Geographical distribution and molecular survey of freshwater snail intermediate hosts of Schistosoma haematobium by DraI/Sh73 PCR in eliminated foci of Morocco

Abstract

Morocco had reached the level of schistosomiasis elimination 16 years ago. However the spread of freshwater snails in several breeding sites, and imported schistosome infection, still exist. Therefore, snail survey is a crucial component to sustain elimination progress. This study aimed to evaluate and to incorporate DraI/Sh73 PCR, for detecting early prepatent Schistosoma haematobium infection in snail host, into epidemiologic surveillance for schistosomiasis, particularly in reportedly eliminated foci where S.bovis overlaps with S. haematobium. The geographical distribution and the density of Bulinus truncatus and Planorbarius metidjensis were monitored for six years (2014–2019) and snail sampling were conducted in Fkih Ben Saleh province. All snails were analyzed in pools by DraI/Sh73 PCR. Results showed absence of Planorbarius metidjensi and none of the collected Bulinus truncatus snails were infected by S. haematobium. DraI/Sh73 PCR using pooled snail extracts is specific, feasible and suitable in routine malacological survey in the post elimination phase of schistosomiasis in Morocco.

Keywords

Elimination DraI/Sh73 PCR Fkih Ben Saleh

Introduction

Urinary schistosomiasis is a tropical parasitic disease.¹ It is regarded as a world health problem as it infects individuals from 76 various countries, particularly in tropical and subtropical areas.² In the last, there has been an observed significant decrease in the prevalence and morbidity of the illness in a lot of these endemic countries.³ Morocco was endemic for only urinary schistosomiasis. The first case was reported in 1914 in Marrakech Province.⁴ The upgrade of irrigation schemes and extension to more arid areas contributed to the spread of the infection.

The control phase started in 1982 and involved case detection and treatment, but also included preventive measures such as health education and snail control. In 1994, morbidity control had been achieved and prevalence of the infection had reduced to less than 1%.⁵ The last autochthonous case was detected in 2004. Five years ago, in 2009, Schisosomiasis national survey showed the absence of antibodies anti S. haematobium in all serum samples of children tested by Enzyme immune transfer blot (HAMA-EITB),⁶ Same result in 2017.^7–9 Molecular malacological survey by simultaneous use of DraI PCR and Sh110/SmSl PCR differentiate S. hametaobium and S. bovis and revealed the absence of S. haematobium infectivity of snails collected from the last endemic areas. Molecular tools using pooled snail extracts to test large sample of snails is specific. However, simultaneous use of two Polymerase Chain Reaction (PCR) takes more time, reagents and required development to be used in the routine survey.¹⁰ Fkih Ben Saleh area in Beni Mellal province is an old focus of schistosomiasis. The last autochthonous case was detected in 2000,¹¹ and the last molecular malacological survey showed that S. haematobium and S. bovis overlap in this area.¹⁰ In the present study, the infectivity of snails using DraI/Sh73 PCR, the geographical distribution and the density of Bulinus truncatus and Planorbarius metidjensis were monitored for a six years period, 2014–2019 in several breeding sites situated in Chorfa, kalkha, Ouelad Zmam and Kourifat localities.

Materials and methods

Study area

Fkih Ben Saleh Province is located in central Morocco, approximately between 32°30 ‘3′N and 6°41′26′ W. The population of this Province is estimated to 124,079 inhabitants in 2014. The climate is continental characterized by intense cold in winter and very hot summer. The mean annual temperature is 18°C with a minimum of 3.5°C and a maximum of 40°C. Rainfall occurs mainly during the first months of the crop year, extends until May, and varies between 100 mm and 600 mm¹² (Figure 1).

Figure 1.

Fkih Ben Salah Province on the map and its districts.

Water contact areas

Human-water contact sites in Fkih Ben Saleh-Beni Mellal district, were identified during a baseline malacological survey carried out in April 2008.¹⁰ A total of 4 human-water contact sites, located in Chorfa, kalkha, Ouelad Zmam and Kourifat localities, were identified that were associated with irrigation canals and human-water contact site (washing and swimming). Breeding sites in Kourifat were endemic of S. bovis in 2008.

Snail sampling

Snail sampling was conducted during six year period (2014–2019), in the former focus of the intermediate host of urinary schistosomiasis as Chorfa, kalkha, Ouelad Zmam and Kourifat localities. The surveys were carried out by 2 trained field investigators, and according to the protocol described in the guide for the fight against schistosomiasis.¹³Sampling time was fixed at 20 min per location and was performed between 10:00 AM and 12:00 AM. At each collection time, snails from each site were appropriately labeled and transported in separate perforated plastic containers to the laboratory, where they were processed. Snails were identified to species level based on shell morphological characteristics using standard keys.¹⁴

For molecular study, snails were collected from kourifate at 3 km of Oued Abdoune in 2014 and were identified in situ by experienced staff. Each 5 to 10 snails were directly placed in ethanol (70%). Samples were sent to the laboratory of Parasitology in the National Institute of Hygiene in Rabat for morphological examination and species identification using molecular techniques.

