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 Table of Contents  
Year : 2014  |  Volume : 7  |  Issue : 2  |  Page : 129-137

Use of nested PCR-RFLP for genotyping of Cryptosporidium parasites isolated from calves and children suffering from diarrhea

Department of Parasitology, Faculty of Medicine, Menoufia University, Menoufia, Egypt

Date of Submission21-Jun-2014
Date of Acceptance09-Sep-2014
Date of Web Publication19-Jan-2015

Correspondence Address:
Gehan S Sadek
10th Ahmed Zaky Street, El Noza El Gedeeda, 11769, Cairo
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/1687-7942.149568

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The vast majority of human cases of cryptosporidiosis worldwide are caused by two species: Cryptosporidium hominis (C. parvum type 1), which causes infection in humans only, and Cryptosporidium parvum (C. parvum type 2), which causes infections in humans and animals. In Egypt, calves carrying the zoonotic C. parvum represent the largest farm animal source of infection for humans. Information on the source of Cryptosporidium spp. contamination is necessary for effective evaluation and selection of management practices for reducing the risk for cryptosporidiosis.
The aim of the study was to genotypically characterize Cryptosporidium spp. in a sample of isolates from calves and children suffering from diarrhea.
Materials and methods
One hundred stool samples were collected from diarrheic calves housed at the Tropical Diseases Clinic, Faculty of Veterinary Medicine, Cairo University. A total of 110 stool samples were also collected from diarrheic children attending the Gastroenteritis Unit, Abo El Reesh Pediatric Hospital, Cairo University. Each stool sample of children or calves was examined microscopically after staining with modified Ziehl-Neelsen stain for the diagnosis of Cryptosporidium spp. Positive samples were then subjected to nested PCR-restriction-fragment length polymorphism (PCR-RFLP) targeting Cryptosporidium oocyst wall protein (COWP) gene for determination of Cryptosporidium genotypes.
Screening by modified Ziehl-Neelsen staining detected Cryptosporidium in stools of 40% of diarrheic calves. Nested PCR-RFLP analysis showed that all positive samples were related to C. parvum genotype 2 (C. parvum). In diarrheic children, screening diagnosed 12/110 (10.9%) positive cases; 9/12 (75%) of them were confirmed positive by nested PCR. RFLP analysis showed that 8/9 (88.9%) samples were C. parvum genotype 1 (C. hominis), whereas one sample was not digested.
Genotype 2 C. parvum is relatively highly prevalent in the sample of calves examined compared with genotype 1 in the sample of children, indicating that transmission of cryptosporidiosis among this sample of children is anthroponotic and not zoonotic. It is advised to include PCR-RFLP technique in routine clinical diagnosis and epidemiological investigations.

Keywords: calves, children, COWP gene, Cryptosporidium, PCR-RFLP

How to cite this article:
Sadek GS. Use of nested PCR-RFLP for genotyping of Cryptosporidium parasites isolated from calves and children suffering from diarrhea. Parasitol United J 2014;7:129-37

How to cite this URL:
Sadek GS. Use of nested PCR-RFLP for genotyping of Cryptosporidium parasites isolated from calves and children suffering from diarrhea. Parasitol United J [serial online] 2014 [cited 2023 Dec 5];7:129-37. Available from: http://www.new.puj.eg.net/text.asp?2014/7/2/129/149568

  Introduction Top

Cryptosporidium is an apicomplexan parasite that infects the microvillus border of the gastrointestinal epithelium in a wide range of vertebrate hosts, including humans and cattle [1] . Infection with Cryptosporidium spp. leads to severe but usually self-limiting diarrhea in immunocompetent individuals but can be life threatening in immunocompromised hosts [2] . The entire life cycle of this parasite takes place in the intestinal epithelium and transmission occurs in the form of environmentally resistant oocysts by the fecal oral route through contaminated food and/or water [3],[4] . Oocysts are even resistant to chlorine disinfection and can survive for days in treated water [5] . This has resulted in major waterborne outbreaks [6] , which together with lack of effective treatment makes cryptosporidiosis a major public health issue and economic problem [7] . The vast majority of human cases of cryptosporidiosis worldwide are caused by two species: Cryptosporidium hominis and Cryptosporidium parvum [8] . C. hominis is known as C. parvum type 1 and C. parvum is known as C. parvum type 2 [9] . However, other species including Cryptosporidium felis, Cryptosporidium meleagridis, Cryptosporidium canis, Cryptosporidium suis, and Cryptosporidium muris can also infect humans [10] . Of the two major genotypes that infect humans, C. hominis causes infections in humans only (anthroponotic infection), whereas C. parvum causes infections in humans and animals (zoonotic infection). Clinically, C. hominis reportedly causes long-term diarrhea and more oocyst excretions, whereas C. parvum causes short-term diarrhea and less oocyst excretion [11] .

