Introduction
Cystic echinococcosis (CE) is a neglected zoonotic disease caused by the larval stage of the tapeworm Echinococcus granulosus sensu lato (s.l.). The parasite is primarily transmitted between canids and, to a lesser extent, felids as definitive hosts and ungulates, including livestock, as intermediate hosts (Thompson, Reference Thompson2017). Adult worms inhabit the intestines of dogs and other definitive hosts and shed eggs in the feces, leading to environmental contamination. Intermediate hosts, including livestock and, occasionally, humans as accidental dead-end hosts, become infected through ingestion of eggs during grazing in livestock and via contaminated food, water or direct contact with infected dogs in humans. After ingestion, the released oncospheres penetrate the intestinal wall and migrate via the bloodstream to various organs, predominantly the liver and lungs, where they develop into hydatid cysts and cause CE. Definitive hosts become infected by ingesting offal containing fertile hydatid cysts from intermediate hosts, typically through access to raw viscera during home slaughter or improper offal disposal, thereby completing the E. granulosus s.l. life cycle (Thompson, Reference Thompson2017).
Globally, CE imposes significant economic losses on livestock production due to the condemnation of infected organs, reduced productivity and weight loss, and it remains a major public-health concern in endemic regions (Budke et al., Reference Budke, Deplazes and Torgerson2006; Kere et al., Reference Kere, Joseph, Jessika and Maina2019). Recognizing its importance, the World Health Organization (WHO) and the Food and Agriculture Organization (FAO) have classified E. granulosus s.l. as a priority food-borne parasite requiring targeted control measures in endemic areas (WHO, 2014, 2020). The disease is globally distributed and remains endemic in most livestock-rearing regions, especially within pastoral and agro-pastoral communities (Deplazes et al., Reference Deplazes, Rinaldi, Alvarez Rojas, Torgerson, Harandi, Romig, Antolova, Schurer, Lahmar, Cringoli, Magambo, Thompson and Jenkins2017; Xiao et al., Reference Xiao, Liu, Du, Tian, Li and Ren2025). In Africa, the disease is widespread in eastern, northern and southern regions but remains relatively uncommon in high-rainfall areas of western and central Africa (Wahlers et al., Reference Wahlers, Menezes, Wong, Zeyhle, Ahmed, Ocaido, Stijnis, Romig, Kern and Grobusch2012; Deplazes et al., Reference Deplazes, Rinaldi, Alvarez Rojas, Torgerson, Harandi, Romig, Antolova, Schurer, Lahmar, Cringoli, Magambo, Thompson and Jenkins2017; Aregawi et al., Reference Aregawi, Levecke, Ashenafi, Byaruhanga, Kebede, Mulinge, Wassermann, Romig, Dorny and Dermauw2024).
Molecular studies have shown that E. granulosus s.l. comprises several cryptic species and genotypes, including E. granulosus sensu stricto (s.s.) (G1, G3, Gomo), E. equinus (G4), E. ortleppi (G5), E. canadensis (G6–G8, G10) and E. felidis. The latter species is restricted to sub-Saharan Africa, reflecting its ecological association with lions as definitive hosts (Nakao et al., Reference Nakao, Yanagida, Konyaev, Lavikainen, Odnokurtsev, Zaikov and Ito2013; Wassermann et al., Reference Wassermann, Woldeyes, Gerbi, Ebi, Zeyhle, Mackenstedt, Petros, Tilahun, Kern and Romig2016; Vuitton et al., Reference Vuitton, McManus, Rogan, Romig, Gottstein, Naidich, Tuxun, Wen and Menezes da Silva2020). All 5 members of E. granulosus s.l. have been reported in Kenya (Romig et al., Reference Romig, Deplazes, Jenkins, Giraudoux, Massolo, Craig, Wassermann, Takahashi and de la Rue2017; Mulinge et al., Reference Mulinge, Zeyhle, Mbae, Gitau, Kaburu, Magambo, Mackenstedt, Romig, Kern and Wassermann2023), indicating diverse transmission cycles shaped by ecological heterogeneity, livestock husbandry systems and parasite genetic diversity (Romig et al., Reference Romig, Ebi and Wassermann2015; Nocerino et al., Reference Nocerino, Pepe, Ciccone, Maurelli, Bosco, Boué, Umhang, Lahmar, Said, Sotiraki, Ligda, Laatamna, Reghaissia, Saralli, Musella, Alterisio, Piegari and Rinaldi2024).