DraI/Sh73 PCR detection of S. haematobium in snails

Molecular study was performed in National Reference laboratory of Schistosomiasis in National Institute of Hygiene in Rabat, Morocco. This technique is performed in 3 steps: Firstly the extraction of DNA with the hexadecyltrimethyl ammonium bromide (CTAB) method as previously described.¹⁰ The second step is amplification by DraI/Sh73 PCR. In fact, PCR was carried out in a volume containing 200 μMdNTPs, 25 pMol of each DraI primers (forward: Sh 73 direct: CCTTGGTCACGTGATTTTCDraI reverse primer: TCACAACGATACGACCAAC), 2.5 units of Taq DNA polymerase, and 5μl of target DNA (not diluted). The final volume for PCR reaction was 50 μl. All PCR assays were carried out by initial denaturation at 95°C for 5 min, 35 cycles of 95°C for 1 min, 58°C for 1 min, 72°C for 1 min, followed by final elongation at 72°C for 10 min, and then held at 4°C. The final step is electrophoresis, the amplified DNA products were loaded and analyzed on agarose gel electrophoresis (1.2%).

Positive and negative controls: In each PCR assay, 10 ng of S. haematobium purified DNA was used as a positive control and negative control was the water. DraI/Sh73 PCR show 200 pband350 pb band in S. haematobium infected snails and 150 pband300 pbbands in S. bovis infected snails.

Results

Epidemiological study

A total of 205 Bulinus truncatus were collected during this period (2014–2019). None of Planorbarius metidjensis snail was found. Analysis of the annual development of Bulinus truncatus harvest numbers shows the presence of bulins in 2014 and 2015 in two positive stations Ouelad Zmam and Kourifat. While, the period from 2016 to 2019 was characterized by the absence of the bulinus in the prospecting stations (Table 1) (Figure 2).

Figure 2.

Distribution of B truncatus in prospect areas.

Table 1.

Distribution of bulins and planorbs from 2014 to 2019 in for different stations in Fkih Bensaleh province.

Station	Years/number of collected snails												GPS coordinates
	2014		2015		2016		2017		2018		2019		GPS coordinates
	B. truncatus	P metidjensis	B. truncatus	P metidjensis	B. truncatus	P metidjensis	B. truncatus	P metidjensis	B. truncatus	P metidjensis	B. truncatus	P metidjensis
Chorfa	0	0	0	0	0	0	0	0	0	0	0	0	3,248,405 6,474,671
Kalkha	0	0	0	0	0	0	0	0	0	0	0	0	32.488503 6.602899
OuledZmam	20	0	50	0	0	0	0	0	0	0	0	0	32,393,936 6.709316
Kourifate	47	0	88	0	0	0	0	0	0	0	0	0	32,462,551 6.729183

GPS, Global Positioning Size marker.

Molecular study

We use 10 ng, 8 ng and 2 ng of S. haematobium positive control to evaluate DraI./Sh73 PCR (Figure 3). Amplification of the DNA of S. haematobium DNA by the DraI./Sh73 primers was successful. It was possible to amplify the DNA of S. haematobium at a lower concentration of 2 ng. The results showed the two bands: 350pb and 200pb with 2 ng of pure S. haematobium DNA. However all Bulinus truncates collected from Fkih Ben Saleh province were negative (Figure 3).

Figure 3.

Agarose gel electrophoresis analysis of DraI/Sh73 PCR amplified products from different collected snails. Lane 1- 3: Positive control, Lane 4: Negative control, and Size marker (base pair), Lane 5–9: Pool of 5 snails was tested per lane, Lane 10: Size marker1000pb.

Discussion

In terms of the distribution of freshwater snail, the data collected during period 2014–2019 highlights a low rate of development of B. truncatus in the prospecting stations in 2014 and 2015, and the absence of freshwater snails from 2016 until 2019. This disappearance of the intermediate host in these cottages would be due to several phenomena namely the unfavorable microclimate, the temporary drying of the canals which impact the development of B. trncatus weeding, the regular cleaning of the canals and the absence of aquatic vegetation. The development of B truncatus is related to the local microclimate of the waters: Temperature (optimal 18–26°C), insolation, low current speed, turbidity, depth (less than 2 m), pH (Optimal 6–7), mineralization (magnesian waters are unfavorable), vegetation and zoophytic associations.^15,16 Planorbarius metidjensis (present) is not found in Fkih Ben Saleh, as it is considered as the probable intermediate host of the schistosome in high altitude regions in Morocco like Agadir.^17,18

In order to properly intervene in the fight against the re-emergence of urinary schistosomiasis, a persistent follow-up of this pathology is indispensable, in particular freshwater snails Bulinus truncatus and planorbarius metidjensis, which are considered as the intermediate host.

