In order to assess the biodynamic and environmental factors of Human enteropathogenic Microsporidia spores present in the groundwater in Mbankomo and Soa municipalities (Center region of Cameroon), a study was carried out on a sample of eight (8) wells and eight ( 8) springs regularly used by the population, from August 2018 to August 2019. The physico-chemical analysis was carried out both in the fields and at the Hydrobiology and Environment laboratory of the University of Yaoundé I. The microsporidian spores observation were made at the 100X objective based on the use of Weber's technique. The physico-chemical parameters show that these waters are slightly acid (6.21 ± 0.09 US) with high values of turbidity (24.88 ± 16.09 FTU). The biological analysis showed variations in shapes within the same species and between species on one hand and identical forms between species on the other hand. In this study, two microsporidian species were characterized with average densities of 193 ± 93 spores/L (45%) and 227 ± 127 spores/L (55%) for Enterocytozoon bieneusi ([1 - 1.6] x [0.7 - 1.2])µm, and Encephalitozoon intestinalis ([1.8 - 2.4 x [1.2-2.0]) µm respectively. Statistical analysis showed significant and positive correlations between the densities of spores and dissolved oxygen (P ˂ 0.05). The Mann-Whitney test revealed significant differences in microsporidian spores densities seasonally with great values on the short rainy season. Furthermore, this study shows that the consumption of untreated water from wells and springs is of health risks and prove the important of Microsporidia to monitor the quality of drinking water.
Research Article
Biodynamic and ecology of Human enteropathogenic Microsporidian spores in groundwater in the municipalities of Mbankomo and Soa in Center Region (Cameroon) and health risks of immunocompromised patient
https://doi.org/10.21203/rs.3.rs-1099832/v1
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Biodynamic
Ecology
Enterocytozoon bieneusi
Encephalitozoon intestinalis
Human enteropathogenic
Health risk
Groundwater
All Microsporidia are indeed able to resist and survive for a long time in the environment in the form of small spores with characteristic organization and similar to the spores of bacteria (Fayer 2004; Alexandra and Monika 2020). They are ubiquitous in the geographical distribution (Weber et al. 1994). Microsporidian spore is coated with a chitin-rich wall and contains a long spirally coiled filament, the sudden extrusion which allows infectious sporoplasmic material to be inoculated into a new host cell. Human Enteropathogenic Microsporidia (HEM) have been recognized as emerging opportunistic agents since the start of the Acquired Immunodeficiency Syndrome (AIDS) epidemic. Almost 50% of cases of chronic diarrhea in individuals infected with Human Immunodeficiency Virus (HIV) are attributable to these two Microsporidia, Enterocytozoon bieneusi and Encephalitozoon intestinalis (Weber et al. 1992 ; Franzen and Muller 2001). Damage to other organs of varying depth have been reported in both immunocompetent and immunocompromised persons (Van Gool et al. 1997). In 1985, Desportes et al. characterized for the first time the presence of Enterocytozoon bieneusi in an AIDS patient presenting with chronic diarrhea, whereas in 1992, the species of Encephalitozoon intestinalis was identified by Orenstein et al. in an AIDS patient. The seroprevalence of Microsporidia in immunocompetent European population has been evaluated between 2 and 22% (Kucerova-Pospisilova et al. 2001; Van Gool et al. 1997) and may be high in Africa with a higher risk in Human from tropical countries (Cornet et al. 1996) specially the immunocompromised patients. The diagnosis of human microsporidiosis often involves the detection of spores directly in the stool mainly, in urine or biopsies of tissue, mucus, respiratory excretions and blood (Alexandra and Monika 2020; Stentiford et al. 2013; Sak et al. 2010). However, little data exists on the biodynamic and ecology of enteropathogenic Microsporidia spores in groundwater in sub-urban areas where the population usually consumed water from wells and springs waters. The work of Deplazes et al. (2000) has shown that children, elderly people and even the immunocompetent are exposed to Microsporidia known as dangerous waterborne pathogens (Kalinová et al. 2015).
Place of study and sampling points in two sub-urbane areas: Mbankomo and Soa of the central region
Mbankomo and Soa are two municipalities of the central region of Cameroun which is the agro-ecological zones of forest with bimodal rainfall. The sub-urban of Mbankomo is located in the department of Mefou-et-Akono, between 3o 47 '31'' North latitude and 11o 24' 13 '' East longitude (Fig.1a). The sub-urban of Soa is located in the department of Mefou-et-Afamba. It extends from 3° 58’ 41’’ North latitude and 11 ° 35’ 35’’ East longitude. (Fig.1b). Mbankomo and Soa are respectively located for about 25 km and 14 km from Yaoundé which is characterized particularly by four seasons climate called the “Yaoundean climate” (Suchel 1972) including: a Long Dry Season (LDS) which extends from mid-November to mid-March, a Short Rainy Season (SRS) which runs from mid-March to the end of May, a Short Dry Season (SDS) from June to August, a Long Rainy Season (LRS) which runs from September to mid-November. Thus, the monthly average temperatures oscillate between 22.4 ° C and 27.2 ° C. The average annual rainfall is 1576mm. The bedrock which constitutes the geological substratum of the Yaounde soils derives from a more or less micaceous quartzo-feldspathic material (Pelletier, 1969), hence the high acidity of its soils with a pH of 4.5 to 5, 5 in the surface layers. The vegetation is of the dense humid semi-deciduous forest type.