Information on the source of Cryptosporidium contamination is necessary for effective evaluation and selection of management practices for reducing the risk for cryptosporidiosis [12] . C. parvum has long been considered zoonotic and the ability of this species to be transmitted from animals to humans is well documented [13] . Contact with infected calves has been implicated as the cause of many cryptosporidiosis outbreaks in veterinary students, research technicians, and children residing in agricultural areas [14] . In Egypt, calves carrying the zoonotic C. parvum may be considered the biggest farm animal source of infection to humans. High prevalence rates of cryptosporidiosis among calves recorded by several studies indicate the potential risk of parasite transmission to humans through contact with infected cattle feces or through milk and food contaminated with oocysts [6] .

Laboratory diagnosis is generally based on microscopic detection of oocysts in stained smears, which offers no information on the infecting species [15] and may prove inadequate in diagnosing a small number of parasites [16] . Besides, it is not suitable for epidemiological investigations [7] . Immunofluorescence assay has been used widely for the detection of Cryptosporidium spp. The use of monoclonal antibodies in immunofluorescence and confocal microscopy has revealed fluorescent staining specifically confined to the oocyst walls of C. muris and C. parvum [17] . Kamal et al. [18] concluded that immunofluorescence is rapid and more sensitive and specific than the conventional acid fast and merthiolate-iodine-formaldehyde concentration for the detection of Cryptosporidium oocysts and Giardia cysts. However, oocyst morphology, host specificity, or preferences in infection sites do not provide sufficient information for the identification of Cryptosporidium spp., genotypes, or subgenotypes [19],[20] . Consequently, molecular analysis used to characterize the genetic structure of Cryptosporidium parasites and to assess their zoonotic significance [21] proved to be a sensitive diagnostic method capable of determining Cryptosporidium spp. with high selectivity in environmental and clinical samples and allowing for genotyping [11] . In recent years, researchers have developed PCR-based techniques for the detection and identification of Cryptosporidium spp. These techniques target the genes of thrombospondin-related adhesive protein 1 (TRAP-C1) [22] , thrombospondin-related adhesive protein 2 (TRAP-C2), polythreonine repeat (Poly-T) [23] , small subunit rRNA [24] , internally transcribed spacer 1 (ITS1) [25] , Cryptosporidium oocyst wall protein (COWP) [26] , dihydrofolate reductase (DHFR) [27] , and undefined genomic sequences among strains that infect humans and most animals [28],[29] . The most commonly used genotyping tool is a small subunit rRNA gene-based RFLP, which has been used effectively for genotyping Cryptosporidium oocysts [30] . Molecular analysis of C. parvum revealed several families among isolates from humans and/or cattle as well as several subtypes within each family [31] .

Although cryptosporidiosis is frequent in humans and livestock in Egypt [32] , little information is available about the genetic diversity of this parasite. Exposition of Cryptosporidium genotypes by molecular assays is required to recognize sources of infections and routes of transmission, facilitating the improvement of risk assessment and measures for prevention and control [6] . Hence, the present work aimed to determine Cryptosporidium genotypes in stool samples of calves and children suffering from diarrhea using nested PCR-RFLP targeting the COWP gene.

  Materials and methods Top

Type of the study

Descriptive analytical study.

The study was conducted from August 2013 to December 2013.

