Cryptosporidium spp. and Giardia duodenalis: A picture in Portugal

9 Cryptosporidium spp. and Giardia duodenalis: A picture in Portugal André Silva Almeida1,2, Sónia Cristina Soares2, Maria Lurdes Delgado1, Elisabete Magalhães Silva2, António Oliveira Castro1 and José Manuel Correia da Costa1 1Center for Parasite Immunology and Biology, CSPGF-INSA, Porto 2Center for the Study of Animal Science, CECA-ICETA, Porto Portugal 1. Introduction Cryptosporidium is an entero-pathogen which causes gastrointestinal disturbs. Primarily this organism infects the microvillous border of the intestinal epithelium, and to lesser extent extra intestinal epithelia, causing acute gastrointestinal disturbs (Fayer, 2004). The duration of infection and the ultimate outcome of intestinal cryptosporidiosis greatly depend on the immune status of the patient. In fact, immunologically healthy patients usually recover spontaneously in a week. The clinical signs can range from asymptomatic to acute, severe and persistent diarrhea and their potential for Cryptosporidium transmission can persist for weeks after symptoms cease (Deng et al., 2004; Fayer, 2004; Hunter and Thompson, 2005). Similar to Cryptosporidium, Giardia is an entero-pathogen, but a non-cell-invasive which causes giardiasis. The most prominent clinical signs of the disease are abdominal pain, nausea, followed by severe watery diarrhea, dehydration, malabsorption (particularly lipids and lipid soluble vitamins) and weight loss. Chronic courses are characterized by recurrent brief or persistent episodes of diarrhoea (Eckmann and Gillin, 2001; Muller and von Allmen, 2005). Due to some particular biological features, Cryptosporidium and Giardia represent an important question of environmental contamination with impact for human and animal health. These are single-celled organisms that belong to the kingdom Protista, and are common food and waterborne protozoa that affect humans and a wide range of domestic and wild animals (Fayer, 2004). They have a low infection dose necessary to infect humans, with possible as few as 10 organisms in some cases (USDA). They have emerged in the last two decades as intriguing microbes with an enormous impact in Animal (including wildlife species) and Human Health, potentiated by the environmental contamination of the infectious stages. Both can cause mild to severe diarrhea. No specific therapy has proven to be effective, but immunocompetent individuals generally recover within a week (USDA). However, immunocompromised individuals may be unable to clear the parasites and, therefore, suffer chronic and debilitating illness. They have been recognized as important pathogens in contaminated drinking water, meaning a problem of environmental contamination, due to two main reasons pointed out: 1) their resistance and biological www.intechopen.com Environmental Contamination 160 viability under conventional drinking water treatment conditions (chlorination and filtration); 2) the occurrence of cryptosporidiosis and giardiasis outbreaks associated with the consumption of contaminated water. This was the case in an outbreak in Milwaukee (Wisconsin, USA) in 1993, the largest waterborne disease outbreak reported all over the World. An estimated 400,000 people were reported ill (USDA). Fig. 1. Life cycle of Cryptosporidium parvum or Cryptosporidium hominis . From CDC (www.cdc.gov). The life cycle of Cryptosporidium comprises an asexual stage and sexual stage; Cryptosporidium has a spore phase named oocyst, which represents the infectious stage of Cryptosporidium and is the resistant form found in the environmental; the oocyst is extraordinary resistant to common disinfectants of water, such as chloride, and the water represents the primary transmission route (Figure 1). The infection begins with the ingestion of oocysts through contaminated water or food, or by fecal-oral contact. After ingestion, the excystation of oocyst is induced by the acidic nature of stomachic lumen and the presence of enzymes. However, this event occurs in the small intestine favored by the presence of the neutral pH, bile salts and fatty acids. Each oocyst contains four sporozoites. They are released and try immediately to infect epithelial cells of the gastrointestinal tract. At the end of this endogenous cycle, sporulated oocysts are formed which, once shed in the