**4. Maintenance of** *Cryptosporidium* **in nature and transmission**

Once excreted into the environment, oocysts can be dispersed from the faecal matrix into the terrestrial environment (**Figure 2**). When present on the soil surface, oocysts may be exposed to high temperatures and desiccation, causing their inactivation. Oocysts are sensitive to desiccation and UV-C irradiation [116]. Reports show that desiccation is lethal to oocysts with only 3 and 5% remaining viable after being air-dried at room temperature for 2 and 4 h, respectively [117, 118]. However, when within the soil column, the oocysts were maintained, protected, and viable [119, 120]. Studies have indicated that oocysts at 4°C recovered from soil column may remain infectious for long periods [119, 121]. These findings suggest that the soil column is a sanctuary for *Cryptosporidium*, protecting it until rainfall events scatter them [120]. Oocysts were able to remain viable and infectious after being frozen at −10°C for up to 168 h, at −15°C for up to 24 h, and at −20°C for up to 8 h [122]. Moreover, *Cryptosporidium* oocysts can be carried in the environment due to interactions with biofilms (surfaceattached microbial communities). They readily attach to biofilms and persist and subsequently separate from it. High concentrations of oocysts in water biofilms that were maintained over several months maintained viable sporozoites [123]. *Cryptosporidium* oocysts in fresh water and marine water can survive at a range of temperatures. Fayer et al. reported that oocysts maintained at 20°C remain infectious for 12 weeks at salinities of 0 and 10 ppt, for 4 weeks at 20 ppt, and for 2 weeks at 30 ppt [124]. Although salinity can have a pronounced effect on oocyst infectivity, they can survive long enough in marine waters to justify their presence in marine animals.

*Cryptosporidium* spp. have a huge impact on both human and veterinary health worldwide, aggravated by the limited diagnosis and current therapeutics. *Cryptosporidium* spp. have a worldwide distribution and the ability to infect a wide range of hosts, including humans, and a broad variety of vertebrate [1, 3]. Humans can acquire cryptosporidiosis through several transmission routes, such as direct contact with infected persons or animals and consumption of contaminated water (drinking or recreational) or food (**Figure 3**).

**339**

**Figure 3.**

*State of the Art and Future Directions of* Cryptosporidium *spp.*

The WHO has categorised *Cryptosporidium* as a reference pathogen for the assessment of drinking water quality [125]. Susceptibility to cryptosporidiosis depends on several factors, including environmental conditions, host immune status, age, geographic location, and contact with infected humans/animals [126]. Animals play an important role in the maintenance, amplification, and transmission of *Cryptosporidium* [127]. In fact, a large range of animals are reservoirs for some species, genotypes, and subtypes, which may infect humans [128–130]. The lack of adequate instruments to continuously monitor animal mobility makes it difficult to study the dynamics of transmission [131, 132]. Also, oocysts are ubiquitous in the environment and easily spread via drinking water, recreational water, and food [3, 133, 134]. The ubiquitousness of the infective oocyst, its resilience to environmental pressures [135], and the low-dose oocyst exposure (ingestion of fewer than 10 oocysts can lead to infection) [136, 137] amplify to outbreaks of *Cryptosporidium* traced to drinking and recreational water. In 1993, the largest *Cryptosporidium* waterborne outbreak was recorded in the United States in Milwaukee, where more than 400,000 people were infected by the drinking water supply [138]. The epidemiology of infection is complex and involves transmission by the faecal-oral route, either by indirect transmission through ingestion of contaminated water or food or by direct human-to-human or animal-to-human transmission [3]. The genus *Cryptosporidium* has about 30 species formally described, as well as various genotypes and subtypes. Some species are relatively promiscuous in terms of host specificity, some of which also infected humans. Currently, a wide range of *Cryptosporidium* species and various genotypes have been recognised as

Human infections predominantly are caused by *C. hominis*, which are considered restricted to humans (anthroponotic transmission), and by *C. parvum*, some of which isolate genotypes and infect ruminants (zoonotic transmission) [18]. However, in recent years, *C. meleagridis*, *C. cuniculus*, and *C. ubiquitum* have also emerged as species relevant to public health, while the other species tend to be associated only with sporadic and rare cases of human infection. Approximately 155

responsible for human cryptosporidiosis (**Table 1**).