In Kenya, CE is well documented in several pastoral regions, with prevalence varying by location and livestock species (Romig et al., Reference Romig, Omer, Zeyhle, Hüttner, Dinkel, Siefert, Elmahdi, Magambo, Ocaido, Menezes, Ahmed, Mbae, Grobusch and Kern2011; Deplazes et al., Reference Deplazes, Rinaldi, Alvarez Rojas, Torgerson, Harandi, Romig, Antolova, Schurer, Lahmar, Cringoli, Magambo, Thompson and Jenkins2017; Aregawi et al., Reference Aregawi, Levecke, Ashenafi, Byaruhanga, Kebede, Mulinge, Wassermann, Romig, Dorny and Dermauw2024). Early investigations from the 1980s in Narok and Kajiado Counties, collectively referred to as Maasailand, reported infection rates of 8.9% (95% Confidence Interval: 7.5–10.4%), 8.1% (95% CI: 6.9–9.5%) and 7.1% (95% CI: 6.0–8.3%) in cattle, sheep and goats, respectively, identifying the area as hyperendemic (Macpherson, Reference Macpherson1985; Romig et al., Reference Romig, Omer, Zeyhle, Hüttner, Dinkel, Siefert, Elmahdi, Magambo, Ocaido, Menezes, Ahmed, Mbae, Grobusch and Kern2011). Approximately 3 decades later, prevalence had increased markedly across Maasailand to 25.8% (95% CI: 22.4–29.4%) in cattle, 16.5% (95% CI: 13.3–20.3%) in sheep and 10.8% (95% CI: 7.2–16.0%) in goats (Addy et al., Reference Addy, Alakonya, Wamae, Magambo, Mbae, Mulinge, Zeyhle, Wassermann, Kern and Romig2012). More recent data from the eastern Maasai region, specifically Kajiado County, reported prevalences of 14.2% (95% CI: 11.1–18.0%), 14.9% (95% CI: 12.3–17.9%) and 15.2% (95% CI: 12.3–18.8%) in cattle, sheep and goats, respectively (Nungari et al., Reference Nungari, Mbae, Gikunju, Mulinge, Kaburu, Zeyhle and Magambo2019). Although these studies indicate temporal variation and overall higher infection levels than early reports, the drivers of these changes remain poorly understood. Moreover, molecular epidemiological data from key endemic areas, such as Maasailand, particularly Narok County, are scarce or outdated, limiting understanding of local transmission dynamics. Against this background of changing CE endemicity and its public-health implications, the present study assessed the current endemic status by determining the prevalence and causative species of E. granulosus s.l. infecting cattle, sheep and goats in Maasailand (Narok County), Kenya.
Materials and methods
Study area
The study was conducted in Maasailand, specifically Narok County in southwestern Kenya, covering approximately 17 944 km2 (Figure 1). The county borders the Serengeti National Park and the Loliondo Game Controlled Area in Tanzania to the south (Mukeka et al., Reference Mukeka, Ogutu, Kanga and Roskaft2018). Maasailand (Narok County) was selected because it is an established endemic area for both livestock and human CE and has substantial historical livestock data available for comparison (Romig et al., Reference Romig, Omer, Zeyhle, Hüttner, Dinkel, Siefert, Elmahdi, Magambo, Ocaido, Menezes, Ahmed, Mbae, Grobusch and Kern2011). The region is predominantly inhabited by a pastoralist community engaged in extensive livestock rearing and dog keeping, practices that promote close interaction between definitive and intermediate hosts, thereby sustaining CE transmission, including zoonotic transmission to humans. Livestock production, tourism and wheat farming form the backbone of the local economy. Its proximity to the Maasai Mara National Reserve, a biodiversity-rich ecosystem where domestic animals frequently graze near wildlife, creates ideal conditions for parasite maintenance and transmission. Domestic animals, often accompanied by dogs, frequently share grazing areas with wildlife, while wild carnivores may prey on or scavenge livestock carcasses near human settlements, thereby facilitating the continued transmission of E. granulosus s.l.
Map of Narok County, Kenya, showing the origins of livestock and the locations of the abattoirs.
Study design, sample collection and preparation
A cross-sectional study was conducted from September 2023 to April 2024. The study population comprised cattle, sheep and goats slaughtered at 4 selected abattoirs in Maasailand (Narok County), with all 3 livestock species represented at each abattoir. Narok Slaughterhouse is located in Narok Town, whereas the other 3 abattoirs (East African, Kerempes and Nkokosh) are located in Ewaso Ng’iro Town (Figure 1). All abattoirs are managed by qualified meat inspectors and were selected because they receive animals from diverse regions across the study area.
Ante-mortem and post-mortem examination
Before slaughter, each animal’s age and sex were recorded. Post-mortem examination involved visual inspection, palpation and incision of visceral organs (liver, lungs, spleen, mesentery and heart) to detect hydatid cysts. All cysts recovered from infected organs were placed in labelled plastic bags and promptly transported to the Kenya Medical Research Institute (KEMRI) for further processing and analysis.
Microscopic examination of cyst
Cyst diameters were measured and classified as small (<4 cm), medium (4–8 cm) or large (>8 cm), following Kebede et al. (Reference Kebede, Hagos, Girma and Lobago2009). Each cyst was examined to determine its fertility status following the standard protocols of Soulsby (Reference Soulsby1982). Cysts were carefully rinsed with water and dissected using a sterile scalpel. Cyst contents were transferred into Petri dishes and examined microscopically at ×40 magnification for the presence of protoscoleces (PS). Portions of cyst tissue or PS were directly preserved in 70% ethanol in individually labelled tubes for subsequent analysis. Cysts were classified as calcified (thick, hardened walls without fluid or PS), sterile (fluid-filled but no PS) or fertile (clear fluid, containing PS).
Polymerase chain reaction, restriction fragment length polymorphism and sequencing
A piece of cyst tissue or a single protoscolex was placed in a 0.2 mL polymerase chain reaction (PCR) tube containing 10 µL of 0.02 m NaOH and lysed at 99 °C for 10 min (Nakao et al., Reference Nakao, Sako and Ito2003). The lysate was used directly as a DNA template for a nested PCR targeting the complete NADH dehydrogenase subunit 1 (nad1) gene following Hüttner et al. (Reference Hüttner, Nakao, Wassermann, Siefert, Boomker, Dinkel, Sako, Mackenstedt, Romig and Ito2008). The primary PCR used primers 5′-TGT TTT TGA GAT CAG TTC GGT GTG-3′ (forward) and 5′-CAT AAT CAA ACG GAG TAC GAT TAG-3′ (reverse), while the nested PCR employed primers 5′-CAG TTC GGT GTG CTT TTG GGT CTG-3′ and 5′-GAG TAC GAT TAG TCT CAC ACA GCA-3′, amplifying a fragment of 1073–1078 bp in length. Each 25 µL PCR reaction contained 2 µL of DNA, 1 × GoTaq® Flexi Buffer (Promega, Madison, USA), 0.2 mM dNTPs (Promega, Madison, USA), 0.25 µM of each primer, 2 mM MgCl₂ and 0.625 U GoTaq® G2 Flexi DNA polymerase (Promega, Madison, USA). For the nested reaction, 2 µL of the primary PCR product was used as the template. The cycling conditions were: initial denaturation at 94 °C for 5 min; 40 cycles of 94 °C for 30 sec, 55 °C for 30 sec, 72 °C for 60 sec and a final extension at 72 °C for 5 min. Secondary PCR products (10 µL) were resolved on 2% agarose gels stained with SYBR Safe DNA gel stain (Thermo Fisher Scientific, Massachusetts, USA).
