This study was conducted in full compliance with international ethical standards for research involving human participants and animals, including the Declaration of Helsinki (for human subjects), the World Organization for Animal Health (WOAH/OIE) Terrestrial Animal Health Code (for animal welfare), and the principles of the 3Rs (Replacement, Reduction, Refinement) for animal use.

A cross-sectional One Health study was conducted from November 2023 to July 2024 to estimate the prevalence of fascioliasis and schistosomiasis in livestock at both farm and abattoir levels, as well as in symptomatic patients attending healthcare facilities within the study area.

Livestock, snail, and human samples were collected simultaneously from the same ecological settings, allowing a cross-species assessment of fascioliasis and schistosomiasis transmission. This design is novel in Rwanda, where previous studies have largely focused on a single host species or isolated settings. In addition, physicochemical water parameters were measured at each sampling site, adding an ecological dimension that is rarely incorporated into regional NTD studies and enabling the evaluation of environmental factors associated with parasite transmission.

This One Health study was carried out in the peri-urban areas of Gasabo and Kicukiro districts in Kigali City, Rwanda. Kigali City is the fifth province of Rwanda and is located near the equator. Gasabo district is situated at latitude -1.8847 and longitude 30.13141. The district experiences a mean annual temperature of 20.9°C and receives approximately 908 mm of rainfall each year. Its landscape features high mountains with an average elevation of 1,800 meters, valleys, and over 30 wetlands. The main river system runs from Lake Muhazi to the Nyabugogo and Nyabarongo rivers, with other significant rivers including Rusumo and Buliza [31].

Kicukiro district is situated in the southeastern part of Kigali City at latitude -2.0039 and longitude 30.1470. Both Kicukiro and Gasabo districts have a tropical climate. In Kicukiro, the average annual temperature ranges from 15.2°C to 24.8°C, humidity ranges from 54% to 86%, annual rainfall varies from 13 to 186 mm, and wind speed averages approximately 10.5 km/h [32].

In Rwanda, part of the marshland is currently used for rice cultivation, representing 1.2% of the main crops grown in Gasabo district. Rice is primarily cultivated in low-lying marshy wetlands. For example, Kabuye marshland, which stretches across Nduba and Jabana sectors in Gasabo district, is used for rice production by smallholder cooperatives, including Cooperative de Riz de Kabuye (CORIKA) in Jabana sector and Cooperative pour la Promotion du Riz (COPRORIZ) in Nduba sector. Additionally, Rugende marshland, covering about 400 hectares across Gasabo, Kicukiro, and Rwamagana, has also been developed for rice production [33]. Freshwater bodies, farms, and health facilities included in this study are located near these marshlands.

The livestock component included both cattle and small ruminants (goats and sheep) and was conducted at farm and abattoir levels. The abattoir-based survey was performed at Société des Abattoirs de Nyabugogo (SABAN) abattoir in Kigali, Rwanda, a modern slaughter facility with a capacity of approximately 200 cattle per day. The farm-based component was carried out in Jabana and Nduba sectors of Gasabo district and Masaka sector of Kicukiro district. A total of 31 farms located near rice plantations, rivers, and water bodies were selected for the study (Figure 2). Human participants were recruited from three health facilities, namely Kibagabaga District Hospital, Masaka Health Center, and Nyacyonga Health Center, all serving peri-urban populations of Kigali City.

To ensure adequate statistical precision, sample sizes for humans, slaughtered animals, and live livestock were calculated using Cochran’s formula. Because no similar previous study had been conducted in the study area, an expected prevalence of 50% was assumed. A precision of 8% was used for slaughtered animals, whereas a precision of 9% was used for humans and live animals.