environment with feces, are ready to infect a new suitable host. The prepatent period, which means the time between the ingestion of infecting oocysts and the excretion of a new generation of oocysts, varies with the host and species of Cryptosporidium, but usually it www.intechopen.com Cryptosporidium spp. and Giardia duodenalis: A picture in Portugal 161 ranges from 4 to 22 days. The patent period, which means the duration of oocyst excretion, ranges from 1 to 20 days. As previously stated, transmission of Cryptosporidium may occur through contact with contaminated water and food. In fact, many reported outbreaks occurred in water parks, pools and day care centers. The life cycle of Giardia is monoxenic, and comprises two stages: the cyst and the trophozoite (Figure 2). The cysts are the resistant form and are approximately 7 to 10 µm length and oval in shape; cysts can be found in feces and are released into the environment where they can survive and remain viable for several months in cool or moist conditions. They are responsible for disease transmission, and they are able to survive under the standard concentrations of chlorine used in water treatment plants. The infection occurs after ingestion of cysts in contaminated water, food, or by the fecal-oral route in the absence of hygienic conditions. The excystation of cysts is induced by the acidic nature of stomachic lumen and the presence of enzymes. However, this event occurs in the small intestine favored by the presence of the neutral pH, and bile salts and fatty acids. Each cyst contains two trophozoites. Trophozoites have two distinct nuclei, four pairs of flagellae, are 12 to 15 µm length. They multiply by asexual reproduction, longitudinal binary fission, and colonize the lumen of the proximal small bowel, attaching to the mucosa of the bowel using a ventral sucking disk. Trophozoites are responsible for the clinical disease in the host. Cysts are excreted in the feces and became immediately infectious, making possible the transmission from person-to-person (http://www.dpd.cdc.gov/dpdx/HTML/Giardiasis.htm). The prepatent period varies with the host and species of Giardia, with a median value of 14 days. Giardia and Cryptosporidium share common characteristics that influence greatly the epidemiology of their infections. They are maintained in a variety of transmission cycles, independently, not requiring interaction between them (Figure 3). Giardia can be maintained in independent cycles involving wildlife or domestic animals, and similarly, Cryptosporidium can be maintained in cycles involving livestock, especially cattle. As observed in Figure 3, the circumstances under which such cycles may interact and where zoonotic transfer occurs are not completely understood (Hunter and Thompson, 2005). Cysts and oocysts are the stage transmitted from an infected host to a susceptible host by the fecal-oral route. Several common transmission routes exist, and include a) person-to-person through direct or indirect contact, where sexual activities may potentiate transmission, b) from animal-toanimal, c) animal-to-human, d) water-borne through drinking water and recreational water, and, e) food-borne (Caccio and Ryan, 2008; Caccio et al., 2005; Fayer et al., 2000; Hunter and Thompson, 2005). The infected hosts, whether human or animals, shed very large numbers of transmissive stages (oocysts and cysts) in their faeces, thereby increasing environmental contamination. The infective dose of both parasites, in human infections, was calculated taking into account statistical data and experimental infection studies: the ID50 varies regarding the isolates, ranging from 9 to 1042 oocysts for Cryptosporidium and 1 to 10 cysts for Giardia (Adam, 2001; Fayer et al., 2000; Okhuysen et al., 1999). These features markedly influence the epidemiology of these infections: a) the infective dose is low for both parasites; b) cysts and oocysts are immediately infectious when excreted in faeces, and possess several transmission routes; c) cysts and oocysts are very stable and can survive for weeks to months in the environment; d) water and food may became contaminated due to the environmental dispersal. The transmission of these infections, either direct or indirect, is favored by several factors such as high population densities and close contact with infected hosts or