*DOI: http://dx.doi.org/10.5772/intechopen.88387*

*Transmission cycles of* Cryptosporidium *infections.*

**Figure 2.** Cryptosporidium *sp. oocysts in safranin-methylene blue staining method.*

*State of the Art and Future Directions of* Cryptosporidium *spp. DOI: http://dx.doi.org/10.5772/intechopen.88387*

*Parasitology and Microbiology Research*

ence in marine animals.

(drinking or recreational) or food (**Figure 3**).

Cryptosporidium *sp. oocysts in safranin-methylene blue staining method.*

takes molecular analyses, mainly DNA sequencing and PCR-related methods, into

Once excreted into the environment, oocysts can be dispersed from the faecal

matrix into the terrestrial environment (**Figure 2**). When present on the soil surface, oocysts may be exposed to high temperatures and desiccation, causing their inactivation. Oocysts are sensitive to desiccation and UV-C irradiation [116]. Reports show that desiccation is lethal to oocysts with only 3 and 5% remaining viable after being air-dried at room temperature for 2 and 4 h, respectively [117, 118]. However, when within the soil column, the oocysts were maintained, protected, and viable [119, 120]. Studies have indicated that oocysts at 4°C recovered from soil column may remain infectious for long periods [119, 121]. These findings suggest that the soil column is a sanctuary for *Cryptosporidium*, protecting it until rainfall events scatter them [120]. Oocysts were able to remain viable and infectious after being frozen at −10°C for up to 168 h, at −15°C for up to 24 h, and at −20°C for up to 8 h [122]. Moreover, *Cryptosporidium* oocysts can be carried in the environment due to interactions with biofilms (surfaceattached microbial communities). They readily attach to biofilms and persist and subsequently separate from it. High concentrations of oocysts in water biofilms that were maintained over several months maintained viable sporozoites [123]. *Cryptosporidium* oocysts in fresh water and marine water can survive at a range of temperatures. Fayer et al. reported that oocysts maintained at 20°C remain infectious for 12 weeks at salinities of 0 and 10 ppt, for 4 weeks at 20 ppt, and for 2 weeks at 30 ppt [124]. Although salinity can have a pronounced effect on oocyst infectivity, they can survive long enough in marine waters to justify their pres-

*Cryptosporidium* spp. have a huge impact on both human and veterinary health worldwide, aggravated by the limited diagnosis and current therapeutics. *Cryptosporidium* spp. have a worldwide distribution and the ability to infect a wide range of hosts, including humans, and a broad variety of vertebrate [1, 3]. Humans can acquire cryptosporidiosis through several transmission routes, such as direct contact with infected persons or animals and consumption of contaminated water

account for the detection and differentiation of *Cryptosporidium* spp.

**4. Maintenance of** *Cryptosporidium* **in nature and transmission**

**338**

**Figure 2.**

The WHO has categorised *Cryptosporidium* as a reference pathogen for the assessment of drinking water quality [125]. Susceptibility to cryptosporidiosis depends on several factors, including environmental conditions, host immune status, age, geographic location, and contact with infected humans/animals [126]. Animals play an important role in the maintenance, amplification, and transmission of *Cryptosporidium* [127]. In fact, a large range of animals are reservoirs for some species, genotypes, and subtypes, which may infect humans [128–130]. The lack of adequate instruments to continuously monitor animal mobility makes it difficult to study the dynamics of transmission [131, 132]. Also, oocysts are ubiquitous in the environment and easily spread via drinking water, recreational water, and food [3, 133, 134]. The ubiquitousness of the infective oocyst, its resilience to environmental pressures [135], and the low-dose oocyst exposure (ingestion of fewer than 10 oocysts can lead to infection) [136, 137] amplify to outbreaks of *Cryptosporidium* traced to drinking and recreational water. In 1993, the largest *Cryptosporidium* waterborne outbreak was recorded in the United States in Milwaukee, where more than 400,000 people were infected by the drinking water supply [138]. The epidemiology of infection is complex and involves transmission by the faecal-oral route, either by indirect transmission through ingestion of contaminated water or food or by direct human-to-human or animal-to-human transmission [3]. The genus *Cryptosporidium* has about 30 species formally described, as well as various genotypes and subtypes. Some species are relatively promiscuous in terms of host specificity, some of which also infected humans. Currently, a wide range of *Cryptosporidium* species and various genotypes have been recognised as responsible for human cryptosporidiosis (**Table 1**).

