Molecular Epidemiology of Cryptosporidium spp. in an Agricultural Area of Northern Vietnam: A Community Survey

Cryptosporidium spp. is a protozoan parasite with worldwide distribution that causes cryptosporidiosis in humans and other animals. In the countryside of northern Vietnam, where free-roaming livestock are widespread, cryptosporidiosis is an important zoonotic disease. However, there have been few studies of cryptosporidiosis in Southeast Asia from the perspective of zoonotic disease epidemiology. The purpose of this study was to investigate the occurrence of Cryptosporidium infection in both humans and animals and to gain an awareness of the potential threat posed by this zoonotic infection in northern Vietnam. We conducted a community survey to collect information about cases of diarrhea in an agricultural area of northern Vietnam. For this study, a total of 2715 samples (2120 human diarrheal samples, 471 non-diarrheal human samples, and 124 animal stool samples) were collected. A direct immunouorescence assay (DFA) was used to detect Cryptosporidium spp. oocysts in concentrated stool samples by observation under a uorescent microscope. DNA extraction, PCR amplication of the three genes (COWP, SSU-rRNA, and GP60), and sequencing analysis were performed to identify Cryptosporidium spp.


Abstract Background
Cryptosporidium spp. is a protozoan parasite with worldwide distribution that causes cryptosporidiosis in humans and other animals. In the countryside of northern Vietnam, where free-roaming livestock are widespread, cryptosporidiosis is an important zoonotic disease. However, there have been few studies of cryptosporidiosis in Southeast Asia from the perspective of zoonotic disease epidemiology. The purpose of this study was to investigate the occurrence of Cryptosporidium infection in both humans and animals and to gain an awareness of the potential threat posed by this zoonotic infection in northern Vietnam.

Methods
We conducted a community survey to collect information about cases of diarrhea in an agricultural area of northern Vietnam. For this study, a total of 2715 samples (2120 human diarrheal samples, 471 non-diarrheal human samples, and 124 animal stool samples) were collected. A direct immuno uorescence assay (DFA) was used to detect Cryptosporidium spp. oocysts in concentrated stool samples by observation under a uorescent microscope. DNA extraction, PCR ampli cation of the three genes (COWP, SSU-rRNA, and GP60), and sequencing analysis were performed to identify Cryptosporidium spp.

Results
Of 2715 samples, 15 samples (10 diarrheal samples, 2 non-diarrheal samples, and 3 animal stool samples) tested positive by PCR for the COWP gene. Three species of Cryptosporidium spp. were detected; C. canis (from six human diarrheal samples, two human non-diarrheal samples, and one dog sample); C. hominis (from four human diarrheal samples); and C. suis (from two pig samples). In terms of C. hominis, the GP60 subtype IeA12G3T3 was detected in all four human diarrheal samples.