contaminated water or food. These factors are dependent on the infecting species, www.intechopen.com Environmental Contamination 162 either in zoonotic and anthroponotic transmissions. Recent studies suggested separated risks for C. hominis (such as travel abroad and contact with infected diarrheic individuals) and C. parvum (contact with cattle) (Caccio et al., 2005; Hunter et al., 2004). In sporadic cryptosporidiosis, risk factors include the age of patients (children under five years of age), travelling, contact with infected individuals and contact with farm animals (Caccio et al., 2005). Furthermore, swimming in public swimming pools or recreational areas represents a risk of infection, as suggested by Australian and US studies (Robertson et al., 2002; Roy et al., 2004). Curiously, authors have postulated that, although Cryptosporidium is transmitted through contaminated food, a small number of parasites in these samples may not induce infection with clinical symptoms but a protective immunity (Meinhardt et al., 1996). Fig. 2. Life cycle of Giardia duodenalis. From CDC (www.cdc.gov). There are no requirements for testing surface waters for the presence of these parasites, although analyses of outbreaks have shown that the pathogens can be shed into recreational waters (Castro-Hermida et al., 2008). Also, similar studies regarding Giardia transmission and sporadic giardiasis performed by authors in UK, reveled as main risks the swallowing of water while swimming, drinking treated tap water, contact with fresh water and easting lettuce (Stuart et al., 2003). The introduction of molecular tools analysis on the epidemiological field can produce useful information allowing to a better understanding about the origin of contamination, the genetic characterization of involved species/genotype/assemblage and their zoonotic potential: a new field in the modern molecular epidemiology. www.intechopen.com Cryptosporidium spp. and Giardia duodenalis: A picture in Portugal 163 The taxonomic and filogenetic relationships of Cryptosporidium and Giardia remain poorly defined; thus, the understanding of their transmission dynamics has been limited. A consensus has been adopted: with molecular techniques, the ability to observe extensive genetic variation within Cryptosporidium and Giardia species is leading to a better understanding of the taxonomy and zoonotic potential of these variants, and the epidemiology of the diseases. Namely, genotyping of samples using molecular analysis at informative loci is necessary to distinguish species and genotypes that are involved and their zoonotic potential. These molecular tools have been helpful to enhance our knowledge and understanding of the taxonomy, host range and transmission routes of Cryptosporidium and Giardia and the epidemiology of human disease. Moreover, these tools are used to understand the public health importance of different environmental routes of transmission, leading toward improved strategies for prevention and surveillance of cryptosporidiosis and giardiasis (Fayer et al., 2000; Jex et al., 2008; Monis and Thompson, 2003; Smith et al., 2006). Some methods rely on the specific in situ hybridization of probes to particular genetic loci within Cryptosporidium oocysts and Giardia cysts, whereas the majority relies on the specific amplification of one or more loci from small amounts of genomic DNA by polymerase chain reaction (PCR). This is particularly helpful in the environmental samples or others where the parasite load is low (Jex et al., 2008; Smith et al., 2006). The present chapter presents a long term study performed in Portugal aiming to assess the prevalence of two Protozoan parasites, Cryptosporidium spp. and Giardia duodenalis, in surface raw water and drinking water samples, and assess the risk that this microbial environmental contamination poses for human and animal health. The study was guided by a multidisciplinary team comprising several Veterinarians, researchers and technicians, and developed in the North region of Portugal. A long term program was established aiming at pinpointing the sources of surface water, drinking water, and environmental contamination, working with the water-supply industry. This program comprised the collection of raw and treated water samples in previously defined areas by using the Method 1623, described by the