Advancing gastrointestinal parasite diagnosis in West African long-legged lambs in Southern Benin: a comparative study of McMaster and Mini-FLOTAC methods

Abstract

Background

Gastrointestinal (GI) parasites remain a significant global challenge to livestock health and farm productivity, particularly in resource-limited regions. Accurate and reliable fecal egg count (FEC) methods are essential for quantifying parasite burden and evaluating anthelmintic efficacy. This study compared the diagnostic performance of the Mini-FLOTAC and the modified McMaster techniques for detecting GI parasites in West African Long-legged (WALL) sheep under field conditions in southern Benin.

Methods

A cross-sectional survey was conducted, during which 200 fresh fecal samples were collected from four-month-old lambs across five representative sheep farms. Each sample was divided and analyzed in parallel using the Mini-FLOTAC (using 2 g of feces diluted in a 1:10 ratio with saturated sodium chloride solution) and the modified McMaster technique (using 3 g of feces in a 1:15 dilution). Diagnostic parameters, including the intensity of infection expressed as eggs/oocysts per gram of feces (EPG/OPG, respectively), the prevalence, and the precision, were statistically analyzed and compared. Method agreement was assessed using Cohen’s kappa coefficient.

Results

The Mini-FLOTAC technique demonstrated superior performance, detecting a broader spectrum of parasites, including Nematodirus spp., Marshallagia spp., and Moniezia spp., which were frequently undetected by McMaster. Agreement between techniques was high for strongylids and Eimeria spp. (κ ≥ 0.76), but poor for other taxa (κ < 0.30). Mini-FLOTAC recorded significantly higher FECs and oocyst per gram of feces (OPG) values across farms (p < 0.05), and consistently exhibited greater diagnostic precision, with lower coefficients of variation (CVs ranging from 12.37% to 18.94%) and higher reproducibility (> 80% precision). Misclassification analysis revealed that the McMaster method underdiagnosed up to 12.5% of infections, especially for low-shedding species.

Conclusions

These findings highlight Mini-FLOTAC as a more sensitive, precise, and operationally robust tool for GI parasite surveillance in small ruminants. Its adoption can improve the reliability of epidemiological monitoring and support sustainable parasite control programs in resource-limited settings.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12917-025-05099-8.

Background

Small ruminants play a crucial role in enhancing livelihoods and food security across much of the developing world. They provide accessible sources of meat, milk, and income for both rural and peri-urban households, and also contribute to crop production through manure, which improves soil fertility [1, 2]. In Benin and throughout West Africa, sheep in particular are culturally and economically important, especially for marginalized communities with limited access to large livestock.

However, despite their significance, small ruminants face numerous health challenges, chief among them being gastrointestinal (GI) parasitic infections. These infections are among the most prevalent and economically damaging diseases affecting small ruminants globally [3]. GI parasites reduce animal productivity by impairing feed intake, growth rate, and reproductive performance, while also increasing susceptibility to other infections. In severe cases, they can lead to premature culling and death, especially in young or immunocompromised animals [1, 4]. The impact of these infections is particularly severe in low-income countries, where inadequate veterinary services, poor nutrition, and unfavorable environmental conditions (e.g., high humidity and continuous grazing) facilitate rapid parasite transmission [2].

In Benin, GI parasitic infections are the most common health problem in small ruminants and are recognized as the second leading cause of mortality in sheep [5, 6]. Young animals are especially vulnerable, typically acquiring infections early in life. Although sheep may develop partial acquired immunity by 8 months of age (and goats between 12 and 18 months), the early stages of life remain critical, as heavy parasite burdens during this period can have lasting impacts on productivity and survival [4].

Moreover, anthelmintic resistance is an emerging global concern. In many parts of Africa, including Benin, the use of dewormers is often empirical and poorly regulated, which accelerates the development of resistance [3]. As the efficacy of conventional treatments declines, there is an urgent need for sensitive and accurate diagnostic methods that can detect low-intensity infections, monitor treatment effectiveness, and support evidence-based parasite control strategies.

