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Dabirzadeh M, Shahraki R, Beheshtizadeh M, Khoshsima Shahraki M. Investigation of Cryptosporidium oocyst contamination in water from Zabol and Zahedan: A molecular and microscopic analysis. mljgoums 2026; 20 (2) :31-35
URL: http://mlj.goums.ac.ir/article-1-1895-en.html
URL: http://mlj.goums.ac.ir/article-1-1895-en.html
1- Department of Parasitology and Mycology, Faculty of Medicine, Zabol University of Medical Sciences, Sistan-Baluchistan Province, Zabol, Iran , mdabirzadeh20002000@gmail.com
2- Department of Parasitology, Medical School, Zabol University of Medical Sciences, Sistan-Baluchistan Province, Zabol, Iran
3- Zabol Medical Plants Research Center, Zabol University of Medical Sciences, Zabol, Iran
2- Department of Parasitology, Medical School, Zabol University of Medical Sciences, Sistan-Baluchistan Province, Zabol, Iran
3- Zabol Medical Plants Research Center, Zabol University of Medical Sciences, Zabol, Iran
Keywords: Cryptosporidium, DNA Sequencing, Polymerase Chain Reaction, Water Pollution, Zabol, Zahedan
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| Abstract (HTML) (5658 Views)
Restriction fragment length polymorphism (RFLP) analysis was conducted on PCR products from samples that tested positive for Cryptosporidium. The enzymes RsaI and AluI were used, as shown in Table 2.
The digested products were subsequently loaded onto a 1.5% agarose gel for electrophoresis to identify the genotype of Cryptosporidium. All procedures adhered to ethical guidelines for environmental sampling and laboratory safety protocols.
Results
Microscopic examination of water samples
A total of 180 water samples were collected from various sources in Zahedan and Zabol. Initial screening was performed using direct microscopic examination, followed by confirmation with modified Ziehl-Neelsen and Trichrome staining methods. Microscopic analysis revealed that 35 samples were positive for Cryptosporidium oocysts, 142 were negative, and 3 samples were classified as suspicious (Figure 1).
DNA extraction results from cryptosporidium
All Cryptosporidium-positive samples yielded successful DNA extraction. The incorporation of glass beads together with repeated freeze-thaw cycles effectively enhanced oocyst disruption and improved DNA yield. Following PCR amplification, the products were electrophoresed on a 1% agarose gel and visualized using a gel documentation system. A clear and distinct single band was observed in all positive samples, falling within the expected size range of approximately 800-900 bp, corresponding to the ITS1/SSU rRNA target region (Figure 2).
The amplified fragment was approximately 824 bp in length, which is consistent with previously reported sizes for Cryptosporidium ITS1/SSU rRNA gene fragments.
Results of enzymatic digestion with AluI and RsaI enzymes
RsaI digestion
No clear restriction fragments were detected following digestion with RsaI. The absence of visible bands is most likely due to the lack of RsaI recognition sites within the amplified ITS1/SSU rRNA region, indicating that this fragment is not cut by RsaI under standard conditions. Therefore, the undigested pattern is considered a normal finding rather than an indication of enzyme failure.
AluI digestion
Digestion with AluI produced a characteristic multi-band pattern. A prominent fragment of approximately 850 bp was observed, representing partial digestion of the original PCR product. Additional fragments of approximately 720 bp, 480 bp, 456 bp, and 410 bp were detected, confirming that AluI cleaved the target sequence at multiple restriction sites. The coexistence of a large undigested band with multiple smaller fragments is consistent with expected RFLP profiles for Cryptosporidium ITS1/SSU rRNA amplicons.
Overall, the observed AluI banding pattern supports the identification of Cryptosporidium spp. (Figure 3).
Selected PCR-positive samples were subjected to Sanger sequencing using a commercial sequencing service (Bioneer Co.). High-quality chromatograms were obtained, and the forward and reverse reads were assembled to generate a consensus sequence. Raw sequence data were inspected and edited using Chromas Lite version 2.1, and the final consensus sequence was compared with reference sequences available in the NCBI GenBank database using the BLASTn algorithm.
The nucleotide sequence generated in this study is provided in Appendix 1. BLAST analysis demonstrated strong and significant sequence identity, with an E-value of 0.0 (Figure 4). Alignment with multiple isolates of Cryptosporidium parvum showed 98.84% identity. These findings confirm that the amplified SSU rRNA fragment corresponds to C. parvum.