Presentation of sampling points of Mbankomo and Soa
The study was carried out in two phases. The first phase was the prospection phases from June 2018 to August 2019. The multiple field trips made it possible to choose the sampling points according to the piezometric level, the maintenance of the well and its close surroundings, the presence or the absence of sources of pollution in the immediate vicinity of the structure and the agreement of the owner. Together, 16 sampling points were chosen in two (2) sub-urban areas, including eight (8) springs and eight (8) (Table 1) wells (Table 2).
Table 1 Characteristics of the sampling points Mbankomo
|
Sampling points |
Caracteristics of wells |
|
Mbankomo well 1 (BP1) |
The BP1 sampling points is characterized by the geographical coordinates positioned between 03° 47'34.6''N, 011° 24'21.4''E and an altitude of 757 m. It is equipped with a pulley system, measure 35m of height, has no coping and situated near the road. |
|
Mbankomo well 2 (BP2)
|
The BP2 sampling points is distinguished by the geographical coordinates of 03° 47'25.0''N, 011° 24'22.6''E and an altitude of 718 m. It measures 1.9m of height, has a partial coping without cover, is located near the fish farming ponds and about 10 m from the toilets. |
|
Mbankomo well 3 (BP3) |
The BP3 station is represented by the geographic coordinates positioned between 03° 47'21.9''N, 011° 24'19.4''E and an altitude of 742m. It measures 2.7 m of height, has a partial coping, without a cover, located near the fish-farming ponds and 10 m from the toilets. |
|
Mbankomo well 4 (BP4) |
The BP4 sampling points has the geographical coordinates located between 03° 47'21.2''N, 011° 24'19.7''E and an altitude of 740 m. This well is located in a housing camp and supplies several households. It measures 10 m of height with coping of 0.5m, the cover, a pulley and a concrete slab around the well. |
|
Mbankomo spring 1 (BS1)
|
The BS1 spring is distinguished by the geographical coordinates between 03° 47'29.93'N, 011° 24'05.3''E and an altitude of 728 m. It is of the holocrene type and is located in the shallows in a marshy area with a low level of surrounding sanitation. This spring receives runoff during the rainy season. |
|
Mbankomo spring 2 (BS2) |
The BS2 spring is illustrated by the geographical coordinates located between 03° 47'36.9'N, 011° 24'06.1''E and an altitude of 724 m. It is of the holocrene type and in lowlands, but with a low level of surrounding sanitation. Its flow is nil and it is invaded by brush. |
|
Mbankomo source 3 (BS4) |
The BS3 spring has geographical coordinates between 03° 47'28.5''N, 011° 24'23.2''E and an altitude of 762 m. It is of the rheocrene type, rises from a rock formation and is situated on a slope and under the canopy of trees. |
|
Mbankomo spring 4 (BS4) |
The BS4 spring is distinguished by its geographical coordinates located between 03° 47'17.5'N, 011° 23'53.1''E and an altitude of 721m. It is of the rheocrene type, this spring is located more than 50 m from the residential areas. In the rainy season, it receives runoff water. |
Table 2 Characteristics of the sampling points in Soa.
|
Sampling points |
Caractéristics of springs |
|
Soa well 1 (PS1) |
This SP1 well has the geographical coordinates of 03° 97'44.9'N, 011° 59'53.6''E and an altitude of 661 m. It measures 15 m of height and protected by 0.83 m high coping. |
|
Soa well 2 (PS2) |
This SP2 well is represented by the geographical coordinates 03° 98'54.1''N, 011° 59'29.6''E and an altitude of 660 m. It measures 13 m of height. This well has a 0.83 m high coping without cover and has a pulley system. |
|
. Soa well 3 (PS3) |
The SP3 well is differentiated by the geographical coordinates between 03° 98'45.1'N, 011° 59'21.0''E and an altitude of 671 m. It measures 15 m of height. It has a 0.83 m high coping with an iron cover. |
|
Soa well 4 (PS4) |
This SP4 well is characterized by the geographical coordinates located between 03° 97'50.1''N, 011° 58'83.9''E and an altitude of 644 m. This well is located near the road. It measures 13 m of height, has a 0.83 m high coping and is without a cover. |
|
Soa spring 1 (SS1) |
This SS1 spring is illustrated by the geographical coordinates between 03° 97'47.8'N, 011° 59'98.9''E and an altitude of 650 m. It is located 5 m from the dam drainage. It is of the rheocrene type with protection system and situated for about 4 m form stream. |
|
Soa spring 2 (SS2) |
This spring SS2 has the geographical coordinates located between 03° 97'51.6''N, 011° 59'51.7''E and an altitude of 652 m. It is overrun with brush and near a river seal of wastewaters. It is of the rheocrene type. |
|
Soa spring 3 (SS3) |
This spring SS3 is materialized by the geographical coordinates between 03° 98'66.4'N, 011° 59'11.9''E and an altitude of 650 m. It is located near the fish farming. It is the rheocrene type and with protection system. |
|
Soa spring 1 (SS4) |
This SS4 spring is characterized by the geographical coordinates located between 03° 97'52.5''N, 011° 58'68.7''E with an altitude of 634 m. It is located for about 100 m from the residential houses. It is of the rheocrene type near wastewater. |
Measurement of physico-chemical parameters of water from wells and springs
The physico-chemical analyzes were carried out both in the field and in the laboratory according to the APHA (1998) method and Rodier et al. (2009). For the parameters measured in the laboratory, the water samples were taken using double-capped polyethylene bottles of 250 mL and 1000 mL, and returned to the laboratory in a refrigerated enclosure.