Positive PCR amplicons were genotyped by restriction fragment length polymorphism (RFLP) as described by Hüttner et al. (Reference Hüttner, Siefert, Mackenstedt and Romig2009). Briefly, 5 µL of PCR product was mixed with 5 U of HphI restriction enzyme, 1× buffer and nuclease-free water to a final volume of 20 µL, and then incubated at 37 °C overnight. Digests were separated on 2% agarose gels and visualized with SYBR Safe DNA gel stain (Thermo Fisher Scientific, Massachusetts, USA). Positive controls for E. granulosus s.s., E. ortleppi, E. canadensis (G6/7) and E. equinus were run alongside test samples. PCR products from isolates that could not be assigned by RFLP, together with selected representatives of each genotype, were purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, Madison, USA) and sequenced at Macrogen Europe BV (Amsterdam, the Netherlands).
Data analysis
Epidemiological data were recorded in Microsoft Excel and analysed in R software (version 4.3.3). Associations between categorical variables were evaluated using the Chi-square (χ2) test. Temporal trends in prevalence were evaluated across 4 time points (1985, 2012, 2019 and 2024) using the Cochran–Armitage trend test, with calendar year treated as the ordinal score variable. Data for 1985, 2012 and 2019 were obtained from previously published studies, whereas the 2024 data were generated from the present study. Statistical significance was set at P < 0.05. DNA sequences were viewed and manually edited using GENtle v1.9.4, and E. granulosus s.l. species were identified through BLAST analysis (Altschul et al., Reference Altschul, Madden, Schäffer, Zhang, Zhang, Miller and Lipman1997). Multiple sequence alignment was performed using the MAFFT online platform (http://mafft.cbrc.jp/alignment/server).
Results
Prevalence of CE in cattle, sheep and goats and temporal trend analysis
A total of 1891 slaughtered livestock were examined for CE during the survey, comprising 667 cattle, 592 sheep and 632 goats. The overall prevalence of CE was 28.7% (543/1891; 95% CI: 26.8–30.8). Prevalence was highest in cattle (37.2%; 248/667; 95% CI: 33.7–41.0), followed by sheep (31.9%; 189/592; 95% CI: 28.4–35.9) and goats (16.8%; 106/632; 95% CI: 14.1–20.0). Across all livestock species, CE prevalence was significantly higher in females (33.3%; 95% CI: 30.9–36.0) than in males (19.0%; 95% CI: 16.1–22.4; P < 0.001). This sex-related difference was significant in cattle (43.9% vs 21.5%; P < 0.001) and goats (20.5% vs 10.8%; P = 0.002), but not in sheep (33.6% vs 27.8%) (P = 0.175) (Table 1). Although CE prevalence varied by age and sex across livestock species, no clear increase was observed with advancing age (Table 2). In cattle, prevalence in females increased marginally from 42.3% at 3–4 years to 45.9% at >6 years, while male prevalence remained lower and showed no age-related pattern. In sheep, prevalence was high across age categories without a consistent age trend. In goats, a moderate increase with age was observed only in females, reaching 25.0% in the 5–6-year group (Table 2).
Overall prevalence of cystic echinococcosis by livestock species and sex in Maasailand, Kenya
Table 1 Long description
The table reports cystic echinococcosis infection counts and prevalence percentages with 95% confidence intervals for cattle, sheep, and goats, overall and by sex, plus a test comparing males and females within each species. Overall prevalence was highest in cattle at 37.2% (248 of 667), followed by sheep at 31.9% (189 of 592), and lowest in goats at 16.8% (106 of 632). In cattle, females had 43.9% prevalence (205 of 467) versus 21.5% in males (43 of 200), and the sex difference was statistically significant with a very small p-value. In goats, females had 20.5% (80 of 391) versus 10.8% in males (26 of 241), also a statistically significant difference. In sheep, females had 33.6% (142 of 423) versus 27.8% in males (47 of 169), and this difference was not statistically significant. Across all animals combined, prevalence was 28.7% (543 of 1,891), with females higher at 33.3% (427 of 1,281) than males at 19.0% (116 of 610), and the overall sex difference was statistically significant. Counts in parentheses indicate how many animals were examined, and confidence intervals show uncertainty around each prevalence estimate.
Values in parentheses indicate the number of animals examined. Prevalence is presented as the percentage of infected animals with 95% CI. The χ2 test compares infection prevalence between males and females within each livestock species. An asterisk (*) indicates a statistically significant difference (P < 0.05).