Phylogenetic analysis
Phylogenetic analysis was performed to evaluate the evolutionary placement of the SSU rRNA sequence obtained in this study. A distance tree was generated using the BLAST pairwise alignment tool, which grouped the sequence (Crypto-F) together with multiple reference isolates of C. parvum. No clustering with C. hominis or other Cryptosporidium species was observed.
The close association of the query sequence with C. parvum isolates, combined with the high sequence identity (98.84%) and high query coverage obtained from BLAST analysis, confirms that the sample belongs to the C. parvum lineage (Figure 5).
In conclusion, the phylogenetic findings indicate that the SSU-rRNA sequence obtained from water samples collected in Zabol and Zahedan shows clear genetic affinity to established isolates of C. parvum. As illustrated in Figure 5, the sequence clusters within the C. parvum lineage and shares conserved regions of the 18S rRNA (SSU-rRNA) gene with these reference isolates. This strong genetic similarity provides robust molecular evidence confirming that the detected organism belongs to C. parvum.
Discussion
Cryptosporidium spp. are major waterborne pathogens with substantial public health implications. Human and animal fecal contamination of environmental sources such as soil, water, and food constitutes a key transmission route for this protozoan, which remains one of the leading causes of diarrheal disease worldwide. The World Health Organization (WHO) has recognized Cryptosporidium as a priority pathogen for global water quality monitoring, highlighting its significance in public health surveillance and waterborne disease management (6). Therefore, the detection and molecular characterization of Cryptosporidium spp. in environmental samples are essential for understanding transmission dynamics and implementing effective control measures.
Genotyping of Cryptosporidium isolates is a critical tool for distinguishing species and genotypes and for elucidating epidemiological patterns. Due to the difficulty of culturing this parasite, an approach that is both time-consuming and susceptible to contamination, PCR-RFLP has become an important molecular method for genotyping (7). This technique differentiates species based on the digestion of PCR-amplified DNA by restriction enzymes that cleave specific nucleotide sequences. The resulting fragment patterns allow comparison with known profiles and facilitate accurate species identification (8). Variations in banding patterns may reflect genetic polymorphisms, particularly in regions where multiple species or genotypes co-circulate.
The epidemiology of Cryptosporidium is further complicated by the coexistence of multiple transmission pathways, including waterborne, foodborne, and zoonotic routes. Genotyping plays an essential role in identifying infection sources and understanding transmission mechanisms by revealing the genetic diversity within Cryptosporidium populations. Previous studies indicate that C. parvum and C. hominis are responsible for the majority of human infections, with C. parvum accounting for approximately 90% of cases in many regions. To date, more than 300 human cases have been genotyped, revealing at least nine infective Cryptosporidium species, including C. andersoni, C. felis, and C. meleagridis, among others (9).
This diversity underscores the need for continuous monitoring and molecular surveillance.
Globally, water contamination remains a major public health concern due to the exceptional resistance of Cryptosporidium oocysts to routine water-treatment processes. Oocysts are highly resistant to chlorine and several commonly used disinfectants, complicating their removal from water supplies and reinforcing the need for sensitive detection methods (10). Given the increasing threat of waterborne diseases worldwide, the development and application of rapid molecular diagnostic techniques have become priorities in water-quality monitoring programs (11).
In the present study, 180 water samples were examined, of which 35 tested positive for Cryptosporidium oocysts. The primers used successfully amplified an approximately 800-900 bp fragment of the SSU rRNA gene, which is consistent with previous studies targeting this region. Microscopic examination and staining techniques initially confirmed the presence of oocysts. Subsequently, subtyping was performed using AluI and RsaI restriction enzymes, followed by DNA sequencing to further characterize the isolates. Consistent with expectations for the SSU rRNA gene, digestion with AluI produced clear fragment patterns suitable for genotyping, whereas digestion with RsaI did not yield visible fragments, likely due to the absence of an appropriate recognition site within the amplified region rather than a technical error.
The combined application of microscopy, staining, RFLP analysis with AluI, and sequencing revealed notable genetic polymorphism among Cryptosporidium isolates from the Zabol and Zahedan districts. This observation indicates substantial genetic diversity among circulating isolates in these geographic regions. The integration of molecular and conventional detection techniques allowed robust subtyping and comparison of isolates, thereby providing a comprehensive picture of the genotypic profiles present in the study area.
