Diagnosis and control of cryptosporidiosis in farm animals

Abstract

Cryptosporidium is a pathogenic protozoan parasite infecting the gastrointestinal epithelium of human and animal hosts. In farm animals, cryptosporidiosis causes significant economic losses including deaths in newborn animals, retarded growth, increased labor involved and high cost of drugs. The detection of Cryptosporidium oocysts in fecal samples is traditionally dependent on examination of stained slides by light microscope or by advanced microscopical tools such as: electron microscopy and phase contrast microscopy. Immunological diagnosis using either antibody or antigen detection could offer high sensitivity and specificity. Examples for these tests are Enzyme Linked Immunosorbent Assay (ELISA), Immunochromatographic tests, Immunochromatographic lateral flow (ICLF), Immunofluorescence assays (IFA) and Flow cytometry coupled with cell sorting. Molecular methods could differentiate species and genotypes of Cryptosporidium and help in studying the epidemiological features of this parasite with rapid, simple and sensitive procedures. Nanotechnology-based platforms could improve the sensitivity and specificity of other detection methods like: ELISA, ICLF, IFA and polymerase chain reaction. As the available prophylactic and therapeutic drugs or natural products treatments are insufficient and no approved vaccines are available, the best approach to control this parasite is by following firm hygienic measures. Many vaccine attempts were performed using hyperimmune colostrum, live or attenuated vaccines, recombinant and Deoxyribonucleic acid vaccines. Also, Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 technology could help in Cryptosporidium genome editing to improve drug and vaccine discovery. Another approach that could be useful for assigning drug targets is metabolomics. Probiotics were also used successfully in the treatment of acute diarrhea and they proved a limiting effect on cryptosporidiosis in animal models. In addition, nanotherapy-based approaches could provide a good strategy for improving the potency of any type of drugs against Cryptosporidium and give good anti-cryptosporidial effects. In conclusion, accurate diagnosis using advanced techniques is the key to the control and prevention of cryptosporidiosis.

Introduction

Cryptosporidium is a pathogenic protozoan parasite infecting gastrointestinal epithelium of the hosts; human and animals (Checkley et al. 2015; Ryan et al. 2018). This parasite was firstly classified within the class Coccidia and then it was reclassified to gregarine on the basis of the evolutionary phases from the process of excision and sequencing of the 18S ribosomal RNA small subunit–SSU (18S rRNA) gene (Cunha et al. 2019).

Molecular characterization combined with experimental data had validated 30 species that infect mammals, birds, fish, reptiles and amphibians (Fayer et al. 2010). Also, a novel additional species is identified and named every year, and more than 40 genotypes were reported (Šlapeta 2013) in which zoonotic cryptosporidiosis plays an important role all over the world (Feng et al. 2018). Among these species, Cryptosporidium parvum (C. parvum) is found to be the most dominant species in farm animals (Tomazic et al. 2018).

Cryptosporidiosis is recognized as one of the major causes of diarrhea in neonatal ruminants (Santín 2020). Its pathogenicity varies with the species of Cryptosporidium and the age, type, and immune status of the host (Feng et al. 2018). The clinical signs observed in infected animals had a wide range from asymptomatic to death (Santín 2013). In cattle, disease is mostly characterized by the acute profuse and watery diarrhea, depression, weakness, loss of appetite (Li et al. 2019) and mortality from dehydration was reported in neonatal calves (Santín and Trout 2008). In small ruminants, cryptosporidiosis is characterized by diarrhea and loss of weight (Santín 2020). It was observed that clinically diseased lambs and kids were mostly infected with C. parvum, while Cryptosporidium xiaoi (C. xiaoi) and Cryptosporidium ubiquitum (C. ubiquitum) were often found in healthy asymptomatic lambs (Papanikolopoulou et al. 2018). In goats, cryptosporidiosis is associated with lower growth rate with or without diarrhea (Jacobson et al. 2018).

The selection of an appropriate diagnostic method depends on some criteria such as: technical expertise, required sensitivity and specificity, available time and financial resources (Chalmers and Katzer 2013). For laboratory diagnosis of cryptosporidiosis: microscopy, antibody and enzyme-based methods, and molecular techniques are used (Cunha et al. 2019; Gerace et al. 2019) and each one has its different sensitivity and specificity (Adeyemo et al. 2018). In case of fecal examination for detection and identification of Cryptosporidium oocysts, although this method is still commonly performed, simple and cost efficient, it lacks sensitivity (McHardy et al. 2014) and its diagnostic accuracy depends mainly on the expertise of the microscopist (Ahmed and Karanis 2018). To overcome this, feces should be subjected to concentration methods, by floatation or sedimentation, and purification procedures, such as immuno-magnetic separation and multiple staining methods, which had been used to increase sensitivity of the microscopic detection (Ryan et al. 2016; Gerace et al. 2019). Immunological tests using either antibody or antigen detection could offer a high sensitivity and specificity, however, few commercial immunochromatographic assays (ICT) offer low sensitivity and specificity (Lichtmannsperger et al. 2019). In addition, these standard methods provide no information about Cryptosporidium at species level or viability of oocysts (Gerace et al. 2019). Molecular methods have changed interest of the diagnostic laboratories towards Polymerase Chain Reaction (PCR) (Xiao and Feng 2017) as they are rapid, more sensitive with the detection range from 1 to 10⁶ oocysts (Smith 2007), allow genetic characterization to identify species, genotypes and subtypes (Santín 2020) and have the major advantage which is understanding Cryptosporidium epidemiological features and transmission routes (Thompson and Ash 2015; Cunha et al. 2019) from fecal or environmental samples (Jex et al. 2008). However, molecular methods are mostly limited to research and specialized laboratories (Santín 2020).

