Abstract: Cryptosporidiosis is mainly a problem in neonatal farm animals. Cryptosporidium parvum is the most commonly found entero-pathogen during the 1st weeks of the life in calves, lambs, foals, piglets and goat kids and is considered to be an important agent in the etiology of the neonatal diarrhea syndrome. The parasite frequently acts alone but the losses are more pronounced when concurrent entero-pathogens are present. Economic losses associated with cryptosporidiosis are retarded growth and mortality and a number of hard to estimate costs resulting from interventions necessitated by diarrheic problems. Especially in small ruminants, the direct losses due to mortality caused by cryptosporidiosis alone were reported to be high. In the current review article, data concerning taxonomy, life cycle, epidemiology and zoonotic importance are discussed.
INTRODUCTION
The genus Cryptosporidium was named at the beginning of the last century but was only recognized as a potential cause of disease in 1955, when it was found to be associated with diarrheic turkeys (Slavin, 1955). Although Cryptosporidium was subsequently found in a broad range of farm animals, its impact was neglected until the early 1980s when it was found to be a common, serious primary cause of outbreaks of diarrhea in calves (Tzipori et al., 1980). The fact that Cryptosporidium was found to infect humans and could cause a life-threatening disease in immuno-deficient people, especially AIDS patients, as well as the association of Cryptosporidium with waterborne-related human outbreaks of diarrhea has certainly given the parasite a more widespread recognition (MacKenzie et al., 1995; Shaapan and Khalil, 2014). It has encouraged the scientific work on Cryptosporidium in many domains, especially in the veterinary field, because animal husbandry is seen as a threatening source of infection for humans by the release of tremendous numbers of resistant oocysts in surface water and environment. Nevertheless, the interest for cryptosporidiosis in the veterinary field arises even more from the fact that it concerns a harmful, difficult to control disease of many farm animals, which result in significant economic losses (Ramirez et al., 2004; Shaapan et al., 2011).
Cryptosporidium are intracellular, extra cytoplasmic protozoan parasites with a monoxenous life cycle. They invade the micro villous border of the gastrointestinal epithelium causing considerable economic losses in livestock. Cryptosporidium parvum is considered to be an important agent in the etiology of the neonatal diarrhea syndrome in different farm animals including calves, lambs, foals, piglets and goat kids. The major economic losses due to cryptosporidial infections are related to diarrhea, dehydration, growth retardation and mortality (Olson et al., 2004). A single oocyst is sufficient to produce infection and disease in susceptible hosts. Environmentally resistant oocysts are transmitted by the fecal-oral route. Transmission can occur from animal-to-animal, animal-to-person and person-to-animal and person-to-person, by ingestion of contaminated water or food or by contact with contaminated surfaces (Smith et al., 2007).
Therefore, the objective of the current review article was to highlight and evaluate data concerning the taxonomic classification, life cycle, epidemiology and zoonotic importance.
Developmental stages of a novel protozoan parasite were first described in the gastric glands of laboratory mice by Tyzzer (1907, 1910). The small parasites appeared to be attached to the surface of the epithelial cells by knob-like projections. Both asexual and sexual developmental stages were described to culminate in the formation of unique spores (or oocysts) containing 4 sporozoites. The parasites were considered to represent a new genus of coccidian like sporozoa which was named Cryptosporidium (meaning hidden sporocysts) and the species named C. muris. Similar parasites were later detected in the small intestines of mice by Tyzzer (1912) but they were considered to be a separate species (named C. parvum) because they were smaller than C. muris and their development was confined to the small intestinal epithelium. Despite further reports on the occurrence of Cryptosporidium infections in various hosts, their pathogenic significance remained unclear until Slavin (1955) described infections by a new species (named C. meleagridis) in the intestines of turkeys in association with acute clinical disease characterized by severe diarrhoea and low mortalities. Infection by Cryptosporidium spp. in cattle was first reported in the early 1970s (Panciera et al., 1971; Meuten et al., 1974). However, because of the association with other viral and bacterial entero-pathogens, the role of Cryptosporidium spp. as primary entero-pathogens was uncertain until 