The measures of national control of this latter must take into account molecular approach detecting early prepatent Schistosoma infection in freshwater snails. However, in 2009, the detection of S. bovis in several freshwater snails collected from Fkih Ben Saleh-Beni Mellal, Chtouka Ait Baha and El Kelaa des Sraghna complicates future transmission monitoring of S. haematobium.⁶ On the other hand pooling individual samples prior to DNA extraction can mitigate the cost of DNA extraction.

DraI PCR can detect infected snails one hour after exposure to miracidia.¹⁰ Regarding DraI / Sh73 PCR, developed by Abassiet al.¹⁹ it's simple and more sensible PCR assay that enables direct discrimination of S. haematobium from related animal schistosomes. The sensitivity of S. haematobium detection was 1pg and could detect snails’ infectivity from very early prepatency.¹⁹ In our study, DraI/Sh73 PCR, detected 2 ng of Schistosoma haematobium DNA, corresponding to DNA concentration of single or two miracidium. Then it s sensible as it can detect early prepatent Schistosoma haematobium infection in snails using pooling individual samples.

Recently, Abbassi et al. clarified the association between DraI, sh and Sh77 repetitive sequences in both S. haematobium and S. bovis genomes.²⁰

Webster et al. present a high-throughput one-step multiplex COX1 PCR diagnostic method to detect and discriminate between S. haematobium and S. bovis. The detection sensitivity of COX1 PCR is 0.8 ng of genomic DNA. However, the estimated DNA concentration of a single miracidium is about 1–2 ng/ml, and taking into consideration that less than a tenth of the extracted DNA is used for amplification.²¹ By consequent, DraI /Sh73 PCR assay is more sensible, useful for malacological survey in post elimination phase of schistosomiasis survey.

In Kenya, Agola et al. and Barber et al. performed a PCR-restriction fragment length polymorphism (PCR-RFLP) method targeting a unique segment of the second internal transcribed spacer (ITS2) region of the ribosomal DNA (rDNA) in the schistosomes. When the control PCR products were subjected to digestion using the Taq 1 restriction enzymes, clear banding patterns were detected and it was possible to distinguish between S. haematobium and S. bovis DNA. Both S. haematobium and S. bovis shared a band at 200 bp. However, S. haematobium had a distinct band at 180 bp while S. bovis had a distinct band at 240 bp. The assay reliably identified and distinguished between S. haematobium and S. bovis cercariae, even when only a few cercariae (5–10) were present in the sample, or when the parasite DNA concentrations were as low as five pico grammes (5pg). DraI/Sh73 PCR has a limit detection of 1pg of S. haematobium. Nevertheless, The outline of PCR-RFLP-ITS2 procedure, using Whatman FTA® technology that simplifies the handling and processing of nucleic acids, is useful in providing a rapid, one-day identification of S. haematobium (and likely S. bovis) cercariae (the infective larval stage) and/or other life cycle stages in a basic molecular biology laboratory.^22,23

Finally, the use of Loop-Mediated Isothermal Amplification (LAMP), will decrease the cost further down and more field friendly.^24–26

Conclusion

DraI/Sh73 PCR using pooled snail extracts to test large sample of snails is feasible, highly sensible, highly specific (discriminated S. bovis and S. haematobium) and suitable in routine malacological survey in post elimination phase of schistosomiasis in Morocco. A future study on a large geographic scale with more Bulinus isolates and more primers might characterize better the B ulinus infectivity, which may have a major impact on management and control of urogenital schistosomiasis.

Footnotes

Data availability

All data generated during the study are included in this article. More details and precision are available from authors via Amarir.fsacc@gmail.com and balahbib.abdo@gmail.com upon reasonable request

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship and/or publication of this article.

ORCID iDs

Fatima Amarir

Abedelaali Balahbib

Abederrahim Sadak

References

Bunnag

Sornmani

Pinithpongse

, et al. Surveillance of water-borne parasitic infections and studies on the impact of ecological changes on vector mosquitoes of Malaria after Dam construction. SoutheastAsian J. Trop. Med. Public Health 1979; 10: 656–660.

Lee

Bacon

Bottazzi

, et al. Global economic burden of chagas disease: a computational simulation model. Lancet Infect. Dis 2013; 13: 342–348.

Tlamçani

Er-Rami

. Schistosomiasis control: moroccan experience compared to other endemic countries. AsianPac. J. Trop. Dis 2014; 4: 329–332.

Gaud

. Revue critique des travaux consacrés à la bilharziose vésicale au Maroc. Bulletin de l’Institut d’Hygiène du Maroc 1951; 11: 69–95.