Cryptosporidium infection is difficult to control in both human and veterinary medicine because of the environmentally stable oocysts (resistant against freezing, drought and water treatment procedures), low infective dose, high levels of excreted infective oocysts and large scale of susceptible hosts. Oocysts are resistant to most disinfectants (Chalmers and Giles 2010) and no treatment or control measures that fully treated or prevented Cryptosporidium infection in humans and animals (Pinto and Vinayak 2021). In addition, Concurrent infections by different Cryptosporidium species are very common and immunity against reinfection is partial (Yang et al. 2021). Many chemical and natural alternatives were tested for treatment of cryptosporidiosis but with limited success (Santín 2020). Also, many vaccine trials have been conducted with numerous candidates against cryptosporidiosis, however, this is very challenging because of the higher incidence of cryptosporidiosis in ungulates (about 18.9%) (Hatam-Nahavandi et al. 2019). A complex cycle of events including various components of innate and adaptive host responses had proved to be important in the control of cryptosporidiosis (McDonald et al. 2013) and while available treatment options are limited (Meganck et al. 2014), fluid therapy remains the most important treatment that diminishes the clinical signs of the disease in humans and animals (Ghazy et al. 2016). Also, successful prevention and control strategies of this disease requires information about the diversity of its species and subtypes (Garcia-R et al. 2020).

Therefore, the aim of the present review is to throw a light on the advances in diagnosis and control of cryptosporidiosis in farm animals.

Diagnosis of cryptosporidiosis in farm animals

Conventional methods followed by immunological or molecular methods were the major diagnostics for cryptosporidiosis in most of the laboratories worldwide (Khurana and Chaudhary 2018).

Microscopical diagnosis

The detection of Cryptosporidium oocysts in feces is traditionally dependent on examination of stained slides by light microscope (Destura et al. 2015). Slides are usually stained with a modified acid-fast staining protocol such as: Modified Ziehl–Neelsen (MZN) or Kinyoun. This is still considered the method of choice as it is rapid, simple and of low cost (Silverlås et al. 2013). The sensitivity of microscopy could decrease in a time and temperature-dependent manner (Kuhnert-Paul et al. 2012). Electron microscopy could provide useful information about ultrastructure of this parasite. Transmission electron microscope (TEM) and scanning electron microscope (SEM) introduced more details about its developmental stages (Ghazy et al. 2015). TEM was used to detect Cryptosporidium and it was a very useful in visualizing the developmental stages of Cryptosporidium, in vitro, with a high ultrastructural resolution (Aldeyarbi and Karanis 2016). SEM also achieved high resolution of the extracellular stages of Cryptosporidium in biofilms (Koh et al. 2014).

Phase contrast microscopy could detect Cryptosporidium oocysts in a fecal sample but the sensitivity of this method is affected by the number of oocysts shed (Ignatius et al. 2016). Lichtmannsperger et al. (2020) confirmed occurrence of zoonotic C. parvum in diarrheic calves using phase-contrast microscopy and nested PCR.

Immunological diagnosis

Immunological diagnosis is based on either antigen detection or antibody detection. Antigens detection indicates current infection, while, antibodies detection might reflect old infection, but it’s useful in seroepidemiological surveys (Khurana and Chaudhary 2018).

