Evidence of Cryptosporidium parvum and Blastocystis hominis transmission from sewage treated to the Cholga mussel

doi:10.21203/rs.3.rs-3195491/v1

Cryptosporidium parvum and Blastocystis hominis are foodborne parasites known for causing diarrhea. They accumulate in mussels grown on contaminated water bodies, due to the discharge of sewage treated from sewage treatment plants (STP). Despite, they are not included in the water microbiology control or monitoring. The goal of this work was to evaluate the transmission of C. parvum. and B. hominis from treated sewage (disinfected by chlorination) to Cholga mussels. Cholga from commercial stores and a sewage treated sample were analyzed. Cryptosporidium spp. was identified by ziehl-neelsen-staining (ZNS) and C. parvum by direct-immunofluorescence-assay (IFA) from ZNS positive samples. B. hominis was identified by PCR using locus SSU rDNA. C. parvum and B. hominis were found in 40% and 45% of Cholga samples, respectively, and both were identified in the sewage treated. SSU rDNA gene alignment from Cholga and sewage treated showed 89% of similarity, indicating that could be the same parasite in both samples. This is the first evidence from treated sewage to Cholga transmission of these parasites in Chile suggesting an environmental contamination with high human risk. Sewage treated microbiological analysis should include these parasites and a combined disinfection systems should be considered to treat sewage when are identified.

Foodborne

sewage treatment plant

parasite

rural zone

fecal contamination

Cryptosporidium parvum and Blastocystis hominis were analyzed in Cholga and sewage treated.
C. parvum and B. hominis were detected in 40% and 45% of Cholga, respectively.
Both parasites were detected in the sewage treated analyzed.
There is a transmission of C. parvum and B. hominis from the sewage treated to the Cholga mussel.

Cryptosporidium parvum and Blastocystis hominis are two parasites transmitted by fecal contamination (Karanis et al., 2007). They can cause intestinal diseases in people with diverse degrees of severity and chronicity (Torgerson et al., 2015). In some cases, they can cause death in people due to severe dehydration (Zahedi and Ryan, 2020).

Cryptosporidium spp. and B. hominis are zoonotic microorganisms, transmitted by fecalism, resist to disinfectants and environmental conditions. (Martín-Escolano et al., 2023). Thus, persist a long time in the environment (Zahedi and Ryan, 2020). They are recognized as waterborne, since they are transmitted mainly through water contaminated with feces (WHO, 2016). The 63% protozoan water outbreak are produced by Cryptosporidium spp. and 8.1% by B. hominis (Efstratiou et al., 2017).

One of the sources of water contamination are sewage treatment plants. This is since the envelope of these parasites presents resistance to certain disinfection treatments, producing differences in the elimination efficiencies. (Nasser, 2016; Suarez et al., 2022). It has been estimated that the prevalence of Cryptosporidium spp. in sewage treated and raw sewage it was 25.7% and 40.1%, respectively (Darei et al., 2021). Therefore, there is a potential risk of transmission of parasite through the discharged sewage treated (WHO, 2016). Australia, the United States and New Zealand, monitoring programs for Cryptosporidium spp. in the discharged sewage treated (King et al., 2017; Petterson et al., 2021).

These programs seek to have preventive control measures in the water treatment processes that allow reducing possible infectious outbreaks in the population avoiding environmental contamination. (King et al., 2017; Zahedi et al., 2018; Petterson et al., 2021).

In Chile, no outbreaks related to these parasites have been reported, but they are the most frequent parasites causing diarrhea in children under five years old (Departamento de Epidemiología, 2018). In addition, Concepción City (Chile) has an estimated prevalence of B. hominis of 74.9% (Cortes et al., 2017). Therefore, its detection in the environment is necessary since it can reach water sources (eg.: river, lakes, and sea), and irrigation systems contaminating vegetables, fruits and seafood. (Zahedi and Ryan, 2020). Moreover, the current regulations for discharges of treated sewage to bodies of surface water consider only the evaluation of fecal or thermotolerant coliforms (Decreto Supremo 90, 2000).

In Chile, one of the main seafood consumed are bivalves (11.9 kg per capita per year), like Cholga mussel, which enhance the gastronomic tourism and economy from different rural zone (Servicio Nacional de Pesca y Acuicultura, 2021).

