An Irish perspective on Cryptosporidium. Part 1

Cryptosporidiosis, a protozoal disease which causes significant morbidity in humans, is one of the chief causes of diarrhoea in neonatal ruminants. Although the parasite poses a significant threat to public health and animal health in Ireland, its epidemiology on the island is only poorly understood. Environmental studies have shown the waterborne parasite to be widespread in some untreated waterbodies around Ireland. The island's hydrogeological situation, combined with high stocking rates of livestock and the absence of filtration from regular water treatment, render it vulnerable to large-scale outbreaks. This review discusses the parasite in the Irish context and underlines the need for a reference facility to provide active surveillance on the island.


Introduction
First described in 1907 (Tyzzer, 1907), Cryptosporidium remained obscure for many decades. During the 1960s and 1970s it was increasingly recognised as an important pathogen of neonatal ruminants (Panciera et al., 1971) and humans (Meisel et al., 1976;Nime et al., 1976), but it was not until the emergence of AIDS in the early 1980s that Cryptosporidium was identified as a life-threatening parasite in immunocompromised patients. Today, it is known to be one of the most serious causes of waterborne diarrhoea (Tzipori and Ward, 2002); it is a most difficult organism to control and it is one of the most heavily researched protozoan parasites. In contrast, we are only beginning to understand the importance of Cryptosporidium on the island of Ireland. This article summarises what we know about the epidemiology of the parasite on the island and how the unique combination of land use, infrastructure and geophysics on the island may affect it.

life-cycle and transmission
peer reviewed An Irish perspective on Cryptosporidium Annetta Zintl 1 , Grace Mulcahy 1 , Theo de waal 1 , Valerie de waele 2 , catherine Byrne 3 , Marguerite clyne 3 , Nicholas Holden 1 and Seamus Fanning 1 Cryptosporidium is an obligate enteric parasite of the phylum Apicomplexa (Figure 1). The disease is transmitted via long-lived, thick-walled oocysts of 3μm to 8μm diameter, excreted in the faeces of infected humans and animals. The infectious dose is dependent on the species and immune status of the host and on the Cryptosporidium species/strain, but generally considered to be very low. Infection experiments in 29 healthy volunteers indicated an infectious dose of 132 oocysts (median value) (DuPont et al., 1995), while mathematical modelling suggested it may be as low as one oocyst (Haas and Rose, 1994). Upon ingestion, invasive stages or sporocysts emerge from the oocysts and invade cells in the microvillous border of the intestinal epithelium and, to a lesser extent, extra-intestinal epithelia ( Figure  2). Situated intracellularly, just beneath the host cell membrane, but extracytoplasmic and enclosed within parasitophorous vacuoles, the parasites multiply through repeated cycles of asexual reproduction and re-invasion, thereby destroying the functional integrity of the brush border. Ultimately, sexual reproduction occurs resulting in the formation of thick-walled oocysts that are shed with the faeces. The oocysts are already sporulated and capable of immediate infection of a new host. Thin-walled oocysts may occur also; these are believed to be capable of excysting within the same host and causing autoinfection in immunodeficient hosts.

