CHAPTER 1
CRYPTOSPORIDIUM: THE ANALYTICAL CHALLENGE
H. V. Smith and A. Ronald
Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, Glasgow G21 3UW, UK. corresponding author. Tel: +44 (0)141 201 3028, Fax: +44 (0)141 558 5508
1 INTRODUCTION
In the last twenty years, the protozoan parasite, Cryptosporidium has been widely recognised as a cause of waterborne disease. Its transmissive stage, the oocyst, is a frequent inhabitant of raw water sources used for the abstraction of potable water and its importance is heightened because, coupled to its low infectious dose, conventional water treatment processes, including chemical disinfection, cannot guarantee to remove or destroy oocysts completely. Furthermore, because of their chlorine insensitivity, the coliform standard cannot be relied upon as an indicator of either the presence or viability of waterborne Cryptosporidium oocysts. For these reasons, robust, sensitive and specific methods are required for the recovery and identification of oocysts in water concentrates.
2 THE PARASITE
Cryptosporidium has a complex life cycle, involving both asexual and sexual reproductive cycles, which it completes within an individual host (monoxenous). Transmission from host to host is via an environmentally robust oocyst which is excreted in the faeces of the infected host. C. parvum is the major species responsible for clinical disease in man and domestic mammals (Current and Garcia, 1991), although infections with species other than C. parvum have been described in both immunocompetent and immunocompromised human hosts (Morgan et al., 2000; Pedraza-Diaz et al., 2000, 2001).
By the nature of its characters, the genus Cryptosporidium (Kingdom, Protozoa; Phylum, Apicomplexa (Sporozoa); Class, Coccidea; Order, Eimeriida) belongs to the Family Cryptosporidiidae. These include: "development just under the surface membrane of the host cell or within its striated border and not in the cell proper. Oocysts and meronts with a knob-like attachment organelle at some point on their surface. Oocysts without sporocysts, with four naked sporozoites. Monoxenous. Microgametes without flagella" (Levine, 1973). Currently, the genus Cryptosporidium contains 10 valid named species. C. parvum, C. muris, C. felis, C. andersoni and C. wrairi infect mammals, C. baileyi and C. meleagridis infect birds, C. serpentis and C. saurophilum infect reptiles and C. nasorum infect fish.
C. parvum infects the epithelia of the intestinal tract (enterocytes) of various mammals. Exposure to the environments of the gastrointestinal tract trigger the poorly understood process of excystation, whereby sporozoites are actively released through the suture of the oocyst. Known triggers include temperature (37°C), acidity (~ pH 2), slight alkalinity (~ pH 7.6) exposure to bile salts and trypsin (Fayer and Leek, 1984; Reducker and Speer, 1985; Hill et al., 1991; Robertson et al., 1993a). During the course of symptomatic infection, up to 1010 oocysts are shed into the environment, which are capable of prolonged survival in moist microclimates. Figure 1 describes the life cycle of C. parvum.
Waterborne transmission is well documented (Smith and Rose, 1990, 1998; Smith and Nichols, 2001), and can affect numerous individuals. Smith and Rose (1998) stated that more than an estimated 427,100 individuals had been affected in 19 documented waterborne outbreaks. The numerous contributors to waterborne oocysts, and the small size, environmental robustness and chlorine insensitivity of oocysts are factors that enhance their presence and likelihood of surviving in water treatment processes. The second report of the UK Joint Group of Experts on Cryptosporidium in water supplies stated that "... absence of Cryptosporidium from drinking water can never be guaranteed ..., criteria for 'best practice' for operating water treatment works could be identified and should be adopted" (Anon., 1995a). Well operated treatment works can achieve better than 2 log.10 of oocyst removal and for disinfectants which could be used within current UK guidelines or legislation, ozone and UV have potential (Hall and Pressdee, 1995; Anon., 1995; Croll and Hall, 1997).
3 GETTING TOUGH ON CRYPTOSPORIDIUM OOCYSTS – A MULTINATIONAL APPROACH
Many requirements, rules and regulations are in place to attempt to address the control of Cryptosporidium which threatens the safety of drinking water. The current European Union 'drinking water' directive (Anon., 1980) requires that 'water intended for human consumption should not contain pathogenic organisms' and 'nor should such water contain: parasites, algas, other organisms such as animalcules'. The proposed revision to this directive, to be implemented in 2003 (Anon., 1995b), in recognising the impracticality of the current zero standard, will make it a general requirement 'that water intended for human consumption does not contain pathogenic micro-organisms and parasites in numbers which constitute a potential danger to health'. No numerical standard for Cryptosporidium is proposed.