Conclusions
Although the number of positive samples was very small, our epidemiological data showed that the emerging pattern of each of the three species (C. canis, C. hominis, and C. suis) was different at this study site. For example, zoonotic transmission of C. canis, between dogs and humans was suspected. Further studies are needed to assess the risk of oocyst contamination in the wider environment, including water, in this study area. Background Worldwide, Cryptosporidium is considered an important protozoan parasite that causes gastroenteritis in a wide range of animals, including humans [1][2][3][4]. Cryptosporidiosis can lead to severe problems in immunocompromised or young hosts, in both animals and humans [5]. In the livestock farming industry, cryptosporidiosis can cause great economic losses due to weight loss and delayed growth in young animals and decreased production in adult animals [1]. In humans, infection with Cryptosporidium spp. can mean not only severe diarrhea but also death in patients with HIV/AIDS, patients who have received an organ transplant, and patients on immunosuppressants [6]. So far, no effective vaccines are available to prevent cryptosporidiosis in humans or in livestock [7]. Prevention and control measures are therefore essential for the protection of vulnerable groups, such as young children and severely immunocompromised individuals, as there are only a few safe and effective therapeutic options available.
In countryside of Vietnam, where livestock live closely with humans, Cryptosporidium transmission to humans can easily occurs via the interaction between animals and humans [8]. People can become infected by ingesting infective Cryptosporidium oocysts through the fecal-oral route, including directly from infected persons (human-to-human) or animals (animal-to-human), or indirectly through the consumption of contaminated drinking water or food [1]. A distinctive pattern of transmission in the countryside of Vietnam may be environmental contamination through infected manure, which is applied to the land. It has been reported that Asia has the highest oocyst load from livestock manure compared with the loads seen on other continents [9]. One of the reasons may be climate. For example, Cryptosporidium oocysts can be maintained for a long time in Vietnam because the climate is warm and humid in the summertime, which is suitable for the survival of oocysts [10]. Also, Vietnam has a rainy season with heavy rainfall, which may facilitate Cryptosporidium oocysts spreading in the environment, leading to infections in humans and animals [11]. Previous studies conducted in Southeast Asia, including in Vietnam, have shown that there is the possibility of human Cryptosporidium infections originating from contaminated environmental sources [12]. To prevent and control cryptosporidiosis in agricultural areas, it is important to improve our understanding of environmental transmission routes of Cryptosporidium. An integrated genotyping approach would help in identifying sources of infection and routes of transmission under Vietnamese agricultural conditions [8].
Currently, many species and genotypes of Cryptosporidium spp. have been described, in a wide range of animals, livestock, and wild animals [2,3,5,12]. It was previously thought that each species of Cryptosporidium had a limited host range and was able to infect only a single host or closely related host species [3]. However, according to the latest data it is likely that each species possesses a very broad range of hosts [3]. Molecular epidemiological surveillance-based studies have revealed an expanded host range of each Cryptosporidium spp. and their geographic distribution [13]. However, there is a scarcity of information about the molecular epidemiology of Cryptosporidium spp. in Vietnam. Therefore, our objective was to investigate the occurrence of Cryptosporidium infections and the potential for transmission of Cryptosporidium spp. between animals and humans in Vietnam.

Methods
Sampling (diarrheal sample collection from humans, non-diarrheal sample collection from humans, and stool sample collection from animals) The study area (12 km 2 ) was a typical agricultural area in northern Vietnam, located about 75 km southeast of Hanoi. A total 2715 samples, comprising both human and animal stools, were collected using three sampling Cross-sectional collection of animal stool samples (August to October 2015) yield 124 animal stool samples from animals (without clinical signs) present in this study area, such as buffalos, dairy and beef cattle, pigs, dogs, and monkey and boar (The detailed information is in Iwashita et al. submitted to Parasite Epidemiol.

Control.).
Procedure of the detection of Cryptosporidium spp.
For all 2715 samples (2120 human diarrheal samples, 471 human non-diarrheal samples, and 124 animal stool samples), the formalin-ether sedimentation technique (406th Medical General Laboratory; MGL) was used to concentrate protozoal oocysts in stool samples [14]. A direct Immuno uorescence assay (DFA) using antibodies tagged with the uorescent markers, DyLight488 (ARK Fluor Ab C/G-DyLight488, ARK Resource Co., Ltd.) was applied to detect oocysts of Cryptosporidium spp. in the concentrated stool samples under a uorescent microscope (Eclipse 90i, Nikon Instruments Inc.) [14]. Using antibodies tagged with uorescent markers, DyLight488 is able to target not only Cryptosporidium spp. but also Giardia spp. Detection by DFA was simultaneously conducted in previous study to detect Giardia spp. using the same 2715 samples (Iwashita et al. submitted to Parasite Epidemiol. Control.). In terms of human stool samples (2120 diarrheal samples and 471 non-diarrheal samples), any samples positive for Cryptosporidium spp. by DFA had their DNA extracted for PCR and sequencing analysis. All 124 animal stool samples, including those positive or negative for Cryptosporidium spp. by DFA, had DNA extracted for PCR and sequencing analysis.