1980, when Tzipori et al. (1980) attributed an outbreak of neonatal diarrhea to cryptosporidial infection alone. In the following years methods to free the infective oocysts from other contaminating pathogens become available, which permitted the experimental demonstration that Cryptosporidium was capable of causing clinical diarrhoea in calves (Tzipori et al., 1983; Heine et al., 1984). In cattle, two species of the genus Cryptosporidium were distinguished, C. parvum is infecting the distal small intestine and C. muris infecting the abomasum. Substantial differences in the size and shape of C. parvum (5.0, 4.5 μm and spherical) and C. muris (7.4, 5.6 μm and ovoid) oocysts enables the two species to be distinguished readily on microscopical examination (Upton and Current, 1985). Only C. parvum has been associated with neonatal diarrhea. Cryptosporidium muris is much less prevalent and was only found in weaned calves or adult cattle. Cryptosporidium muris infection is considered to be clinically mild, affecting weight gain and milk production (Anderson, 1987; Esteban and Anderson, 1995; Bukhari and Smith, 1996). In sheep, infection by Cryptosporidium was first described in Australia in 1-3 week old lambs with diarrhea (Barker and Carbonell, 1974). Their roles as a primary etiological agent was confirmed in the early 1980s in studies on experimental infections in the absence of other enter pathogenic agents (Angus et al., 1982; Snodgrass et al., 1984). Since then, Cryptosporidium has been attributed an increasingly important role in neonatal diarrhea syndrome in this domestic species and is currently associated with high morbidity rates and mortality, depending on environmental conditions and the presence of other intestinal pathogens (Munoz et al., 1996). In goats, infection by this agent was also first described in Australia in 2 week old kids with diarrhea (Mason et al., 1981) Since then, the infection has been diagnosed in outbreaks of diarrhoea in goat kids in several countries and is now considered to be one of the principal enteropathogens in these animals (Munoz et al., 1996; Noordeen et al., 2012). Cryptosporidium parvum has been associated with diarrhea in small ruminants. In a recent work, a bovine isolate of C. muris did not produce infection in goats (Koudela et al., 1998). Cryptosporidium infection was first reported in pigs by Bergeland (1977) and Kennedy et al. (1977). Thereafter, naturally occurring cryptosporidiosis in pigs has been described worldwide (De Graaf et al., 1999). In humans, Cryptosporidium infections were reported in 1976 in two patients with severe watery diarrhea (Meisel et al., 1976; Nime et al., 1976). Additional cases were reported intermittently over the next few years, most involving immunocompromised patients who had congenital immunological disorders or were undergoing immunosuppressive therapy (Ungar, 1990; Bouzid et al., 3013). Further medical interest was generated in 1982 when an outbreak of cryptosporidiosis was reported in healthy immunocompetent individuals who had been in close contact with infected calves (CDC., 1982a; Current et al., 1983) and when chronic infections were associated with several mortalities in patients infected with Human Immunodeficiency Virus (HIV) who had developed acquired immunodeficiency syndrome (AIDS) (CDC., 1982b). Infections have since been associated with mild to severe clinical disease in both immunocompromised and immunocompetent patients throughout the world (Ungar, 1990; Current and Garcia, 1991). Currently about 30 species of the genus Cryptosporidium are proposed as valid. Humans and cattle are the hosts for 14 and 13 out of 30 named species, respectively. There are at least nine species that are shared between humans and cattle (Slapeta, 2013).
TAXONOMIC CLASSIFICATION
The genus Cryptosporidium has been classified in the phylum Apicomplexa, class Sporozoasida, subclass Coccidiasina, order Eucoccidiida, suborder Eimeriina, family Cryptosporidiidae (O'Donoghue, 1995). Cryptosporidium has been classified together with other enteric coccidian parasites in the order Eucoccidiida on the basis of many similarities in their morphological characteristics and life cycles (Table 1). Most coccidian parasites occur in the gastrointestinal tracts of vertebrates (Levine, 1985) and many genera have monoxenous life cycles involving one host species, where all stages of parasite development take place. At least 22 species of Cryptosporidium have been named based on host occurrence, parasite morphology, host predilection and site of infection (Table 2). However, only 13 species are considered valid by most investigators (Table 3). Cryptosporidium parvum is the major species responsible for disease in humans and domestic animals such as cattle, horses, sheep, goats and pigs (De Graaf et al., 1999).