The temperature and the electrical conductivity were measured in situ using a multiparameter of the HANNA HI 9829 brand. The electrode of the device is immersed 2/3 in the sample and the values have been read on the display screen. The results were expressed in °C (degrees Celsius) and in µS/cm (microSiemens per centimeter) respectively. Turbidity and suspended solids were measured in the laboratory using a HACH DR/3900 spectrophotometer. The values were expressed respectively in mg/L and in FTU (Formazin Turbidity Unit). The measurements of the pH of the water, expressed in Conventional Units (CU), were carried out in situ using a multiparameter brand HANNA HI 9829. The pH reflects the degree of acidity or alkalinity (basicity) water. Measurements of the nitrates and orthophosphates content in water were made by colorimetry with a HACH DR/3900 spectrophotometer. The nitrates (NO3-) and orthophosphates (PO4 3-) contents were measured on 10 mL of water sample with as reagents Nitraver V for nitrates and Phosver III for orthophosphates, at the respective wavelengths of 507 nm and 530 nm. The results were expressed in mg/L of NO3- (nitrates) and PO4 3- (orthophosphates). Percentage saturation of dissolved oxygen was measured in the field using a HANNA HI 9829 brand multiparameter. The electrode of the device is immersed at 2/3 in the sample and the value of dissolved O2 is read on the display screen. The results were expressed as percentage O2 saturation (%). Alkalinity was determined by volumetric analysis. 50 mL of sample of water was titrated against sulphuric acid N/50, in the presence of the green-red methyl bromo-cresol indicator. The results were expressed in mg/L of HCO3- . Oxidability was measured by volumetric analysis method. Into a 500 mL conical flask was introduced 200 mL of our water sample was introduced into a 500 mL conical flask, 2 mL of sodium hydrogen carbonate was added to the contents of the flask which was left to boil. During boiling, 20 mL of KMnO4 N/80 was introduced into the conical flask. Ten minutes later, the conical flask containing the solution will be cooled under a running tap and 5 mL of H2SO4 25% and 20 mL of ammonium ion (II) sulfate was added simultaneously. The constituted solution was titrated against N/80 potassium permanganate solution until the persistence of the pink coloration. The results were expressed in mg/L of O2 gas.
Human enteropathogenic Microsporidia (HEM) analysis
Sample collection methodology for microsporidia analysis
Water samples for the identification of Human enteropathogenic Microsporidian spores were carried out using water from wells and springs. Samples were collected directly from wells and springs using a bucket after homogenization. The water samples thus collected were immediately placed in sterile 1000 mL polyethylene bottles and then transported to the laboratory in a cooler. In the laboratory, the samples were measured and stored in a test tube for immediately analysis.
Observation of enteropathogenic microsporidian spores: Weber staining technique
Among the different Trichrome techniques (Weber, Ryan, Kokoskin and Deluol for Weber staining) (1992), the Weber technique appears to be the most suitable for specifically distinguishing spores from Microsporidia. It is characterized by good specificity, allows satisfactory parasitological screening both in terms of specificity, sensitivity, and reliability (Sparfel et al., 1997). It is the most widely used technique, and is considered the reference method (Dei-cas, 1994). After homogenization of the sample in 1L, 5 mL of the pellet are taken and introduced into a test tube. To this, 1 mL of 10% formalin was added to ensure the fixation of the organisms and 3 mL of 33% zinc sulfate was successively added for flotation (Faust et al., 1938). The mixture obtained was brought to centrifugation at 500 turns/min for 10 min using a MEDIFRIGER brand centrifuge. With the help of a syringe, 4 mL of the supernatant is removed and spread on the slides at a rate of 1 mL per slide. After drying in air for 24 hours, the slides are then stained and immersed in the trichrome solution for 90 minutes at room temperature [Composition of the trichrome: chromotrope 2 R: 6 g; light green: 0.15 g; phosphotungstic acid: 0.7 g; 3 mL of glacial acetic acid; wait 30 minutes; gradually add 100 mL of distilled water to a 125 mL flask]. The slides were rinsed in acetic alcohol for 10 seconds to differentiate the structures of the microsporidian spores (5 mL of acetic acid + 995 mL of 90° alcohol), then quenched successively in 95° ethanol for 30 seconds; in absolute ethanol for 10 minutes and in Xylene for 10 minutes to dehydrate. The reading was taken first at the 40X objective and then at the 100X oil immersion objective. The enteropathogenic microsporidian spores, usually oval and ellipsoidal in shape, appear fuchsia pink to trichrome red, and exhibit a consistent and characteristic colorless eccentric vacuole. Measurements were taken using a micrometer integrated into the lens and photographs were taken using an Xploview brand photographic camera connected to the computer.