Infected and examined livestock by age and sex in Maasailand, Kenya
Table 2 Long description
The table reports, for cattle, sheep, and goats, how many animals were infected out of how many examined, broken down by sex and four age bands. In cattle, females have consistently higher prevalence than males, rising from 44.4 percent at 2 years and under to 45.9 percent over 6 years, while males range from 0 percent at 2 years and under to about 21 to 24 percent in older groups. In sheep, prevalence is moderate in most groups, with the highest value in males aged 5 to 6 years at 45.1 percent; the oldest sheep groups show 0 percent but are based on very small samples. In goats, prevalence is generally lower than in cattle and sheep, with females higher than males in each age band; the oldest goat groups include very small samples and show 0 percent in males and 50.0 percent in females. Totals across species show females higher than males in every age band, increasing from 22.0 percent in females aged 2 years and under to 45.4 percent in females over 6 years, while males range from 13.6 percent to 24.0 percent. Comparisons for the oldest age band, especially in sheep and goats, should be interpreted cautiously because the numbers examined are very small.
The number of infected animals/number of animals examined, with the percentage prevalence shown in parentheses for each age-sex category.
A Cochran–Armitage trend test applied across all 4 time points revealed a highly significant linear trend in CE prevalence over time (Z = 6.07, P < 0.001), indicating a substantial overall increase from 8.0% in 1985 to 28.7% in 2024. However, the trend was not strictly monotonic, as prevalence declined from 20.1% in 2012 to 14.8% in 2019 before rising sharply in 2024.
Cyst conditions and organ distribution in cattle, sheep and goats
Based on morphological characteristics, cysts were classified as fertile, sterile or calcified (Figure 2). Sheep exhibited the highest cyst fertility rate (11.6%), followed by goats (7.6%) and cattle (6.7%) (Table 3). Across all livestock species, the lungs were the most frequently infected organ (52.9%), followed by the liver (45.6%), while infection of the heart (0.7%), spleen (0.6%) and mesentery (0.2%) was rare. Single-cyst infections were the most common across all slaughtered livestock, whereas multiple-cyst infections were observed less frequently (Table 4).
Macroscopic appearance of cyst conditions in livestock from Maasailand, Kenya: (A) fertile, (B) sterile and (C) calcified.
Figure 2 Long description
The map shows Narok County in Kenya, highlighting towns, abattoirs, roads and wards. Towns are marked with black triangles, including Narok Township, Narok and Ewaso Ngiro. Abattoirs are indicated with red triangles, located in Narok and Ewaso Ngiro. Roads are depicted as red lines, connecting various parts of the county. The wards are shaded in light green, with names such as Narok, Ewaso Ngiro, Nkareta and Maji Moto/naroosura. A scale bar at the bottom left indicates distances in kilometers. An inset map shows the location of Narok County within Kenya, with a blue outline highlighting the county's position. A compass rose at the top right indicates north. The legend at the bottom right explains the symbols used for towns, abattoirs, roads and wards.
Organ distribution, number and condition of cysts in cattle, sheep and goats from Maasailand, Kenya
Table 3 Long description
The table reports counts of cysts by livestock species and organ, and breaks each organ total into fertile, sterile, and calcified conditions with percentages within that organ. Overall, 1,828 cysts were recorded across cattle, sheep, and goats; most were sterile, with calcified next and fertile least common. Cattle had the most cysts at 1,196, mainly in lungs (676) and liver (503); lungs were mostly sterile (489, 72.3 percent) while liver had more calcified cysts (269, 53.5 percent). Sheep had 407 cysts, concentrated in liver (223) and lungs (176); liver was mostly calcified (128, 57.4 percent) and lungs were mostly sterile (129, 73.3 percent). Goats had 225 cysts, mainly in lungs (115) and liver (108); lungs were mostly sterile (86, 74.8 percent) and liver was mostly calcified (62, 57.4 percent). Fertile cysts were a small share overall (144, 7.9 percent), but were relatively higher in sheep lungs (24, 13.6 percent) and goat lungs (16, 13.9 percent) than in most other organs. Some organs had very small totals, such as hearts, spleen, and mesentery, so their percentages can appear large despite few observations. Dashes indicate that no cysts of that condition were observed for that organ and species.
The number of cysts, with the percentage relative to the total number of cysts in the corresponding organ, is shown in parentheses. The symbol ‘—’ indicates that no cysts of that condition were observed.
Cyst load among infected cattle, sheep and goats in Maasailand, Kenya
Table 4 Long description
The table reports how many infected cattle, sheep, and goats fall into cyst-load categories from one cyst through five or more cysts, with counts and within-species percentages. Among cattle, 97 of 248 infected animals have one cyst (39.1 percent) and 67 have five or more cysts (27.0 percent), the highest high-burden share of the three species. Sheep show a stronger concentration at low burden: 106 of 189 have one cyst (56.1 percent) and 15 have five or more (7.9 percent). Goats are similar to sheep at the low end, with 63 of 106 having one cyst (59.4 percent) and 10 having five or more (9.4 percent). Across all infected animals combined, one cyst is most frequent at 266 of 543 (49.0 percent), while five or more cysts occurs in 92 (16.9 percent). Overall, sheep and goats tend to have fewer cysts per infected animal than cattle, but the percentages are calculated only among infected animals within each species.
The numbers in parentheses indicate the total number of infected animals examined for each livestock species and overall. Values represent the number of animals with the indicated cyst load, with percentages in parentheses calculated relative to the total number of infected animals within each species.
Genotyping of E. granulosus s.l. in cattle, sheep and goats
Of the 1828 cysts isolated, 857 were subjected to nested PCR, of which 850 were successfully amplified. PCR–RFLP and sequencing identified E. granulosus s.s. in 841 samples (98.9%), E. ortleppi in 8 (0.9%) and E. canadensis (G6/7) in 1 (0.1%) (Table 5). Cyst fertility varied by host species, E. granulosus s.s. exhibited the highest fertility in sheep (47/158; 29.7%), followed by goats (17/109; 15.6%) and cattle (76/574; 13.2%). All 8 E. ortleppi cysts were identified in cattle, of which 4 were fertile, whereas the single E. canadensis (G6/7) cyst detected in sheep was sterile (Table 5). Mixed infections were identified in 2 cattle, each co-infected with E. granulosus s.s. and E. ortleppi. No mixed infections were observed in sheep or goats.