The phylogenetic tree (Figure 5), constructed using SSU rRNA sequences, demonstrates distinct clustering of C. parvum isolates according to geographic origin and host specificity. Similar findings have been reported previously, showing that C. parvum isolates from different regions often form independent clades, suggesting geographic structuring and restricted gene flow (12,13). These genetic markers are essential for tracking outbreaks and distinguishing between zoonotic and anthroponotic transmission routes. Certain C. parvum genotypes may exhibit host adaptation, emphasizing the importance of recognizing host-associated variants for public health surveillance.
Genetic analyses of C. parvum also provide insights into potential environmental and animal reservoirs, helping to identify sources of contamination in water systems. Such information is vital for designing targeted interventions, enhancing disease surveillance, and improving our understanding of transmission routes. Molecular epidemiology using SSU rRNA markers therefore plays a crucial role in monitoring and controlling cryptosporidiosis.
Worldwide, the prevalence of C. parvum in diverse water sources continues to present major challenges for water safety. Studies have reported prevalence rates of 46.9% in wastewater, 45.3% in surface water, and 31.6% in untreated raw water (14). These findings reflect the persistence of Cryptosporidium oocysts in the environment and their resistance to conventional treatment methods, particularly chlorine-based disinfection (10). In Iran, C. parvum is the dominant species, accounting for 83.3% of human cases, followed by C. hominis with 8.3% (6). In livestock, particularly cattle, C. parvum is also the predominant species, representing approximately 36% of infections, with genotypes similar to those identified in countries such as France (15,16). These data further highlight the importance of zoonotic transmission in regional epidemiology. Globally, C. parvum remains one of the most frequently reported pathogens in waterborne outbreaks, with contaminated water serving as a major transmission route (10).
Conclusion
In conclusion, the persistence of C. parvum in water sources in Iran remains a considerable public health challenge. The detection and molecular characterization of C. parvum from environmental water samples provide essential information for assessing the risk of waterborne cryptosporidiosis. Addressing contamination requires improvements in water-treatment infrastructure, enhanced monitoring systems, and effective intervention strategies. The findings of this study, particularly the genotypic and phylogenetic characterization of isolates, contribute valuable insights into the transmission dynamics of Cryptosporidium and underscore the importance of molecular tools in surveillance and outbreak prevention.
Acknowledgement
The authors would like to thank all the staff of the Department of Parasitology and the Vice Chancellor for Research and Technology of the University of Medical Sciences.
Funding Sources
Not applicable.
Ethical Statement
This study involved harvested oocysts from water and did not include unethical practices. It was approved by the University Research and Ethics Committee (Code: http://ethics.research.ac.ir/IR.ZBMU.RECRYPTOSPORIDIUM1398.159), and all procedures followed institutional guidelines for the care and use of humans and animals.
All participants in this study provided informed consent before participation. They were fully informed about the study’s purpose, procedures, potential risks, and benefits.
This consent process ensured that participants were aware of and agreed to the terms of their involvement in the study.
Conflicts of Interest
There are no conflicts of interest.
Author Contributions
Mansour Dabirzadeh supervised and designed the study. Reza Shahraki and Mahdi Khoshsima Shahraki collected and analyzed the samples. Mohammadreza Beheshtizadeh performed the molecular analysis and approved the final manuscript.
Data Availability Statement
Upon reasonable request, the corresponding author will share the datasets of this study.
Use of Artificial Intelligence
No artificial intelligence tools were used in the preparation of this manuscript.
Full-Text: (258 Views)
Introduction
The primary aim of this study is to investigate the prevalence and genotypic diversity of Cryptosporidium in drinking and surface water sources in the Zabol and Zahedan districts. By employing PCR-RFLP techniques for molecular characterization, this research seeks to fill existing knowledge gaps regarding the origins and transmission pathways of Cryptosporidium.
Cryptosporidium is a significant enteric parasite responsible for widespread outbreaks of diarrheal illness globally, affecting both immunocompromised and immunocompetent individuals. Transmission through contaminated drinking and surface water has become a critical public health concern, particularly in regions where water-quality management is inadequate. Recent studies have highlighted the increasing prevalence of Cryptosporidium infections, underscoring the urgent need for comprehensive research to understand its epidemiology and transmission dynamics. This study focuses on investigating the prevalence and genotypic diversity of Cryptosporidium in the Zabol and Zahedan districts of Southeast Iran, an area where such outbreaks have not been officially reported. This research is vital for developing targeted intervention strategies that can mitigate the risks associated with waterborne transmission of this pathogen, especially in vulnerable populations (1).