The Unites States Environmental Protection Agency (USEPA) has issued several rules. One goal of the Surface Water Treatment Rule (USEPA, 1989) was to minimize waterborne disease transmission to levels below an annual risk of 10-4 by reducing Giardia cysts and viruses by 99.9% and 99.99%, respectively through filtration and disinfection requirements. The Rule lowered the acceptable limit for turbidity in finished drinking water to a level not to exceed 0.5 nephelometric turbidity unit (NTU, established by a standard haze created chemically in water and measured by light scatter) in 95% of four-hour measurements. The Enhanced Surface Water Treatment Rule (USEPA, 1994) includes regulation of Cryptosporidium, and in order to implement it, the Information Collection Rule (a national database on the occurrence of oocysts in surface and treated waters) had to be enacted.
In England and Wales, the Water Supply (Water Quality) Regulations 2000, SI No. 3184 and Water Supply (Water Quality) (Amendment) Regulations 1999, SI No. 1524, require water undertakers to determine whether there is a significant risk from Cryptosporidium oocysts in water supplied from waterworks and to comply with the requirement for treating the water intended to be supplied so that the average number of Cryptosporidium oocysts per 10 litres of water is less than one. Within the wording of the regulation, contravention of the numerical standard or the monitoring (sampling and analysis) requirements is an offence. To implement the regulations, which came into force on 30th June 1999, an information letter from the UK Drinking Water Inspectorate (DWI) (DWI Information letter 10/99) identified the Protocol containing Standard Operating Protocols (SOPS) for monitoring Cryptosporidium oocysts in water supplies. Similar directives are in place in Scotland and Northern Ireland. From data reported up to March 2001 under this regulation, 3,840 (8.8 %) of 43,740 samples taken from 178 water treatment works contained oocysts. Oocysts were detected in 43% of water treatment works which came under the regulation. Of the 3,840 positive samples, 500 (1.2 %) contained > 1 oocyst 1001-1 and 10 (0.023%) contained > 10 oocysts 100-1 (Drury, 2001).
In addition, the Cryptosporidium problems in Australia (McClellan, 1998) also highlight the need for robust methods, fit for propose, including detection, identification, enumeration and assessment of viability / infectivity of waterborne Cryptosporidium oocysts.
4 WHAT IS AN OOCYST?
The laboratory diagnosis of Cryptosporidium in faeces and its detection in environmental samples (e.g. water and food) is dependent upon demonstrating oocysts in the sample by microscopy. Here, oocysts must be distinguished from other, similarly shaped, contaminating bodies present in the sample, and the microscopic identification of oocysts is dependent upon morphometry (the accurate measurement of size) and morphology. The morphometric and morphological features of C. parvum oocysts viewed in suspension by Nomarski differential interference contrast (DIC) microscopy are as follows: spherical or slightly ovoid, smooth, thick walled, colourless and refractile, measuring 4.5 - 5.5 µm, containing four elongated, naked (i.e. not within a sporocyst(s)) sporozoites and a cytoplasmic residual body within the oocyst. Each sporozoitecontains one nucleus. Not all four sporozoites may be visible at once. The size ranges of oocysts of various Cryptosporidium species are presented in Table 1.
Detection in both clinical and environmental laboratories is performed on samples that have been dried down onto microscope slides or membranes. The drying process causes oocysts to collapse, which leads to shape distortion and the mechanical release of sporozoites (Figure 2). Commercially available, fluorescein isothiocyanate (FITC)-labelled monoclonal antibodies (mAbs; FITC-C-mAb), assist in the detection and measurement of oocysts in environmental samples by binding to surface exposed oocyst epitopes, hence defining shape (Rose et al., 1989; Smith et al., 1989a, Smith, 1995, 1996, 1998). The use of the fluorogenic dye 4'6 diamidino-2-phenyl indole (DAPI), which highlights sporozoite nuclei by fluorescing sky blue when intercalated with sporozoite DNA, provides supportive evidence (Grimason et al., 1994; Smith, 1995, 1996, 1998).
Occurrence studies of waterborne Cryptosporidium oocysts in numerous countries (Smith and Rose, 1998) have been performed using a limited (c. 3-4) number of commercially available (diagnostic) FITC-C-mAbs, which is indicative of the fact that the epitopes recognised by these FITC-C-mAbs are not only commonly expressed in Cryptosporidium species oocysts, but are also environmentally robust. For many in the water industry, interest in Cryptosporidium oocysts is rarely required to extend beyond their identification in water concentrates, where reliance is placed upon the reactivity of FITC-C-mAb, DAPI, knowledge of oocyst morphology and experience, yet a clearer understanding of oocyst form and function may lead to better detection and destruction methods which, in turn could reduce the risk associated with waterborne oocysts.