Sample size formula: n = Z²P(1 − P)/d²

For the abattoir component:

n = (1.96² × 0.5 [1 − 0.5])/(0.08)²= (3.8416 × 0.25)/0.0064= 0.9604/0.0064= 150.06 ≈ 150

At a 95% confidence level, Z was 1.96. With an expected prevalence of 50% and a precision of 8%, the minimum required sample size for slaughtered animals was 150. The selected abattoir receives cattle, goats, and sheep from more than 24 districts across the country. Accordingly, 100 cattle, 30 goats, and 20 sheep were sampled at the abattoir during three visits per week.

For humans and live animals:

n = (1.96² × 0.5 [1 − 0.5])/(0.09)²= (3.8416 × 0.25)/0.0081= 0.9604/0.0081= 118.56 ≈ 120

Therefore, the study ultimately included 120 human participants (40 from each health facility) and 120 farm animals from 31 farms. The farm animals consisted of cattle (n = 91), goats (n = 15), and sheep (n = 14) from peri-urban areas of Gasabo and Kicukiro districts. These two districts were chosen because of the presence of livestock farms, water bodies, and rice plantations, and because they had been underrepresented in previous studies. A stratified random sampling method was used to select patients attending the health facilities. In this study, four strata were considered: patients from the three health facilities, farm livestock, slaughtered livestock, and snails.

This comprehensive One Health analysis, which combines data from snails, livestock, and humans, highlighted localized transmission hotspots and emphasized the importance of integrated surveillance.

Out of 100 slaughtered cattle examined, 36.0% (95% CI: 25.6–45.4) tested positive for Fasciola spp., followed by goats at 30.0% (95% CI: 13.6–46.4) and sheep at 25.0% (95% CI: 6.0–44.0). Female animals showed a higher positivity rate for Fasciola spp. (39.4%, 95% CI: 27.6–51.2) compared to males (28.6%, 95% CI: 18.9–38.2). Younger animals (≤2 years for cattle and ≤8 months for small ruminants) had a higher positivity rate (36.8%, 95% CI: 25.3–48.2) than adults (>2 years for cattle and >8 months for small ruminants), which had a positivity rate of 30.5%. Crossbred animals showed a higher positivity rate (37.0%, 95% CI: 26.5–47.6) than local breeds (29.9%). Animals slaughtered from the Northern Province had the highest positivity rate for Fasciola spp. (42.4%, 95% CI: 29.8–55.0), followed by those from the Southern Province (40.0%) and Eastern Province (26.4%, 95% CI: 16.2–36.6). None of the slaughtered animals tested positive for Schistosoma spp. (Table 2).

Fasciola trematodes were macroscopically present in 36.0% of cattle (36/100, 95% CI: 26.6–45.4), 16.6% of goats (9/30, 95% CI: 13.6–46.4), and 25.0% of sheep (5/20, 95% CI: 6.0–44.0). Lesions specific to schistosomiasis were not documented in this study.

Of the 31 farms included in the study, 22 (70.9%, 95% CI: 54.9–86.9) tested positive for Fasciola eggs, while only 2 (6.5%, 95% CI: 0.0–15.1) tested positive for Schistosoma eggs. A total of 120 fecal samples were collected from Jabana, Masaka, and Nduba sectors (40 samples per sector). The overall prevalence of Fasciola spp. was 40.8% (49/120, 95% CI: 32.0–49.6), and the prevalence of Schistosoma spp. was 7.5% (9/120, 95% CI: 2.8–12.2). One animal (0.8%) tested positive for both Fasciola and Schistosoma. The prevalence of Fasciola infection was highest in Nduba and Jabana sectors, both at 45.0% (18/40, 95% CI: 29.6–60.4), compared to 32.5% (13/40, 95% CI: 18.0–47.0) in Masaka sector. Notably, all Schistosoma-positive cases were found on farms 25 and 26 in Masaka sector, resulting in a sectoral prevalence of 22.5% (9/40, 95% CI: 9.6–35.4) (Table 3).

Table 4 displays the univariate analyses of the seven explanatory variables. Of the seven initially assessed factors, only three variables—breed, animal species, and body condition score—had p < 0.25 in the univariate analysis for Fasciola, while only sector and breed met this criterion for Schistosoma. The variables “breed” and “animal species” were collinear (Cramer’s V = 0.713); therefore, breed was eliminated, and only animal species was further analyzed for Fasciola infection (Table 4).