C o n t a m i n a t i o n o f t h e e n v i r o n m e n t w i t h Cryptosporidium
Cryptosporidium is a waterborne pathogen and its epidemiology is determined by environmental factors. According to the Geological Survey of Ireland, in many parts of the island there is only a shallow layer of soil and subsoil over karst limestone (Daly, 2003). In other areas, the General Soil Map of Ireland (Gardiner and Radford, 1980)  indicates heavy soils that can be prone to either rapid surface runoff after rainfall or channeling of water along large pores, known as preferential flow. In all these situations, instead of being filtered by slow passage through a substantial volume of soil, rainwater that may hold a significant contaminant load rapidly reaches surface water reservoirs or groundwater. A study carried out on Lough Owel, County Westmeath, confirmed this threat. The water level in the lake was shown to rise rapidly after rainfall events, indicating that areas within the catchment have extremely poor permeability, leading to excessive overland flow and facilitating direct ingress of contaminants into the lake (Report, Trinity College Dublin, 1999). Incidentally, three years later the lake water, which is used as a source for drinking water, was linked to one of the largest recorded outbreaks of cryptosporidiosis in the Republic of Ireland. Cryptosporidium oocysts are extremely resilient and remain viable for long periods in the environment (Kato et al., 2004). Long survival periods in the environment, combined with quick passage to surface and ground waters, mean that many of the oocysts are still viable when they reach reservoirs of drinking water. Several studies investigating waterbodies and associated shellfish, on the island of Ireland found that contamination with oocysts was chiefly restricted to more densely populated areas (Belfast, Dublin, Sligo) and the River Shannon, where low levels of oocyst contamination were widespread (Chalmers et al., 1997;Finn et al., 2003;Graczyk et al., 2004;Lowery et al., 2001a, b;Skerrett and Holland, 2000). This widespread occurrence of oocysts in Irish waterbodies needs to be considered in view of the fact that the chief disinfection treatment used for drinking water in Ireland is chlorination, which does not kill Cryptosporidium. Filtration, the only reliable method to remove the parasite from drinking water, is not included in regular water treatment (Report of Waterborne Cryptosporidiosis Subcommittee of the Scientific Advisory Committee, 2004). This fact, combined with the high stocking rates that are carried on dairy farms in Ireland and the agricultural practices, soils and hydrology found on the island, could mean that Ireland is highly vulnerable to large-scale outbreaks should, for whatever reason, environmental contamination with oocysts increase.