Enumeration of Human enteropathogenic Microsporidian spores
The resulting preparation was placed on the stage of the Olympus CK2 brand inverted microscope for spore observation after adjustment to 40X or 100X magnifications. The identification of these enteropathogenic Microsporidian spores was carried out according to several criteria including size, shape of the WHO Bench Aids for the Diagnosis of Intestinal Parasites (1994) and appropriate books. In addition, the size of the spores was measured using the ocular micrometer. Thus, the spore count was performed using the formula below. The number (x) of spores contained in 1L of sample is obtained by the formula below:
With Vx = the volume of sample in 1L, Vy = the volume of sample used to observe (10 mL), y the number of spores observed in Vy. The results are given in number of spores. The measurements are expressed at the 100X and 40X objectives according to the ratio below.
[F (2/5)] [u x v] (100x) = [u’ x v’] (40x) = Sr
u and v are the dimensions of the spore in length and width respectively at the 100X objective while u’ x v’ are the dimensions of the spore in length and width respectively at the 40X objective; Sr is the real size of the spore and [F (2/5)] represents the conversion factor.
Physico-chemical of water from wells and springs
Spatial and seasonal variations in temperature and hydrogen potential (pH)
The water temperature values ranged from 23.5 ± 0.27 °C in SS4 sampling point during LRS to 26.5 ± 1.72 °C at BP2 in LDS. The mean temperature values are 25.12 ± 0.27 °C at Mbankomo and 24.53 ± 0.39 °C at Soa (Fig. 2).
The pH of the waters studied changed little. The lowest value was recorded at sampling point SP1 during SRS (4.72 ± 0.57 CU) and the highest value at sampling point BP1 during SRS (7.28 ± 0.29 CU) (Fig. 3). The average pH values are 6.36 ± 0.34 CU at Mbankomo and 6.03 ± 0.40 CU at Soa.
Suspended solids and Turbidity
The suspended solids content oscillates between 1 ± 5.68 mg/L (SS2 station during LDS) and 71.00 ± 47.17 mg/L (BS1 sampling point during LRS) with the mean values of 12.62 ± 20, 71 mg/L at Mbankomo and 10.06 ± 2.94 mg/L at Soa (Fig. 4a). Statistical tests show significant differences seasonally for this parameter.
For turbidity, the maximum value was recorded at sampling point OS1 during SRS (90.5 ± 37.39 FTU) and the minimum value noted at stations SP1 during SDS (2 ± 27.09 FTU), with averages values of 33.43 ± 13.47 FTU and 20.97 ± 9.00 FTU respectively at Mbankomo and Soa (Fig. 4 c). Statistical tests showed significant differences seasonally for this parameter.
Dissolved Oxygen, Oxydability and Alkalinity
For dissolved oxygen, the maximum value was recorded at SS2 sampling point during SRS (66.9 ± 7.46%) and the minimum value noted at BP1 sampling point during LDS (48.05 ± 8.94 %), with the mean values of 61.64 ± 1.18 % and 61.90 ± 1.00 % respectively at Mbankomo and Soa (Fig. 5a). Statistical tests showed significant differences seasonally for this parameter.
The oxydability values vary between 0.59 ± 1.03 mg/L of KMnO4 and 5.23 ± 1.54 mg/L of KMnO4 respectively at the BS4 stations in LDS and SS2 during the same season. The mean values of 2.63 ± 0.40 mg/L and 2.22 ± 0.64 mg/L respectively at Mbankomo and Soa (Fig. 5b).
The alkaline ion contents vary from sampling points to sampling points, with a maximum value of 28 ± 13.40 mg/L of HCO3- at the BP4 sampling point during SRS and a minimum value of 1.4 ± 3.38 mg/L of HCO3- at the BS3 station during LDS. The mean values of 9.82 ± 4.93 mg/L and 5.78 ± 5.43 ± 1.20 mg/L respectively at Mbankomo and Soa (Fig. 5c). Significant differences were noted seasonally.
Electrical conductivity
The electrical conductivity variation profile shows a minimum of 28.5 ± 5.49 µS/cm at sampling point BS4 during SRS, and a maximum of 549.5 ± 137.94 µS/cm recorded at sampling point SP1 during LDS with an average value of 43.50 ± 60.60 µS/cm and 157.98 ± 188.33 µS/cm respectively at Mbankomo and at Soa (Fig. 6).