Study design and collection of samples

One hundred samples were collected from diarrheic calves at the Tropical Diseases Clinic, Faculty of Veterinary Medicine, Cairo University. Fifty-two samples were from calves aged less than 1 month (group A) and 48 samples were from calves aged 1-2 months (group B). A total of 110 samples were also collected from diarrheic children attending the Gastroenteritis Unit, Abo El Reesh Pediatric Hospital, Cairo University. The children were aged 3 months to 8 years and were of both sexes. They were classified according to their age into four groups: group 1 included children aged 3-6 months (23 cases); group 2 included children aged 7-12 months (33 cases); group 3 included children aged 1-5 years (41 cases); and group 4 included children aged 6-8 years (13 cases). Parents were asked about the duration of diarrhea, presence of vomiting, abdominal colic, and fever. The consistency of stool of every child was also recorded. Each stool sample was divided into two parts. The first was subjected to concentration and microscopic examination and the second was kept at −20°C for molecular assay.

Microscopic examination of samples

The first part of each sample was concentrated twice by formalin ether sedimentation method [33] , and smears were stained by modified Ziehl-Neelsen (MZN) stain [34] . Stained smears were examined through ×40 objective and then ×100 objective.

Polymerase chain reaction

DNA extraction

Molecular assay was performed at the Diagnostic and Research Unit of Parasitic Diseases, Medical Parasitology Department, Kasr Al-Ainy School of Medicine, Cairo University. Samples proved positive by microscopic examination were subjected to DNA extraction. Before extraction, each sample was subjected to eight cycles of freezing in liquid nitrogen for 1 min, followed by thawing at 98°C for 1 min to disrupt the oocyst wall [6],[29],[35],[36] . DNA extraction was performed with the Favor Stool DNA Spin Columns Isolation Mini Kit (cat. no. FAST1; Favorgen Biotech Corporation, Taiwan) following the manufacturer's instructions. Incubation was performed at 56°C for 10 min and then the temperature was raised to 95°C for 1 h. This gave a better yield of extracted DNA with excellent DNA/protein ratio, indicating that the proteinase K in the extraction buffer eliminated all or most of the protein.

Nested polymerase chain reaction of the Cryptosporidium oocyst wall protein gene

According to Spano et al. [37] and Pedraza-Díaz et al. [38] , nested PCR of the COWP gene included two consecutive PCR reactions. The first reaction amplified the 769 bp fragment by using an external pair of primer sets - BCOWPF: 5´-ACC GCT TCT CAA CAA CCA TCT TGT CCT C-3´; and BCOWPR: 5´-CGC ACC TGT TCC CAC TCA ATG TAA ACC C-3´. The second reaction contained two nested primers internal to the first primer pair and delimits a 553 bp fragment. These were nest Cry-15: 5´-GTA GAT AAT GGA AGA GAT TGT G-3´ and Cry-9: 5´-GGA CTG AAA TAC AGG CAT TAT CTT G-3´. The larger fragment produced by the first reaction was used as a template for the second reaction. Oligonucleotide primers were synthesized by Fermentas (Fermentas UAB, Lithuania). Amplification in each reaction was done on the PCR mix, which consisted of 12.5 μl PCR Master Mix (product no. K1081; Thermo Scientific, UK), 1 μl (200 nmol/l) of each forward and reverse primer, 2.5 μl of template DNA, 0.1 μl Taq polymerase (5 U/μl) (product no. EP0701; Thermo Scientific), and 7.9 μl of sterile distilled water to complete a total volume of 25 μl. Reactions were performed in a gradient thermal cycler (professional thermocycler, Biometra; Applied Biosystems, California, USA) after adjusting the thermal profile to initial denaturation at 95°C for 4 min, followed by 30 cycles of amplification consisting of denaturation at 94°C for 60 s, annealing at 65°C for 60 s, and extension at 72°C for 60 s. Final elongation was performed at 72°C for 10 min. The second-round PCR was identical to the first-round PCR except for denaturation at 94°C for 50 s, annealing at 54°C for 30 s, and extension at 72°C for 50 s. The amplified PCR products were separated by electrophoresis on 2% agarose gel in TBE buffer and visualized under a transilluminator after staining with ethidium bromide.