Interest in Cryptosporidium research has increased among scientists and public health practitioners, driven by the growing recognition of the parasite as a leading cause of waterborne disease. This has prompted extensive investigations into its genetic diversity and transmission pathways. Recent advancements in molecular techniques, such as PCR-RFLP, have facilitated more accurate identification of Cryptosporidium species and genotypes, enhancing epidemiological tracking (2).
Furthermore, the emergence of new strains and their potential zoonotic implications have heightened awareness of the need for continuous surveillance and research. Despite substantial progress, several challenges persist. One major issue is the organism’s ability to survive harsh environmental conditions and resist conventional disinfection methods, which complicates its control (3). Additional gaps remain concerning the specific sources of contamination and ecological factors contributing to outbreaks. However, recent technological advancements, including high-throughput sequencing and improved culture methods, offer new opportunities to address these challenges by providing deeper insights into Cryptosporidium biology and transmission. Current literature also reveals several areas requiring further exploration. Although previous studies have contributed valuable data on the epidemiology of Cryptosporidium (4,5), many aspects remain understudied, particularly regional variations in prevalence and genetic diversity. Limited data are available on the specific genotypes circulating in Southeast Iran and their potential reservoirs within local wildlife or livestock populations. Addressing these gaps is essential for developing a comprehensive understanding of Cryptosporidium dynamics in this region and for improving preventive public health measures.
Methods
Sample preparation
Water samples were collected between April 2021 and August 2022 from various locations in Zahedan and Zabol cities, including surface water, tap water, bottled water, well water, and wastewater sources. A total of 180 samples were systematically collected (80 from Zabol and 100 from Zahedan), and each sample was labeled for identification during subsequent analyses for Cryptosporidium parasites.
Sampling sites were precisely delineated, and random sampling protocols were followed to ensure representativeness. Health and safety protocols were strictly observed, including the use of personal protective equipment (PPE), biosafety cabinets, and disinfectants. A minimum of 10 liters of water was collected per site according to EPA Method 1623.1.
After collection, samples were filtered through membranes with a pore size of 2-3 µm using the centrifugal vacuum filtration method. Pellets trapped in the filter were removed and centrifuged to concentrate the oocysts. The sediment was excised from the filters using a scalpel and transferred into 1.5 ml microtubes. To preserve sample integrity, the samples were stored at 4°C in a preserving solution of 2.5% potassium dichromate.
DNA extraction
DNA extraction was performed using the YEKTA-TAJHIZ DNA Extraction Kit according to the manufacturer’s instructions. Briefly, a freeze-thaw cycle was applied to lyse the cells, followed by washing with PBS to remove residual contaminants. Glass bead powder was then added to enhance mechanical cell disruption during vortexing. After incubation to ensure complete lysis, DNA was isolated according to the kit protocol, eluted in elution buffer, and stored at −20°C until further analysis.
The quality of the extracted DNA was evaluated by agarose gel electrophoresis. Approximately 5 µl of extracted DNA, mixed with loading dye, was loaded onto a 1% agarose gel containing ethidium bromide and electrophoresed at 75 V for 60 minutes. The gel was visualized using a gel documentation system, and the resolved DNA bands were compared with a standard molecular weight marker.
PCR method
The PCR reaction mixture consisted of 5 µl of extracted DNA, 12.5 µl of master mix (Brand/Type), 4.5 µl of sterile distilled water, and 1 µl of each primer. PCR amplification was performed in an Eppendorf thermocycler under the following conditions: initial denaturation at 94°C for 5 minutes, followed by denaturation at 94°C for 45 seconds, annealing at 55°C for 45 seconds, elongation at 72°C for 60 seconds, and a final elongation step at 72°C for 7 minutes, for a total of 35 cycles. The primer sequences and PCR product size are shown in Table 1.
The primary aim of this study is to investigate the prevalence and genotypic diversity of Cryptosporidium in drinking and surface water sources in the Zabol and Zahedan districts. By employing PCR-RFLP techniques for molecular characterization, this research seeks to fill existing knowledge gaps regarding the origins and transmission pathways of Cryptosporidium.