Anti-Cryptosporidium Immunoglobulin G (IgG), Immunoglobulin M (IgM), or both can be detected by Enzyme Linked Immunosorbent Assay (ELISA). The sensitivity was found to be 66–100% and specificity of 93–100% (De 2013). Detection of antibodies in serum or fecal samples is an indirect diagnostic method for the evidence of infection or exposure (Khurana and Chaudhary 2018). Aboelsoued et al. (2020a) compared the copro-microscopic examination of calves’ fecal samples and ELISA using two types of antigens and recorded a higher prevalence of cryptosporidiosis using ELISA than microscopy. Also, Fereig et al. (2016) detected the prevalence of C. parvum in cattle using ELISA. The purified C. parvum recombinant protein (CP23) was used in an indirect ELISA to detect antibodies against C. parvum. It was easy to prepare than conventional techniques and was a good candidate for detection of cryptosporidiosis in field animals (Wang et al. 2009). ELISA offered higher sensitivity and specificity than microscopy with a detection limit of 10³–10⁴ oocysts/ml (Ghoshal et al. 2018). It can detect either antigen or antibody in a sample as it is easy, fast, sensitive and specific but it is unable to identify Cryptosporidium species or subtypes, which is important for studying transmission dynamics or outbreaks, especially in case of zoonotic species (Ahmed and Karanis 2018). Enzyme immunoassays (EIA) based on the detection of oocyst antigens were used widely (Tomazic et al. 2018). It can be automated and is more sensitive and specific than microscopy (Giadinis et al. 2012). Commercial EIA kits’ results were not influenced by sample storage or temperature (Kuhnert-Paul et al. 2012).

Immunochromatographic assays (ICT) could enable the detection of Cryptosporidium antigens in unconcentrated feces from diarrheic calves and can be used in the field (Cho and Yoon 2014). The ICT assays give fast results, and this led to its expanded use for Cryptosporidium detection (Khurana and Chaudhary 2018). The specificity is reported to be high (98–100%) (Smith 2007), however, they have poor sensitivity (75–88%), high false positives and false negatives with a few numbers of oocysts (Ghallab et al. 2016). Also, it was shown that the sensitivity and specificity was dependent on the kit and species (Agnamey et al. 2011). ICT can detect and differentiate between Giardia and C. parvum in fecal sample extracts (fresh, frozen or formalin fixed). Crypto-Giardia antigen rapid test detected 86.7% while MZN staining technique detected 73.3% positive Cryptosporidium infected cases (Zaglool et al. 2013). Immunochromatographic lateral flow (ICLF) for Cryptosporidium qualitatively detects Cryptosporidium antigen in feces. It is a dipstick that uses a monoclonal antibody (mAb) sandwich design to detect Cryptosporidium oocyst wall antigen. Fleece et al. (2016) evaluated rapid ICLF technique using ELISA as the reference standard for antigen detection and ICLF gave 100% sensitivity and 94% specificity. ICLF had the advantage of being easily performed without specialized equipment (Tomazic et al. 2018). Test strips ICT follow the lateral flow concept in which the antigen-specific antibodies are bound to a membrane and use the capillary flow to move the labeled antigen–antibody complex (El-Moamly and El-Sweify 2012).

Immunofluorescence assay (IFA), involving staining of the oocyst walls with fluorescein isothiocyanate-conjugated anti-Cryptosporidium mAbs or Auramine O, is a method of diagnosis of cryptosporidiosis in fecal samples of human and animals as it makes a sensitive, specific, and easier detection of Cryptosporidium oocyst than conventional staining methods (Chalmers and Katzer 2013; Ryan et al. 2016). The high sensitivity of these methods is due to the stability of the parasite antigens in the fecal preparations and that cross reaction with other organisms does not happen (Jex et al. 2008). Also, IFA allows visualization of the intact parasites giving a definitive diagnosis (Ahmed and Karanis 2018) with sensitivity from 96 to 100% and specificity from 98.5 to 100% (Mirhashemi et al. 2015; Roellig et al. 2017) and low concentration of oocysts that makes it the best choice in the prevalence studies of cryptosporidiosis in human or animals (Ahmed and Karanis 2018). However, in poor areas, it is difficult to find the required microscope and the expertise to work on it (Ryan et al. 2016).

Animal species (cattle, horse or sheep) played an important role in selecting IFA to be used as alone or if it needs to be combined with other technique. IFA combined with Kinyoun’s staining was usually suitable for sheep and cattle samples (Mirhashemi et al. 2015), while, IFA combined with PCR was used for screening subclinical horse samples. When linked with mAb, IFA had proved to be useful in detecting Cryptosporidium oocysts in fecal samples. This provides an excellent screening technique and offers valuable data for epidemiological studies. This monoclonal based fluorescence assay had a higher sensitivity and specificity in oocysts detection when compared with ELISA (Adeyemo et al. 2018; Cunha et al. 2019). These mAbs recognize the oocysts’ surface of epitopes (Khurana and Chaudhary 2018). Most of the commercially available mAbs are raised against C. parvum, and no antibody preparation is available for the specific epitopes on human or animal pathogenic Cryptosporidia (Khurana and Chaudhary 2018). Therefore, species and genotypes Cryptosporidium which differ in the oocyst epitopes expression will have a less intense fluorescence. So, the negative samples should be confirmed by another method (Smith 2007).