Human infections predominantly are caused by *C. hominis*, which are considered restricted to humans (anthroponotic transmission), and by *C. parvum*, some of which isolate genotypes and infect ruminants (zoonotic transmission) [18]. However, in recent years, *C. meleagridis*, *C. cuniculus*, and *C. ubiquitum* have also emerged as species relevant to public health, while the other species tend to be associated only with sporadic and rare cases of human infection. Approximately 155


#### **Table 1***.*

*Currently recognised species of* Cryptosporidium *spp. associated with human infections.*

species of mammals have been reported as non-human hosts of *C. parvum*, indicating that the parasite is adapting and developing in many hosts [176].

The human-to-human spread is particularly well described within families (often secondary cases after a primary outbreak infection) in childcare nurseries, nursing homes, and hospitals [42, 177, 178]. In addition, contact with production animals, mainly cattle, that are the main hosts of *C. parvum* can potentially infect humans [40, 178, 179]. To date, studies in developing countries have shown a predominance of *C. hominis* in HIV-positive children and adults. These findings are also valid in the United States, Canada, Australia, and Japan. In Europe and New Zealand, several studies have shown a similar prevalence of *C. parvum* and *C. hominis* in immunocompetent and immunocompromised individuals. Thus, in most developing countries, the anthroponotic transmission of *Cryptosporidium*

**341**

*State of the Art and Future Directions of* Cryptosporidium *spp.*

plays an important role in human cryptosporidiosis [18, 180], while in Europe, New Zealand, and rural areas of the United States, there are both anthroponotic and zoonotic transmissions. In Middle Eastern countries, children are mainly infected with *C. parvum*, but the significance of this occurrence is not clear [181]. An

exception is *Cryptosporidium* infections in HIV-positive patients in Ababa, Ethiopia, where *C. parvum* is highly endemic and where contact with calves is an important

In developing countries, most *C. parvum* infections in HIV-positive children and adults are caused by subtype IIc, with IIa largely absent, indicating that anthroponotic transmission of *C. parvum* is common in these areas. Conversely, families of subtype IIa are commonly diagnosed in humans in industrialised regions, where their occurrence is often associated with contact with calves. Another family of *C. parvum* subtypes commonly found in sheep and goats, IId, is dominant in humans in Middle Eastern countries and is occasionally found in humans in some European countries, such as Sweden, where it is commonly diagnosed in dairy calves. A systematic review of the anthroponotic transmission of *Cryptosporidium* concluded that subtype IIc predominates in low-income countries with poor sanitation and in HIV-positive individuals, unlike in higher-income countries, where it is rarely evident. Lacking effective treatment or vaccine, intervention to improve basic sanitation in these regions is the best option. This prophylactic action certainly may reduce the anthropogenic and zoonotic transmission of cryptosporidiosis, reducing the damage to human health. It is important to emphasise the importance of personal hygiene practices to minimise cryptosporidiosis, in addition to other

**5. Genome of** *Cryptosporidium***: new insight and future challenges**

Recent years have seen impressive progress of next-generation sequencing technologies in genome assembly and annotation methodologies, mainly by advancements in the fields of molecular biology and technical engineering and by reducing cost. *Cryptosporidium* has been the subject of genome sequencing projects, which have provided valuable insights into the species, biology, and host-parasite relationships. The genomic data of multiple *Cryptosporidium* species are available and accessible in a *Cryptosporidium*-dedicated database, CryptoDB (http://cryptodb.org/cryptodb/) [182], and in the GenBank database (www.ncbi.nlm.nih.gov). Comparative analyses have shown that *Cryptosporidium* genomes are highly compact, containing 8.50–9.50 megabase pairs (Mbp), a total gene count ranging from 3769 to 7610, and coding sequence composition (75–77.6%). Moreover, in general,