The Cholga, like other mussels, tends to accumulate diverse type of contaminants, such as parasites, due to their feed system (filtration of nutrients in water) (Giangaspero et al., 2019; Li et al., 2023). In this context, Cryptosporidium spp. was detected in different species of mussel with a prevalence of 26.7 to 38.9% (Mariné Oliveira et al., 2016), and B. hominis in 5.1% (Słodkowicz-Kowalska et al., 2015).

Due to the high prevalence in Chile of these parasites, it is necessary to evaluate and monitor C. parvum and B. hominis in the environment, especially, on extractive zones of bivalves where STP sewage treated is discharged. Moreover, these parasites are not included in the effluent microbiological analysis. Therefore, the objective of this research was to evaluate the transmission of C. parvum. and B. hominis from sewage treated (disinfected by chlorination) to Cholga mussels.

2.1 Parasite in Mussel

2.1.1 Sampling

A total of 73 Cholga mussels were obtained from three commercial stores (CS-1, CS-2, and CS-3) located on a rural cove ubicated in Concepcion Bay, 28.6 km from the city of Concepción, Chile. The samples were transported in cold to the Parasitology Laboratory (University of Concepcion). The identification of Cholga mussel (Aulacomya ater species) was based on their morphological characteristics (Aldea and Valdovinos, 2005). These mussels were randomly grouped obtaining 5, 7, and 8 groups from commercial stores (CS)CS-1, CS-2, and CS-3, respectively.

2.1.2 Processing

The Cholga were washed externally with sterile distilled water. Valves were removed and the hepatopancreas and gills were extracted (Cazeaux et al., 2022). Both organs were homogenized in phosphate buffer saline (PBS) with a porcelain mortar and pestle. The homogenized organs were filtered through a strainer. The filtrate was allowed to settle in a 50 mL tube (12 h at 4 °C) and the sediment was dissolved in 70% (v/v) of ethanol and stored at 4 °C for parasite detection.

2.2 Parasite in treated sewage

2.2.1 Sampling

Twelve liters of the sewage treated were taken from the disinfection effluent. The sewage treated was transported to the parasitology laboratory (University of Concepcion) in sterile glass bottles with screw caps at 4 °C. In supplementary material S1 is described the sewage treatment plant (STP).

2.2.2 Processing

For the parasite extraction, the sewage treated was filtered through a cellulose membrane (3.0 µm pore size). A volume of 25 mL PBS with 1% of Tween (PBS-T) was added to the membrane, and subjected to ultrasound extraction (40KHz ultrasonic bath, 10 min at 25 °C) to release the parasite. Then, the solution was centrifuged (1500xg, 15 min at 4 °C). The supernatant was discarded, and the pellet was divided into two equal parts to detect C. parvum. and B. hominis.

2.3 Detection of C. parvum

The detection of C. parvum oocysts in Cholga samples was performed by the modified ZNS method (Mead and Arrowood, 2020) combined with an immunofluorescence assay (IFA) according to Giangaspero et al. (2005). Only positive sample for ZNS were analyzed with the IFA. For the detection by IFA, anti-Cryptosporidium parvum antibody conjugated to FITC (J27E, from Thermofisher, USA) was used according to the manufacturer's instructions. An inverted fluorescence microscope was used at 40x magnification (Olympus, IX81) to visualize C. parvum oocysts detected by IFA, and the images were processed with the Cell-IR software version 2.0.

In the case of the sewage treated, before using ZNS and IFA techniques, the Cryptosporidium spp. oocysts were isolated by immunomagnetic separation (IMS) according to the manufacturer's instructions (Dynabeads^TManti-Cryptosporidium kit from Thermofisher, USA).

2.4 Detection of B. hominis

2.4.1 Conventional PCR

The SSU rDNA gene was used to identify B. hominis by conventional PCR according to Böhm-Gloning et al. (1997) in the sewage treated and Cholga samples. The genomic DNA extraction was performed with the DNeasy Powersoil® DNA extraction kit (Qiagen, USA) according to the manufacturer's instructions. The conventional PCR was performed using the DreamTaq DNA polymerase (Thermofisher, USA). The PCR conditions were as follows: 95 °C for 4 min; 40 cycles of 95 °C for 30 sec, 54 °C for 30 sec, and 72°C for 30 sec; and a final extension of 72 °C for 5 min. The PCR products (̴ 500 pb) were visualized in electrophoresis agarose gel (1.5%).