Organ distribution, cyst condition and frequency of molecularly analysed E. granulosus s.l. in cattle, sheep and goats in Maasailand, Kenya
Table 5 Long description
The table reports, by livestock species and organ, how many hydatid cysts were examined and how many were assigned to E. granulosus sensu stricto, E. ortleppi, or E. canadensis G6/7, including cyst condition categories. Cattle contributed the most cysts, mainly from lungs, and nearly all typed cysts were E. granulosus sensu stricto, with smaller numbers of E. ortleppi and no E. canadensis detected. In cattle, lungs dominated the organ distribution, followed by liver, while heart and spleen had very few cysts. Sheep had fewer cysts than cattle, again mostly from lungs and liver, with E. granulosus sensu stricto predominant and a single E. canadensis G6/7 cyst recorded in the lung. Goats had the fewest cysts, concentrated in lungs and liver, and all typed cysts were E. granulosus sensu stricto with some E. ortleppi and no E. canadensis detected. Across species, sterile cysts were more common than fertile cysts, and calcified cysts were reported only within the E. granulosus sensu stricto counts. Percentages shown in the table are within each parasite species for each livestock species, so they should not be compared as overall prevalence between species without considering the different totals examined.
Values in parentheses represent the percentage relative to the total number of cysts identified for that parasite species within each livestock species. ‘Number of cysts’ indicates the total number of cysts examined in the corresponding organ.
Complete 894-bp nad1 gene amplicons were sequenced from E. granulosus s.s. isolates with inconclusive RFLP patterns that required confirmation. All 24 suspected E. granulosus s.s. isolates were confirmed, and 10 nad1 haplotypes were identified (Table 6). Haplotype 1 (Hap1) was predominant, comprising 13 sequences that were 100% identical to each other and to several global GenBank reference sequences (e.g. MN199128). Haplotypes 2 and 3 were each represented by 2 identical sequences; Hap2 showed complete identity (100%) with a reference sequence previously reported from cattle in Kenya (MZ736690), whereas Hap3 differed by a single nucleotide substitution from multiple global reference sequences (99.9% identity). The remaining haplotypes (Hap4–Hap10) occurred as singletons and exhibited 99.6–99.9% identity with GenBank references. Overall, sequence variation was observed at 12 nucleotide positions within the nad1 gene, comprising 6 synonymous and 6 nonsynonymous substitutions (Table 6). Sequences representing E. granulosus s.s. haplotypes have been deposited in GenBank under accession numbers PX508169–PX508178. Similarly, 5 of the 8 isolates identified as E. ortleppi were successfully sequenced. All 5 sequences were identical and showed 100% similarity with several reference sequences available in GenBank, including an isolate from Kenyan cattle (accession number KX010904), representing the most common African haplotype, Eo01 (Addy et al., Reference Addy, Wassermann, Banda, Mbaya, Aschenborn, Aschenborn, Koskei, Umhang, DE LA Rue, Elmahdi, Mackenstedt, Kern and Romig2017). A representative sequence from this group has been deposited in GenBank under accession number PX508179. The only E. canadensis (G6/7) isolate in this study yielded a low-quality sequence that was trimmed to 482 bp. The trimmed sequence showed 98.9% identity with several reference sequences in GenBank, including Kenyan camel isolates (accession numbers MT525967 and KX010873). This sequence has been deposited in GenBank under accession number PX508180.
Nucleotide substitutions identified in 894-bp mitochondrial nad1 gene sequences of E. granulosus sensu stricto isolates from cattle, sheep and goats in Maasailand, Kenya
Table 6 Long description
The table lists nucleotide bases at 12 variable positions in an 894 base-pair mitochondrial nad1 sequence for multiple Echinococcus granulosus sensu stricto isolates, compared with a reference pattern. A dot indicates the isolate matches the reference base at that position, while letters mark substitutions. The reference row MN199128 provides the baseline bases across all positions. Several isolates differ only at position 256, where MZ736690 and Hap2 show guanine instead of adenine. Other haplotypes each carry one or a few additional substitutions: Hap3 changes position 400 to guanine, Hap4 changes position 480 to cytosine, and Hap5 changes positions 375 and 477 to cytosine. Variation is more concentrated in the later positions for some haplotypes: Hap7 has substitutions at positions 512, 521, 527, and 531, and Hap9 shows multiple changes from positions 519 through 527. Hap6 differs at position 541, and Hap10 differs at position 527. Positions marked with an asterisk are nonsynonymous sites, so substitutions there may affect the encoded protein, but functional impact cannot be determined from this table alone.
Substitution sites are numbered from the initiation codon. A indicates adenine, T indicates thymine, G indicates guanine, C indicates cytosine and • indicates consensus nucleotide base with the reference sequence.
a Nonsynonymous substitution.