Overall, gene content and genomic organisation among intestinal occurrences of the species are well conserved, with *Cryptosporidium* gene clusters encoding putative secreted proteins. Comparison of the *Cryptosporidium* genomes has identified a core set of proteins commonly studied, as well as major differences in particular gene families, which could be involved in biological differences between species and genotypes [114, 183–185]. Gene encoding proteins that are associated with invasion processes, e.g. protein kinases and thrombospondin-related adhesive proteins (TRAPs), insulinase-like peptidases, MEDLE secretory proteins, and mucin glycoproteins, are observed in genome *Cryptosporidium* spp. [32]. However, some of them differ in copy number variations of genes. Comparative genomic analysis revealed that one of the primary features differentiating *Cryptosporidium* species is the sequence diversity present in major secreted protein families, MEDLE, and insulinase-like proteases [184]. This is consistent with transcriptomic studies

*DOI: http://dx.doi.org/10.5772/intechopen.88387*

risk factor for cryptosporidiosis [174].

pathogens transmitted by water and food.

they share a comparable GC percentage (**Table 2**).

*State of the Art and Future Directions of* Cryptosporidium *spp. DOI: http://dx.doi.org/10.5772/intechopen.88387*

*Parasitology and Microbiology Research*

*Cryptosporidium* **spp. Major host References** *C. andersoni* Cattle [139–144] *C. baileyi* Chickens and turkeys [145] *C. bovis* Cattle [146, 147] *C. canis* Dogs [148–150] *C. cuniculus* Rabbits [151–155] *C*. *fayeri* Marsupials [141, 156] *C. felis* Cats [93, 157, 158]

*C. hominis\** Humans [18]

*C. muris* Rodents [161–163] *C. parvum\** ruminants, especially calves [18] *C*. *scrofarum* Pigs [164]

*C. tyzzeri* Rodents, snake [168]

*C*. *viatorum* Humans [93, 170] *C. erinacei* Hedgehogs and horses [171, 172] *C. wrairi* Guinea pigs [173, 174] *C. xiaoi* Sheep and goat [174]

*C. suis* Pigs [139, 160, 165–167]

*C*. *ubiquitum* Sheep and cervids [152, 154, 157, 158, 169]

Rodents [93]

Minks [175]

Monkey [152]

Skunk [152, 153]

Horses [152, 153]

[18, 93, 153, 159, 160]

humans

*C. meleagridis\** Turkeys, chickens,

*Cryptosporidium* Chipmunk genotype

*Cryptosporidium* Horse genotype

*Cryptosporidium* Mink genotype

*Cryptosporidium* Monkey genotype

*Cryptosporidium* Skunk genotype

**Table 1***.*

*\*The most prevalent species.*

**340**

species of mammals have been reported as non-human hosts of *C. parvum*, indicat-

The human-to-human spread is particularly well described within families (often secondary cases after a primary outbreak infection) in childcare nurseries, nursing homes, and hospitals [42, 177, 178]. In addition, contact with production animals, mainly cattle, that are the main hosts of *C. parvum* can potentially infect humans [40, 178, 179]. To date, studies in developing countries have shown a predominance of *C. hominis* in HIV-positive children and adults. These findings are also valid in the United States, Canada, Australia, and Japan. In Europe and New Zealand, several studies have shown a similar prevalence of *C. parvum* and *C. hominis* in immunocompetent and immunocompromised individuals. Thus, in most developing countries, the anthroponotic transmission of *Cryptosporidium*

ing that the parasite is adapting and developing in many hosts [176].

*Currently recognised species of* Cryptosporidium *spp. associated with human infections.*

plays an important role in human cryptosporidiosis [18, 180], while in Europe, New Zealand, and rural areas of the United States, there are both anthroponotic and zoonotic transmissions. In Middle Eastern countries, children are mainly infected with *C. parvum*, but the significance of this occurrence is not clear [181]. An exception is *Cryptosporidium* infections in HIV-positive patients in Ababa, Ethiopia, where *C. parvum* is highly endemic and where contact with calves is an important risk factor for cryptosporidiosis [174].