Antigen detection kits have the advantage of good specificity of 98–100%, and many samples could be processed easily and quickly (Ghoshal et al. 2018). EIA and ICT kits are available for individual pathogens or in combination with Giardia and/or Entamoeba histolytica. These tests are suitable to be performed on fresh, frozen or formalin-preserved samples (Khurana and Chaudhary 2018). Geurden et al. (2008) evaluated two ELISA tests and an ICT assay (dipstick) for Cryptosporidium diagnosis in calves. Despite a lower sensitivity, the dipstick assay provided a practical alternative to laboratory diagnosis of cryptosporidiosis in calves. Danišova et al. (2018) compared specificity of immunological methods used for detection of Cryptosporidium parasites in diarrheic pigs, calves and lambs using RIDASCREEN^® Cryptosporidium EIA test, Cryptosporidium second Generation (ELISA) and RIDAQUICK^® Cryptosporidium ICT. The prevalence of cryptosporidiosis in the entire group determined by EIA, ELISA and ICT were 34.17%, 27.84% and 6.33%, respectively, and different results obtained indicated difference in the copro-antigens produced by Cryptosporidium species. Therefore, some of the commercially available tests involve a combination of antibodies specific for 2 or 3 antigens (Agnamey et al. 2011; Goñi et al. 2012).

Flow cytometry coupled with cell sorting is used to detect oocysts with high sensitivity (Simjee 2007). This technique offers a fast, simple and economic method to quantify C. parvum burden in intestine and fecal samples of mice and other animal hosts (Sonzogni-Desautels et al. 2019). However, it is expensive, needs technical expertise and its sensitivity with the asymptomatic carriers had not been estimated (Ahmed and Karanis 2018).

Molecular diagnosis

PCR-based techniques

The PCR technique is an automated and repeatable technique (Adeyemo et al. 2018). It had a significant advantage of increased sensitivity and specificity but the sensitivity of its detection from fecal materials is affected by the quality and purity of Deoxyribonucleic acid (DNA) (Checkley et al. 2015). Sometimes, molecular detection of cryptosporidiosis is difficult in calves with inapparent or subclinical infection (Wang et al. 2021). The 18S rRNA is the most commonly used locus for detecting Cryptosporidium as it is able to detect all species of Cryptosporidium, and also, contains a hypervariable region for differentiating species and genotypes (Xiao and Feng 2017). Phylogenetic analysis of the amplified and sequenced 18S rRNA gene fragment could allow determining the species or genotype (Šlapeta 2013). In addition to 18S rRNA, molecular epidemiological studies identified more valuable genes including 70-kDA heat shock protein (HSP70), internal transcriber region-1 (ITS-1) and Cryptosporidium oocyst wall protein (COWP). For example, 60-kDa glycoprotein gene (gp60) is commonly used to provide details about possible transmission routes (Thompson and Ash 2015), zoonotic transmission of disease (Mahfouz et al. 2014).

Nested PCR is used to determine Cryptosporidium spp. in samples (Koehler et al. 2017). This is a two-step PCR with two sets of primers to confirm specific binding of DNA template where the common gene targets are COWP, gp60, 18S rRNA and HSP70 (Ahmed and Karanis 2018). Uppal et al. (2014) showed that nested PCR had the ability to detect 17.78% more positives than MZN staining and ELISA. Previously established nested PCR protocols were the best techniques to screen oocysts in fecal samples from different hosts regarding their ability in detection of cryptosporidial 18S rRNA in animal fecal samples (sheep, horses, buffaloes and cattle) (Abdelrahman et al. 2015; Mirhashemi et al. 2015; Abu El Ezz et al. 2018; Essendi et al. 2022). However, combining three nested PCR tests could provide a better knowledge about species’ diversity in livestock and to diagnose mixed infections (Ahmed and Karanis 2018).

PCR-restriction fragment length polymorphism (PCR–RFLP) improved the diagnosis of Cryptosporidium spp. as it helps to analyze PCR products after amplification of genomic DNA. PCR–RFLP could detect and identify the species and subtypes of this parasite using specific primers and restriction enzymes but it is time-consuming (Vanathy et al. 2017). Also, this approach could help investigating the molecular basis of the pathogenesis and virulence of Cryptosporidium (Wu et al. 2003). PCR- RFLP after nested PCR could detect and identify species in livestock animals (Helmy et al. 2015).

Quantitative PCR (qPCR) offers a quantification of many genetic targets over a wide range when compared with classic PCR (Ahmed and Karanis 2018). The qPCR is generally accepted as the most sensitive laboratory test for detecting and enumerating Cryptosporidium oocysts in fecal samples from many different hosts (Yang et al. 2015). Compared with nested PCR, qPCR is rapid, accurate and cost-effective for identification and quantification of Cryptosporidium spp. It could detect C. parvum and C. hominis separately or mixed without cross reactivity (Yang et al. 2013).

Droplet digital PCR (ddPCR) can give an absolute quantitation of DNA and species specific detection of parasite load without calibration curves as qPCR (Xiao and Feng 2017). ddPCR gives a higher precision in quantitation of Cryptosporidium DNA based on two loci (18S rRNA and actin) for most fecal samples of humans, sheep and cattle. Although ddPCR is more expensive than qPCR, it can provide absolute quantitation without using reference to external standard and with low sensitivity to PCR inhibitors (Yang et al. 2014).