After the stepwise elimination process during multivariable logistic regression, only body condition score was retained as a significant factor associated with Fasciola infection, while only sector was retained for Schistosoma infection (Table 5). The odds of Fasciola infection were significantly higher in animals with lower body condition scores, and the model demonstrated acceptable goodness-of-fit (area under the curve = 0.644). For Schistosoma infection, animal origin (sector) had a significant effect on infection occurrence, with all infected animals (n = 9) coming from Masaka and none from Nduba or Jabana (Tables 4 and 5). The goodness-of-fit for the Schistosoma model was also good (AUC = 0.86).

A total of 11 freshwater snail species were identified across the study sites, with three species observed shedding cercariae, marking the first report of cercarial shedding in these snails in Rwanda. Overall, 222 snails were collected from three rice-growing wetlands in peri-urban Kigali City: Kajevuba, Kabuye, and Masaka. The identified species included Biomphalaria pfeifferi, Biomphalaria sudanica, Physa acuta, Lymnaea natalensis, Biomphalaria sp. 2, Biomphalaria sp. 3, Bulinus spp., Physa spp., Pila ovata, Melanoides spp., and Lymnaea columella. The most abundant species were Lymnaea natalensis and B. sudanica (Figure 5). Only 1.4% (3/222) of the snails released cercariae: one B. pfeifferi released a S. mansoni cercaria, one B. sudanica released a Fasciola spp. cercaria, and one L. natalensis released 21 Fasciola spp. cercariae. All infected snails were found in Kajevuba wetland, Nduba sector (Figures 2 and 6). No snail released more than one type of cercaria.

The measured water parameters included NaCl, pH, electrical conductivity, temperature, and redox potential. The pH, temperature, electrical conductivity, redox potential, and salinity values peaked at point 1, showing clear variation in water chemistry across the sampling locations (Figure 7). However, these differences did not appear to influence snail abundance or cercarial presence. Redox potential was positive at points 1 and 5 and did not impact snail presence. Overall, the other water parameters showed minimal variation and no clear effect on snail occurrence. Spearman’s correlation indicated that pH, temperature, electrical conductivity, NaCl, and redox potential were all very weak predictors of cercarial count (|r| < 0.2); however, temperature was significantly linked to cercarial number (p = 0.0045), while all other predictors were not significantly associated (p > 0.05). Conversely, there was no strong linear relationship between snail species and water parameters, including redox potential (r = 0.165, p = 0.014), pH (r = 0.134, p = 0.047), NaCl (r = 0.091, p = 0.176), and temperature (r = 0.070, p = 0.300).

In contrast, no human cases were detected during the survey; however, this likely reflects underdiagnosis due to low or intermittent egg shedding rather than a true absence of infection.Most participants were aged 18–35 years [41.2% (59/119), 95% CI: 32.3–50.0], 56.3% (67/119, 95% CI: 47.4–65.2) were female, and 55.5% (66/119, 95% CI: 46.5–64.4) had primary education. Crop farming was the most common economic activity in the study area, accounting for 38.7% (46/119, 95% CI: 29.9–47.4). Livestock rearing represented 11.8% (14/119, 95% CI: 7.0–16.7). In this One Health study, none of the participants (0%) tested positive for schistosomiasis or fascioliasis by egg detection in stool or urine.

Fasciola and Schistosoma infections continue to pose significant threats to livestock productivity and public health globally, yet remain overlooked in many developing African countries [1, 41]. In this study, eggs of Fasciola and Schistosoma were found in fecal samples from cattle, sheep, and goats, and infected snail vectors were also identified. These results confirm the ongoing circulation of trematode infections at the livestock-snail-environment interface. Since definitive hosts, including humans and livestock, become infected through contact with contaminated water and ingestion of contaminated aquatic plants or food, understanding the epidemiology of these trematodiases is crucial for reducing infection risk and enhancing prevention strategies, especially through improved livestock management and decreased exposure to intermediate hosts [42].