C. parvum oocysts afford a stable and resistant covering for infective sporozoites, providing protection from external influences (adverse temperatures, desiccation, salinity, disinfection processes and other environmental insults) yet remaining sensitive to triggers known to initiate excystation and the active release of sporozoites. Knowledge of the ultrastructure, physiology, biochemistry, biophysics and antigenicity of the oocyst can assist our understanding of this environmentally robust, protective structure. In addition, a clearer understanding of the nature and structure of the oocyst can also provide insight into improved methods for identification and sub-typing by targeting novel markers, and for disinfection, by identifying structures and topographies sensitive to disinfectants. For these reasons we require a fuller understanding of the biology of waterborne oocysts. Here, we present information on Cryptosporidium oocysts obtained both from published data and experiments performed at the SPDL over the last ten years, with particular reference to the oocyst wall.
5 OOCYST SIZE AND MORPHOLOGY
Morphometry and morphology frequently provide the basis for the decision as to whether an object is an oocyst. From Table 1 we can see that there is overlap between oocyst size ranges for many of the species presented, which presents a significant limitation to our ability to determine the presence of C. parvum oocysts at the light microscope level.
To determine the degree of overlap between similarly sized oocysts of Cryptosporidium species likely to contaminate UK waters, we obtained stocks of C. paivum (cervine / ovine isolate, MD, Moredun Animal Health, Edinburgh, UK), C. baileyi (Belgium strain, LB 19; Dr. K. Webster, Veterinary Laboratories Agency, Weybridge, UK) and C. muris (RN 66, Dr. V. McDonald, London School of Hygiene and Tropical Medicine, London, UK) oocysts and stored them in reverse osmosis (RO) water containing antibiotics at 4°C. We measured oocysts suspended in RO water, using an Olympus BH2 microscope equipped with DIC optics at a total magnification of x1250 (12.5 eyepieces x 100 oil objective). Forty oocysts each of C. parvum, C. muris and C. baileyi were measured (Table 2).
Given a maximum size range of 5.35 x 5.9 for C. parvum oocysts in this study, analysis of the minimum size of C. muris oocysts (6.25 x 7.5 pm) indicate that they are larger than the maximum size range for C. parvum oocysts. Therefore, based on morphometry, intact C. muris oocysts can be distinguished readily from C. parvum oocysts in water concentrates. The minimum size of C. baileyi oocysts detected was 5.0 x 5.0 µm which falls within the size range for C. parvum oocysts and analysis of our data indicate that 40% (16/40) of the C. baileyi oocysts measured fall within the maximum size of C. parvum oocysts.
If we adopt the morphometric guidelines for C. parvum oocysts identified in the provisional recommended UK methods and the UK Regulatory method (Anon., 1990, 1999a,b; 4-6 µm in size) then 45% (18/40) of our C. baileyi oocysts fall within this size range. Using the dimensions of Tyzzer (1912) and Upton and Current (1985) (4.5 x 5.0 µm), then 10% (4/40) of our C. baileyi oocysts fall within this size range. Immaterial of which size range we adopt, there is overlap between the measured sizes of C. parvum and C. baileyi oocysts. These data indicate that morphometric measurements will not always be sufficient to discriminate between oocysts of C. parvum and C. baileyi, therefore better discriminatory methods are required. The occurrence of other Cryptosporidium spp. oocysts which are similar in size to C. parvum oocysts (Table 1) in the aquatic environment, including human-derived C. meleagridis oocysts, further compromises size-based detection methods.
6 C. PARVUM GENOTYPES
Recent genetic analyses have raised doubt about the validity of current species, and previously accepted criteria including oocyst morphology, host specificity and parasite location may not be sufficiently discriminatory (Tzipori and Griffiths, 1998). Analysis of several polymorphic sites in at least 6 different genetic loci of the C. parvum genome (e.g. Cryptosporidium oocyst wall protein (COWP), dihydrofolate reductase, Cryptosporidium thrombospondin related adhesive protein-1 & -2, ribonuclease reductase and the internal transcribed spacer 1 of the 18s rRNA gene) indicate that C. parvum is composed of two distinct genotypes: genotype 1, which infects humans, primarily, and genotype 2,which infects both humans and other mammals, particularly ruminants and rodents.
7 OOCYST WALL STRUCTURE
The environmental robustness of Cryptosporidium oocysts is well documented. At the host-oocyst-environment interface is the oocyst wall, which is often regarded as a near-inert protective covering.