DNA extraction
Stool samples concentrated using the MGL technique were washed twice with sterile water and then ten freezethaw cycles (freezing in liquid nitrogen for 5 min and heating at 95℃ for 5 min) were performed to disrupt the oocysts. DNA was then extracted using a PowerSoil DNA Isolation Kit (MoBio Laboratories Inc., Carlsbad, Calfolnia) following the manufacturer's instructions and stored at -20℃ prior to use. To avoid crosscontamination between animal and human samples, both samples was separately treated. PCR analysis of the three genes (COWP, SSU-rRNA, GP60) Fragment of the oocyst wall protein (COWP) gene [15] and the small-subunit ribosomal RNA (SSU-rRNA) gene [16,17] were ampli ed using nested PCR protocols for the identi cation of Cryptosporidium species (Table 1). Fragments of the 60-kDa glycoprotein (GP60) gene was also ampli ed using nested-PCR protocols [18,19] for further subtyping of Cryptosporidium species, especially C. homins and C. parvum (Table 1). PCR was performed using a MyCycler thermal cycler (Bio-Rad, Hercules, USA). Each 25 µL reaction mixture contained GoTaq Green Master Mix (containing Go Taq® DNA Polymerase, dNTP mixture, Green Go Taq Reaction Buffer, MgCl2; Promega) with 5% dimethyl sulfoxide (Sigma-Aldrich, USA) and 0.4 mg/ml BSA (Sigma-Aldrich, USA).
Two types of nested-PCR protocols were used to prepare the SSU-rRNA [16,17] and GP60 genes [18,19] ( Table 1). All samples were ampli ed using three nested-PCR protocols targeting the COWP [15] and SSU-rRNA genes [16,17] (Table 1) and all second PCR products were evaluated by a 1.5% agarose gel electrophoresis. In terms of the COWP gene [15], both the rst and second PCR products were additionally evaluated to identify longer sequences. For all the PCR reactions, Cryptosporidium-positive DNA and distilled water were used as positive and negative controls, respectively. Sequencing and phylogenetic analyses.
All positive PCR products were puri ed using an MonoFas DNA Puri cation Kit (GL Sciences, Tokyo, Japan) and sequenced in both directions (with forward and reverse primers) using an ABI 3730xl DNA analyzer (Applied Biosystems, Foster City, CA, USA). All sequences including SSU-rRNA, COWP and GP60 were analyzed using the BLAST program (http://blast.ncbi.nih.gov/BLAST.cgi) for homology searches. Cryptosporidium species reference sequences were obtained from GenBank to ensure accurate species/genotype and subtype identity, and reports of human cases were searched [20][21][22][23][24][25][26] (Additional le 1: Table S1). Phylogenetic analyses of the COWP and SSU-rDNA sequences of different Cryptosporidium species and genotypes were performed using MEGA (Molecular Evolutionary Genetic Analysis) 6.0 software (http://www.megasoftware.net/).