Restriction-fragment length polymorphism analysis

RFLP analysis was performed according to the manufacturer's instructions by digesting 10 μl of nested PCR product (target DNA) with 1 μl of RsaI (product no. ER1121; Thermo Scientific) in 2 μl green buffer and adding 17 μl nuclease-free water to reach a final volume of 30 μl. Gentle mixing was done followed by spinning down for a few seconds and then incubation at 37°C for 5 min. Cryptosporidium genotypes were detected by using electrophoresis in 3.2% typing-grade agarose gels containing ethidium bromide, and gels were recorded by UV transillumination. By using restriction enzyme RsaI for digestion of nested PCR products targeting the COWP gene, we detected two genotypes of Cryptosporidium: genotype 1 (C. hominis), when four bands appeared at 34, 106, 125, and 285 bp; and genotype 2 (C. parvum), when three bands appeared at 34, 106, and 410 bp [37],[38] .

Statistical analysis

Data were collected, tabulated, and statistically analyzed by means of a computer using SPSS (version 16; SPSS Inc., Chicago, Illinois, USA); two types of statistical analyses were performed. Descriptive statistics including qualitative data expressed in number and percentage and quantitative data expressed to measure the central tendency of data and diversion around the mean (X) and SD. The Mann-Whitney test was used to compare between two quantitative variables not normally distributed. The χ2 - test was used to compare categorical outcomes, and Fischer's exact test was used to compare categorical outcomes of 2 × 2 tables if one of the cells is less than 5. P values less than or equal to 0.05 were considered statistically significant.

  Results Top

Examination of MZN-stained fecal samples of children and calves revealed Cryptosporidium oocysts as round or ovoid 2-6 μm bright red structures against a green background. They had distinct thick walls and various inner structures, as four dark sporozoites could be seen in some oocysts ([Figure 1] and [Figure 2]). This preliminary screening detected 40% of diarrheic samples from calves positive for Cryptosporidium. Nested PCR-RFLP analysis showed that all of these positive samples were of C. parvum genotype 2 (C. parvum) ([Figure 3]), 25/52 were from group A (48.07%) and 15/48 (31.25%) were from group B. There was no statistically significant difference between the two groups (P>0.05) ([Table 1]). Screening of 110 children's fecal smears revealed 12 positive Cryptosporidium samples (10.9%). By nested PCR, the COWP gene was successfully amplified in only 9/12 (75%) samples ([Figure 3]). RFLP analysis showed that 8/9 (88.9%) samples were positive by PCR, classified as genotype 1 (C. hominis), whereas one sample was not digested (11.1%) ([Figure 3]).
Figure 1 Cryptosporidium oocysts ↑ in feces of an infected child (modified Ziehl– Neelsen stain, ×800).

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Figure 2 Cryptosporidium oocyst ↑ in stool of an infected calf (modified Ziehl– Neelsen stain, ×1000).

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Figure 3 Agarose gel electrophoresis for the products of the nested PCR targeting the Cryptosporidium oocyst wall protein (COWP) gene of Cryptosporidium at 553 bp and restriction-fragment length polymorphism products after digestion with RsaI endonuclease. Lane 1: 100 bp DNA marker ladder. Lanes 2, 3, and 4: Cryptosporidium parvum genotype 2 digestion products at 34, 106, and 410 bp (34 band is very small, faint, and difficult to see). Lanes 5 and 6: Cryptosporidium hominis genotype 1 digestion products at 34, 106, 125, and 285 bp. Lanes 7 and 8: product of nested PCR targeting the COWP gene at 553 bp.

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Table 1 Results of examination of 100 fecal samples from two age groups of diarrheic calves by modified Ziehl– Neelsen stain

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On correlating the age of infected children with Cryptosporidium infection, 2/12 (16.7%) positive cases were found to be from group 1 (3-6 months), two (16.7%) cases from group 2 (7-12 months), five (41.6%) cases from group 3 (1-5 years), and three (25%) cases from group 4 (6-8 years). The difference between these four age groups regarding Cryptosporidium infection was not statistically significant (P > 0.05) ([Table 2]). Correlation between sex and infection in the 12 positive cases showed equally positive occurrence in male and female children (50%). The difference between the two sexes was not significant (P > 0.05) ([Table 2]). The relation between consistency of children's stools and infection showed that 6/12 (50%) positive cases had formed stool, four (33.3%) cases had loose stool, and two (16.7%) cases had watery stool. There was no statistically significant correlation between consistency of stool and Cryptosporidium infection (P > 0.05) ([Table 2]).
Table 2 Correlation between age groups, sex, consistency of stool, duration of diarrhea, clinical manifestations, and Cryptosporidium infection in examined children