The coprological findings from slaughtered animals in this One Health study showed that the prevalence of Fasciola infection was broadly similar to the 20% previously reported in Nyagatare district, Rwanda [13], and the 20.9% reported in slaughtered livestock in Tanzania [4]. However, the prevalence seen in this study was lower than that reported in the Imbo region of Burundi (47.7%) and at Lira municipal abattoir in northern Uganda (65.7%) [43]. Such variation across regions may be due to differences in environmental suitability for intermediate hosts, seasonal effects, and livestock management practices [24].

Postmortem examination further confirmed the presence of Fasciola trematodes in the livers of cattle (40.0%), goats (16.6%), and sheep (20.0%). These findings align with earlier reports from Rwanda showing that fascioliasis is a major cause of liver condemnation at slaughter. Previous postmortem studies documented infection rates of 41.1%, 90.0%, and 65.5% in cattle slaughtered in Bugesera, Gicumbi, and Rwamagana districts, respectively [30]. Another report from Nyabugogo abattoir indicated that 78.7% of liver condemnations were due to fascioliasis [15]. Collectively, these findings highlight the significance of fascioliasis as a source of direct economic loss through organ condemnation and decreased livestock value. Strengthening fascioliasis prevention and control in Rwandan livestock would likely improve animal productivity and indirectly reduce human exposure.

In live farm animals, the overall prevalence of trematode infections (Fasciola and Schistosoma) was 47.5%, significantly higher than the 3.9% reported in Ethiopia [44]. The prevalence of Fasciola infection in this study (40.0%) exceeded that in Tanzania (21.7%) but was lower than in the Imbo region of Burundi (47.0%) [45]. Additionally, the prevalence of fascioliasis was greater than that of schistosomiasis (6.7%), aligning with findings from Ethiopia reporting prevalences of 20.5% for Fasciola and 6.3% for Schistosoma in various livestock species [46]. This variation may stem from biological and ecological differences between the two parasites. Fasciola has a broader host range, widespread snail intermediate hosts, and infective metacercariae that encyst on vegetation and are easily ingested during grazing. Conversely, Schistosoma requires more specific intermediate hosts, has a narrower host range, and depends on direct skin penetration by its short-lived infective stages, which reduces transmission opportunities in livestock [47].

The present study also identified important host-related risk factors. Animals with a body condition score of 2 were more susceptible to fascioliasis than animals with a body condition score of 3. Poor body condition is often linked to inadequate nutrition and weakened immune function, which can reduce the animal’s ability to resist parasitic infections [48]. Additionally, Friesian and crossbred cattle had higher odds of infection compared to local breeds. This likely reflects the better adaptation of local breeds to endemic parasitic pressures, while exotic and crossbred animals are generally considered less resistant to helminth infections [49].

The current findings show that snail intermediate hosts for fascioliasis and schistosomiasis are found across the studied wetlands. However, infected snails were only detected at a few sites, with three infected snails found in the Kajevuba wetland. B. pfeifferi and B. sudanica were linked to S. mansoni, while one Lymnaea natalensis was infected with Fasciola spp. The overall rate of snails shedding trematode cercariae was 1.4%. This is lower than what has been reported along the Mara River in Kenya and Tanzania [42]; however, even a small percentage of infected snails can maintain transmission because infected snails can stay infectious for life and may release large numbers of cercariae [8]. Snails that did not shed cercariae might have been uninfected, affected by environmental factors, or harboring immature parasite stages that were not yet ready for shedding [8, 50]. These results highlight the need for broader studies across different ecological zones and seasons.