Cryptosporidium is a significant enteric parasite responsible for widespread outbreaks of diarrheal illness globally, affecting both immunocompromised and immunocompetent individuals. Transmission through contaminated drinking and surface water has become a critical public health concern, particularly in regions where water-quality management is inadequate. Recent studies have highlighted the increasing prevalence of Cryptosporidium infections, underscoring the urgent need for comprehensive research to understand its epidemiology and transmission dynamics. This study focuses on investigating the prevalence and genotypic diversity of Cryptosporidium in the Zabol and Zahedan districts of Southeast Iran, an area where such outbreaks have not been officially reported. This research is vital for developing targeted intervention strategies that can mitigate the risks associated with waterborne transmission of this pathogen, especially in vulnerable populations (1).
Interest in Cryptosporidium research has increased among scientists and public health practitioners, driven by the growing recognition of the parasite as a leading cause of waterborne disease. This has prompted extensive investigations into its genetic diversity and transmission pathways. Recent advancements in molecular techniques, such as PCR-RFLP, have facilitated more accurate identification of Cryptosporidium species and genotypes, enhancing epidemiological tracking (2).
Furthermore, the emergence of new strains and their potential zoonotic implications have heightened awareness of the need for continuous surveillance and research. Despite substantial progress, several challenges persist. One major issue is the organism’s ability to survive harsh environmental conditions and resist conventional disinfection methods, which complicates its control (3). Additional gaps remain concerning the specific sources of contamination and ecological factors contributing to outbreaks. However, recent technological advancements, including high-throughput sequencing and improved culture methods, offer new opportunities to address these challenges by providing deeper insights into Cryptosporidium biology and transmission. Current literature also reveals several areas requiring further exploration. Although previous studies have contributed valuable data on the epidemiology of Cryptosporidium (4,5), many aspects remain understudied, particularly regional variations in prevalence and genetic diversity. Limited data are available on the specific genotypes circulating in Southeast Iran and their potential reservoirs within local wildlife or livestock populations. Addressing these gaps is essential for developing a comprehensive understanding of Cryptosporidium dynamics in this region and for improving preventive public health measures.
Methods
Sample preparation
Water samples were collected between April 2021 and August 2022 from various locations in Zahedan and Zabol cities, including surface water, tap water, bottled water, well water, and wastewater sources. A total of 180 samples were systematically collected (80 from Zabol and 100 from Zahedan), and each sample was labeled for identification during subsequent analyses for Cryptosporidium parasites.
Sampling sites were precisely delineated, and random sampling protocols were followed to ensure representativeness. Health and safety protocols were strictly observed, including the use of personal protective equipment (PPE), biosafety cabinets, and disinfectants. A minimum of 10 liters of water was collected per site according to EPA Method 1623.1.
After collection, samples were filtered through membranes with a pore size of 2-3 µm using the centrifugal vacuum filtration method. Pellets trapped in the filter were removed and centrifuged to concentrate the oocysts. The sediment was excised from the filters using a scalpel and transferred into 1.5 ml microtubes. To preserve sample integrity, the samples were stored at 4°C in a preserving solution of 2.5% potassium dichromate.
DNA extraction
DNA extraction was performed using the YEKTA-TAJHIZ DNA Extraction Kit according to the manufacturer’s instructions. Briefly, a freeze-thaw cycle was applied to lyse the cells, followed by washing with PBS to remove residual contaminants. Glass bead powder was then added to enhance mechanical cell disruption during vortexing. After incubation to ensure complete lysis, DNA was isolated according to the kit protocol, eluted in elution buffer, and stored at −20°C until further analysis.
The quality of the extracted DNA was evaluated by agarose gel electrophoresis. Approximately 5 µl of extracted DNA, mixed with loading dye, was loaded onto a 1% agarose gel containing ethidium bromide and electrophoresed at 75 V for 60 minutes. The gel was visualized using a gel documentation system, and the resolved DNA bands were compared with a standard molecular weight marker.