Multiplex real-time PCR protocols can detect Cryptosporidium species and other enteroprotozoans as they proved to be valuable with mixed infections (Nurminen et al. 2015) with more sensitivity than microscopy (Ögren et al. 2016). For Cryptosporidium, the sensitivity ranged from (95–100%), while, specificity ranged from (99.6–100%) but it’s still expensive than conventional PCR (Ryan et al. 2017).

Loop-mediated isothermal amplification (LAMP)

LAMP is a low cost, accurate, simple, and highly sensitive method that detects Cryptosporidium spp. with the possibility to differentiate six distinct regions of the target gene (Plutzer et al. 2010). Using the loop primers accelerates reaction and this increases LAMP specificity. In addition, amplification product could be visualized by naked eye as turbidity or fluorescence (Mori et al. 2001). LAMP isn’t like PCR as it involves only an incubator not a thermocycler and so, it is less time consuming compared to PCR-based methods (Nagamine et al. 2001). Plutzer et al. (2010) found that the LAMP was superior to nested PCR for detecting Cryptosporidium species as an efficient tool for epidemiological studies that can diagnose low numbers of Cryptosporidium oocysts shed from apparently healthy animals even if these oocysts are very low below the detection limit of conventional PCR. Domingo et al. (2018) detected C. parvum DNA in fecal samples of cattle and water buffaloes in the Philippines by LAMP using specific primers and determined a probable zoonotic transmission of it between the farmers and their animals.

Aptamers

Aptamers are short and single-stranded DNA or RNA which have a unique three-dimensional structure allowing them to differentiate a specific molecular target with a high affinity and specificity (Zhang et al. 2019). They might serve as tools for diagnosis, therapy, biosensors, food risks or toxins detection, nanoparticle markers and drug carriers (Ospina-Villa et al. 2018). Aptamers are considered to be promising therapeutics as they had the ability to compete with protein ligands and small molecules to inhibit their targets (Hwang et al. 2012). Also, they can stimulate target receptors or act as carriers for the delivery of therapies to the target cells or tissue (Zhou and Rossi 2017). To date, “Pegaptanib” is the only aptamer which is approved by the Food and Drug Administration (FDA) for commercial use (Ospina 2020). The use of aptamers to study Cryptosporidium had not been widely reported, however, Iqbal et al. (2015) selected DNA aptamers against C. parvum oocysts using Systematic Evolution of Ligands by Exponential enrichment (SELEX) to design a sensitive and specific method to detect Cryptosporidium in food as one of the main sources of infection. Ospina-Villa et al. (2018) recognized a few promising aptamers with very high affinity for C. parvum oocysts as alternatives to mAbs in the detection of C. parvum oocysts. This method might be a good alternative to IFA as it offers a low limit of oocyst detection. Detection of Cryptosporidium oocysts by DNA aptamers could be promising in overcoming the challenge of better sensitivity and specificity, especially in case of contaminants (Hassan et al. 2021).

Nanotechnology-based platforms

These highly reduced-size particles provide high sensitivity, early diagnosis of diseases and genetic dispositions using accurate imaging methods and simple, rapid and inexpensive tests (Jackson et al. 2017). The nanotechnology-based platforms were utilized to improve sensitivity and specificity of other detection methods like: ELISA, lateral flow assays, IFA and PCR (Checkley et al. 2015). Another application of nano-materials in diagnosis of cryptosporidiosis is using oligonucleotide nanoparticles (NPs) for molecular detection of cryptosporidiosis without the need for amplification of nucleic acids (Weigum et al. 2013). Some assays were conducted using dual-labeled gold NPs (alkaline phosphatase and anti-oocyst mAb) such as: electrochemical-based sandwich enzyme-linked immunosensor (Thiruppathiraja et al. 2011a), rapid immunodot blot assay (Thiruppathiraja et al. 2011b), and amplification-free detection systems (Weigum et al. 2013). The sensitivity of electrochemical immunosensor increased with a limit of detection of three oocysts/ml in the least processing time (Thiruppathiraja et al. 2011a).

Control of cryptosporidiosis in farm animals

Control of cryptosporidiosis requires continued efforts to interrupt its transmission through water, food and contact with infected animals (Yoder and Beach 2010). Cryptosporidium loads in livestock manure were estimated to be around 3.2 × 10²³ oocysts/year, with cattle being the predominant source (Vermeulen et al. 2017). Oocysts could be transported in manure via run-off into surface waters and become a source of infection. Asia has the highest oocyst load from livestock manure, followed by Africa, South America and Europe (Vermeulen et al. 2017). Thousands of therapeutic agents has been tested against cryptosporidiosis in vitro and in vivo (Shahiduzzaman and Daugaschies 2012) but with limited success (Santín 2020).