2.4.2 PCR product Sequencing

The PCR products were cut off from the agarose gel and sent to the AUSTRAL-omics (Universidad Austral, Chile) laboratory for agarose DNA extraction and sequencing. The sequences were analyzed in the Bioedit Sequence Alignment Editor 7.05.3 program (Hall, 1999). A BLASTn was performed to identify and compare the B. hominis sequences with the NCBI database of sewage treated and Cholga samples.

2.5 Physicochemical and microbiological characterization of the raw sewage and sewage treated

The raw sewage and sewage treated were physiochemically and microbiologically characterized according to the Standard Methods protocols (Baird et al., 2017). For this, 200 ml of raw or sewage treated was taken for analysis. In both samples were determined in situ the temperature, pH, and turbidity parameters with an OAKTON multiparameter meter (model PC650-4800485). Also, only for the effluent, was determined the residual chlorine concentration through the Checker®HC colorimetric meter for residual chlorine (model HI701). To determine the parameters of total suspended solids (TSS), volatile suspended solids (VSS) and chemical oxygen demand (COD), the samples were transported in cold to the laboratory of the Environmental Engineering and Biotechnology Group (GIBA) of the University of Concepción, where they were processed.

For the microbiological quantification of total and fecal colfiorms a volume of 100 ml of raw sewage and sewage treated samples were transported in cold in less than 2 hours to the laboratory Total and fecal coliforms were determined by the most probable number method. The samples were cultured in aerobic lauryl sulfate lactose medium at 35°C for 18h.

3.1 C. parvum and B. hominis in Cholga

Table 1 and Figure 1 show the results of the detection of C. parvum and B. hominis from Cholga sample, which were acquired from three different Commercial Stores on the same rural zone.

Cryptosporidium spp. was detected in a 65% (13 groups) of Cholga by the ZNT and then 40% (8 groups) correspond to the zoonotic Cryptosporidium parvum specie detected by IFA.

B. hominis SSU rDNA gene was detected in CS-2 and CS-3 stores (Table 1). For CS-2, from a total of seven groups of samples, four were positive. In the CS-3, from eight sample groups analyzed, five were positive. Overall, 45% (9 groups) of Cholga sample groups were positive to B. hominis. The sequence analysis by BLASTn match with high identity (99%) to the sequence deposited in GenBank (KU719568.1) confirming B. hominis as the identified parasite.

3.2 C. parvum and B. hominis in sewage treated

To determine the source of fecal contamination, the sewage treated of a STP from the rural zone was analyzed. This effluent is discharged into a rain channel up to the beach near to the extractive Cholga zone (Figure S1).

The physicochemical and microbiological (fecal coliforms) parameters of this sewage treated sample (12 L) agreed with the Chilean discharge norm (Table 2; Decreto Supremo 90). The sewage treated was positive to Cryptosporidium spp. by ZNS. In addition, the IFA positivity indicates that the zoonotic C. parvum species was present in the sewage treated.

Respect to B. hominis, the SSU rDNA gene was positive in the analyzed sewage treated by PCR. The PCR product analyzed by BLASTn match with high identity (89%) to the deposited in GenBank (KM213462.1) confirming B. hominis as the detected parasite. Moreover, the sequence alignment between SSU rDNA PCR product from Cholga and sewage treated presented 88.9% of identity (E-value = 2x10^-132) indicating that could be the same parasite in sewage treated and Cholga (Figure 2).

The result indicates that this rural zone presents parasite in the STP sewage treated that is discharged to the beach near to the Cholga extractive zone. Therefore, these parasites are presented in the sea water reaching the Cholga mussels, which are commercialized.