Nitrates and Orthophosphates
Speaking of nitrates, they are in the form of nitrogen. In fact, nitrates vary from 0.05 mg/L (during SRS at sampling point BS2) to 7.5 ± 3.12 mg/L (during SRS at sampling point BS2) with an average value of 1.80 ± 1.60 mg/L and 1.26 ± 1.20 mg/L respectively at Mbankomo and Soa (Fig. 7a). However, nitrate fluctuations show significant differences from sampling point to sampling point and season to season (Kruskal-Wallis H test; p> 0.05).
On the other hand, in these groundwater, the maximum mean orthophosphates is recorded in the SS4 wells (1.49 ± 0.72 mg/L) during the LDS while the smallest values are recorded in the BP4 sampling point (0.015 ± 0.33 mg/L) during LDS with an average value of 0.49 ± 2.27 mg/L and 0.64 ± 0.27 mg/L respectively at Mbankomo and Soa (Fig. 7b).
Morphological description of enteropathogenic microsporidian spores identified in the samples of water from wells and sources
Morphological characterization of Human enteropathogenic Microsporidia (HEM)
The spores of Human enteropathogenic Microsporidia (HEM) may be ellipsoidal in shape. In this case, they are usually oval shapes with symmetrical poles. They have a straight shape and slightly curved at their ends with rounded tips (Fig. 8). As for the oval in shapes, the posterior and anterior poles are asymmetrical (Fig. 10). The spores can also have a pyriform shape with one end rounded and the other slightly tapered at the pointed end (Fig. 9). The anterior pole is less rounded than the posterior pole with a slightly thin spore wall at the apex of the spore through which the sporoplasm extrusion takes place. The thickness of the contours are slightly reduced at the forelegs and the contours are uniform and refractive. The Enterocytozoon bieneusi spores are smaller than the Encephalitozoon intestinalis spores which measure ([2.5-4] x [1.8-3]) µm and ([4.5-5.5] x [3-4]) respectively µm at the 100x objective whose real size is given by the convection factor [F (2/5)], corresponding to the size ([1 - 1.6] x [0.7 - 1. 2]) µm and ([1.8-2.2] x [1.2-1.6]) µm at the 40X objective which represents the real size (Sr) of the spores. The various forms of microsporidian spores below were generally described by Asi and Ajeagah (2020a) and are characterized as HEM.
[F (2/5)] [2.5- 4 x 2- 3] (100x) = [1- 1.6 x 0.8- 1.2] (40x) = Sr1
[F (2/5)] [4.5-5.5 x 4- 3] (100x) = [1.8- 2.2 x 1.2- 1.6] (40x) = Sr2
With Sr1 ˂ Sr2
Drawings of the forms of some spores of Human enteropathogenic Microsporidia observed
however, enteropathogenic microsporidia spores were characterized by three (03) identified forms. These are the pyriform (A), ellipsoidal (B) and oval (C) shapes shown below (Fig. 11).
Spatial and seasonal variations in the densities of enteropathogenic microsporidian spores in groundwater
Seasonally, the minimum density of enteropathogenic Microsporidia spores in wells and springs during the seasons (0 spores/L) and the maximum value were obtained in SP4 sampling point in LRS (750 ± 333 spores/L for (Enterocytozoon bieneusi) (Fig. 12a) and in the SP3 sampling point in LRS (1300 ± 104 spores/L for Encephalitozoon intestinalis) (Fig. 12b). The mean densities are 207 ± 110 spores/L at Mbankomo and 210 ± 103 spores/L at Soa. moreover, the highest densities of microsporidian spores are obtained during SRS. Statistical tests showed significant differences between seasons and between sampling points for some spores. However, some sampling points are characterized with great densities of spores during the dry seasons (BS1, BS2, SS4, SS1, SS2, BP3 for Enterocytozoon bieneusi and SS2, BP3, BP4, for Encephalitozoon intestinalis).
Spatio-temporal variation in the densities of enteropathogenic microsporidian spores in groundwater
Spatio-temporally, the minimum enteric spore density varied from 63 ± 94 spores/L (Enterocytozoon bieneusi) to 525 ± 127 spores/L (Encephalitozoon intestinalis). The mean spore densities are 193 ± 93 mg/L (45 %) and 227 ± 127 mg/L (55 %) for Enterocytozoon bieneusi and Encephalitozoon intestinalis, respectively. Additionally, the microsporidian spores of Encephalitozoon intestinalis were relatively more abundant with a difference of 34 ± 34 spores/L (Fig. 13) in groundwater during the study period.
Correlations between physicochemical variables and spore densities
No significant positive and negative correlations were found during the study between suspended solids, turbidity and electrical conductivity and spore densities. On the other hand, the dissolved oxygen value as a percentage showed positive and significant correlations with the densities of spores. Positive and significant correlations have been revealed between species. Significant differences were noted seasonally. The Mann-Whitney test revealed significant differences (p ≤ 0.05) seasonally between LRS and SRS (Enterocytozoon bieneusi); LDS and SRS; SRS and SDS (Encephalitozoon intestinalis).
Comparation of Enterocytozoon bieneusi and Encephalitozoon intestinalis
This table compares the size, distribution of Microsporidia in the intestine (prevalence) in human and the abundance of Human enteropathogenic Microsporidia during our study (Table 3).