Hygienic measures

As the available prophylactic and therapeutic drugs or natural products treatments are insufficient and no approved vaccines are available, the best approach to control cryptosporidiosis is by following firm hygienic measures. In animal farms, this approach could be challenging as animals are able to disseminate large number of oocysts. Using concrete flooring in animal housing areas, steam-cleaning of animal houses, increasing the depth of the bedding, daily feeding with fermented milk, treatment by antibiotics (Delafosse et al. 2015), frequent removal of the feces and contaminated bedding from the calving areas (Thomson et al. 2017) and making sure newborns receive adequate quantities of colostrum in the first few hours of their lives (Meganck et al. 2014). Disinfection of soil, maternity pens, calf housing and feeding equipment could be useful (Tomazic et al. 2018) with disinfectants containing hydrogen peroxide which were found to be effective (Thomson et al. 2017). Björkman et al. (2018) suggested that disinfection of animal pens with hydrated lime could reduce the onset and severity of Cryptosporidium in calves. Also, implementation of water treatment procedures to decrease the spread of Cryptosporidium oocysts in the environment is critical as one of the major sources of infection is contaminated water (Ghazy et al. 2016).

Vaccination

Two strategies have been used to protect animals against Cryptosporidium infection. The first is to induce hyperimmune colostrum containing a high titer of antibodies for neonatal calves by inoculating cattle with Cryptosporidium antigens and the other strategy is to immunize the neonatal calves directly with Cryptosporidium antigens (Wyatt 2000). Also, a strategy of vaccinating dams against some other pathogens that cause a diarrheal disease, such as: Escherichia coli (E. coli), rotavirus and coronavirus, could help in generating specific immunity in the dam passing it to its calf through the colostrum (Innes 2011).

Bovines have a syndesmochorial placenta and the calf doesn’t take immunoglobulins from the dam via transplacental transmission. Passive transfer of these immunoglobulins is carried out by feeding of the first milking colostrum which provides the quickest form of protection (Tomazic et al. 2018). Many studies evaluated colostrum or mAb produced against multiple specific antigens which are involved in cryptosporidial attachment or invasion of host cells. Bovine colostrum induced by immunization with C. parvum recombinant protein rC7 gave a protection against cryptosporidiosis in neonatal calves (Perryman et al. 1999). A mixture of C. parvum antigens generated hyperimmune colostrum in calves and they were partially protected showing less clinical symptoms and a shorter time of oocysts shedding than controls (Wyatt 2000). Also, cattle vaccinated with rCP15/60 produced a significantly higher antibody response compared to controls and this response was found to be strongly associated with the level of colostral antibody (Burton et al. 2011). After challenge with hyperimmune bovine colostrum, generated by immunization of pregnant cows with a recombinant form of C. parvum protein (p23), neonatal calves showed protection presented by absence of diarrhea and almost no oocysts shedding was recorded (Askari et al. 2016). Also, using an appropriate antigen for generating colostrum gave a complete protection from this disease (Wyatt 2000).

Development of vaccine candidates carrying Cryptosporidium parasites which are incapable of causing disease, through genetic engineering or irradiation, is considered a promising approach for development of potent vaccine strains (Mead 2014). Vaccination of calves with gamma-irradiated Cryptosporidium oocysts gave a protection against subsequent challenge and prevented generation of the clinical disease (Jenkins et al. 2004). Attenuation by irradiation is a challenge as too much irradiation could kill the parasite and too little irradiation could introduce complete life cycle stages (Mead 2014). In pigs, C. hominis specific immunity completely protected against subsequent challenge by the same species (Sheoran et al. 2012). Also, the primary infection of another group with C. hominis followed by a challenge with C. parvum oocysts gave a partial cross-protective immunization with moderate symptoms and lower oocysts shedding than the infected controls (Sheoran et al. 2012). Antibody response against many dominant Cryptosporidium antigens, which might play a role in Cryptosporidium attachment and invasion of the host cells, such as: Cp15, Cp23 and Gp15 that were considered the major protective antigens, could be important as vaccine candidates (Mead 2014). Riggs et al. (2002) hypothesized that the targeting of apical complex and surface antigens Gp25-200, CSL and P23 could immunize against Cryptosporidium infection. Also, using an attenuated Salmonella strain holding specific C. parvum antigens as a vaccine vector offered a protection against C. parvum infection (Benitez et al. 2009).