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The relation between duration of diarrhea and infection showed that 5/12 (41.7%) positive cases had diarrhea for 1-5 days, whereas seven (58.3%) cases had diarrhea for more than 5 days, with nonsignificant difference (P > 0.05) between these two groups ([Table 2]). The clinical manifestations of the examined children recorded the occurrence of vomiting in 2/12 (16.7%) positive cases and 54/98 (55.1%) negative cases. Abdominal colic occurred in 9/12 (75.5%) positive cases and in 91/98 (92.9%) negative cases. In addition, 6/12 (50%) positive cases suffered from fever versus 83/98 (84.7%) negative cases. Statistical analysis showed that there was no significant correlation between these three clinical manifestations and occurrence of Cryptosporidium infection (P > 0.05) ([Table 2]).

  Discussion Top

Cryptosporidiosis is a cause of diarrhea in children and immunocompromised patients worldwide [29] . It also frequently affects domestic animals, with a strong relation found between cryptosporidiosis infection and diarrhea among Egyptian buffalo calves [39] . In Egypt, several studies have been devoted to ascertain cryptosporidiosis in humans [18],[40] and animals [6],[39] . The application of genotyping techniques has provided a better understanding of the epidemiology of cryptosporidiosis [41] , including its different sources of transmission [38],[42] . At present, a variety of genotypic markers are available to differentiate species and subspecies within the genus Cryptosporidium [10] . Nested PCR has proven more beneficial than primary PCR and produces more positive results [36] . Hence, the present work aimed to determine Cryptosporidium genotypes in stools of calves and children suffering from diarrhea using nested PCR-RFLP targeting the COWP gene.

Cryptosporidium oocysts detected in this study were morphologically similar to those detected in many previous studies [6],[43] . Morgan-Ryan et al. [44] cited that morphometric measurement of oocysts represents the cornerstone of Cryptosporidium taxonomy and is one of the requirements for establishing a new species; however, it is not adequate by itself and other parameters must be used.

In the present study, the infection rate of cryptosporidiosis in examined calves was 40%. This result is not unexpected as traditionally cattle have been considered the primary nonhuman species impacted by cryptosporidiosis and the major source of Cryptosporidium for human infections [45] . Having targeted diarrheic calves, our recorded rate proved higher than that of previous studies in Egypt, in which the prevalence was 30.2% in dairy calves [39] and 21.7% in buffalo calves [46] . It is also higher than the 26% recorded in Brazil [1] . However, it is lower than the 54.4% recorded in diarrheic calves from Behira locality in Egypt [6] , and in the USA (95%) [47] . Several factors may be responsible for the differences in infection rates between the present and previous studies, such as the breeding conditions of the calves, husbandry and management system, exact age and nursing conditions of the calves, season of the sample collection, and the sanitary conditions inside and around the farms. Some of these factors may act individually or collectively to increase the risk factors associated with transmission and prevalence of Cryptosporidium between calves [6] . In the present study, the infection rate of cryptosporidiosis in calves aged less than 1 month was higher than that in calves aged 1-2 months (48.07 and 31.25%, respectively). This result is consistent with that of Hassanain et al. [6] in Egypt but was contradictory to that of the Brazilian study [1] in which the lowest prevalence was in calves aged less than 1 month. However, another study stated that only preweaned calves are important sources of zoonotic cryptosporidiosis in humans [48] . All of our positive fecal samples from calves were classified as genotype 2 (C. parvum) by RFLP analysis. This result is in agreement with the majority of worldwide studies, which have reported C. parvum as the most prevalent and widely distributed species in neonatal and preweaned dairy cattle [1] .