Table 3 Comparation of size and distribution of Human enteropathogenic Microsporidia
|
Comparation |
Enterocytozoon bieneusi |
Encephalitozoon intestinalis |
Illustrations |
|
Size of Human enteropathogenic Microsporidia |
1-1.6 x 0.7-1 µm (Small) |
1.7-2.2 x 0.8-1.2 µm (Big) |
Weber et al. (1994) ; WHO (1994) ; Birkhead et al. (2017) |
|
Prevalence In the intestine |
Higher in human (90%) |
lower in human (less than10%) |
Henriques et al. (2010); Stentiford et al. (2016) |
|
Abundance of spores observed in water |
lower (45%) |
Higher (55%) |
Authors |
Evaluation of physico-chemical parameters in the water studied
Physico-chemical analysis revealed that groundwater has a slightly acidic pH (6.20 ± 0.40 UC). These waters are weakly mineralized (124.47 ± 129.93 µS/cm), poor in organic matter (2.43 ± 0.56 mg/L), in nitrates (1.53 ± 1.40 mg/L), alkalinity (7.63 ± 4.13 mg/L) and Suspended Solids (15.39 ± 10.42 mg/L). The values of turbidity (27.21 ± 12.80 mg/L) are higher compared to WHO standards value for water intended for drinking (4 FTU). orthophosphate (0.57 ± 0.27 mg/L) are high proven the anthropogenic impact. the temperature is 24.82 ± 0.45 °C suitable for water consumption. The average values in percentage saturation of dissolved oxygen is moderated (61.63 ± 1.18 mg/L) for a satisfactory drinking water. The physico-chemical results show the groundwater of the medium is poorly disturbed with low pollution and weakly anthropized. In comparative, the wells and springs of Mbankomo are characterized by high turbidity and suspended solids (Fig. 4) while the wells and springs of Soa are characterized by high values of electrical conductivity (Fig. 6). In regard to the physico-chemical results, the research of Ajeagah et al. (2020c) had proven that the values of pH in wells water in the sub-urban areas are slightly acidic and high level of turbidity and also Ajeagah et al. (2016) in groundwater of Yaounde. According to Nola et al. (1998) the pH of water is influence by the pH of the soil (with an average 5.52 ± 1.00 UC of four sub-urbans areas of the Central Region) depending of nature and texture of the sol.
Morphological characterization of Enterocytozoon bieneusi and Encephalitozoon intestinalis spores
The results of this work showed that the spores of enteropathogenic Microsporidia vary in size and shape (Fig. 8-10). Observations of Microsporidia spores showed variations in shapes within the same species and between species on the one hand and identical shapes between species on the other. In this study, two species of Microsporidia were characterized, Enterocytozoon bieneusi and Encephalitozoon intestinalis. According to Agnew et al. (2003) some species produce only one type of spores while others produce up to four types. While according to Vávra et al. (1976), the variations in size and shape of microsporidian spores allow species to be characterized. The size of Enterocytozoon bieneusi varied from ([1 - 1.6] x [0.7-1.2]) µm while that of Encephalitozoon intestinalis varied from ([1.8-2.2] x [1.2-1.6]) µm. These two species occur in pyriform (Fig. 11a), ellipsoidal (Fig.11B) and oval (Fig. 11C) shapes with a predominance of the ellipsoidal shapes observed during our study. According to Weber et al. (1994), WHO (1994) and Didier et al. (2004) Enterocytozoon bieneusi spores measure approximately 0.7-1.0 x 1.08-1.64 μm and these shapes are generally ellipsoidal and oval (Birkhead et al. 2017). According to Weber et al. (1994), WHO (1994) and Birkhead et al. (2017) mature spores of Encephalitozoon intestinalis measure 2.0-2.2 x 1.2 μm and are generally ellipsoidal in shape. Compared to the probable identified species of Human enteropathogenic Microsporidia, Enterocytozoon bieneusi is characterized by its smaller size than Encephalitozoon intestinalis.