The environmental data further indicate that water chemistry may influence transmission suitability. Parameters such as pH, electrical conductivity, and redox potential appear relevant to the presence and abundance of coexisting Bulinus spp. and Biomphalaria spp. Wetlands, irrigation channels, and muddy semi-oxygenated soils are known to provide favorable conditions for Fasciola transmission and the survival of its intermediate host snails [51, 52]. In this study, the mean pH (7.3), temperature (29.1°C), electrical conductivity (234.9), NaCl concentration (114.8), and redox potential (-29.75) generally align with conditions suitable for the survival of Fasciola and its intermediate host snails. These snails tend to prefer neutral to slightly alkaline pH and moderate redox conditions [23]. Similarly, Schistosoma transmission is promoted in stable freshwater environments with neutral to alkaline pH and moderate to high redox potential [23]. Thus, environmental conditions in the study area may directly affect infection risk by supporting both snail survival and parasite development.

The absence of Fasciola and Schistosoma eggs among sampled human participants does not necessarily mean there is no disease in the study area. Previous studies in Rwanda have documented Fasciola infection in livestock [13, 30] and S. mansoni and S. haematobium infections in humans [23, 28]. The 0% prevalence observed in this study aligns with findings from similar settings, such as a community-based study in Malawi that also reported 0% prevalence of human fascioliasis among 538 participants [53]. Overall, these results may suggest a truly low disease burden in some populations, but they could also be influenced by methodological and sampling limitations, including small sample size, health facility-based recruitment, effective deworming programs, parasite biology, and imperfect diagnostic sensitivity.

One explanation is that the identified transmission hotspots, especially rice-growing wetlands, were near health facilities. However, few participants attending these facilities were likely rice workers or people with intense wetland exposure. Higher rates of human fascioliasis and schistosomiasis are more often reported in rural farming communities where humans and livestock share water sources and where raw or undercooked freshwater plants may be eaten [42], conditions that might not fully reflect the habits of the sampled population. Additionally, previous studies have shown that stool egg detection is often more successful in community-based surveys than in health facility-based sampling [54]. This is partly because infected patients may visit health facilities during the acute phase, usually 1-3 weeks after infection, when eggs are not yet present in stool [55]. Egg shedding generally begins later, around 8-13 weeks after infection, often when individuals are asymptomatic [22]. Therefore, community-based sampling around hotspots like farms and schools is more likely to give a more accurate estimate of the actual burden of fascioliasis and schistosomiasis [54]. Moreover, symptoms such as abdominal pain, hepatomegaly, and diarrhea are nonspecific and do not reliably identify infected individuals.

A second explanation relates to the biology of Schistosoma. Eggs may hatch quickly after contact with freshwater, which can lower the chances of recovery in some field conditions, and the human participants in this study were mainly adults, while schistosomiasis is often more prevalent in children [4]. Human trematode transmission is heavily affected by behaviors such as open urination, defecation, livestock grazing, farming, and swimming in contaminated water bodies [51].

A third possible explanation is the impact of ongoing control efforts. Rwanda has been running a control program since 2008, which includes routine deworming and mass administration of praziquantel, albendazole, and mebendazole in high-risk populations [23]. These measures may have lowered human infection rates in the current setting. However, other epidemiological studies still report persistent Schistosoma transmission and the emergence of new foci in Rwanda [23, 25, 29]. Therefore, the lack of detected human cases in this One Health study should not be seen as proof that transmission has completely stopped.

Finally, diagnostic limitations should be taken into account. The current study employed Flukefinder® along with direct microscopy to enhance detection; however, both methods have reduced sensitivity in cases of light infections. Although Flukefinder® is effective at detecting the large, heavy eggs of Fasciola, both techniques may miss infections when egg counts are low [38]. Additional research using rapid serological or antigen-detection assays combined with molecular methods would help clarify the true prevalence of human fascioliasis and schistosomiasis in the region.