C. parvum. and B. hominis are foodborne that cause intestinal disease in the population, mainly acute and chronic diarrhea, when food is not well cooked or sanitized (Badparva and Kheirandish, 2020; Bouzid et al., 2013). Nevertheless, the source of transmission to seafood, such as Cholga mussels, has not been reported in Chile. In this context, these parasites have been detected in sewage treated in worldwide (WHO, 2016), which could represent a contamination source to Cholga and consequently to humans.

In this work, both parasites were detected in Cholga. This mussel can concentrate microorganism from the seawater due to their filtration feeding process (Ben-Horin et al., 2015), but they can depurate them from their system when the water body is without or in low levels of these parasites (Willis et al., 2013; Ben-Horin et al., 2015). Nevertheless, our results showed high presence of these parasite in Cholga indicating water contamination. In addition, the climate change generated in the bay of Concepcion a low renew rate of sea water due to a change in the marine currents (González-Saldía et al., 2019) increasing the probability of parasite bioaccumulation (Willis et al., 2013; Ligda, et al., 2020).

These results are in agreement with Giangaspero et al., (2005), who identify C. parvum in 23 groups of Chamelea gallina clam taken from the coasts of the Adriaco sea (Abruzzo region). Also, Słodkowicz-Kowalska et al. (2015) detected 15.4% and 5.1% positive samples of Cryptosporidium spp. and B. hominis, respectively, among other parasitic agents, in Anodonta anatine and Unio tumidus mussels extracted from the municipal reservoir of Lake Malta (Poland). Both works concluded that there is fecal contamination that could become a public health problem. Recently, Ligda et al. (2020) detected the presence of Cryptosporidium spp. in three STP sewage treated, corresponding to 22% of positive samples, which discharge near the seafarm of Mytilus galloprovincialis mussel on the Thermaikos Gulf (North Greece). But Cryptosporidium spp. was not detected in the mussel samples. The high detection limit of IFA (5.0 - 5 x10³ oocyst mL^-1 Kuczynska et al., 2002) used to detect the parasite and the low contamination pressure were attributed to this result.

To identify specific sources of contamination, we evaluated the STP that discharge its sewage treated to the beach near the extractive zone of Cholga mussels (Figure S1). It is well known that the STP contribute to the environmental contamination of both parasites (WHO, 2016; Ligda et al., 2020). The STP from the studied zone consist of a primary (grid), secondary (vermifilter) and disinfection (chlorination) processes. The STP recollects the raw sewage from 200 habitants.

This agrees with the reported disinfection efficiency (< 3.2%) of Cryptosporidium spp. (Suarez et al., 2022). In addition, C. parvum was identified by IFA in effluent and Cholga suggesting a transmission pathway. As for C. parvum, the same species of B. hominis was identified in sewage treated and Cholga samples.

The most efficient disinfection process to lower Cryptosporidium spp. is the ultrafiltration combined with other technologies such as chlorination or UV radiation (Nasser et al., 2016). However, the monitoring of the sewage treated in the discharges must consider microorganisms resistant to disinfection, such as Cryptosporidium spp. and B. hominis, that are dangerous for the population.

In countries like the United States, New Zealand, Australia, and England, Cryptosporidium sp. water outbreaks in people were reported (Ma et al., 2022; Garcia et al., 2023). Therefore, in those countries, Cryptosporidium sp. is included in the microbiological water quality regulation, mainly in drinking water (EPA, 2001; Noke, 2008; Health Canada, 2019). The regulations established the monitoring of drinking water, surface water, and even groundwater (EPA, 2001; Noke, 2008; Health Canada, 2019). When Cryptosporidium sp. is above the allowed limits, as occurred by chlorination disinfection, the reduction efficiency must be continuously monitored up to values below the 3log depending on the concentrations of the place (Noke, 2008; Health Canada, 2019). Moreover, most countries do not have regulations considering the presence of these parasites in mussel, clams, and shellfish collection areas, and only fecal coliforms are considered as a water quality parameter (EPA, 2001; Noke, 2008; Health Canada, 2019).

Our work provides the first report of C. parvum and B. hominis in Cholga and STP sewage treated on a Chilean rural zone. These parasites and other fecal contamination microorganisms resistant to the disinfection treatment must be monitored to perform corrective actions on the STP and also be considered in sewage-treated discharges' regulations. In future studies we will extend the sample zones, consider seasonal variation during the sampling, and people infection.