| Table 1: | Taxonomic classification of Cryptosporidium |
| *Based on classification system proposed by Levine (1985) | |
| Table 2: | List of the currently recognized species of Cryptosporidium |
| Smith et al. (2007) | |
| Table 3: | Valid taxonomic nomenclature of Cryptosporidium species and their host range |
| Ramirez et al. (2004) | |
Although, phenotypic differences were traditionally used to distinguish between strains of C. parvum isolated from several host species, molecular epidemiological studies have demonstrated the existence of at least two unique and separate C. parvum genotypes, human (genotype 1) and bovine (genotype 2) (Sulaiman et al., 1998). The human genotype has recently been re-classified as a different species and named Cryptosporidium hominis (Morgan-Ryan et al., 2002) for several important reasons. Cryptosporidium hominis infects mainly humans while bovine C. parvum infections have been reported in numerous animal species including cattle, sheep, goats, pigs, mice and humans (Table 3). Morphologically, the two species are nearly indistinguishable; however, genetic differences have been demonstrated (Morgan et al., 1998; Xiao et al., 1999). In addition, C. parvum and C. hominis showed distinctly different pathological disease patterns in gnotobiotic pigs and failed to exchange genetic material during dual infections (Pereira et al., 2002). Cryptosporidium's unusual location within the host cell, sequestered between the cell cytoplasm and cell membrane, its ability to autoinfect, its innate antimicrobial resistance and its general lack of host specificity (especially C. parvum) are unique features that distinguish it from other enteric protozoa. Moreover, the taxonomy remains controversial and the epidemiology of different species is unclear, although implementation of newer molecular tools that have recently become available should help clarify Cryptosporidium diversity and improve our understanding of the parasite. Further studies are required to confirm the validity of Cryptosporidium species not only for taxonomic purposes but also for adequate diagnosis and treatment. By recent molecular and biological studies, it was found that Cryptosporidium is more closely related to gregarine parasites rather than to coccidians. Whereas, the identification of gregarine like gamont stages and the ability of Cryptosporidium to complete its life cycle in the absence of host cells further confirm its relationship with gregarines (Ryan and Hijjawi, 2015).
LIFE CYCLE
Cryptosporidium spp. have monoxenous life cycles, where all stages of development (asexual and sexual) occur within one host (Fig. 1-3). Thick-walled oocysts are excreted from the infected host in fecal material and represent the infective stage of the parasite.
| Fig. 1: | Life cycle of Cryptosporidium in farm animals (Smith et al., 2007) |
| Fig. 2: | Cryptosporidium life cycle (O'Donoghue, 1995) |
Infection of Cryptosporidium in a new host results from the ingestion of these oocysts. Once they are ingested, oocysts excyst in the gastrointestinal tract releasing the infective sporozoites. Excystation has been reported to be triggered by various factors including reducing conditions, carbon dioxide, temperature, pancreatic enzymes and bile salts (Robertson et al., 1993). The sporozoites escape through a slit-like opening created at one end of the oocyst by dissolution of a special suture in the oocyst wall (Fayer et al., 2000). The freed sporozoites attach to epithelial cells where they become enclosed within parasitophorus vacuoles and develop attachment organelles (stages generally referred as trophozoites). The trophozoites then undergo asexual proliferation by merogony (previously called schizogony). Cell division occurs by endopolygeny where multiple daughter cells are formed by internal budding within the mother meront (Fig. 2). Most studies performed on Cryptosporidium spp. have described sequential development involving two types of meronts (Fayer et al., 1997; Spano and Crisanti, 2000). Type I meronts form 8 merozoites which are liberated from the parasitophorus vacuole when mature. The merozoites then invade other epithelial cells where they undergo another cycle of type I merogony or develop into type II meronts. The type II meronts form 4 merozoites which do not undergo further merogony but produce sexual reproductive stages (called gamonts). Sexual reproduction occurs by gametogony and both microgamonts (male) and macrogamonts (female) are formed (O'Donoghue, 1995; Fayer et al., 1997; Collinet-Adler and Ward, 2010; Huang et al., 2014). Microgamonts develop into microgametocytes which produce up to 16 non-flagellated microgametes. Macrogamonts develop into uninucleate macrogametocytes which are fertilized by mature microgametes. The resultant zygotes undergo further asexual development (sporogony) leading to the production of sporulated oocysts containing 4 sporozoites (Fig. 1-3). Most oocysts (80%) are thick-walled and are excreted from the host in faecal material. Some oocysts (20%), however, there are thin-walled and have been reported to excyst within the same host animal leading to a new cycle of development (autoinfection).
