis, Kimura, Igarashi, Nagasawa and Suzuki2008) as they suggested that they could have been aged sporozoites rather than trophozoites or merozoites which resulted from extracellular development, leading to a wrong conclusion. This interpretation would, however, contradict Levine (Reference Levine1984) and Fayer (Reference Fayer, Fayer and Xiao2008), who stated that the sporozoites are initially slender and bow-, crescent- or boomerang-shaped and that they then later become oval-shaped during the early internalization stage in epithelial cells. It is believed that the sporozoite stage of C. parvum is motile and certainly short-lived in vitro (Tetley et al. Reference Tetley, Brown, McDonald and Coombs1998; Widmer et al. Reference Widmer, Klein and Bonilla2007). Consequently, due to the typically brief lifespan of sporozoites after excystation and the rapid physiological and biochemical events that occur soon after excystation (King et al. Reference King, Hoefel, Lim, Robinson and Monis2009), the continued existence of sporozoites used in the Petry et al. (Reference Petry, Kneib and Harris2009) study, which were 24 h old after the start of excystation, were truly unusual. It is noteworthy that most excysted sporozoites in cell-free cultures are typically transformed into oval/circular trophozoites soon after excystation and they then develop into meronts of different sizes (Hijjawi et al. Reference Hijjawi, Estcourt, Yang, Monis and Ryan2010). The definition of an established culture system implies the survival of sub-cultures and consistent harvest yields from specific inocula within a designated period of incubation. It is clear that further work is required to satisfy the above-mentioned criteria. In pursuit of a practical and reproducible axenic in vitro culture system, different strains of the parasite should be included. Zhang et al. (Reference Zhang, Sheoran and Widmer2009) reported a qPCR method to measure changes in the C. parvumDNA level in a cell-free culture. With this molecular approach to analyse C. parvum growth in the cell-free culture, the authors measured an increase in the concentration of C. parvum DNA of approximately 5·6-fold over a 5-day culture period. These results were quite like the findings of Hijjawi et al. (Reference Hijjawi, Estcourt, Yang, Monis and Ryan2010), who reported a 6·3-fold increase in DNA over 9 days for cultures inoculated with excysted oocysts and a 5·9-fold increase in DNA for cultures inoculated with excysted sporozoites using qPCR analysis. Immunolabelling of cultured organisms revealed morphologically distinct stages, only some of which reacted with Cryptosporidium-specific monoclonal antibodies. Hijjawi et al. (Reference Hijjawi, Estcourt, Yang, Monis and Ryan2010) suggested that the use of the Cryptosporidium-specific polyclonal antibody Sporo-GloTM and the Cry1 FISH probe, which is specific for Cryptosporidium, could provide strong evidence that the visualized life-cycle stages are indeed Cryptosporidium, also Nomarski DIC was sufficient to identify the life cycle stages. What we require is an established culture that has surviving subcultures, yet also yields consistent harvests from a specific inoculum within a designated period of incubation. The stages can simply be visualized and counted under the microscope, however, further work is required in order to prove the above.

During the complete development and multiplication phases of C. hominis in a cell-free culture (Hijjawi et al. Reference Hijjawi, Estcourt, Yang, Monis and Ryan2010), a process similar to syzygy and the formation of Cryptosporidium stages (trophozoites and meronts) were observed inside of oocysts without excystation. qPCR analysis revealed 5- to 6-fold amplification of parasite DNA. Additionally, the results from this study confirmed the possibility of the in vitro development of Cryptosporidium; however, the culturing model was again unable to provide effective propagation of the parasite stages. Impressive images of different stages (Karanis et al. Reference Karanis, Kimura, Igarashi, Nagasawa and Suzuki2008), after incubation in excystation solution, with all the life cycle stages of C. parvum HNJ-1 strain, without any development in culture medium has been observed. The observations were made using confocal laser scanning microscopy (CLSM) immediately after simple excystation in non-pretreated oocysts decontaminated with bleach. During excystation, 1-month-old oocysts were excysted in a freshly prepared, filter-sterilized medium composed of acidic H₂O containing 0·50% trypsin as well as 0·75% taurocholate and they were then incubated at 37 °C for 40 min, this indicated that we need to question the maturation of the Cryptosporidium ‘material’ inside of the oocyst and the development of the life cycle stages. These findings were very clear in detailed structures (see Fig. 1 in Karanis et al. Reference Karanis, Kimura, Igarashi, Nagasawa and Suzuki2008) and fundamental in their simplicity.