Distribution of Human enteropathogenic Microsporidia
Statistical tests showed significant differences between sampling points. The mean densities are and 207 ± 110 spores/L at Mbankomo and 210 ± 103 spores/L at Soa. In contribution Human source of contamination, the abundance of microsporidian spores in wells and springs may also be due to uncontrollable animal husbandry and agriculture activities at the sampling points in relation to a low level of sanitation and hygiene. In the same way, Desportes (2005) notes that spores are also found in companion animals and livestock (dogs, cats, pigs, goats, donkeys, cattle and rabbits) which would thus be reservoirs of spore transmission. The abundance of Encephalitozoon intestinalis (55%) is greater than the abundance of Enterocytozoon bieneusi (45%) ((Fig. 13; Table 3). In fact, the high abundance of Encephalitozoon intestinalis may be linked to its great size of which would allow them to better resist environmental stress and due to the intensive zoonotic character unlike the Enterocytozoon bieneusi species which may be more capable of hiding in the environment due to its small size. According to Delage et al. (2005) Microsporidia Encephalitozoon intestinalis has a large capacity for dissemination to other organs and is liable to contaminate the external environment through or even in the urine in addition to faeces (Orenstein et al., 1992). The high values of the spores densities of Human enteropathogenic Microsporidia in the groundwater (BS1, SS2 and SP4) prove that the contamination may be of faecal origin due to the poor sanitation and hygiene (Fig. 13). These high values of spores densities mean waters analyses are therefore over polluted and unfit for consumption despite their physico-chemical analysis. These results reveal that the abundance of Enterocytozoon bieneusi in the waters studied is opposed to its prevalence in the host. Epidemiological studies have shown that Enterocytozoon bieneusi is responsible for almost 90% of gastrointestinal infections while the genus Encephalitozoon including Encephalitozoon intestinalis, Encephalitozoon cuniculi and Encephalitozoon hellem account for the remainder of 10% (Henriques et al. 2010). Australia (42 HIV + patients) who manifested abdominal pain, anorexia, nausea and diarrhea revealed a prevalence of Encephalitozoon intestinalis of 31% versus 69% Enterocytozoon bieneusi (Leder et al. 1998). The great rate of Encephalitozoon intestinalis (55%) also prove the faecal contamination of wells and springs is mainly originating from the animals (Table 3). Both parasites are known as dangerous waterborne pathogens and to cause aggressive forms of gastro-intestinal disease special on patient with a rate of T CD4 <50/mm3 (Weber et al. 1994 ; Sarfati et al. 2001; Kalinová et al. 2015) while population are living around and use the untreated water for drinking and others activities. It is also possible that the prevalence of this infection is high not only in humans but also in animals (Malčeková et al., 2011; Sak et al., 2010). Enterocytozoon bieneusi and Encephalitozoon intestinalis were detected in both, ground and surface waters (Kalinová et al. 2015) and also an epidemiological study has shown the direct correlation between the use of groundwater, well water and Encephalitozoon intestinalis infections (Enriquez et al. 1997).
Impacts of physico-chemical factors on the distribution of spore densities of Human enteropathogenic Microsporidia
Statistical tests have shown positive correlations between certain physico-chemical parameters and the densities of the spores. The densities of Enterocytozoon bieneusi and Encephalitozoon intestinalis showed positive correlations with percent dissolved oxygen. Despite non-significant correlations between the densities of spores, turbidity and the mater which likely promote the dissemination of spores in an aquatic environment, the high values of these parameters would not only promote the dissemination of the spores but also their concealment making it difficult for them to identify them on optical microscopes. These results are illustrated by the positive and significant correlations between the densities of the spores and the values of dissolved oxygen showing that the high rate of dissolved oxygen may poorly promote the dissimulation of the spores in mater. The positive and significant correlations between the spore densities of Enterocytozoon bieneusi and the densities of Encephalitozoon intestinalis showed a possibility of spores co-habitation in the environment with a significant risk of co-infection. The most exposed people are children and the elderly who directly consume this water without prior treatment in sub-urban and rural areas. In the same way, the research of Deplazes et al. (2000) has shown that children, elderly people and even immunocompetent people are exposed to Microsporidia and that diarrhea is the most frequent health problem caused, mainly in immunocompromised people (Izquierdo et al. 2011).
Seasonal influence on the distribution of spore densities of Human enteropathogenic Microsporidia
Statistical tests showed significant differences between seasons. however, spores densities ranged from 0 spores/L to 1300 spores/L with the average density of 210 ± 103 spores/L. Biological analysis have shown the presence of spores in groundwater during all seasons. Seasonally, the highest spores densities were observed during the short rainy season (Fig. 12). This could be explained by the fact that in the rainy season, the fecal matter and urine drained by runoff would favor the contamination of wells and springs by the opening of wells and springs or by infiltration process of spores. Also, the formation of muds due to rainstorm may contribute to contamination of groundwater (Ajeagah et al. 2019). This water would also drain the waste rich in organic matter in the water reservoirs (springs and wells) which are devoid of copings and covers. The influence of the rainy season on the increase in spore density has been proven by Tumwine et al. (2002) following their epidemiological work in Africa and corroborate on the seasonally variation of Microsporidian spores in groundwater in sub-urban areas. The low densities observed during the short dry season compared to those recorded in SRS would be due to the scarcity of runoff water during this season and the importance of the mineralizing activity of microorganisms. Indeed, organic matter stored in well and spring water is gradually transformed into mineral elements which could constitute stress for the survival of spores. These high concentrations of mineral elements could increase the inactivation of resistance forms of Microsporidia. However, the high spore values observed at the sampling points BS1, BS2, BS4, SS2 and SS2 for Enterocytozoon bieneusi as well as SS2 and BP3 for Encephalitozoon intestinalis during LDS may be due to the wind action. In fact, these two spores are small in size or of low molecular weight; during the dry seasons the wind may transport the spores and contaminate vulnerable wells and springs. According to the review, the presence of the spores in the air may also favor the contamination of Human by inhalation. The transmission routes indicated are via airborne, person-to-person, zoonotic, and waterborne means (Didier et al. 2004; Graczyk et al. (2007c). The high spores values observed during all seasons at some sampling points could explain the position of de Wuhit et al. (1994), Conteas et al. (1998) and Anane et al. (2010) who suggest that there would be no seasonal influence on the prevalence of intestinal Microsporidia in Human and that the contamination is due to a constant presence of Microsporidia in the environment rather than an increase in the contamination of water supplies during the rainy season. However, the densities of microsporidian spores were higher in the rainy season, showing that the rains may influence the density of spores in the environment, highlighting the significant external contribution.