| Fig. 3: | Life cycle of Cryptosporidium in humans (Fayer et al., 2000) |
The presence of these auto-infective oocysts and recycling merozoites of type I meronts are believed to be the means by which persistent chronic infections may develop in hosts without further exposure to exogenous oocysts (Current and Garcia, 1991; Ramirez et al., 2004).
The entire life cycle of the parasite may be completed in as little as 2 days in many hosts and infections may be short-lived or may be persisting for several months. The prepatent period (time between infection and oocyst excretion) ranges from 1-3 weeks, whereas the patent period (duration of oocysts shedding) can range from several days to months or years, demonstrating the potential of this infection to persist. Many factors may influence the longevity of infections but the most important appear to involve the immunocompetency of the host and the parasite species involved (O'Donoghue, 1995). Compared with Eimeria spp., the life cycle of Cryptosporidium is characterized by a number of peculiarities, some of them of major importance for the establishment and spread of the infection and for the treatment of the disease: (1) Exposure of Cryptosporidium oocysts to reducing conditions, pancreatic enzymes and bile salts results in a high percentage of excystation. However, in contrast to most other Coccidia, Cryptosporidium oocysts can liberate their sporozoites in warm aqueous solutions without any of the aforementioned special stimuli (De Graaf et al., 1999). This spontaneous excystation explains the ability of Cryptosporidium to infect tissues other than the intestine, such as the conjunctiva of the eye and the respiratory tract (Mascaro et al., 1994; Baskin, 1996), (2) The parasite develops inside the epithelial cell of the digestive or respiratory tract, although on the edge of the host cell cytoplasm and separated from it by a feeder organelle membrane. This intracellular extra-cytoplasmic location is unique for the coccidia and might play a major role in the failure of many antimicrobial agents to inhibit the growth of Cryptosporidium (Tzipori and Griffiths, 1998), (3) Two stages can cause autoinfection: the recycling merozoites of type I meronts and the thin-walled oocysts. Consequently, in the absence of a protective immune response, Cryptosporidium may persist inside a single host even without further exposure to exogenous oocysts and (4) The thick-walled oocysts are already fully sporulated when they leave the body with the feces and are therefore immediately infectious. Thus, Cryptosporidium seems to have an extraordinary reproductive ability. In addition, the oocysts can travel a considerable distance following runoff can survive for a relatively long time in an aqueous environment and are infections to a wide range of animals, thus having many potential excretors. As a result, this parasite undoubtedly has an exceptional capacity to disseminate (Mawdsley et al., 1996; Tzipori and Griffiths, 1998).