The ‘secret life’ of Cryptosporidium and the similarities with gregarines

It seems that Cryptosporidium has another ‘secret life’, and the ultra-structural similarities between gregarines have been described (Aldeyarbi and Karanis, Reference Aldeyarbi and Karanis2016c). The issue with gregarines has been previously mentioned and the similarities between Cryptosporidium and gregarines have been supported by molecular, genomic, biochemical and microscopic data (see more details in Aldeyarbi and Karanis, Reference Aldeyarbi and Karanis2016c). Cavalier-Smith (Reference Cavalier-Smith2014) provided the revision of gregarine higher classification and the evolutionary diversification of Sporozoa using the gregarine site-heterogeneous 18S rDNA trees. Cryptosporidium has been reclassified, and it has already moved from the subclass Coccidia, class Coccidiomorphea, to a new subclass, Cryptogregaria, within the class Gregarinomorphea (Cavalier-Smith, Reference Cavalier-Smith2014). It is the sole member of Cryptogregaria and it is presented as an epi-cellular parasite of vertebrates possessing a gregarine-like feeder organelle, however, it lacks an apicoplast (Cavalier-Smith, Reference Cavalier-Smith2014). Cryptosporidium is now officially a gregarine based on its reclassification by Cavalier-Smith (Reference Cavalier-Smith2014) and also according to the International Code of Zoological Nomenclature (ICZN) (http://www.iczn.org/iczn/index.jsp) (Ryan et al. Reference Ryan, Paparini, Monis and Hijjawi2016).

In in vitro axenic culture, Cryptosporidium developed gregarine-like zoites with an epimerite-like part (Fig. 1 in Aldeyarbi and Karanis, Reference Aldeyarbi and Karanis2016c) and a protomerite-like part with apparent separation. It resembled the mature trophozoite of the eugregarine Gregarina steini and the granular-like vesicles that filled the plasm of the epimerite (Valigurová et al. Reference Valigurová, Hofmannová, Koudela and Vávra2007; Aldeyarbi and Karanis, Reference Aldeyarbi and Karanis2016c). Epicytic-life folds have been observed to cover the surface of free zoites, similar to what has been described in the trophozoites of the seven other gregarine species (Valigurová et al. Reference Valigurová, Hofmannová, Koudela and Vávra2007; Aldeyarbi and Karanis, Reference Aldeyarbi and Karanis2016c). The specific structure, called the mucron (epimerite-like structure), recalls similar findings described by Leander (Reference Leander2008) for Lecudinid eugregarines and it was confirmed with ultra-structural observations by Aldeyarbi and Karanis (Reference Aldeyarbi and Karanis2016c, see details). Such epi-cytic folds might play a certain role in the attachment of stages during syzygy as well as in the gliding movements of the stages. Perhaps Cryptosporidium can combine the fine structural characteristics of Coccidia and Gregarina as the phylogenetic link between both genera, however, it was not possible to determine this link in the past because the classical coccidian life cycle of C. parvum was generated close to the 1990s, first in 1986 by Current and Reese (Reference Current and Reese1986).

Short history of the last decade's highlights and future perspectives

Although major efforts in culturing Cryptosporidium in vitro have been exerted since 1984, infections could only be maintained for a few days, and only the asexual phase of the parasite life cycle was observed. Clearly, what is required in the field of Cryptosporidium now is continuous culture. Karanis and Aldeyarbi (Reference Karanis and Aldeyarbi2011) placed details on the subject, including many physiological and biological parameters, supplement's contribution, etc. Independent of the culture systems approaches, it is recommended to take the following into consideration, it is, however, not a prerequisite: (a) physiological and biological parameters, (b) supplements, (c) genotyping of isolates, (d) relation between Cryptosporidium and gregarines, (e) new reported in vitro systems. These elements are part of the ‘holistic approach’ to develop an in vitro system either axenic, in cells, advanced systems, and or in organoids (see below). It is not yet clear though as to what extent such aspects will contribute to the development of the Cryptosporidium's functional system of its continuous culture.