Discussion
This study provides compelling evidence of a marked increase in CE prevalence among livestock in Maasailand (Narok County), Kenya. The prevalences observed in cattle (37.2%), sheep (31.9%) and goats (16.8%) substantially exceed those reported by Macpherson (Reference Macpherson1985) and represent a continued escalation from levels documented by Addy et al. (Reference Addy, Alakonya, Wamae, Magambo, Mbae, Mulinge, Zeyhle, Wassermann, Kern and Romig2012) and Nungari et al. (Reference Nungari, Mbae, Gikunju, Mulinge, Kaburu, Zeyhle and Magambo2019) in the same region, and are higher than those reported in other endemic areas of Kenya (Njoroge et al., Reference Njoroge, Mbithi, Gathuma, Wachira, Gathura, Magambo and Zeyhle2002; Mbaya et al., Reference Mbaya, Magambo, Njenga, Zeyhle, Mbae, Mulinge, Wassermann, Kern and Romig2014; Odongo et al., Reference Odongo, Tiampati, Mulinge, Mbae, Bishop, Zeyhle, Magambo, Wasserman, Kern and Romig2018). The Cochran–Armitage trend test was performed to assess whether the observed increase in prevalence represented a statistically significant trend over time. The test confirmed a highly significant overall increase in CE prevalence between 1985 and 2024 (Z = 6.07, P < 0.001). Notably, however, the trend was not strictly monotonic, with a transient decline observed in 2019 before rising sharply again, suggesting that transmission intensity may be subject to temporal fluctuations within the broader upward trajectory. These findings indicate a trend towards intensifying transmission within this pastoral system. The specific drivers of this rising prevalence remain unclear. CE transmission is generally facilitated by close livestock–dog interactions, poor slaughter practices, inadequate offal disposal, limited dog deworming and the presence of free-roaming dogs (Craig et al., Reference Craig, Hegglin, Lightowlers, Torgerson and Wang2017; Nocerino et al., Reference Nocerino, Pepe, Ciccone, Maurelli, Bosco, Boué, Umhang, Lahmar, Said, Sotiraki, Ligda, Laatamna, Reghaissia, Saralli, Musella, Alterisio, Piegari and Rinaldi2024). All of these factors are present in the study area and likely contribute to ongoing transmission. However, these parasites have long been present in the region and therefore do not readily explain the pronounced upward trend observed over the past 4 decades. Over the same period, Narok County’s human and livestock populations increased approximately 3-fold since 1989 (KNBS, 1991, 2001, 2010, 2019), which may enhance transmission by increasing the density of both definitive and intermediate hosts. A positive association between host density and parasite prevalence has been documented for other cestodes, for example, E. multilocularis in Europe (Romig et al., Reference Romig, Deplazes, Jenkins, Giraudoux, Massolo, Craig, Wassermann, Takahashi and de la Rue2017).
Sex- and age-related differences in infection were evident, with females exhibiting significantly higher CE prevalence than males across livestock species, except in sheep, consistent with reports from other endemic regions (Khan et al., Reference Khan, Syed, Shahnawaz, Syed and Nazir2010; Haleem et al., Reference Haleem, Niaz, Qureshi, Ullah, Alsaid, Alqahtani and Shahat2018; Abdelghani et al., Reference Abdelghani, M’rad, Chaâbane-banaoues, Taoufik, Charfedine, Zemzemi, Kamoun, Babba and Oudni-m’rad2025). The higher CE prevalence observed in females possible reflects immune modulation during pregnancy and lactation (Houdijk, Reference Houdijk2008; Beasley et al., Reference Beasley, Kahn and Windon2010). When considering all livestock species combined, CE prevalence increased with age, plausibly reflecting the cumulative probability of exposure to infective eggs over time (Torgerson et al., Reference Torgerson, Oguljahan, Muminov, Karaeva, Kuttubaev, Aminjanov and Shaikenov2006; Ibrahim et al., Reference Ibrahim, Thomas, Peter and Omer2011; Li et al., Reference Li, Quzhen, Xue, Han, Chen, Yan, Li, Quick, Huang, Xiao, Wang, Wang, Zuoga, Bianba, Ma, Gasong, Niji, Zheng, Wu and Zhou2019; Tamarozzi et al., Reference Tamarozzi, Legnardi, Fittipaldo, Drigo and Cassini2020). However, species-specific analyses showed that this age-related pattern was consistently observed only in goats. In sheep and cattle, the pattern was heterogeneous and, where increases occurred, they were modest rather than pronounced. Notably, relatively high prevalence levels were already present in younger animals and were comparable to those observed in older age groups. This suggests high infection pressure in the Maasai region, resulting in substantial infection early in life, while older animals may acquire partial immunity through repeated exposure.
In this study, cysts were more frequently detected in the lungs than in the liver, a pattern consistent with several reports from Kenya and other East African endemic regions (Njoroge et al., Reference Njoroge, Mbithi, Gathuma, Wachira, Gathura, Magambo and Zeyhle2002; Getaw et al., Reference Getaw, Beyene, Ayana, Megersa and Abunna2010; Kebede et al., Reference Kebede, Gebre-egziabher, Tilahun and Wossene2011; Mbaya et al., Reference Mbaya, Magambo, Njenga, Zeyhle, Mbae, Mulinge, Wassermann, Kern and Romig2014; Kere et al., Reference Kere, Joseph, Jessika and Maina2019; Tamarozzi et al., Reference Tamarozzi, Legnardi, Fittipaldo, Drigo and Cassini2020). Although some Kenyan studies have identified the liver as the primary site of infection (Addy et al., Reference Addy, Alakonya, Wamae, Magambo, Mbae, Mulinge, Zeyhle, Wassermann, Kern and Romig2012; Odongo et al., Reference Odongo, Tiampati, Mulinge, Mbae, Bishop, Zeyhle, Magambo, Wasserman, Kern and Romig2018; Nungari et al., Reference Nungari, Mbae, Gikunju, Mulinge, Kaburu, Zeyhle and Magambo2019; Omondi et al., Reference Omondi, Gitau, Gathura, Mulinge, Zeyhle, Kimeli and Bett2020), lung cysts are more often fertile, as reported in livestock and camels from Kenya and Ethiopia (Addy et al., Reference Addy, Alakonya, Wamae, Magambo, Mbae, Mulinge, Zeyhle, Wassermann, Kern and Romig2012; Kebede et al., Reference Kebede, Gebre-egziabher, Tilahun and Wossene2011; Omondi et al., Reference Omondi, Gitau, Gathura, Mulinge, Zeyhle, Kimeli and Bett2020). This lung predominance and higher cyst viability likely reflect host physiological factors, including the lungs’ softer parenchyma and capillary architecture, which facilitate oncosphere lodging and cyst development (Kebede et al., Reference Kebede, Gebre-egziabher, Tilahun and Wossene2011), together with genotype-specific organ tropism, whereby E. granulosus s.s. infects both liver and lungs, while E. ortleppi and E. canadensis (G6/7) show a marked preference for lung tissue (Addy et al., Reference Addy, Alakonya, Wamae, Magambo, Mbae, Mulinge, Zeyhle, Wassermann, Kern and Romig2012; Romig et al., Reference Romig, Deplazes, Jenkins, Giraudoux, Massolo, Craig, Wassermann, Takahashi and de la Rue2017).