Environmental Protection Agency (US – EPA). Fig. 3. The most important cycles of transmission for maintaining Giardia and Cryptosporidium. Besides direct transmission, water and food may also play a role in transmission. The frequency of interaction between cycles is not known. From (Hunter and Thompson, 2005) www.intechopen.com Environmental Contamination 164 2. Material and methods In the north of Portugal there are 5 major hydrographical basins forming the most important water resources of the country. These hydrographical basins are named after the main rivers; Minho, Lima, Cávado, Ave, and Douro (Figure 4, 5). Cávado and Ave rivers run entirely inside national (Portuguese) borders, while Minho, Lima, and Douro are international rivers, with sources in Spain. Global features of the area, based on data provided by CCDRN, are as follows: The resident population in 2006 is 3,744,341 inhabitants; animal husbandry is an important economic activity; there are poorly developed sanitation infrastructures with wastewater plants discharging into these hydrographical basins; 64% of resident population has access to sanitation and 84% has access to treated drinking water; the region has the highest population density of the country, reaching 176 inhabitants per km2, there are a high number of recreational areas, river beaches as well as pumping areas for drinking water plants. Fig. 4. Geographic location of the North of Portugal and its 5 hydrographical basins into Iberian Peninsula Water sample collection Raw water collection Raw water samples were collected twice a year between January 2004 to December 2006 from 97 sources, including main rivers and respective affluents, from upriver to downriver. The volume of each sample ranged from 25 to 100 L. Samples were filtered through FiltaMax filters (IDEXX Laboratories, Inc., Westbrook, Maine, USA) with a pump on the inlet side of the filter according to the recommendation of the manufacturer. Intact filters were kept in refrigerated containers and transported immediately to the laboratory. The filter was taken from the container and processed with the aid of a Filta-Max Manual Wash Station (IDEXX Laboratories, Inc.) for further elution and concentration process, which consisted of 40 kms Portugal Spain www.intechopen.com Cryptosporidium spp. and Giardia duodenalis: A picture in Portugal 165 decompression of the filter, passing the sample through a membrane, and centrifugation. A sample pellet (around 2 ml) was obtained and transferred to a Leighton tube for subsequent immunomagnetic separation (IMS). Fig. 5. Location of the 5 hydrographical basins in the North of Portugal. 1. Minho; 2. Lima; 3. Cávado; 4. Ave; 5. Douro Drinking water collection As previously described for raw water, drinking water samples were collected twice a year from January 2004 to December 2006 in 43 sampling points in the drinking water treatment plants, from upriver to downriver, from all the 5 hydrographical basins (Figure 4 and 5). Always as possible, drinking water sampling points were derived from the previous related raw water collection points. This was not always possible due to legal impositions from the water supply companies. The volume of each sample ranged from 80 to 100 L. Samples were collected locally, filtered through Filta-Max filters (IDEXX Laboratories, Inc., Westbrook, ME, USA) as previously described for raw water processing. Parasite detection The IMS procedure was performed according to the USEPA method 1623 [USEPA]. Briefly, anti-Giardia and anti-Cryptosporidium magnetic beads were mixed with SL Buffer A and SL Buffer B in each Leighton tube containing the sample concentrate (Dynabeads GC-Combo, Invitrogen Dynal, A. S., Oslo, Norway) and incubated 1 hr at room temperature. Then, using 2 magnetic particle concentrators, beads were collected, washed, and transferred into a 1.5 2 1 3 4 5 www.intechopen.com Environmental Contamination 166 ml tube. Fifty-l of 0.1 N HCl were added to each sample to dissociate beads from the target organisms, the beads were rejected, and the suspension was transferred to the wells of the slides containing 5 l of 1.0 N NaOH. The samples were air-dried overnight and stained with FITC-conjugated anti-Cryptosporidium spp. and anti-Giardia spp. monoclonal antibodies, according the manufacturer’s instructions (Crypto/Giardia Cells, Cellabs, Australia). The