In developing countries, most *C. parvum* infections in HIV-positive children and adults are caused by subtype IIc, with IIa largely absent, indicating that anthroponotic transmission of *C. parvum* is common in these areas. Conversely, families of subtype IIa are commonly diagnosed in humans in industrialised regions, where their occurrence is often associated with contact with calves. Another family of *C. parvum* subtypes commonly found in sheep and goats, IId, is dominant in humans in Middle Eastern countries and is occasionally found in humans in some European countries, such as Sweden, where it is commonly diagnosed in dairy calves. A systematic review of the anthroponotic transmission of *Cryptosporidium* concluded that subtype IIc predominates in low-income countries with poor sanitation and in HIV-positive individuals, unlike in higher-income countries, where it is rarely evident. Lacking effective treatment or vaccine, intervention to improve basic sanitation in these regions is the best option. This prophylactic action certainly may reduce the anthropogenic and zoonotic transmission of cryptosporidiosis, reducing the damage to human health. It is important to emphasise the importance of personal hygiene practices to minimise cryptosporidiosis, in addition to other pathogens transmitted by water and food.

### **5. Genome of** *Cryptosporidium***: new insight and future challenges**

Recent years have seen impressive progress of next-generation sequencing technologies in genome assembly and annotation methodologies, mainly by advancements in the fields of molecular biology and technical engineering and by reducing cost. *Cryptosporidium* has been the subject of genome sequencing projects, which have provided valuable insights into the species, biology, and host-parasite relationships. The genomic data of multiple *Cryptosporidium* species are available and accessible in a *Cryptosporidium*-dedicated database, CryptoDB (http://cryptodb.org/cryptodb/) [182], and in the GenBank database (www.ncbi.nlm.nih.gov). Comparative analyses have shown that *Cryptosporidium* genomes are highly compact, containing 8.50–9.50 megabase pairs (Mbp), a total gene count ranging from 3769 to 7610, and coding sequence composition (75–77.6%). Moreover, in general, they share a comparable GC percentage (**Table 2**).

Overall, gene content and genomic organisation among intestinal occurrences of the species are well conserved, with *Cryptosporidium* gene clusters encoding putative secreted proteins. Comparison of the *Cryptosporidium* genomes has identified a core set of proteins commonly studied, as well as major differences in particular gene families, which could be involved in biological differences between species and genotypes [114, 183–185]. Gene encoding proteins that are associated with invasion processes, e.g. protein kinases and thrombospondin-related adhesive proteins (TRAPs), insulinase-like peptidases, MEDLE secretory proteins, and mucin glycoproteins, are observed in genome *Cryptosporidium* spp. [32]. However, some of them differ in copy number variations of genes. Comparative genomic analysis revealed that one of the primary features differentiating *Cryptosporidium* species is the sequence diversity present in major secreted protein families, MEDLE, and insulinase-like proteases [184]. This is consistent with transcriptomic studies


#### **Table 2.**

*Genomic features of* Cryptosporidium *spp.*

of *C. parvum*, which have demonstrated MEDLE proteins in different subcellular locations that may perform their functions in distinct stages of the invasion and development process [33]. Moreover, a reduction in the number of genes encoding secreted MEDLE and insulinase-like proteins was observed in *C. ubiquitum* and *C. andersoni*, whereas the mucin-type glycoproteins are highly divergent between the gastric *C. andersoni* and intestinal *Cryptosporidium* species [184]. Unlike most other apicomplexans, *Cryptosporidium* spp. have no apicoplast or mitochondrial genomes but have remnant ones, the so-called mitosomes. However, *Cryptosporidium* species disagree from each other mostly in mitosome metabolic pathways. *C. parvum, C. hominis*, and *C. andersoni* present more aerobic metabolism

**343**

cryptosporidiosis.

**6. Conclusions and future perspectives**

*State of the Art and Future Directions of* Cryptosporidium *spp.*

and a conventional electron transport chain [114], whereas *C. ubiquitum* has further reductions in ubiquinone and polyisoprenoid biosynthesis and has lost both the conventional and alternative electron transport systems, unlike *C. muris* genome encoding core enzymes for the Krebs cycle and a functional ATP synthase. Thus, the mitosome of *C. muris* functions essentially as a peculiar mitochondrion [186]. However, the loss of biosynthetic pathways is a common feature observed in *Cryptosporidium* spp. genomes, e.g. the cytochrome-based respiratory chain and main de novo synthetic pathways for amino acids, nucleotides, fatty acids, and the Krebs cycle [32, 183]. Conversely, families of transporters to acquire nutrients from the host were expanded, including transporters for amino acids, sugars, and ATPbinding cassettes (ABCs) that drive the transport of various metabolites, lipids/ sterols, and drugs [32]. Although these genomic sequences provide valuable data, the genome analyses have revealed contradictory data and inconsistencies between the annotated gene models and transcriptome evidence [31, 36]. Notoriously, those findings are related to sequencing platforms, which have been applied to having different strengths and weaknesses and the use of different strategies and stringen-