Pathology and prevalence in the human population
The most common clinical signs in humans are diarrhoea, abdominal cramps, fever, nausea and vomiting, which may result in weight loss and dehydration. Sometimes, headache, weakness, fatigue, myalgia and inappetence are also reported. In immunocompetent patients, the primary site of infection is the distal small intestine (Chen et al., 2002). These patients usually recover within one to two weeks; however, shedding of oocysts may continue for several weeks after symptoms cease and clinical relapses may occur up to 14 days after resolution of the initial symptoms (MacKenzie et al., 1994). In contrast, immunocompromised persons suffer chronic enteritis, lasting as long as the immune impairment. In these patients, the disease may go through cycles of resolution and recurrence, or it may be persistent and become life-threatening (Fayer et al., 1997). In immunocompromised hosts, the parasite can also infect other organs, resulting in hepatitis, pancreatitis, cholecystitis, cholangitis and conjunctivitis (Fayer et al .,1997). Respiratory infections are associated with coughing, wheezing, croup, hoarseness and shortness of breath Over the last number of years, there have been several outbreaks of cryptosporidiosis on the island of Ireland. Between April 2000 to April 2001, there were three outbreaks associated with drinking-water in the greater Belfast area and neighboring communities, involving at least 476 cases in total (Smyth, 2001;Communicable Disease Surveillance Centre, 2000;Glaberman et al., 2002). Officials blamed Northern Ireland's antiquated system of water pipes (Birchard, 2000), which allowed the ingress of human sewage from a septic tank and of wastewater from a blocked drain (Glaberman et al., 2002). In the 0 50 1 9 9 2 1 9 9 3 1 9 9 4 1 9 9 5 1 9 9 6 1 9 9 7 1 9 9 8 1 9 9 9 2 0 0 0  1 9 9 0 1 9 9 1 1 9 9 2 1 9 9 3 1 9 9 4 1 9 9 5 1 9 9 6 1 9 9 7 1 9 9 8 1 9 9 9 2 0 0 0 2 0 0 1  (Carson, 1989;Corbett-Feeney, 1987) (Figure 3). According to estimates made by the Health Protection Surveillance Centre for the period before 2004, Cryptosporidium accounted for approximately 8% of laboratory-confirmed cases of gastroenteritis in children under two years old. The number of cases reported in the Republic of Ireland since January 2004 can be viewed on the HPSC website (www.ndsc.ie/NotifiableDiseases). In both jurisdictions, the majority of cases involve children under five years of age. In common with the UK, there is a significant urban-rural divide with a higher incidence amongst the rural population, indicating farmers and animal health workers as occupational risk groups. In addition, private water schemes, more common in remote areas, are known to be at higher risk from contamination than publicly managed water supplies in urban areas (Environmental Protection Agency, 2003). There is also a marked seasonal pattern with a peak in spring/ early summer and often a further smaller rise in the autumn (Carson, 1989;Corbett-Feeney, 1987;Moore et al., 2002;Communicable Disease Surveillance Centre, 2000;Garvey and McKeoan, 2004;Lowery et al., 2001a). Cryptosporidiosis in animals is not a notifiable disease in either jurisdiction. In all, there are currently 15 recognised species of Cryptosporidium Ryan et al., 2004;Fayer et al., 2005). The main species infecting humans are C. hominis and C. parvum. Reports of natural infections with C. hominis have been restricted to humans so far, with the exception of two cases identified last year in Scottish cattle (Smith et al., 2005). Experimental infections with C. hominis have been carried out in neonatal pigs and lambs (Morgan-Ryan et al., 2002). In contrast, C. parvum infects most, if not all, mammals including humans and is a major pathogen of calves. Recent studies suggest that a number of other zoonotic species, particularly the avian pathogen, C. meleagridis, also cause diarrhoea in humans. Morphologically indistinguishable, the various species are most easily discriminated genetically, mostly by polymerase chain reaction (PCR) combined with restriction fragment length polymorphism (RFLP) analysis (Leng et al., 1996) (Figure 4). Most work to date on the biology and pathogenicity of Cryptosporidium in relation to human infection has been done using C. parvum. Therefore, compared to C. parvum, little is known of the biology of invasion of the human-restricted C. hominis. Recent studies carried out in the Children's Research Centre, Our Lady's Hospital for Sick Children, Crumlin, Dublin, indicate that there is diversity in the mechanisms used by C. parvum and C. hominis to infect cells of different origin (Hashim et al., 2004;Hashim et al., 2006). More specifically, it was shown that host cell invasion by the two species was mediated by different receptor-ligand interactions. For instance, while a galactose-N-acetylgalactosamine (Gal/GalNAc)-specific sporozoite epitope is crucial for host cell invasion by C. parvum, (Chen et al., 2000), Hashim et al.(2004) found that in vitro, C. hominis interacted with potential host cells via a Gal/GalNAc-independent mechanism. These data have important implications for understanding the pathogenesis of cryptosporidiosis and for improved chemotherapy.
The distribution of C. hominis, C. parvum and C. maleagridis in the human population is dependent on the geographical region. For example, in North and South America C. hominis appears to be more prevalent (Widmer, et al., 1998;Peng, et al., 1997;Xiao, et al., 2001;Harp, 2003), while in Europe zoonotic infections with C. parvum are more common (McLauchlin et al., 1999). In Latin America, the prevalence of C. meleagridis may exceed that of C. parvum (Xiao et al., 2001;Cama et al., 2003). On the other hand, in the UK this  species accounted for only 0.3% of clinical cases (McLauchlin et al., 2000). Generally, it is thought that the prevalence of zoonotic species reflects the importance and type of farming and agriculture in a region. Farming practices, such as pasture grazing, the storage and spreading of slurry/manure, or events such as lambing and calving, all contribute to environmental contamination with oocysts which may enter surface waters either directly or through surface runoff. In fact, it has been suggested that C. parvum is more common in areas where a greater proportion of surface water is used in the public water supply (Lowery et al., 2001a). The spring peak of cryptosporidiosis cases is usually attributed to C. parvum, as it coincides with the calving and lambing seasons. In contrast, C. hominis, a strictly human parasite, is significantly more common in patients infected during the late-summer/autumn peak and in those with a history of foreign travel (McLauchlin et al., 2000). Infections with this parasite are due to contamination with human faeces originating from leaking waste water pipes or septic tanks. Other common routes of transmission are recreational facilities, particularly swimming pools (Report of Waterborne Cryptosporidiosis Subcommittee of the Scientific Advisory Committee, 2004).
Considering the important role of the agricultural sector on this island and the pronounced spring peak, one would assume that C. parvum is the most prevalent species in the human population here. Unfortunately, species has been determined in only a very small number of cases. A study of 39 positive samples collected in the greater Belfast area during 1998 reported that C. parvum accounted for the greatest number of sporadic cases: 87.2% (Lowery et al., 2001a). However, two of the three outbreaks in the same geographical area between April 2000 and 2001 were ascribed to C. hominis and only one to C. parvum (Glaberman et al., 2002). To date, there has been no report of C. meleagridis infection in Ireland. Clearly, in order to improve our understanding of the epidemiology of the parasite, there is an urgent need for a reference facility to provide active surveillance, including routine typing.