Results
A total of 2591 human stool samples (2120 human diarrheal samples and 471 human non-diarrheal samples) were screened with DFA. Of these, 77 human diarrheal samples and 11 human non-diarrheal samples were positive for Cryptosporidium spp. DNA was extracted from these samples for PCR analysis. All 124 animal stool samples, regardless of whether they were positive or negative for Cryptosporidium spp. by DFA had DNA extracted for PCR analysis (Fig. 1).
Through our sampling methods, only fteen samples (ten human diarrheal samples, two human non-diarrheal samples, and three animal stool samples) were found to be positive for Cryptosporidium spp. by PCR ( Table 2). The patterns of Cryptosporidium infection caused by C. canis, C. hominis and C. suis at our study site became clear. Cryptosporidium canis was detected from human non-diarrheal (Fig. 2) and diarrheal samples (Fig. 3), and dog stool samples (Fig. 4 ), while C. suis was only detected in pigs (Fig. 4 ). Cryptosporidium hominis was only detected in human diarrheal samples (Fig. 3). Through our prospective diarrhea sampling, four samples that were positive for C. homins were coincidentally detected from two neighboring households at the same time (Fig. 3 ). The six samples positive for C. canis, on the other hand, were sporadically detected without any obvious patterns of occurrence. The C. hominis-positive cases were aged 11, 15, 37 and 70 years (Table 2). There were no samples from children aged less than 5 years, the group most vulnerable to severe diarrhea. On the other hands, the age of C. canis-positive cases were 1, 2, 5, 6, 7, and 26 years for diarrheal samples and 2 years for the two non-diarrheal samples ( Table 2). No samples from buffalos or dairy and beef cattle were positive for Cryptosporidium spp. Primers targeting the COWP gene had a higher detection rate (15/15) than primers targeting the SSU-rRNA gene (9/15) ( Table 2). Samples in which C. canis and C. suis were identi ed by PCR using the COWP gene and/or the SSU-rRNA gene, failed to amplify any DNA using primers targeting the GP60 gene, because the GP60 primers were more speci c for C. hominis. In terms of the COWP and SSU-rRNA genes, the phylogenetic tree constructed using MEGA software is shown in Figs. 5 and 6. Bootstrap values were obtained using 1000 pseudo-replicates; those > 50% are shown on nodes. The evolutionary distances were computed using the Kimura-2 parameter method [27]. GenBank accession numbers are shown before the species names in Figs. 5 and 6 and Additional le 1: Table S1.
Molecular diagnosis using the COWP gene Good quality sequencing data of COWP gene were available for the 15 isolates. Phylogenetic analysis could identify three species of Cryptosporidium spp. (Fig. 5). Two isolates (from pig stool samples) assigned to C. suis, exhibited 100% identity with the reference sequence AF266270.1, between position 359 to 712. Four isolates (four human diarrheal samples) assigned to the C. hominis, exhibited 100% identity with the reference sequence AF266265.1, while three isolates comprised 506 bp between positions 359 to 864 and one isolate (CDS_1788) comprised 354 bp between positions 359 to 712. Nine isolates (one dog sample, six human diarrheal samples, and two human non-diarrheal samples) were assigned to C. canis. One of them exhibited 100% identity with the reference sequence AF266274.1, between 17 to 370 (Ani_140). The remaining eight isolates differed by one or two single nucleotide polymorphisms (SNPs) with it, although the ampli ed product sizes were different: CDS_263 and CDS_449 were 354 bp, while the others are 506 bp ( Table 3). The unique sequences described here have been deposited in the GenBank database under accession numbers LC503969 to LC503972 (Table 3). Details of the results using COWP gene are shown in Additional le 2: Table S2.
Molecular diagnosis using the SSU-rRNA gene Good-quality sequencing data of SSU-rRNA gene were available for the nine isolates. Phylogenetic analysis could clearly distinguish three species of Cryptosporidium spp., the same as the analysis based on the COWP gene (Fig. 5, 6). Two isolates (from pig stool samples) assigned to C. suis exhibited 100% identity with the reference sequence AB449824, between position 98 to 638. Three isolates (three human diarrheal samples) assigned to the C. hominis exhibited 100% identity with the reference sequence AF108865.1, while two isolates comprised 543 bp between positions 313 to 855 and one isolate (CDS_1788) comprised 735 bp between 241 to 975. Four isolates (one dog sample, two human non-diarrheal samples, and one human diarrheal sample) assigned to C. canis showed 100% identity with the reference sequence AB210854.1, between 312 to 846 (535 bp). Details of the results using the SSU-rRNA gene are shown in Additional le 3: Table S3.