Notwithstanding its novelty, the major challenges for the generation of whole genomes of *Cryptosporidium* are the quality and the yielding of limited DNA. Indeed, this is a critical step, as it is hard to recover enough quantity of DNA (2.5 × 10−5 highly purified oocysts correspondent approximately 10 μg) from faeces from natural infections. A theoretical estimate of the DNA content of one oocyst is of 40 fg [187]; therefore, it is tricky and arduous to recover enough quantity of DNA (2.5 × 10−5 highly purified oocysts correspondent approximately 10 μg) from unculturable samples with the quality necessary for high-throughput sequencing. Non-cultured samples may introduce a level of uncertainty and possess limited metadata. The lower the quality of the initial genome sequence, the higher the likelihood of yielding a missing or misassembled genome. A recent study evaluated an alternative method of preparing faecal samples using the combination of salt flotation, immunomagnetic separation (IMS), and surface sterilisation of oocysts prior to DNA extraction. The method has shown promise when used for the genome sequencing of samples of *C. parvum* and *C. hominis* [36]. This challenging issue can be resolved using a novel approach of *Cryptosporidium* cell-free culture and new long-read sequencing techniques, which will likely be beneficial for improving data. Increases in the quality of the target DNA boost the depth of coverage of the genome in higher levels, so base calls can be made with a higher degree of confidence. Also, the ability to culture relevant *Cryptosporidium* isolates in vitro, the development of novel gene-editing tools (knockout genes, CRISPR/ Cas9, and RNAi), and 'omic' research (genomics, transcriptomics, and proteomics) represent essential paths towards significant advancements in the control of

*Cryptosporidium* is a major cause of diarrhoeal disease in humans worldwide, yet an effective therapy to eradicate the parasite is not available. Also, the diagnosis options remain limited in developing countries, which harm the surveillance and understanding of the epidemiology in resource-poor settings. In developed countries, large waterborne outbreaks in drinking and recreational water continue to occur, emphasising the need for better regulation and for improvements of drinking water treatment processes and control guidelines. However, in recent years, significant improvements have been achieved in understanding the key concepts

*DOI: http://dx.doi.org/10.5772/intechopen.88387*

cies in gene prediction.

