Transmission sites for Schistosoma haematobium and Schistosoma bovis identified in localities within the Athi River basin of Kenya using a PCR-RFLP assay

Abstract

Background: The epidemiology of human urinary schistosomiasis caused by Schistosoma haematobium can be complicated by the presence of ruminant schistosomiasis caused, primarily by S. bovis. The two schistosome species may be transmitted by the same Bulinus species, they may occur sympatrically in the same habitat, and their cercariae are very similar in morphology and therefore, difficult to tell them apart. Screening of snails collected from freshwater habitats for schistosome infections is often used to identify transmission sites or to evaluate success or failure of interventions. However, pin-pointing sites involved in S. haematobium transmission can be complicated by the presence of other mammalian schistosomes such as the bovine schistosome, which is a fairly common parasite. A PCR-RFLP method targeting a unique segment of the second internal transcribed spacer (ITS2) region of the ribosomal DNA (rDNA) in the schistosomes was used to identify mammalian schistosome cercariae shed by bulinid snails collected from endemic freshwater habitats located within Machakos county in south-eastern Kenya, with the aim to identify the transmission sites and assess the distribution each of the parasite species in the study area.

Results: A total of 5,034 bulinid snails were collected from 41 different sites and screened for schistosome infections, and out of these, 43 (<1%) were found to be shedding mammalian schistosome cercariae. On analysis using the Polymerase chain reaction- Restriction Fragment Length Polymorphisms (PCR-RFLP) assay, cercariae from 32 snails were identified as S. haematobium while cercariae from 11 snails turned out to be S. bovis. Only two sites out of 40 namely Kisukioni and Katiwa, were active transmission sites. Both sites were active transmission sites for both S. haematobium and S. bovis. 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). The FTA® paper offered a more reliable way of collecting, transporting and storing DNA material, and the samples.

Conclusion: The PCR-based assay can potentially be used to support schistosomiasis control efforts, in epidemiological studies of urinary schistosomiasis, or in transmission ecology studies of S. haematobium and S. bovis.

Keywords: Athi River Basin; Kenya; PCR-RFLP; Schistosoma bovis; Schistosoma haematobium; Schistosomiasis; Transmission sites.

Figure 1

Bar graph Shows monthly abundance of snails (Data from all study sites).

Figure 2

(a) Shows the abundance of snails collected within the Katiwa study site by month for the 10 months that sampling was carried out. (b): Shows the abundance of snails and the infection status of Kisukioni sampling site. Kisukioni recorded the highest number of infected snails collected during the 10 months sampling period between March 2012 and Janary 2013.

Figure 3

Shows PCR product of DNA amplification following extraction using the FTA paper and the HotShot methods. Lane 1 and 10 is 100bp marker, lane 2–4 are PCR product from DNA extracted from 5, 10, 20 cercariae extracted using the FTA paper, and Lane 5–8 PCR product from DNA extracted from 5, 10, 20 cercariae extracted using Hotshot method. Lane 9 is the negative control.

Figure 4

Showing the banding pattern of the PCR products following amplification with the ITS2 primers. Lane 1 and 6 are the 100bp molecular markers, lane 2 and 4 are DNA amplified from control S. bovis worms (5fg and 10fg concentrations for lane 2 and 4 respectively) and Lane 3 and 5 are DNA amplified from control DNA extracted from S. haematobium worms (5fg and 10fg concentrations for lane 2 and 4 respectively).

Figure 5

Shows the RFLP product following digest of the PCR product with Taq 1 resetriction enzyme. Lane 1 and 10 shows 100 bp marker, lane 2 is the control undigested S. bovis control, lane 9 is the control undigested S. haematobium control, lanes 3–6 are the control digested S. bovis DNA, lane 7 and 8 are the control digested S. haematobium DNA.

Figure 6

Shows the banding patterns of RFLP product of unknown DNA extracted from cercariae shed by Bulinus sp snails collected from snail habitats, following digestion with Taq 1 restriction enzyme. Lane 1 and 10 shows the 100bp marker, lane 2 is control undigested S. bovis, lane 3–6 are the digested unknown mammalian schistosome cercarial DNA, lane 9 is control undigested S. haematobium DNA.