This study confirms the predominance of E. granulosus s.s. in livestock from Maasailand, accounting for 98.94% of infections, while E. ortleppi and E. canadensis (G6/7) occur only sporadically. The widespread occurrence of E. granulosus s.s. across cattle, sheep and goats, and its detection in multiple organs, underscores its ecological adaptability and efficient transmission in pastoral systems, consistent with previous Kenyan and global studies (Addy et al., Reference Addy, Alakonya, Wamae, Magambo, Mbae, Mulinge, Zeyhle, Wassermann, Kern and Romig2012; Wachira et al., Reference Wachira, Bowles, Zeyhle and McManus1993; Dinkel et al., Reference Dinkel, Njoroge, Zimmermann, Wälz, Zeyhle, Elmahdi, Mackenstedt and Romig2004; Mbaya et al., Reference Mbaya, Magambo, Njenga, Zeyhle, Mbae, Mulinge, Wassermann, Kern and Romig2014; Deplazes et al., Reference Deplazes, Rinaldi, Alvarez Rojas, Torgerson, Harandi, Romig, Antolova, Schurer, Lahmar, Cringoli, Magambo, Thompson and Jenkins2017; Romig et al., Reference Romig, Deplazes, Jenkins, Giraudoux, Massolo, Craig, Wassermann, Takahashi and de la Rue2017; Odongo et al., Reference Odongo, Tiampati, Mulinge, Mbae, Bishop, Zeyhle, Magambo, Wasserman, Kern and Romig2018; Nungari et al., Reference Nungari, Mbae, Gikunju, Mulinge, Kaburu, Zeyhle and Magambo2019). Its concurrent predominance in both livestock and dogs from the same region further supports the persistence of an active livestock–dog transmission cycle in Maasailand (Mulinge et al., Reference Mulinge, Magambo, Odongo, Njenga, Zeyhle, Mbae, Kagendo, Addy, Ebi, Wassermann, Kern and Romig2018), reaffirming E. granulosus s.s. as the most widespread and zoonotically significant genotype in Kenya (Romig et al., Reference Romig, Deplazes, Jenkins, Giraudoux, Massolo, Craig, Wassermann, Takahashi and de la Rue2017).
The low-frequency detection of E. ortleppi and E. canadensis (G6/7) in this study indicates the coexistence of multiple, but unevenly sustained, transmission cycles in Maasailand (Romig et al., Reference Romig, Deplazes, Jenkins, Giraudoux, Massolo, Craig, Wassermann, Takahashi and de la Rue2017). Echinococcus ortleppi was detected exclusively in cattle, predominantly in the lungs, reinforcing its strong host and organ specificity, as previously reported (Addy et al., Reference Addy, Alakonya, Wamae, Magambo, Mbae, Mulinge, Zeyhle, Wassermann, Kern and Romig2012; Romig et al., Reference Romig, Deplazes, Jenkins, Giraudoux, Massolo, Craig, Wassermann, Takahashi and de la Rue2017). In contrast, E. canadensis (G6/7) was identified only as a sterile lung cyst in sheep, consistent with earlier findings from Maasailand and neighbouring regions that suggest limited parasite viability and possible host adaptation constraints in the absence of camels. Goats have so far been the only alternative hosts shown to occasionally act as competent hosts (Addy et al., Reference Addy, Alakonya, Wamae, Magambo, Mbae, Mulinge, Zeyhle, Wassermann, Kern and Romig2012; Romig et al., Reference Romig, Deplazes, Jenkins, Giraudoux, Massolo, Craig, Wassermann, Takahashi and de la Rue2017; Nungari et al., Reference Nungari, Mbae, Gikunju, Mulinge, Kaburu, Zeyhle and Magambo2019). To date, the only reported wild intermediate host of E. canadensis (G6/7) is the Oryx antelope in Namibia; however, this species does not occur in the present study area (Aschenborn et al., Reference Aschenborn, Aschenborn, Beytell, Wachter, Melzheimer, Dumendiak, Rüffler, Mackenstedt, Kern, Romig and Wassermann2023; Romig and Wassermann, Reference Romig and Wassermann2024). Consequently, how the transmission cycle is maintained in this region remains largely unknown. Mixed infections were rare and confined to cattle, where the co-occurrence of E. granulosus s.s. and E. ortleppi suggests ecological overlap and shared transmission niches, consistent with previous reports from Kenya (Addy et al., Reference Addy, Alakonya, Wamae, Magambo, Mbae, Mulinge, Zeyhle, Wassermann, Kern and Romig2012; Mbaya et al., Reference Mbaya, Magambo, Njenga, Zeyhle, Mbae, Mulinge, Wassermann, Kern and Romig2014; Odongo et al., Reference Odongo, Tiampati, Mulinge, Mbae, Bishop, Zeyhle, Magambo, Wasserman, Kern and Romig2018; Nungari et al., Reference Nungari, Mbae, Gikunju, Mulinge, Kaburu, Zeyhle and Magambo2019; Omondi et al., Reference Omondi, Gitau, Gathura, Mulinge, Zeyhle, Kimeli and Bett2020; Mulinge et al., Reference Mulinge, Zeyhle, Mbae, Gitau, Kaburu, Magambo, Mackenstedt, Romig, Kern and Wassermann2023).