DNA immunization was utilized to stimulate antigen specific responses of T and B cells in many infection models (Hong-Xuan et al. 2005). DNA encoding the 15/60-kDa antigen could evoke immune response in sheep (Jenkins et al. 1993). Immunization with Cp15-DNA induced a long lasting and specific production of anti-Cp15 IgA in the intestinal secretions and specific IgG in the sera of mice which continued for up to one year after the first DNA inoculation (Sagodira et al. 1999). Recombinant fusion protein CP15-23 could be utilized as a candidate antigen for protection against cryptosporidiosis (Hong-xuan et al. 2005). Mice immunized with Cp23-DNA gave a partial protection against C. parvum infection with more than 60% reduction in the shedding of oocysts after challenge (Ehigiator et al. 2007). Also, administration of a DNA vaccine encoding C. parvum Cp23 and Cp15 induced of T-helper 1 immune responses and increased the resistance to infection (Wang et al. 2010). A recombinant DNA vaccine consisting of Cp15 antigen immunized pregnant goats and protected offspring (Roche et al. 2013). Trotz-Williams et al. (2007) used E. coli as a vaccine in calves and it was strongly protective against oocysts shedding while Trotz-Williams et al. (2008) and Díaz et al. (2018) found that this vaccine didn’t offer immunization against cryptosporidiosis. Sobati et al. (2017) cloned gp40/15 surface antigen in E. coli successfully suggesting that it can be used in developing recombinant vaccines or diagnostic kits.

Molecular biology

Recently, it was found that manipulation of specific genes could help to investigate the function of genes in the parasite (Innes et al. 2020) and many advances were achieved in the genetic engineering of Cryptosporidium parasites (Pawlowic 2017). For example, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology, which enables Cryptosporidium genome editing, helped in the generation of reporter strains in order to improve drug and vaccine discovery (Vinayak et al. 2015; Innes et al. 2020). Also, including protein tagging and subjected knockdown, it could improve knowledge about its biology and host pathogen interactions (Pawlowic 2017).

Another approach that could be useful for biomarkers discovery, assigning drug targets and improving diagnostic techniques is metabolomics. It concerns studying intracellular and extracellular metabolites which are consumed or produced as a consequence of biological activity (Ryan and Hijjawi 2015). Biochemical data and genome sequencing revealed that Cryptosporidium parasite derives nutrients from its host or environment and lacks the ability for synthesis of nucleosides, amino acids, and fatty acids (Xu et al. 2004). Lower metabolite levels were detected after metabolomics analysis of fecal samples of experimentally infected mice (Ng Hublin et al. 2013). Differences in fecal metabolite profiles among different hosts were previously recorded by Saric et al. (2008). Huang et al. (2020) found an imbalance of many metabolites in important metabolic pathways after infection when assessed the metabolic changes in healthy and diarrheic calves after infection with different types of pathogens including parasites. In another study, metabolomic procedures with statistical chemometric analyses of viable and irradiated oocysts of Cryptosporidium and some key metabolites was identified and this allowed differentiation between viable and non-viable oocysts (Beale et al. 2013). Many studies recorded a noticeable change in the metabolic activity according to the status of the Cryptosporidium infection and so, an investigation using a collection of all ‘omics approaches should be performed (Pinto et al. 2022).

Probiotics

Few studies evaluated the effect of probiotics (live bacterial cell supplements) on parasitic infections (Travers et al. 2011). Probiotics were used successfully in treatment of the acute diarrhea and they had also a limiting effect on cryptosporidiosis in animal models (Pickerd and Tuthill 2004). Probiotics reduced the duration and number of oocysts shedding in experimentally infected mice (Alak et al. 1997) and also, reduced oocyst viability in vitro (Foster et al. 2003). Guitard et al. (2006) failed to find a significant activity of mixtures containing Lactobacillus casei against cryptosporidiosis in experimental conditions, however, a more rapid clearance of oocysts from probiotics-treated rats than controls was reported. Also, the fact that Cryptosporidium parasites need an alkaline medium to excyst (Smith et al. 2005) and the acidification induced by lactic acid bacteria could hinder this process by reducing its viability (Del Coco et al. 2016).

Nanotechnology

Nanomaterials have been widely used in various sectors. It was found that Nanotherapy-based approaches provide a general strategy that could be useful for improving the efficacy of any type of drug targeting Cryptosporidium parasite and achieve good antiparasitic activities (Mukerjee et al. 2015). Many types of NPs, varying in their non-toxic properties, cost effectiveness, simplicity in preparation, stability and biodegradability, displayed an effective anti-cryptosporidial action such as: silver (Cameron et al. 2016) and gold NPs (Jain et al. 2019) which were promising against Cryptosporidium as they were assumed to have the ability to break the oocyst wall. Also, artesunate loaded polymeric nanofibers offered a promising activity in reducing C. parvum in experimentally infected mice with minor toxic effects (Abdelhamed et al. 2019). Bondioli et al. (2011) encapsulated the drug Indinavir in biodegradable NPs and conjugated their surface with an anti-Cryptosporidium IgG polyclonal antibody and this formula was able to target C. parvum offering a good strategy for cryptosporidiosis control. In addition, chitosan NPs suspension was evaluated against cryptosporidiosis in experimentally infected mice and proved an anti-cryptosporidial effect (Ahmed et al. 2019).