excess of FITC-mAb was removed by adding 100 l of PBS to each well, leaving the slides for 5 min, and aspirating the excess of PBS. A 50-l aliquot of 4’-6’- diamino-2-phenylindole (DAPI) solution (0.4 mg/ml in PBS) was introduced into each well. The slides were left at room temperature for 15 min, and excess DAPI solution removed by washing the slides in PBS. Slides were examined by epifluorescence microscope. Giardia cysts and Cryptosporidium oocysts were indentified and counted based on their shape and size using a Nikon Optiphot fluorescence microscope (Nikon Corporation, Tokyo, Japan). The number of cysts and oocysts per each well was recorded and concentrations extrapolated per 10 L of sample. Positive and negative controls were performed as indicated by the manufacturer and recommended in the Method 1623. The mean recovery percentages of oocysts of Cryptosporidium spp. and cysts of Giardia spp. using the Filta-Max system and IMS procedures from water samples is, according to the manufacturer, 50 ± 13% and 41 ± 79%, respectively (McCuin and Clancy, 2003). DNA extraction, PCR and sequencing PCR analysis was performed in the samples with the highest density of infectious stages of both parasites detected by DFA. The criterion utilized was the detection of a minimum of 50 cysts/oocysts of any parasite in the total sample volume. In this context, the genetic characterization was executed in 80 samples derived from raw water and 4 samples derived from treated drinking water. The cover slip was separated from the slide and with the aid of cotton swab soaked with 100 l of distillated water, the surface of the slide was scraped in order to collect the sample. It was confirmed, under microscope observation that the slide had no remaining cysts or oocysts. The tip of the cotton swab was cut and placed in a 1.5 ml tube for subsequent DNA extraction with a QIAamp DNA Mini Kit (QIAGEN GmbH, Hilden, Germany), according to the manufacturer’s instructions. For determining the species of Cryptosporidium and Giardia present in the samples, PCR analyses were performed. A 2-step nested PCR was performed to amplify a portion of the small subunit (SSU) ribosomal RNA gene of Cryptosporidium spp. (Xiao et al., 1999). For the molecular typing of Giardia spp., a semi-nested PCR was performed to amplify a portion of the b-giardin gene (Caccio et al., 2002). For all PCR reactions, negative and positive controls were prepared, with sterile water and reference DNA, respectively. The PCR products were analyzed in agarose gel (1.4%) stained with ethidium bromide under UV light. Images were captured with a gel documentation system (GelDoc2000, BioRad, Hercules, California, USA). The PCR products of the successful reaction were purified by Wizard SV Gel and PCR Clean-up System (Promega, Madison, Wisconsin, USA) and sequenced in both strands by an external laboratory (EUROFINS MWG OPERON, Ebersberg, Germany). Chromatograms were examined with the software ChromasPro (http://www.technelysium.com.au/ ChromasPro.html) and the sequences with the software ProSeq (http://www.biology.ed.ac.uk/ research/institutes/evolution/software/filatov/ proseq.htm). Sequences were compared with the GenBank database with the tool BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and deposited in the database Zoop-Net of the Med-Vet-Net network (http://www. medvetnet.org). www.intechopen.com Cryptosporidium spp. and Giardia duodenalis: A picture in Portugal 167 3. Results IMS and DFA detection of infectious stages of Cryptosporidium spp. and G. duodenalis in raw and drinking water samples The number of validated raw water samples in this study was 283. Environmental stages of the protozoa were detected in 72.8% (206 of 283) of the water samples, including 15.2% (43 of 283) positive for cysts of G. duodenalis, 9.5% (27 of 283) for oocysts of Cryptosporidium spp., and 48.1% (136 of 283) for both parasites (Table 1). In Figure 6, the percentages of positive and negative samples from the 5 hydrographical basins are shown individually. The Ave basin showed the highest percentage of positive samples; 90.2% of the samples were positive. Minho basin showed the lowest percentage of positive samples, even though this value was more than 64%. In all the 5 hydrographic basins, the co-presence of Cryptosporidium and Giardia counted for the majority