Attempts for continuous culture of Cryptosporidium have been focused in the last decade and are divided into three categories: cell lines, axenicaly and advanced systems.

The Bill and Melinda Gates Foundation (BMGF) contribution in the Cryptosporidium research

Cryptosporidium has reached the top priority level for funding and research support by the BMGF, like malaria and tuberculosis previously have. The Global Strategy has already recognized how a multidimensional approach to disease eradication must also include the general improvement of standards of living. This approach was possible for Cryptosporidium due to the invaluable studies of Kotloff et al. (Reference Kotloff, Nataro, Blackwelder, Nasrin, Farag, Panchalingam, Wu, Sow, Sur, Breiman, Faruque, Zaidi, Saha, Alonso, Tamboura, Sanogo, Onwuchekwa, Manna, Ramamurthy, Kanungo, Ochieng, Omore, Oundo, Hossain, Das, Ahmed, Qureshi, Quadri, Adegbola, Antonio, Hossain, Akinsola, Mandomando, Nhampossa, Acacio, Biswas, O'Reilly, Mintz, Berkeley, Muhsen, Sommerfelt, Robins Browne and Levine2013) and Checkley et al. (Reference Checkley, White, Jaganath, Arrowood, Chalmers, Chen, Fayer, Griffiths, Guerrant, Hedstrom, Huston, Kotloff, Kang, Mead, Miller, Petri, Priest, Roos, Striepen, Thompson, Ward, Van Voorhis, Xiao, Zhu and Houpt2015) thanks to the funding support of BMGF. These are large achievements so far, for a parasite with a short history of recognition due to its water significance and involvement in an environment of political and regulatory forces.

The strategy that BMGF focuses on is the advancement of the development of safe, affordable and effective vaccines for the leading causes of diarrhoeal and enteric diseases in low- and lower-middle-income earning countries. The foundation also invests in research to improve the case management and delivery of appropriate treatment to children with diarrhoea in high-burden countries, it is exploring new ways to prevent and reverse growth stunting. It funds research on the global and regional burdens of these diseases in order to be able to make decisions on when and how to deploy new interventions and how to expand the use of existing ones. Cryptosporidium was found to be a significant cause of moderate to severe diarrhoea in the GEMS study in South Asia and Africa. Its approach is two-fold: first, it invests in research to better understand the epidemiology and health consequences of the Cryptosporidium parasite; second, it pursues drug development by supporting preclinical tools and models and by screening the existing compound libraries for potential drugs against Cryptosporidium (http://www.gatesfoundation.org/What-We-Do/Global-Health/Enteric-and-Diarrheal-Diseases). So far, according to the statements from the home page of the BMGF for Grand Challenges and Accelerate Development of New Therapies for Childhood Cryptosporidium Infection and Strategy Leadership for fighting infectious diseases, the entire world will be forever thankful to BMGF for increasing the allocation of resources and in this case, for Cryptosporidium research, as well as its major philanthropic actions, including highly appreciated donations that support the poor and assist in fighting the burden of infectious diseases.

In the last two years, BMGF has funded several projects related to Cryptosporidium research to accelerate the development of new therapies for the childhood Cryptosporidium infection. ‘This call is soliciting new tools and technologies that have the potential: (i) to overcome the technical barriers in working with Cryptosporidium that have historically hampered progress and (ii) to improve our ability to develop and translate active compounds into effective therapies for the treatment of paediatric cryptosporidiosis. The goal of this call is to broadly develop the applicable approaches that can be used to accelerate development of therapeutic interventions, rather than to support the development of specific interventions themselves’ (BMGF homepage citation 2016).

The BMGF and the scientific community are certainly and undeniably interested in having the value of the currently proposed approach recognized as well as any other contributions from other individuals that display a keen interest in contributing to the success of the efforts of the Bill and Melinda Gates Foundation Enteric and Diarrheal Diseases (BMGF EDD) on behalf of children and affected humans globally. If opportunities exist, such as for assisting in understanding the details of various approaches, assisting in the review of alternative proposals, or contributing to the development of strategic alternatives, a larger number of real experts in the field can participate.