Anthropogenic action on the distribution of Microsporidia densities
Although Soa municipality is more anthropogenic than Mbankomo, the difference of spores densities varied very little (The average densities are 207 ± 110 spores/L at Mbankomo and 210 ± 103 spores/L at Soa). In fact, wells in Soa are very deep with a high protected system (˃13m) except SP4 (with low protected system) and the springs are Rheocrene type at the proximity of stream or fish ponds while in Mbankomo the wells are not deep except BP1 (35m) without any a protected system except BP4 and are at the proximity of toilets ( BP2 and BP3). the springs are holocrene type except BS3 and BS4 and used for agricultural activities and fish production (Table 1 and Table 2). With regard to this, in Soa wells may be contaminated mainly by poor hygiene and by infiltration in springs. While in Mbankomo, it may be contaminated mainly by poor hygiene, sanitation and the agricultural activities around well and springs, runoffs and infiltration during the rainy season. This may prove that contamination of groundwater may not only depend of Human anthropogenic actions but the combination with other factors (the nature and texture the soil, agriculture activities, proximity of stream and toilets, hydrogeology factors, infiltration and recharge, drainage and runoffs, state of wells and springs and sources of pollution). This study, also showed that contamination of groundwater may not only depend on anthropogenic action of the areas (quarter, rural, sub-urban and urban areas) but meanly the pressure done on the point (well or sping) depending of the level of protection. The wells and springs of Mbankomo is characterize by high turbidity and suspended solids while the wells and springs of Soa is characterize by high values of electrical conductivity. According to Ajeagah et al. (2016) turbidity and suspended solids may favor dissemination of Protozoa in water while electrical conductivity may favor their inactivation.
Bioindicator base on Human enteropathogenic Microsporidia
Enterocytozoon bieneusi and Encephalitozoon intestinalis are released in the environment through feacal. The present of their spores in groundwater may expose the population to a health risk living around the areas. The detection of microsporidian spores illustrates a poor quality of water independent of the physico-chemical factors. These observations were already indicated by Izquierdo et al. (2011) that Microsporidia can be added as further evidence to support that new and appropriate control and regulations for drinking water, wastewater and recreational water should be established to avoid health risks. These Microsporidia are considered as dangerous waterborne pathogens (Kalinová et al. 2011) and can also survive for a long time in the environment and more than one year in aquatic system (Cali et al. 2016). For these reasons Microsporidia were included by United States Environmental Protection Agency on the list of microbial contaminants of drinking water, safe drinking water act in the years 1998 (EPA). The Human enteropathogenic Microsporidia can be used in Africa particularly in Tropical areas as good indictor to evaluate the quality of water for drinking specially in sub-urban and rural areas where population frequently consumes untreated groundwater.
Ultimately, this study revealed that groundwater was contaminated by the Enterocytozoon bieneusi and Encephalitozoon intestinalis spores. Their dissemination and resistance in the environment would be influenced by seasonal factors with increased abundance in the rainy season without neglecting the wind action and physico-chemical parameters. Statistical analysis showed positive and significant correlations between dissolved oxygen and spore densities indicating poor microsporidian spore concealment in water. The diversity of forms within the same species and between species is characteristic of polymorphism in Microsporidia and would promote their dissemination as well as their resistance in the environment. The results of this study show the importance of clinical diagnosis of Microsporidiosis in people with diarrhea, especially in immunocompromised (AIDS patient). The presence of these spores in the environment indicates contamination from fecal origin and would provide information on its quality. Contamination of these waters by spores may expose the population to health risks. This study shows the need to sensitize the population exposed aware of the health risks of intestinal microsporidiosis and the need to treat wells and springs water before consumption. In addition, the importance of extending or strengthening drinking water supply networks sub-urban and rural areas. It is then necessary to integrate neglected tropical diseases special Microsporidia as a priority in the world heath strategy and development in prescription of WHO recommendation.
Code availability Source codes are available on request.
Author contribution Asi Quiggle Atud: Conceptualization, project administration, data collection, formal analysis, writing—review & editing, data curation, writing—original draft, Writing—review & editing, validation. Ajeagah Gideon Aghaindum: Conceptualization, methodology, supervision. Ngakomo Ananga Rose Pulcherie: Conceptualization, methodology, Mboumbouo Mama: Data collection, writing—original draft & analysis data. Okoa Amougou Thérèse Nadège: Data collection, writing—original draft & analysis data.
Funding The authors acknowledge financial support from Authors.
Data availability Data are available on request.
Ethical approval and consent to participate Not applicable.
Consent for publication Not applicable.
Conflict of interest The authors declare no competing interests.
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