For first time the transmission of C. parvum and B. hominis from a STP sewage treated to the Cholga was described in a rural zone of Concepción City, (Chile) indicating an environmental contamination. This finding makes it clear that it is relevant to review the regulations for discharges into surface ecosystems. Particularly, in the case of effluents from sewage treated by chlorination, in addition to the indicator of thermotolerant coliform bacteria, the evaluation of parasites should be considered.

Acknowledgements

This work was supported by ANID/FONDAP/15130015. P. Suarez thanks to ANID/Scholarship Program/DOCTORADO BECAS CHILE 2021-21210338 for her scholarship and thanks to Postdoctoral FONDECYT-ANID (3210523).

Author contributions

Suarez, P.: recopilation, data analysis, validation, investigation, methodology, writing original Draft, editing. Vallejos-Almirall, A.: validation, methodology, investigation, editing Alonso, J.L: methodology, conceptualization, and editing. Gonzalez-Chavarría, I.: methodology Fernandez, I.: methodology. Vidal, G.: conceptualization, resources, original draft, editing, visualization, supervision, project administration.

Funding sources

This work was supported by ANID/FONDAP/15130015, ANID/Scholarship Program/BECAS DOCTORADO NACIONAL CHILE/2021-21210338 [2021] and to the Facultad de Ciencias Biologicas, Universidad de Concepción, Chile.

Ethical Approval. Not applicable.

Consent to participate. Not applicable.

Consent for publication. All authors gave explicit consent to submit the work.

Competing interests. The authors declare no competing interests.

Availability of data and materials. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Table 1: Positive sample for C. parvum and B. hominis in Cholga from different commercial store and in sewage treated from sewage plant.

Sample	Total sample	*Cryptosporidium* sp.		*Cryptosporidium* *parvum*	*Blastocystis hominis*
Sample	Total sample	ZNS²	IFA²		PCR²
CS-1¹	5	3 (60.0%)	1 (20.0%)		0 (0.0%)
CS- 2¹	7	4 (58.0%)	3 (43.0%)		4 (57.0%)
CS-3	8	6 (75.0%)	4 (50.0%)		5 (62.5%)
Commercial store (Total)	20	13(65%)	8 (40%)		9 (45)
Sewage treated	12 L	positive	positive		positive

(¹): Sample taken in the same day. (2): Detection method. ZNS: Ziehl Neelsen staining; IFA: Immunofluorescences Assay; PCR: Polymerase Chain Reaction.

Table 2: Physicochemical and microbiological characterisation of raw sewage and sewage treated from sewage plant

Parameters	Raw Sewage	Treated Sewage
Temperature (°C)	16.1	15.4
Turbidity (NTU)	326.0	30.5
pH	7.95	3.85
TSS (mg L^-1)	613.0	37.0
VSS (mg L^-1)	533.0	30.0
COD (mg L^-1)	624.0	143.7
Residual chlorine (mg L^-1)	-	0.61
Total Coliforms (CFU/100ml)	3.1x10⁷	1x10⁰
Fecal Coliforms (CFU/100ml)	6.4x10⁶	1x10⁰

TSS: Total Suspended Solid; VSS: Volatile Suspended Solid; COD: Chemical Oxygen Demand

Supplementary Material S1 and Figure S1 are not available with this version.

No competing interests reported.

Evidence of Cryptosporidium parvum and Blastocystis hominis transmission from sewage treated to the Cholga mussel

Status:

Version 1

Abstract

Figures

Highlights

1. Introduction

2. Materials And Methods

2.1 Parasite in Mussel

2.1.1 Sampling

2.1.2 Processing

2.2 Parasite in treated sewage

2.2.1 Sampling

2.2.2 Processing

2.3 Detection of C. parvum

2.4 Detection of B. hominis

2.4.1 Conventional PCR

2.4.2 PCR product Sequencing

2.5 Physicochemical and microbiological characterization of the raw sewage and sewage treated

3. Results

3.1 C. parvum and B. hominis in Cholga

3.2 C. parvum and B. hominis in sewage treated

4. Discussion

5. Conclusion

Declarations

References

Tables

Supplementary Materials

Additional Declarations

Status:

Version 1