EPIDEMIOLOGY AND INCIDENCE IN EGYPT
Among cattle, calves are susceptible to infection shortly after birth and remain so for several months. Infection in dairy calves is most often detected between 8 and 15 days of age, whereas, infection in beef calves most often occurs between 1 and 2 months of age (Garber et al., 1994; Ramirez et al., 2004). Infection in lambs and goat kids is more common in animals under 1 month old (Ortega-Mora and Wright, 1994). Fathia (1993) found that the infection rate in diarrheic calves less than 1 month was 65.5% while it was 33.3% in calves aged 1-2 months. Infection can be spread animal to animal by the fecal-oral route, usually when animals are housed together in an overcrowded environment but contamination of udder and water supplies by feces is another common source of transmission in livestock. Daily excretion of oocysts by infected lambs can exceed 2×109. Infected newborn calves excrete oocyst numbers of the order of 106-107 g–1 of feces and were considered to be a more dangerous source of infection. Moreover, adult sheep and cattle can act as asymptomatic carriers shedding small numbers of oocysts to the environment which was shown to increase in number in the perinatal period and contribute to maintaining the infection between parturition periods (Hill et al., 1990; Garber et al., 1994; Xiao and Herd, 1994). Between 75-100% of lambs and calves born in this environment become infected in the first few weeks of life (De Graaf et al., 1999). Rodents can be potential reservoirs of infection for livestock enterprises. Mechanical transmission via birds, insects and man may also be involved (Martin and Aitken, 2000). Other potential risk factors are herd size and season. In a Canadian study of beef calves, higher prevalence was found in winter and spring, the period related to calving season and consequently the period with the greatest number of calves in the high risk group (1-3 weeks old). However, in American dairy farms, where calving tend to be year-round and environmental contamination level is less subjected to fluctuations, cryptosporidiosis was more prevalent in the summer (Garber et al., 1994). In Egypt, several authors reported that the highest infection rate of Cryptosporidium among calves and lambs was in summer and spring and the lowest was in autumn and winter (Abdel-Salam et al., 1993; Fathia, 1993; Lubna, 1993; Aboul-Khir, 1996). Milk can be contaminated through mechanism of poor udder hygiene and recent outbreaks of human cryptosporidiosis associated with drinking unpasteurized milk have been reported (Harper et al., 2002). Furthermore, the large number of oocysts excreted during infection helps to ensure a high level of environmental contamination. Cattle facilities are frequently blamed when Cryptosporidium is found in surface water. Thus, cattle living in close proximity to rivers should be considered potential causes of waterborne contamination, as surface run-off does transport Cryptosporidium oocysts in soils to water sources. Cryptosporidium oocysts have been recovered from untreated surface water (rivers, streams and reservoirs), untreated and treated sewage effluents, filtered swimming pool water and most importantly from treated drinking water supplies (Richardson et al., 1991). Another risk factor identified for increasing probability of calves shedding oocysts is frequent bedding changes as personnel and equipment used for removal of the bedding can actually become a vehicle for spreading the infection (Sischo et al., 2000). In Egypt, cryptosporidiosis was reported in farm animals by many authors in different localities. In Sohag, Abdel-Salam et al. (1993) reported a high prevalence level of 33.9% in diarrhoeic calves less than one month age. In Ismailia, Abou-Eisha (1994) found that 36.6, 24.0 and 21.3% of diarrhoeic buffalo-calves, lambs and goat kids, up to one month age were infected with Cryptosporidium oocysts, respectively. Moreover, Abou-Eisha (1994) detected Cryptosporidium oocysts for the first time in Egypt from a calf camel aged less than one month. Abou El-Hassan (1996) in Giza, found that 16.5% of diarrhoeic goat kids were positive for Cryptosporidia. Ghazy et al. (2004) revealed a prevalence level of 46.3% in foals suffering from emaciation, anemia, recurrent colic and chronic intermittent diarrhea.
INFECTION IN HUMANS AND ZOONOTIC IMPORTANCE
Contaminated water represents the major source of Cryptosporidium infections for humans (Fig. 3). Several waterborne outbreaks of cryptosporidiosis have been reported implicating contaminated drinking water and recreational water (Kramer et al., 1998). The most severe and largest human waterborne outbreak occurred in Milwaukee in 1993, where more than 400,000 people were infected (MacKenzie et al., 1995). In the search for sources of waterborne outbreaks of cryptosporidiosis, livestock have often been implicated as the origin of contaminating isolate (Olson et al., 2004). Cryptosporidiosis in humans typically manifests itself as a self-limiting disease with a median duration of 9-15 days, resulting in total recovering in healthy individuals. The major symptoms are watery diarrhea associated with abdominal cramps, anorexia, weight loss, nausea, vomiting, fatigue and low-grade fever (Marshall and LaMont, 1997). Symptoms are similar in children and adults, although cryptosporidiosis acquired during infancy may have permanent effects on growth and development (Molbak et al., 1997). However, it is in the immunocompromised host (due to a variety of causes including but not limited to HIV infection and AIDS, drugs, organ transplantation, cancer chemotherapy, etc.) that the infections are most chronic and debilitating (Farthing, 2000). Patients can have chronic diarrhea that can last for more than 2 months, shed oocysts in stool during the entire period, contributing to severe dehydration, weight loss and malnutrition, extended hospitalization and mortality (Ramirez et al., 2004). Thus, the severity and duration of illness depends on the host’s immune status. The groups implicated with higher risks of infection include children and staff in day care centers, farmers and animal handlers and health care workers. Travelers are at risk when they travel from developed to developing countries with high prevalence of the disease.