Detecting Fasciola and Schistosoma in livestock, along with identifying snails shedding cercariae, signals an ongoing risk of exposure for humans, especially those working in or near rice farms. These findings support the need for integrated, multisectoral control strategies within a One Health framework. Such strategies should include regular livestock deworming, water, sanitation, and hygiene interventions for humans, and targeted snail control [1]. Regular livestock deworming can reduce reservoir infections and decrease environmental contamination [56]. Snail control through habitat management, combined with molluscicides when appropriate, remains one of the most effective ways to interrupt the transmission cycle of fascioliasis and schistosomiasis [57]. Additionally, expanding access to safe water, reducing open urination and defecation, and improving hygiene practices through WASH programs can lower snail infection rates and consequently decrease transmission to both animals and humans [58].

Some limitations should be considered when interpreting these results. The deworming history of slaughtered livestock could not be traced because livestock owners and other stakeholders in the meat value chain often lacked reliable records. The sedimentation method used in livestock may also have missed some infections due to parasite biology; for example, Schistosoma eggs may hatch within 20 min after exposure to freshwater at ambient temperature. In the human component, sampling patients at health facilities during symptomatic presentation likely underestimated infections because egg shedding usually occurs later during asymptomatic stages. These factors may have reduced the detection sensitivity and led to an underestimation of the true burden.

An especially notable finding of this One Health study was the close spatial connection between infected farm livestock and snails shedding Fasciola and Schistosoma cercariae in Kajevuba wetland, Shango village, Nduba sector. Snail sampling locations were within or near Fasciola-infected farms (Figure 2). One L. natalensis snail released 21 Fasciola cercariae, one B. sudanica released one Fasciola cercaria, and one B. pfeifferi released one Schistosoma cercaria. This localized overlap between infected livestock and infected snail hosts strongly indicates active transmission potential and emphasizes the risk of ongoing exposure, maintenance, and persistence of fascioliasis and schistosomiasis in Nduba sector.

To the best of our knowledge, this One Health investigation is the first study in Rwanda to simultaneously sample human participants, livestock, and freshwater snails, along with assessing environmental water parameters and epidemiological risk factors. This integrated approach offers a comprehensive One Health perspective on the transmission of fascioliasis and schistosomiasis in peri-urban ecosystems. The finding of infected snail intermediate hosts in areas with high human activity highlights potential transmission hotspots and demonstrates the epidemiological connection between snail habitats, livestock grazing areas, and shared water sources.

Previous studies in Rwanda have mainly focused on human schistosomiasis [26, 28] or fascioliasis in livestock based on farm or abattoir surveys [13, 30], without simultaneously investigating the human–livestock–snail interface. Therefore, this study provides new insights into cross-species transmission dynamics and highlights the importance of integrated surveillance for snail-borne trematode infections within the One Health framework. These findings establish baseline data that can support the development of coordinated control strategies targeting both animal and human health in high-risk environments.

A major strength of this study is its integrated One Health approach, which includes livestock, humans, and snail intermediate hosts within the same ecological settings. This multidisciplinary design enables the identification of localized transmission hotspots and offers valuable information for policymakers to prioritize peri-urban wetlands and agricultural areas for targeted control efforts, such as vector control, livestock deworming, and water, sanitation, and hygiene initiatives aimed at reducing Fasciola and Schistosoma infections.

However, several limitations should be considered when interpreting the findings. First, the number of human participants included in the study was relatively small, which may have reduced the likelihood of detecting positive cases of schistosomiasis or fascioliasis. Second, sampling was conducted over a limited period during the wet season, which may not fully reflect seasonal variations in snail density, parasite transmission, or the potential effects of climate variability on disease occurrence. Third, identification of snail species was based on shell morphology using standard taxonomic keys, which could lead to misclassification of closely related species. Therefore, more precise molecular techniques should be used in future studies to improve species identification.

Additionally, molecular diagnostic methods would be useful for distinguishing zoonotic species such as Fasciola gigantica and Fasciola hepatica, as well as for differentiating S. mansoni from Schistosoma bovis and other related species [59]. Implementing these methods would enable a more precise understanding of transmission pathways and host–parasite relationships, thereby enhancing future One Health surveillance and control programs.