Given that E. granulosus s.s. is the principal cause of human CE in Kenya and globally (Wachira et al., Reference Wachira, Bowles, Zeyhle and McManus1993; Casulli et al., Reference Casulli, Zeyhle, Brunetti, Pozio, Meroni, Genco and Filice2010; Mutwiri et al., Reference Mutwiri, Magambo, Zeyhle, Mkoji, Wamae, Mulinge, Wassermann, Kern and Romig2013; Alvarez Rojas et al., Reference Alvarez Rojas, Romig and Lightowlers2014; Romig et al., Reference Romig, Deplazes, Jenkins, Giraudoux, Massolo, Craig, Wassermann, Takahashi and de la Rue2017), its dominance in Maasailand underscores a persistent and escalating public-health threat in pastoral communities, where close contact between livestock, dogs and humans facilitates transmission. The likelihood of human infection through contaminated food, water or direct contact with infected dogs (Eckert and Deplazes, Reference Eckert and Deplazes2004) may increase, as the rising prevalence of CE in livestock suggests greater environmental contamination and potential transmission from infected dogs. This concern is further reinforced by recent evidence of human CE infections reported from the same region (Zeyhle, unpublished data). These findings highlight the urgent need for integrated One Health control strategies that prioritize genotype-specific surveillance, improved slaughter hygiene, safe offal disposal, regular dog deworming and sustained community education to disrupt transmission and reduce the CE burden in endemic settings.
Limitations
This study was limited by its cross-sectional design and sampling from only 4 abattoirs, which may not fully capture infection patterns across all pastoral settings. In addition, limited livestock demographic information, particularly regarding animal origin within Maasailand and factors influencing the sale of female versus male animals, constrained a more detailed interpretation of risk patterns. Future longitudinal and One Health-oriented studies integrating animal and human data are recommended to better elucidate CE transmission dynamics (Romig et al., Reference Romig, Deplazes, Jenkins, Giraudoux, Massolo, Craig, Wassermann, Takahashi and de la Rue2017; Tamarozzi et al., Reference Tamarozzi, Legnardi, Fittipaldo, Drigo and Cassini2020).
Conclusion
This study provides updated epidemiological and molecular evidence confirming E. granulosus s.s. as the predominant cause of CE in livestock in Maasailand, Kenya. The high prevalence across species, coupled with elevated cyst fertility in sheep and documented human CE in the region, indicates sustained and intense transmission within pastoral systems. The additional detection of E. ortleppi and E. canadensis (G6/7) further suggests the coexistence of multiple transmission cycles and a continuing public-health risk. These findings underscore the need to better understand local drivers of transmission to inform strengthened and scaled-up One Health-based prevention and control strategies in this endemic region.
Data availability statement
All data supporting the findings of this study are available within the article. The nucleotide sequence data generated in this study have been deposited in GenBank under accession numbers PX508169–PX508180.
Acknowledgements
This manuscript is published with the permission of the Director General of KEMRI. The authors express their gratitude to the Director of Veterinary Services and technical staff in Narok County, and to the traders and butchers in all participating slaughterhouses, for their valuable support in this study. Authors acknowledge the use of ChatGPT-5 on 18.3.2026 to assist in the design and generation of the graphical abstract. The authors reviewed and edited the output and take full responsibility for the final content. The free version can be accessed through https://www.chatgpt.com.
Author contributions
Conceptualization: L.G., T.T., C.M., E.Z., E.M., T.R. and M.W. Data curation: L.G., E.Z., E.M., T.R. and M.W. Formal analysis: L.G., E.M. and M.W. Funding acquisition: C.M., J.M., P.K., T.R. and M.W. Investigation: L.G., E.Z., E.M., Z.G. and B.S. Methodology: L.G., E.M., Z.G. and B.S. Supervision: C.M., T.T. and M.W. Validation: L.G., E.M., T.R. and M.W. Writing original draft: L.G. All authors revised the manuscript and approved the final version for publication.
Financial support.
This study was supported by the Deutsche Forschungsgemeinschaft (DFG) (grant numbers RO 3753/9-1: KE 282/12-1) and the German Academic Exchange Service (DAAD; personal reference number 91871802), which funded the first and corresponding author.
Competing interests
All authors listed in this manuscript declare that they have no competing interests related to the submission or publication of this article.
Ethical standards
Institutional approval was obtained from the Department of Animal Biology, University of Dschang. Ethical clearance was granted by the KEMRI Scientific and Ethics Review Unit (SERU) and the Animal Care and Use Committee (ACUC) under reference number KEMRI/SERU/CMR/P00262-010-2023/4938. A research license was granted by the National Commission for Science, Technology and Innovation (NACOSTI) under reference number NACOSTI/P/26/4183129 and the Ministry of Agriculture, Livestock and Fisheries, Department of Veterinary Services, Narok County.
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