Treatment

The preferred procedure in farm animals is supportive treatment by replacement of fluid and electrolytes, nutritional support and the administration of antidiarrheal drugs (Tomazic et al. 2018). In farm animals, many therapeutic drugs were tested against cryptosporidiosis and offered good effect on reducing oocyst shedding, severity and duration of diarrhea including: coccidiostatic drugs such as: Halofuginone lactate, which had been licensed for the control of cryptosporidiosis in livestock (Santín 2013; Brainard et al. 2021), however it’s not effective once cryptosporidiosis diarrhea was established (Trotz-Williams et al. 2011), and Decoquinate (Ortega-Mora et al. 2005), antiparasitic drugs like: nitazoxanide (Chavez and White 2018) and antibiotics like: paromomycin (Grinberg et al. 2002), azithromycin (Nasir et al. 2013), boromycin (Abenoja et al. 2021), as well as starch derivatives α- and β-cyclodextrin (Castro-Hermida et al. 2004) and chitosan oligosaccharide preparation in calves (Alam et al. 2012) and lambs (Aydogdu et al. 2019).

Also, some protease inhibitors (indinavir, nelfinavir and ritonavir) demonstrated anti-cryptosporidial activity (Rossignol 2010). Bumped kinase inhibitors, targeting Cryptosporidium calcium-dependent protein kinase-1, reduced oocyst shedding and clinical signs in calves (Lendner et al. 2015; Van Voorhis et al. 2021) and piglets (Lee et al. 2018).

Also, some plant extracts were evaluated against this parasite, such as: garlic, which was effective in prophylaxis and treatment of cryptosporidiosis (Abdel Megeed et al. 2015), curcumin (Asadpour et al. 2018), cinnamon and onion (Abu El Ezz et al. 2011), black seed (Nasir et al. 2013), moringa (Aboelsoued et al. 2019a), ginger, ginseng, and sage (Aboelsoued et al. 2020b) and pomegranate (Al-Mathal and Alsalem 2013; Aboelsoued et al. 2019b). Also, it was found that propolis (bee glue) extracts have an activity on cryptosporidiosis (Soufy et al. 2017; Asfaram et al. 2020). In addition, yeast (Saccharomyces cerevisiae) fermentation products were used as a natural alternative against bovine cryptosporidiosis (Vélez et al. 2019).

Conclusions

The selection of an appropriate diagnostic method depends on technical expertise, required excellent sensitivity and specificity, available time and financial resources. Advances in diagnosis of cryptosporidiosis and adoption of standardized methods could allow better correlations between animal, human, and environmental data. Molecular biology could offer a simple and fast approach in diagnosis and control of cryptosporidiosis. Nanotechnology-based platforms could improve the efficacy of drugs targeting Cryptosporidium and obtain potent antiparasitic activities.

Recommendations

Combination of microscopical and molecular diagnosis could be helpful for detecting cryptosporidiosis and differentiating the parasite species in farm animals. Understanding host–parasite interactions could aid in the development of successful immunotherapies or vaccines. Application of better management practices including routine cleaning and disinfection of farms, diagnostic surveys, isolation of infected animals, potential waste-water treatment processes and implementation of periodic health education training to the farm owners and staff could help to avoid infection and limit the hazards of environmental contamination and economic losses in animal wealth.

Abbreviations

C. hominis

Cryptosporidium hominis

C. parvum

Cryptosporidium parvum

C. ubiquitum

Cryptosporidium ubiquitum

C. xiaoi

Cryptosporidium xiaoi

COWP

Cryptosporidium Oocyst wall protein

CRISPR

Clustered Regularly Interspaced Short Palindromic Repeats

ddPCR

Droplet digital PCR

DNA

Deoxyribonucleic acid

E. coli

Escherichia coli

EIA

Enzyme immunoassay

ELISA

Enzyme linked immunosorbent assay

FDA

Food and Drug Administration

gp60

60 kDA Glycoprotein

HSP70

Heat shock protein 70

ICLF

Immunochromatographic lateral flow

ICT

Immunochromatographic test

IFA

Immunofluorescence assay

IgG

Immunoglobulin G

IgM

Immunoglobulin M

ITS-1

Internal transcriber region-1

LAMP

Loop-mediated isothermal amplification

mAb

Monoclonal antibody

MZN

Modified Ziehl–Neelsen

NPs

Nanoparticles

PCR

Polymerase chain reaction

PCR-RFLP

PCR-restriction fragment length polymorphism

qPCR

Quantitative PCR

SELEX

Systematic evolution of ligands by exponential enrichment

SEM

Scanning electron microscope

spp.

Species

TEM

Transmission electron microscope

18S rRNA

18S ribosomal RNA

Funding

This research has not received any funding.

Declarations

Conflict of interest

The authors declare that they have no conflicts or competing interests.