#### *State of the Art and Future Directions of* Cryptosporidium *spp. DOI: http://dx.doi.org/10.5772/intechopen.88387*

*Parasitology and Microbiology Research*

**Organism/name Strain Bio-**

*C. hominis* — SAMEA

*C. hominis* TU502\_2012 SAMN

*C. hominis* UKH1 SAMN

*C. hominis* 30,976 SAMN

*C. parvum* Iowa type II SAMN

*C. andersoni* 30,847 SAMN

*C. meleagridis* UKMEL1 SAMN

*C. meleagridis* UKMEL4 SAMN

*C. meleagridis* UKMEL3 SAMN

*C. ubiquitum* 39,726 SAMN

*Cryptosporidium* sp. 37,763 SAMN

*C. baileyi\** TAMU 09Q1 SAMN

*C. cuniculus* UKCU2 SAMN

*C. muris* RN66 SAMN

*C. viatorum* UKUIA1 SAMN

*Genomic features of* Cryptosporidium *spp.*

LX-2015

*Cryptosporidium* sp. Chipmunk

*C. hominis* TU502 — PRJNA

**sample**

3496639

02382005

02382004

02862040

02952908

04417240

02666797

08383028

08383027

02768023

SAMN 03281121

10623052

02382006

08383019

02953683

10107889

**Bioproject**

PRJEB 10000

13200

PRJNA 222836

PRJNA 222837

PRJNA 252787

PRJNA 144

PRJNA 354069

PRJNA 222838

PRJNA 315503

PRJNA 315502

PRJNA 534291

PRJNA 272389

PRJNA 511361

PRJNA 222835

PRJNA 3154496

> PRJA 19553

PRJA 492837

**Size (Mb)** **GC %**

9.10 30.1 3818 3817

8.74 30.9 3949 3885

9.10 30.1 3796 3745

9.15 30.1 3769 3718

9.06 30.1 3995 3959

9.10 30.2 7774 7610

9.09 28.5 3897 3876

8.97 31.0 3806 3753

8.79 30.9 — —

8.70 31.0 — —

8.97 30.8 3766 3766

9.51 31.9 — —

9.05 32.0 — —

8.50 24.2 — —

9.18 25.8 — —

9.25 28.5 — 3934

9.26 31.1 — —

**Gene Protein**

**342**

*\**

**Table 2.**

*Draft genome.*

of *C. parvum*, which have demonstrated MEDLE proteins in different subcellular locations that may perform their functions in distinct stages of the invasion and development process [33]. Moreover, a reduction in the number of genes encoding secreted MEDLE and insulinase-like proteins was observed in *C. ubiquitum* and *C. andersoni*, whereas the mucin-type glycoproteins are highly divergent between the gastric *C. andersoni* and intestinal *Cryptosporidium* species [184]. Unlike most other apicomplexans, *Cryptosporidium* spp. have no apicoplast or mitochondrial genomes but have remnant ones, the so-called mitosomes. However, *Cryptosporidium* species disagree from each other mostly in mitosome metabolic pathways. *C. parvum, C. hominis*, and *C. andersoni* present more aerobic metabolism and a conventional electron transport chain [114], whereas *C. ubiquitum* has further reductions in ubiquinone and polyisoprenoid biosynthesis and has lost both the conventional and alternative electron transport systems, unlike *C. muris* genome encoding core enzymes for the Krebs cycle and a functional ATP synthase. Thus, the mitosome of *C. muris* functions essentially as a peculiar mitochondrion [186]. However, the loss of biosynthetic pathways is a common feature observed in *Cryptosporidium* spp. genomes, e.g. the cytochrome-based respiratory chain and main de novo synthetic pathways for amino acids, nucleotides, fatty acids, and the Krebs cycle [32, 183]. Conversely, families of transporters to acquire nutrients from the host were expanded, including transporters for amino acids, sugars, and ATPbinding cassettes (ABCs) that drive the transport of various metabolites, lipids/ sterols, and drugs [32]. Although these genomic sequences provide valuable data, the genome analyses have revealed contradictory data and inconsistencies between the annotated gene models and transcriptome evidence [31, 36]. Notoriously, those findings are related to sequencing platforms, which have been applied to having different strengths and weaknesses and the use of different strategies and stringencies in gene prediction.

Notwithstanding its novelty, the major challenges for the generation of whole genomes of *Cryptosporidium* are the quality and the yielding of limited DNA. Indeed, this is a critical step, as it is hard to recover enough quantity of DNA (2.5 × 10−5 highly purified oocysts correspondent approximately 10 μg) from faeces from natural infections. A theoretical estimate of the DNA content of one oocyst is of 40 fg [187]; therefore, it is tricky and arduous to recover enough quantity of DNA (2.5 × 10−5 highly purified oocysts correspondent approximately 10 μg) from unculturable samples with the quality necessary for high-throughput sequencing. Non-cultured samples may introduce a level of uncertainty and possess limited metadata. The lower the quality of the initial genome sequence, the higher the likelihood of yielding a missing or misassembled genome. A recent study evaluated an alternative method of preparing faecal samples using the combination of salt flotation, immunomagnetic separation (IMS), and surface sterilisation of oocysts prior to DNA extraction. The method has shown promise when used for the genome sequencing of samples of *C. parvum* and *C. hominis* [36]. This challenging issue can be resolved using a novel approach of *Cryptosporidium* cell-free culture and new long-read sequencing techniques, which will likely be beneficial for improving data. Increases in the quality of the target DNA boost the depth of coverage of the genome in higher levels, so base calls can be made with a higher degree of confidence. Also, the ability to culture relevant *Cryptosporidium* isolates in vitro, the development of novel gene-editing tools (knockout genes, CRISPR/ Cas9, and RNAi), and 'omic' research (genomics, transcriptomics, and proteomics) represent essential paths towards significant advancements in the control of cryptosporidiosis.