Laamrani

Mahjour

Madsen

, et al. Schistosoma haematobium in Morocco: moving from control to elimination. Parasitol Today 2000; 16: 257–260.

Amarir

El Mansouri

Fellah

, et al. National serologic survey of Haematobium Schistosomiasis in Morocco: evidence for elimination. Am. J. Trop. Med. Hyg 2011; 84: 15–19.

Balahbib

Amarir

Corstjens

, et al. Selecting accurate post-elimination monitoring tools to prevent reemergence of urogenital schistosomiasis in Morocco: a pilot study. Infect Dis Poverty 2017; 6: 1–9.

Balahbib

Amarir

Bouhout

, et al. Retrospective study on imported schistosomiasis in Morocco between 2005 and 2017. Trop Dr 2020; 50: 317–321.

Balahbib

Amarir

Bouhout

, et al. Review of the urinary schistosomiasis control in Morocco (1960–2018). Interdisciplinary Perspectives on Infectious Diseases 2020; 2020: 7. https://doi.org/10.1155/2020/3868970

10.

Amarir

Sebti

Abbasi

, et al. Schistosoma haematobium detection in snails by DraI PCR and Sh110/Sm-Sl PCR: further evidence of the interruption of schistosomiasis transmission in Morocco. Parasit Vectors 2014; 7: 1–8.

11.

Organisation Mondiale de la sante. Élimination de La Schistosomiase Au Maroc: Une Réalité et Un Sucées Apres Trois Décennies de Lutte. 2012. https://apps.who.int/iris/bitstream/handle/10665/119958/emropub_2012_860.pdf?sequence=1&isAllowed=y (accessed 15 June 2021).

12.

Salah

. DIRECTION REGIONALE BENI MELLAL – KHENIFRA. 76. https://www.hcp.ma/regiondrta/docs/Publications/Monographie%20Fkih%20ben%20saleh.pdf (accessed 26 June 2021).

13.

LAAZIRI, M. et BENNOUNA, M. Guide de la lutte contre la schistosomiase. Basel: Ciba-Geigy, for the Ministry of Public Health, Morocco, 1982.

14.

Brown

. Escargots d’eau douce d’Afrique et leur importance médicale. Londres: Taylor et Francis, 2 ans, 1994.

15.

Anderson. note la rareté des bulins pendant les mois de mars et d’octobre dans les canaux d’irrigation des oasis tunisiennes. 1923.

16.

Laamrani

Ecologie de

. Truncatus Dans Un Foyer de Bilharziose Uro-Génitale : Attaouia (Province KelaâSraghna). 1987, P60.

17.

Zekhnini

Yacoubi

Moukrim

, et al. Effect of a short period of desiccation during the patent period on cercarial shedding of Schistosoma haematobium from PlanorbariusMetidjensis. Parasitol. Res 2002; 88: 768–771.

18.

Gaud

. Rythmes biologiques Des mollusques vecteurs Des Bilharzioses. Bull. World HealthOrgan 1958; 18: 751–769.

19.

Abbasi

King

Sturrock

, et al. Differentiation of Schistosoma haematobium from related schistosomes by PCR amplifying an inter-repeat sequence. Am. J. Trop. Med. Hyg 2007; 76: 950–955.

20.

Abbasi

Webster

King

, et al. The substructure of three repetitive DNA regions of Schistosoma haematobium group species as a potential marker for species recognition and interbreeding detection. Parasit Vectors 2017; 10: 1–9.

21.

Webster

Rollinson

Stothard

, et al. Rapid Diagnostic Multiplex PCR (RD-PCR) to Discriminate Schistosoma haematobium and S. Bovis. 2010.

22.

Agola

Mwangi

Maina

, et al. Transmission sites for Schistosoma haematobium and Schistosoma bovis identified in localities within the Athi river basin of Kenya using a PCR-RFLP assay. Heliyon 2021; 7: e06114.

23.

Barber

Mkoji

Loker

. PCR-RFLP analysis of the ITS2 region to identify Schistosoma haematobium and S bovis from Kenya. Am J Trop Med Hyg 2000; 62: 434–440.

24.

Rong

Zhang

, et al. Sensitive and rapid detection of Schistosoma japonicum DNA by Loop-Mediated Isothermal Amplification (LAMP). Int. J. Parasitol 2010; 40: 327–331.

25.

Hamburger

Abbasi

Kariuki

, et al. Evaluation of loop-mediated isothermal amplification suitable for molecular monitoring of schistosome-infected snails in field laboratories. Am. J. Trop. Med. Hyg 2013; 88: 344–351.

26.

World Health Organization. Elimination of schistosomiasis from low-transmission areas: report of a WHO informal consultation, Salvador, Bahia, Brazil, 18–19 August 2008. World Health Organization, 2009.