The infection rate in the present sample of diarrheic children was 10.9%. A higher rate of 17% was recorded in Egypt by Abdel-Messih et al. [49] , whereas similar rates of 10.3% were recorded in Pakistan by Mojarad et al. [29] and in a cross-sectional study of cryptosporidiosis in Saudi diarrheal children in whom the overall prevalence was 9.6% [50] . The discrepancy in infection rates is expected, because of differences in the living conditions of children or differences in their immune status. In the present study, the COWP gene was successfully amplified in 75% of samples that were positive on MZN staining. Another report noted lower positivity of PCR as opposed to microscopy [51] . This may be attributed to failure of amplification of some fecal samples derived from a diminished quantity of DNA, either due to its degradation in time or due to the presence of some PCR inhibitors such as lipids, hemoglobin, bile salts, polysaccharides from mucus, bacteria, and food degradation products [52] . As indicated, PCR inhibitors may act at three levels: interference in cellular lysis, degradation or uptake of nucleic acids, and inactivation of thermostable polymerases [53] . It was proved that the QIAamp DNA Stool Mini Kit removes these inhibitors by absorbing them in the Inhibitex matrix [48] . Naeini et al. [5] stated that the inhibitors may be removed by repeated washing of collected oocysts with PBS buffer, but with the disadvantage of possible decrease in the recovery rate of oocysts and consequent reduction in the final DNA concentration and sensitivity of PCR. However, in another report by Coupe et al. [7] PCR-RFLP was applied to routine clinical samples, with 98.5% detection rate of infection in patients with positive microscopic results and in two other stool samples that had not been found positive on microscopic examination. The same authors added that they believed those results were not PCR false positives but rather they reflected the higher sensitivity of PCR for low-level infection. This group of researchers [7] used the previously mentioned QIAamp DNA Stool Mini Kit for DNA extraction, that removes DNA inhibitors by absorbing them in the Inhibitex matrix [54] , which may explain the high diagnostic yield obtained in that study. However, in our study, following the manufacturer's instructions, no extra measures were taken for removing DNA inhibitors as it was mentioned in the kit manual that inhibitors are removed. Besides, Yu et al. [15] stated that although microscopy was useful for identification of infection, this tool is relatively insensitive when a small number of oocysts are excreted or the period of oocyst shedding is short. Therefore, many infections may escape microscopic detection. Moreover, Silva et al. [1] claimed that their results and those of previous studies indicate that the detection of Cryptosporidium oocysts in fecal smears by microscopy is less sensitive than the detection of DNA by PCR. Furthermore, Nikaeen et al. [35] mentioned that the nested PCR procedure described in their study further enhanced the sensitivity of PCR, so that they could detect as few as one oocyst in purified samples. Bairami Kuzehkanan et al. [36] reported that nested PCR was better than primary PCR as it detected one more case that was negative on both microscopy and primary PCR. Another advantage over microscopy is the ability of PCR tools to differentiate Cryptosporidium spp. by incorporating RFLP analysis as well as DNA sequencing or other molecular procedures that allow the differentiation of species or genotypes [55] .

In the current work, RFLP analysis classified 88.9% of the children's positive fecal samples as genotype 1 (C. hominis), whereas one (11.1%) sample was not digested. None of the samples were classified as genotype 2 (C. parvum). These results are in agreement with previous studies reporting the predominance of C. hominis species in some nations such as Brazil [56] , Malawi [57] , the USA [58] , Thailand [59] , Japan [60] , and South Africa [61] . In France, Coupe et al. [7] obtained 98.5% of stool samples from patients with microscopically proven cryptosporidiosis and two other stool samples positive by PCR and had them genotyped by PCR-RFLP analysis as 47.7% related to C. hominis, 34.1% related to C. parvum, 13.6% related to C. meleagridis, and 4.5% to C. felis. Interestingly one of their negative PCR samples that had microscopically shown rare oocysts proved to contain a potent inhibitor that could not be efficiently removed. Also, Gatei et al. [62] in Kenya found that 87% of the PCR-positive cases were C. hominis, 9% were C. parvum, and the remaining 4% were C. canis, C. felis, C. meleagridis, and C. muris. Moreover, Feng et al. [63] reported that C. hominis was the predominant species in wastewater in Shanghai (China). They indicated that anthroponotic transmission is important in cryptosporidiosis epidemiology in that area, and commented that C. hominis is responsible for far more infections than C. parvum in humans in developing countries where genotyping studies were conducted. It was suggested that anthroponotic transmission of Cryptosporidium spp. in outbreaks that had occurred in swimming pools might be associated with fecal contamination from a single infected person (especially in toddler pools), in which case a single genotype is recovered from the patients [38] . However, our results contradict those obtained by some previous authors in the Netherlands and UK where C. parvum was more predominant than C. hominis in humans [31],[64] . Several authors previously stressed on the zoonotic transmission of Cryptosporidium spp. Pedraza-Díaz et al. [38] mentioned that patients reporting contact with animals or farms were almost all infected by genotype 2 (C. parvum). Also Mojarad et al. [29] found that C. parvum infection predominated in children in Iran (83.4%), indicating the probability of high prevalence of bovine cryptosporidiosis in that area. Moreover, Usluca and Aksoy [11] stated that the majority of cases diagnosed in their study infected with C. parvum and C. meleagridis species lived and bred animals in rural areas. They also mentioned that lack of hygiene, poor living conditions, and direct contact with farm animals in which cryptosporidiosis has a high prevalence account for the spread of infection. Pedraza-Díaz et al. [38] in their study on 1300 sporadic cases indicated that patients reporting contact with animals or farms were almost all infected by genotype 2, which was apparently also exclusively responsible for the spring peak. Genotype 1 was significantly more common in patients infected during the late summer-autumn peak and in those with a history of foreign travel. There were also distinct geographical variations in the distribution of genotypes.