of positive samples, with the exception of Minho basin in which Giardia positive samples were slightly more than Cryptosporidium and Giardia positive samples. In the cases where both parasites were present in the same sample, the number of G. duodenalis cysts always outnumbered Cryptosporidium spp. oocysts. We also found no correlation between the concentrations of both parasites, meaning that, when the concentration of Giardia cysts is high, Cryptosporidium oocysts are not necessarily high (data not shown). Furthermore, the range of the concentrations of G. duodenalis cysts was much higher than Cryptosporidium spp. oocysts (0.17-50,000 cysts per 10 L and 0.2-726.1 oocysts per 10 L, respectively). In positive water samples, no empty (i.e., without internal characteristics, or ghosts) or DAPI negative Cryptosporidium spp. oocysts or G. duodenalis cysts were found. In all cases, it was possible to observe an increase of parasite load from upriver to downriver. The majority of water samples from the international rivers (Minho, Lima, and Douro) collected at the border with Spain was negative. Table 1. Overall prevalence of infectious stages of Cryptosporidium spp. and Giardia duodenalis in raw and drinking water samples collected. Similar to this, the number of validated drinking water samples in this study was 167. In Figure 6 the percentage and concentration of isolates from the 5 hydrographical basins are shown. Negative results, for both protozoa, were obtained in 124 out of 167 drinking water samples. Infectious stages of the protozoa were detected in 25.7% (43 out of 167) of the water samples. Among them, 8.4% (14 out of 167) of the samples were with cysts of Giardia, 10.2% (17 out of 167) with oocysts of Cryptosporidium, and 7.2% (12 out of 167) with both parasites (Table 1). In positive water samples, no empty (i.e., without internal characteristics, or ghosts) or DAPI negative oocysts of Cryptosporidium spp. or cysts of G. duodenalis were found. Furthermore, the mean concentrations of G. duodenalis cysts were much higher than those of the Cryptosporidium spp. oocysts (0.1-108.3 cysts per 10 L and 0.1-12.7 oocysts per 10 L, respectively). www.intechopen.com Environmental Contamination 174 Xiao, L., Morgan, U.M., Limor, J., Escalante, A., Arrowood, M., Shulaw, W., Thompson, R.C., Fayer, R., Lal, A.A., 1999, Genetic diversity within Cryptosporidium parvum and related Cryptosporidium species. Appl Environ Microbiol 65, 3386-3391. www.intechopen.com Environmental Contamination Edited by Dr. Jatin Srivastava ISBN 978-953-51-0120-8 Hard cover, 220 pages Publisher InTech Published online 29, February, 2012 Published in print edition February, 2012 InTech Europe University Campus STeP Ri Slavka Krautzeka 83/A 51000 Rijeka, Croatia Phone: +385 (51) 770 447 Fax: +385 (51) 686 166 www.intechopen.com InTech China Unit 405, Office Block, Hotel Equatorial Shanghai No.65, Yan An Road (West), Shanghai, 200040, China Phone: +86-21-62489820 Fax: +86-21-62489821 Nature minimizes the hazards, while man maximizes them. This is not an assumption, but a basic idea of the findings of scientists from all over the world. The last two centuries have witnessed the indiscriminate development and overexploitation of natural resources by man causing alterations and impairment of our own environment. Environmental contamination is the result of the irrational use of resources at the wrong place and at the wrong time. Environmental contamination has changed the lifestyle of people virtually all over the world, and has reduced the extent of life on earth. Today, we are bound to compromises with such environmental conditions, which was not anticipated for the sustenance of humanity and other life forms. Let us find out the problem and its management within this book. How to reference In order to correctly reference this scholarly work, feel free to copy and paste the following: André Silva Almeida, Sónia Cristina Soares, Maria Lurdes Delgado, Elisabete Magalhães Silva, António Oliveira Castro and José Manuel Correia da Costa (2012). Cryptosporidium spp. and Giardia duodenalis: A picture in Portugal, Environmental Contamination, Dr. Jatin Srivastava (Ed.), ISBN: 978-953-51-0120-8, InTech, Available from: http://www.intechopen.com/books/environmental-contamination/cryptosporidium-sppand-giardia-duodenalis-a-picture-in-portugal