At least nine molecularly different types of Cryptosporidium have been found to infect humans (Table 4 and Fig. 4). Cryptosporidium hominis and C. parvum are responsible for the majority of infections in humans (Ryan and Hijjawi, 2015). Excluding C. parvum pig genotype and C. muris, infection with these species have been reported not only in HIV positive individuals but also in immunocompetent children and adults (Katsumata et al., 2000; Morgan et al., 2000a; Xiao et al., 2001, 2002; Morgan-Ryan et al., 2002; Mallon et al., 2003; Palmer et al., 2003). In the US, most human cryptosporidiosis cases are caused by C. hominis (>75%). However, in the UK bovine C. parvum is responsible for 61.5% of human cases and 37.8% of human cryptosporidiosis was caused by C. hominis (Peng et al., 1997; Sulaiman et al., 1998; McLauchlin et al., 2000). These differences may be due to the obvious separation between urban and rural populations in the US, compared to the UK where communities are closely related to agricultural sources (Hunter et al., 2003; Smerdon et al., 2003). The human genotype is also notably predominant in Australia, Kenya, Guatemala and Peru, since it is responsible for 85-92% of the human infections (Xiao et al., 2000). In Egypt, Habib (1985) and Mabrouk (1986) found that C. parvum were presented in 9-11.7% of diarrhoeic children. Out of which 60% were coming from urban area and 40% from rural area. They added that 17.2% of the positive cases had a history of contact with animals. Selim (1995) identified Cryptosporidium oocysts from 4.5% of humans in contact with infected animals (sheep, goats, dogs and cats).
| Table 4: | Cryptosporidium species capable of causing infections in humans (Rimhanen-Finne, 2006) |
| Fig. 4: | Transmission cycles of human Cryptosporidium infections. Dotted cycle demonstrates rare transmission (Rimhanen-Finne, 2006) |
Humans have close interactions with companion animals, sharing their living space and consequently, sharing microorganisms that may cause disease. Most commonly, people have cats, dogs and birds but fish, lizards, snakes, ferrets and other exotic animals are also frequent household pets. The occurrence of Cryptosporidium in dogs, cats and other pets has been described in the study of Morgan et al. (2000b) and Fayer et al. (2001). Cryptosporidium infection in dogs is generally asymptomatic. Dogs less than 6 months of age are affected more often than the adults. By comparison, in a report of Cryptosporidium in cats, 50% of infected cats showed diarrhea symptoms (Hill et al., 2000). Zoonotic cryptosporidiosis from exposure to pets has not been documented in healthy adults but transmission of (bovine) C. parvum from companion (cats, dogs) to HIV-infected persons has been reported (Meisel et al., 1976). In addition, other Cryptosporidium species such as C. felis, C. canis and C. meleagridis (Fig. 4) have also been recorded to infect healthy children and adults (Pedraza-Diaz et al., 2001a, b; Xiao et al., 2001). The concern of acquiring cryptosporidiosis from pets is more serious for children, the elderly and immunocompromised individuals; however, one report suggests that pets are not a major risk factor (Glaser et al., 1998). Veterinarians are the best-suited professionals to provide accurate advise to persons about the risk of Cryptosporidium infection from their pets and what measures should be taken to minimize the occurrence of cryptosporidiosis in animals, especially to owners of high risk (i.e. immunocompromised) (Irwin, 2002).
CONCLUSION
The genus Cryptosporidium has been classified in the phylum Apicomplexa, class Sporozoasida, subclass Coccidiasina, order Eucoccidiida, suborder Eimeriina, family Cryptosporidiidae. Cryptosporidium spp. has monoxenous life cycles, where all stages of development (asexual and sexual) occur within one host. Infection in dairy calves is most often detected between 8 and 15 days of age, whereas infection in beef calves most often occurs between 1 and 2 months of age. Infection in lambs and goat kids is more common in animals under 1 month old. Rodents can be potential reservoirs of infection for livestock enterprises. Mechanical transmission via birds, insects and man may also be involved.