Molecular diagnosis using the GP60 gene
In this study, all four C. hominis-positive samples using the COWP and/or SSU-rRNA genes were successfully subtyped using the GP60 gene (Table 2). One subtype from all four isolates, IeA12G3T3 was identi ed according to the scheme described by Xiao [28]. Other Cryptosporidium-positive samples using COWP and/or SSU-rRNA gene were not ampli ed with primers for the GP60 gene. Details of the results using the GP60 gene are shown in Additional le 4: Table S4.

Discussion
Although Cryptosporidium infection was not highly prevalent among human or animal samples tested from this study site, the potential for zoonotic transmission of C. canis from dogs to humans and vice versa was apparent. We found no positive samples in cattle or buffalos we tested, although zoonotic transmission from these species has been reported by researches in other areas studied, such as China [29]. Instead, in our study area, there were samples positive for Cryptosporidium in dogs, pigs and humans. Although most cases of cryptosporidiosis caused by C. canis globally have been reported in dogs, some cases have been reported in humans, according to other studies and our results [30,31]. For example, in Siem Reap, northwest Cambodia, human stool samples collected from ve patients attending the out-patient clinic or admitted to hospital were positive for C. canis [32]. The dogs at this study site were free roaming, as is commonly the case in the countryside of Vietnam, and had close relationship with humans. The infection may occur either inside or outside of households. Human and animal feces can easily contaminate the environment, including with Cryptosporidium spp., due to a lack of a sewage system at the study site.
In previous studies conducted in Vietnam and surrounding countries, Cryptosporidium infections have been identi ed in animals and/or humans using methods such as microscopic analysis, immuno uorescence, enzyme-linked immunosorbent assays, and PCR [32,33,[42][43][44][45][46][47][48][49][50][34][35][36][37][38][39][40][41]. Most studies applied molecular methods to identify species and genotypes of Cryptosporidium (Table 4). In Vietnam and surrounding countries or areas (e.g., Laos, Cambodia, the southern provinces of China), the following species of Cryptosporidium were detected (with host animals in parentheses): C. andersoni (dairy cattle) [46]; C. bovis (beef cattle) [33]; C. canis (human) [32]; C. hominis (human, monkey) [32,47]; C. meleagridis (human) [32]; C. muris (monkey) [47]; C. parvum (human) [32]; C. ryanae (cattle) [33]; C. scrofarum (pig) [49]; C. suis (pig, human, environmental sample) [32,35,38]; C. ubiquitum (human) [32]; Cryptosporidium avian genotype II (ostrich) [37]; Cryptosporidium bat genotype (bat) [45]; and Cryptosporidium pig genotype II (pig) [35] (Table 4). In Cambodia, Moore et al. detected many species of Cryptosporidium from symptomatic children in a hospital [32]. The species reported in their study were C. canis, C. hominis, C. meleagridis, C. parvum, and C. suis, all of which have zoonotic potential [31]. Cryptosporidium hominis and C. canis were detected in human samples collected at our study site, but C. suis was not. Cryptosporidium canis and C. suis were detected in animal samples collected at our study site, but C. hominis was not. All species detected in this study area could easily contaminate the environment and/or water and/or food through the excretion of stools that contain oocysts, and there is always a risk of a spillover of disease transmission to humans. Table 4 gave us the opportunity to estimate the potential for transmissions between animals and humans by different species of Cryptosporidium in Vietnam and surrounding countries. It should be noted that the majority of patients with HIV in the studies listed in Table 4 were infected with C. hominis and C. parvum. This is consistent with a previous study of children infected with HIV in Kenya [51,52]. Although C. hominis is widely considered to be a human-speci c Cryptosporidium species, it has increasingly been reported in animals from all over the world except Vietnam [53][54][55].
Cryptosporidium hominis is known to cause severe to moderate infections in humans [56]. More than ten subtypes of C. hominis have been identi ed, based on sequence analysis of the GP60 gene [30]. It has been reported that GP60 analysis has discriminatory power to determine transmission dynamics and source of infection [31].
In our study, only one subtype, IeA12G3T3, was detected from all human diarrheal samples identi ed as having C. hominis based on COWP and/or SSU-rRNA gene analysis. This IeA12G3T3 subtype has also been identi ed in various samples in other countries, such as stool specimens from individuals with HIV infection in Jamaica [57], water samples in Shanghai, China [58], stool specimens from Filipino immigrants in Qatar [59] and stool specimens from an immunocompromised patient in Slovakia [60]. Fortunately, this subtype has never been found to be responsible for an outbreak, and its infectivity or degree of virulence have yet to be reported. This is different from a notorious subtype, such as IbA10G2, which is widely distributed and was responsible for outbreaks in Europe, Australia, and the USA [31]. Following our investigation using GP60 subtyping for C. hominis, it became clear that one subtype (IeA12G3T3) was dominant at our study site. We suspect that the transmission route might have been the same in all cases, occurring in neighboring households.
Our study has several limitations. First, the number of positive Cryptosporidium spp. cases was very small, comprising ten human diarrheal samples, two human non-diarrheal samples, and three animal stool samples.
As the different sampling methods and timing for collecting these three kinds of samples could not be perfectly matched, it was very di cult to accomplish our ambitious goal to determine transmission routes using these samples.