PCR method
The PCR reaction mixture consisted of 5 µl of extracted DNA, 12.5 µl of master mix (Brand/Type), 4.5 µl of sterile distilled water, and 1 µl of each primer. PCR amplification was performed in an Eppendorf thermocycler under the following conditions: initial denaturation at 94°C for 5 minutes, followed by denaturation at 94°C for 45 seconds, annealing at 55°C for 45 seconds, elongation at 72°C for 60 seconds, and a final elongation step at 72°C for 7 minutes, for a total of 35 cycles. The primer sequences and PCR product size are shown in Table 1.
|
Table 1. The SSU-rRNA primer sequences and PCR product
.PNG) |
Restriction fragment length polymorphism (RFLP) analysis was conducted on PCR products from samples that tested positive for Cryptosporidium. The enzymes RsaI and AluI were used, as shown in Table 2.
|
Table 2. The restriction enzymes used and their related cutting sites
.PNG) |
The digested products were subsequently loaded onto a 1.5% agarose gel for electrophoresis to identify the genotype of Cryptosporidium. All procedures adhered to ethical guidelines for environmental sampling and laboratory safety protocols.
Results
Microscopic examination of water samples
A total of 180 water samples were collected from various sources in Zahedan and Zabol. Initial screening was performed using direct microscopic examination, followed by confirmation with modified Ziehl-Neelsen and Trichrome staining methods. Microscopic analysis revealed that 35 samples were positive for Cryptosporidium oocysts, 142 were negative, and 3 samples were classified as suspicious (Figure 1).
.PNG) Figure 1. Cryptosporidium oocyst samples isolated from water and stained by the modified Ziehl-Neelsen method |
DNA extraction results from cryptosporidium
All Cryptosporidium-positive samples yielded successful DNA extraction. The incorporation of glass beads together with repeated freeze-thaw cycles effectively enhanced oocyst disruption and improved DNA yield. Following PCR amplification, the products were electrophoresed on a 1% agarose gel and visualized using a gel documentation system. A clear and distinct single band was observed in all positive samples, falling within the expected size range of approximately 800-900 bp, corresponding to the ITS1/SSU rRNA target region (Figure 2).
.PNG) Figure 2. Agarose gel electrophoresis (1% agarose, ethidium bromide staining) of the PCR products amplified from the ITS1-rRNA gene. Lane L: 100 bp DNA ladder; Lane 1: positive control (Cryptosporidium parvum) ; Lane 2: PCR-positive water sample; Lane 3: negative sample. |
The amplified fragment was approximately 824 bp in length, which is consistent with previously reported sizes for Cryptosporidium ITS1/SSU rRNA gene fragments.
Results of enzymatic digestion with AluI and RsaI enzymes
RsaI digestion
No clear restriction fragments were detected following digestion with RsaI. The absence of visible bands is most likely due to the lack of RsaI recognition sites within the amplified ITS1/SSU rRNA region, indicating that this fragment is not cut by RsaI under standard conditions. Therefore, the undigested pattern is considered a normal finding rather than an indication of enzyme failure.
AluI digestion
Digestion with AluI produced a characteristic multi-band pattern. A prominent fragment of approximately 850 bp was observed, representing partial digestion of the original PCR product. Additional fragments of approximately 720 bp, 480 bp, 456 bp, and 410 bp were detected, confirming that AluI cleaved the target sequence at multiple restriction sites. The coexistence of a large undigested band with multiple smaller fragments is consistent with expected RFLP profiles for Cryptosporidium ITS1/SSU rRNA amplicons.
Overall, the observed AluI banding pattern supports the identification of Cryptosporidium spp. (Figure 3).
.PNG) Figure 3. SM (Sample marker)100 genetic marker (In the right). PCR-RFLP analysis ITS1 gene Band 1 to 3 on the right side of the marker cut with Alu1 restriction enzyme. Enzymes Alu1 yielded profile banding sizes of 410-457-480-760-850 bp. |
Selected PCR-positive samples were subjected to Sanger sequencing using a commercial sequencing service (Bioneer Co.). High-quality chromatograms were obtained, and the forward and reverse reads were assembled to generate a consensus sequence. Raw sequence data were inspected and edited using Chromas Lite version 2.1, and the final consensus sequence was compared with reference sequences available in the NCBI GenBank database using the BLASTn algorithm.
The nucleotide sequence generated in this study is provided in Appendix 1. BLAST analysis demonstrated strong and significant sequence identity, with an E-value of 0.0 (Figure 4). Alignment with multiple isolates of Cryptosporidium parvum showed 98.84% identity. These findings confirm that the amplified SSU rRNA fragment corresponds to C. parvum.