It must be mentioned that waterborne cryptosporidiosis and giardiasis are significant public health concerns [42] . Cryptosporidium and Giardia spp. are threats in water supplies because their infecting stages are resistant to chlorine disinfection; moreover, they have a low infectious dose and they are harbored by many animal species [65] . Consequently, numerous waterborne outbreaks of cryptosporidiosis and giardiasis have been documented [66] . Cryptosporidium and Giardia spp. are reportedly the two major pathogens specifically identified in drinking water regulations in industrialized countries [30] . The incorporation of flow cytometry improved the sensitivity of detection of Giardia cysts and Cryptosporidium oocysts and the accurate estimation of their quantity and viability in water samples from Alexandria (Egypt) [67] . Oocysts of both genotypes 1 and 2 are water transmissible, and in one report of seven drinking waterborne outbreaks four were almost exclusively due to genotype 1 and three due to genotype 2 [38] . Thus, controlling agricultural and human sewage discharge is important in watershed protection [55] .

In the present work, the highest percentage of infection was recorded in children aged 1-5 years and the lowest in children under 1 year. This result is in accordance with reports by Wellington et al. [68] and Yoder et al. [69] . Children under 1 year may be protected by immunoglobulins conferred by breast feeding or by unexposure to contaminated water. Although it is generally believed that a high percentage of infected children complain of watery stools [70] , we found fewer number of positive children with watery diarrhea. This may be explained by a difference in the immune status of examined children or a difference in the method of their selection. In addition, we found no significant relation between duration of diarrhea and cryptosporidiosis, an observation consistent with that of Usluca and Aksoy [11] . Our recorded clinical manifestations in infected children, such as vomiting, abdominal colic, and fever, are consistent with previous studies [11],[70] . However, it was found that there was no significant statistical difference between negative and positive cases regarding these clinical manifestations.

  Conclusion Top

Hence, it can be concluded that C. parvum (genotype 2) is relatively highly prevalent in the sample of calves examined. All infected children showed C. hominis (genotype 1). Thus, it seems that transmission of cryptosporidiosis in these children was anthroponotic and not zoonotic. Attention must be given for periodic determination of Cryptosporidium spp. in watersheds or sources of water, which in turn will be helpful in the development of strategies for their scientific management and protection. Nested PCR-RFLP is an added advantage for the determination of Cryptosporidium genotype in suspected cases. Information generated from this study is useful not only in identifying the sources of contamination but also in helping public healthcare systems in the prevention and management of cryptosporidiosis through development of precautionary measures.

  Acknowledgements Top

The author expresses her gratitude to Professor Dr. Ayman A. El-Badry, Head of Diagnostic and Research Unit of Parasitic Diseases, Medical Parasitology Department, Kasr Al-Ainy School of Medicine, Cairo University, for his assistance in performing nested PCR-RFLP.

Conflicts of interest

There are no conflicts of interest.

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