Second, we are concerned that the PCR analyses used in our study might have underestimated the prevalence of Cryptosporidium spp. This could be due to one or more of the following reasons: DNA degradation [61], insu cient DNA [37], inhibition of PCR [62], and prolonged storage of the stool samples [63]. We were also concerned about cross-reactions with non-target organisms, such as algae, when we apply DFA [64], meaning we could not count the number of oocysts. False positive microscopy results were also not ruled out. Although our study objectives did not include estimating the exact prevalence or incident of cryptosporidiosis, such low detection of Cryptosporidium spp, was likely to miss several transmission routes and affect the objective of this study. In terms of storage of the samples, it was di cult to exactly match the conditions of storage time until DNA extraction for all samples collected over three years. As we followed the experimental process described in Fig. 1, the time until DNA extraction varied depending on the time to complete detection by DFA using microscopy, which was time consuming and laborious. In fact, all our stool samples were stored in the suitable condition for prolonged storage, according to the conditions noted by Jongwutiwes et al. [65]. In addition, each primer has a different detection rate when identifying Cryptosporidium spp. The primers targeting the COWP gene have higher detection rates than the primers targeting the SSU-rRNA region. The GP60 gene is usually used as a marker for the detection of C. parvum and C. hominis [28]. Therefore, we did not succeed using PCR to identify GP60 for the samples already detected as C. suis and C. canis by COWP and RRU-rRNA. Moreover, we did not detect any C. parvum-positive samples. Generally, in agricultural area, the risk of infection may be greater for larger livestock, such as buffalos and cattle, which are the main hosts of C. parvum. We suspected that zoonotic transmission was occurring from cattle and buffalos to humans. However, false negative PCR result was not ruled out, as mentioned above. In agricultural area of Vietnam, where there are many freeroaming livestock and no sewage systems, water is considered to be an important mechanism in the transmission of Cryptosporidium spp. If contamination of water from a particular population of animals is suspected, investigation of the water itself is needed to verify the risk of Cryptosporidium spp. to public health. In fact, contamination of water with Cryptosporidium spp. from pig farms at our study site might represent a growing problem unless sanitary conditions are improved.
Third, it is very di cult to determine the pathogenicity and virulence of Cryptosporidium. In terms of human cases at our study site, we suspect that C. hominis was more pathogenic than C. canis, even though we could not address this risk through our study design. Four cases of C. hominis, of the same subtype, were detected at the same time from human diarrheal samples. On the other hand, C. canis were detected sporadically from many kinds of samples, such as human diarrheal and non-diarrheal samples and dog stool samples, which were spread throughout our study site. The pattern of C. hominis and C. canis throughout our study were quite different and we suspect that Cryptosporidium spp. virulence or pathogenicity also differs. There have been reports of differences in clinical manifestations among Cryptosporidium species and subtypes [66][67][68][69][70][71]. In adults and children infected with HIV, for example, it has been suggested that C. hominis is mainly associated with diarrhea, nausea, vomiting, and malaise, whereas C. parvum, C. meleagridis, C. canis, and C. felis are associated with diarrhea only [66,67]. Another report, of the medium-to long-term impact of cryptosporidiosis, suggested that C. hominis infection is mainly associated with fatigue and abdominal pain greater than that seen with C. parvum infection [69,70]. In addition, different subtypes of C. hominis have been linked to variable clinical outcomes [71]. However, the etiology of diarrhea itself is very complicated, and we could not rule out pathogens except Cryptosporidium spp. as the cause of diarrhea. In fact, there was some additional information about other diarrheal pathogens from the human stool samples used in this study. These other pathogens were as follows: rotavirus, norovirus GI and norovirus GII, Aeromonas spp., Campylobacter spp., Clostridium di cile, Enterotoxigenic Escherichia coli, enteroaggregative E coli, Salmonella spp., Shigella spp., Vibrio spp., Giardia spp., Entamoeba histolytica. The presence of these pathogens was investigated using the same samples already detected Cryptosporidium spp. in this study. Coincidentally, four samples which were positive for C. hominis were also positive for enteroaggregative E coli (unpublished data). In cases of mixed infection with various diarrheal pathogens, it is very di cult to identify the etiology of diarrhea.
To control cryptosporidiosis, a "One Health approach" can be applied; this is a collaborative approach among public and veterinary health professionals. Although the distribution patterns of Cryptosporidium spp. vary from country to country and even from one region to another, we believe that even our small quantity of local molecular epidemiological data could contribute to improve the knowledge around the transmission of this parasite. In Vietnam, there have still been few studies focused on both public and veterinary health, as was the case with our study. Although our results are limited to one small area, our information could form part of a network of molecular-based surveillance systems. We believe that the accumulation of each local reports has a possibility to help reduce disease incidence in country level.