Phylogenetic analysis
Phylogenetic analysis was performed to evaluate the evolutionary placement of the SSU rRNA sequence obtained in this study. A distance tree was generated using the BLAST pairwise alignment tool, which grouped the sequence (Crypto-F) together with multiple reference isolates of C. parvum. No clustering with C. hominis or other Cryptosporidium species was observed.
The close association of the query sequence with C. parvum isolates, combined with the high sequence identity (98.84%) and high query coverage obtained from BLAST analysis, confirms that the sample belongs to the C. parvum lineage (Figure 5).
In conclusion, the phylogenetic findings indicate that the SSU-rRNA sequence obtained from water samples collected in Zabol and Zahedan shows clear genetic affinity to established isolates of C. parvum. As illustrated in Figure 5, the sequence clusters within the C. parvum lineage and shares conserved regions of the 18S rRNA (SSU-rRNA) gene with these reference isolates. This strong genetic similarity provides robust molecular evidence confirming that the detected organism belongs to C. parvum.
.PNG) Figure 4. BLAST output showing a strong alignment between the SSU rRNA sequence obtained in this study and reference Cryptosporidium parvum isolates, with 98.84% sequence identity and an E-value of 0.0. .PNG) Figure 5. Phylogenetic tree generated using the BLAST distance-tree method based on pairwise sequence alignments of the SSU-rRNA gene. The sequence obtained in this study (Crypto-F / Query_3448041) clustered closely with Cryptosporidium parvum isolates, confirming its identity as C. parvum. |
Discussion
Cryptosporidium spp. are major waterborne pathogens with substantial public health implications. Human and animal fecal contamination of environmental sources such as soil, water, and food constitutes a key transmission route for this protozoan, which remains one of the leading causes of diarrheal disease worldwide. The World Health Organization (WHO) has recognized Cryptosporidium as a priority pathogen for global water quality monitoring, highlighting its significance in public health surveillance and waterborne disease management (6). Therefore, the detection and molecular characterization of Cryptosporidium spp. in environmental samples are essential for understanding transmission dynamics and implementing effective control measures.
Genotyping of Cryptosporidium isolates is a critical tool for distinguishing species and genotypes and for elucidating epidemiological patterns. Due to the difficulty of culturing this parasite, an approach that is both time-consuming and susceptible to contamination, PCR-RFLP has become an important molecular method for genotyping (7). This technique differentiates species based on the digestion of PCR-amplified DNA by restriction enzymes that cleave specific nucleotide sequences. The resulting fragment patterns allow comparison with known profiles and facilitate accurate species identification (8). Variations in banding patterns may reflect genetic polymorphisms, particularly in regions where multiple species or genotypes co-circulate.
The epidemiology of Cryptosporidium is further complicated by the coexistence of multiple transmission pathways, including waterborne, foodborne, and zoonotic routes. Genotyping plays an essential role in identifying infection sources and understanding transmission mechanisms by revealing the genetic diversity within Cryptosporidium populations. Previous studies indicate that C. parvum and C. hominis are responsible for the majority of human infections, with C. parvum accounting for approximately 90% of cases in many regions. To date, more than 300 human cases have been genotyped, revealing at least nine infective Cryptosporidium species, including C. andersoni, C. felis, and C. meleagridis, among others (9).
This diversity underscores the need for continuous monitoring and molecular surveillance.
Globally, water contamination remains a major public health concern due to the exceptional resistance of Cryptosporidium oocysts to routine water-treatment processes. Oocysts are highly resistant to chlorine and several commonly used disinfectants, complicating their removal from water supplies and reinforcing the need for sensitive detection methods (10). Given the increasing threat of waterborne diseases worldwide, the development and application of rapid molecular diagnostic techniques have become priorities in water-quality monitoring programs (11).
In the present study, 180 water samples were examined, of which 35 tested positive for Cryptosporidium oocysts. The primers used successfully amplified an approximately 800-900 bp fragment of the SSU rRNA gene, which is consistent with previous studies targeting this region. Microscopic examination and staining techniques initially confirmed the presence of oocysts. Subsequently, subtyping was performed using AluI and RsaI restriction enzymes, followed by DNA sequencing to further characterize the isolates. Consistent with expectations for the SSU rRNA gene, digestion with AluI produced clear fragment patterns suitable for genotyping, whereas digestion with RsaI did not yield visible fragments, likely due to the absence of an appropriate recognition site within the amplified region rather than a technical error.