Conclusions
As cryptosporidiosis takes the largest toll on the health of vulnerable populations, such as patients who are immunode cient and young children living in low-income settings, it is important to prevent infection by strictly minimizing the number of oocysts in the environment. Although the detection rate of Cryptosporidium spp. in our study was not high, it was certain that at least three species of Cryptosporidium were present. Particularly with C. canis, zoonotic transmission between dogs and humans was suspected. Cryptosporidium spp. is an environmentally ubiquitous protozoan parasite. Our study only used stool samples only, although Cryptosporidium spp. can also be found in water and environmental samples. As our study site is a typical agricultural area where there are many free-roaming livestock and no sewage systems, environmental surveillance would be helpful in avoiding outbreaks of cryptosporidiosis. Not only animal stools but also human stools could contaminate water with Cryptosporidium spp. at our study site, due to no sewage systems. This study was approved by the Ethical Committee of the Graduate School of International Health and Development, Nagasaki University and the Institutional Review Board of NIHE in Vietnam. Written informed consent was obtained from participants, who were the head of the household for each household. A verbal consent statement was obtained from livestock owners prior to the collection of fecal samples from their private land.
As a major consideration, no patients who were immunocompromised or had AIDS/HIV were included in the study. Although the participants were noti ed and understood that the test results of their samples would not be available to assist in any treatment, they would be monitored for clinical manifestations of Cryptosporidium infection and other diarrheal symptoms trained health workers. The participants were advised to report any abnormal health status to the local physician in the Hien Khanh Commune Health Station, which was staffed with three physicians, one pharmacist, one midwife, and one nurse. If any manifestations of cryptosporidiosis occurred, all participants with diarrhea were treated at the community health center, according to Ministry of Health guidelines. In cases of severe diarrhea, participants were referred to the district or the provincial hospital for appropriate laboratory testing and treatment.

Consent for publication
Not applicable Availability of data and materials Not applicable

Competing interests
The authors declare that they have no competing interests.