The combined application of microscopy, staining, RFLP analysis with AluI, and sequencing revealed notable genetic polymorphism among Cryptosporidium isolates from the Zabol and Zahedan districts. This observation indicates substantial genetic diversity among circulating isolates in these geographic regions. The integration of molecular and conventional detection techniques allowed robust subtyping and comparison of isolates, thereby providing a comprehensive picture of the genotypic profiles present in the study area.
The phylogenetic tree (Figure 5), constructed using SSU rRNA sequences, demonstrates distinct clustering of C. parvum isolates according to geographic origin and host specificity. Similar findings have been reported previously, showing that C. parvum isolates from different regions often form independent clades, suggesting geographic structuring and restricted gene flow (12,13). These genetic markers are essential for tracking outbreaks and distinguishing between zoonotic and anthroponotic transmission routes. Certain C. parvum genotypes may exhibit host adaptation, emphasizing the importance of recognizing host-associated variants for public health surveillance.
Genetic analyses of C. parvum also provide insights into potential environmental and animal reservoirs, helping to identify sources of contamination in water systems. Such information is vital for designing targeted interventions, enhancing disease surveillance, and improving our understanding of transmission routes. Molecular epidemiology using SSU rRNA markers therefore plays a crucial role in monitoring and controlling cryptosporidiosis.
Worldwide, the prevalence of C. parvum in diverse water sources continues to present major challenges for water safety. Studies have reported prevalence rates of 46.9% in wastewater, 45.3% in surface water, and 31.6% in untreated raw water (14). These findings reflect the persistence of Cryptosporidium oocysts in the environment and their resistance to conventional treatment methods, particularly chlorine-based disinfection (10). In Iran, C. parvum is the dominant species, accounting for 83.3% of human cases, followed by C. hominis with 8.3% (6). In livestock, particularly cattle, C. parvum is also the predominant species, representing approximately 36% of infections, with genotypes similar to those identified in countries such as France (15,16). These data further highlight the importance of zoonotic transmission in regional epidemiology. Globally, C. parvum remains one of the most frequently reported pathogens in waterborne outbreaks, with contaminated water serving as a major transmission route (10).
Conclusion
In conclusion, the persistence of C. parvum in water sources in Iran remains a considerable public health challenge. The detection and molecular characterization of C. parvum from environmental water samples provide essential information for assessing the risk of waterborne cryptosporidiosis. Addressing contamination requires improvements in water-treatment infrastructure, enhanced monitoring systems, and effective intervention strategies. The findings of this study, particularly the genotypic and phylogenetic characterization of isolates, contribute valuable insights into the transmission dynamics of Cryptosporidium and underscore the importance of molecular tools in surveillance and outbreak prevention.
Acknowledgement
The authors would like to thank all the staff of the Department of Parasitology and the Vice Chancellor for Research and Technology of the University of Medical Sciences.
Funding Sources
Not applicable.
Ethical Statement
This study involved harvested oocysts from water and did not include unethical practices. It was approved by the University Research and Ethics Committee (Code: http://ethics.research.ac.ir/IR.ZBMU.RECRYPTOSPORIDIUM1398.159), and all procedures followed institutional guidelines for the care and use of humans and animals.
All participants in this study provided informed consent before participation. They were fully informed about the study’s purpose, procedures, potential risks, and benefits.
This consent process ensured that participants were aware of and agreed to the terms of their involvement in the study.
Conflicts of Interest
There are no conflicts of interest.
Author Contributions
Mansour Dabirzadeh supervised and designed the study. Reza Shahraki and Mahdi Khoshsima Shahraki collected and analyzed the samples. Mohammadreza Beheshtizadeh performed the molecular analysis and approved the final manuscript.
Data Availability Statement
Upon reasonable request, the corresponding author will share the datasets of this study.
Use of Artificial Intelligence
No artificial intelligence tools were used in the preparation of this manuscript.
Research Article: Original Paper |
Subject:
Parasitology
Received: 2024/12/14 | Accepted: 2025/03/1 | Published: 2026/04/28 | ePublished: 2026/04/28
Received: 2024/12/14 | Accepted: 2025/03/1 | Published: 2026/04/28 | ePublished: 2026/04/28
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