The McMaster technique is currently the most widely used coprological method for estimating fecal egg counts (FEC) in veterinary practice in Sub-Saharan Africa [7]. Its widespread adoption is due to its simplicity, cost-effectiveness, and minimal equipment requirements [8, 9]. However, the technique suffers from reduced sensitivity, particularly when infection intensities are low. This limitation compromises its reliability for precision diagnosis and may hinder accurate monitoring of anthelmintic efficacy [10, 11].

To address these shortcomings, the FLOTAC technique was introduced as a more sensitive alternative, employing centrifugation and larger flotation chambers to improve egg recovery [11, 12]. Nevertheless, its use in field settings has been limited due to the need for specialized equipment and technical expertise. As a response to these constraints, the Mini-FLOTAC was developed. This simplified method uses passive flotation, fewer processing steps, and does not require electricity or centrifugation, making it well-suited for application in low-resource and rural environments [12, 13].

Despite the advantages of Mini-FLOTAC, its use remains limited in Sub-Saharan Africa, particularly in veterinary parasitology. This gap is especially critical given the susceptibility of local sheep ecotypes to parasitic infections. Notably, the West African Long-legged (WALL) sheep, which are increasingly being raised in the humid and sub-humid zones of southern Benin due to their larger frame and higher market value, are more sensitive to gastrointestinal parasites than the indigenous West African Dwarf (WAD) sheep [14]. This increased susceptibility, likely linked to genetic differences and reduced adaptation to local parasites, necessitates improved diagnostic capacity for effective health management in WALL sheep populations [6].

Therefore, the present study aimed to evaluate and compare the diagnostic performance of the McMaster and Mini-FLOTAC techniques for detecting helminth and protozoan infections in sheep under field conditions in southern Benin. By focusing on the context of extensive management systems and emphasizing the diagnostic needs of more susceptible breeds like the West African Long-legged sheep, this work seeks to inform context-appropriate and sustainable strategies for parasite control in small ruminant production systems across West Africa.

Materials and methods

Animal collections and geoclimatic characterizations

The study was conducted from May to July 2024 across five West African Long-legged (WALL) sheep farms located in five of the eight townships of the Atlantic Department in southern Benin (Fig. 1): Abomey-Calavi (1 farm with 253 sheep, including 203 lambs), Allada (1 farm: 173 sheep with 107 lambs), Sô-Ava (1 farm: 123 sheep with 94 lambs), Toffo (1 farms: 105 sheep with 71 lambs) and Tori-Bossito (1 farm: 271 sheep with 203 lambs). Geographically, the Atlantic Department lies between 6°18′ and 6°58′ N latitude and 1°56′ and 2°30′ E longitude. It is bordered by the Atlantic Ocean to the south, Mono and Couffo Departments to the west, Zou to the north, and Ouémé to the east. The region experiences a tropical climate with an average annual rainfall of approximately 1,200 mm and mean monthly temperatures ranging from 27 °C to 31 °C. Extensive agricultural expansion in the area has led to significant degradation of natural vegetation, resulting in a landscape now dominated by fallows and croplands. Within this context, small ruminant farming plays an important role in rural livelihoods.

Fig. 1.

Fecal samplings’ locations (sheep farms) in the Atlantic department

Preliminary survey

The study sites were further characterized through a structured questionnaire administered to 5 farm managers and 15 shepherds (n = 20). The survey covered general farm practices, pasture management, feeding strategies, and herd health programs.

All respondents (100%) confirmed operating under traditional extensive systems, where animals grazed on natural pastures with minimal supplemental feeding. Due to rapid urbanization in the Atlantic department, 90% of interviewees reported a significant decline in natural grazing areas. As a result, 85% of herds left for grazing around 8:00 a.m. and returned by 4:00 p.m., often covering distances greater than 5 km. None of the farms practiced systematic pasture rotation or rest.

Regarding feeding practices, 80% of respondents acknowledged using supplemental feeding, primarily with root and tuber by-products such as cassava peelings (65%), yam peelings (45%), sweet potato peels (30%), and cassava chips (25%). All farms (100%) relied on natural water sources, including rainwater tanks (40%), piped sources (30%), runoff (20%), and ponds (10%).

On herd hygiene, 90% of the sheepfolds were built with rudimentary materials and lacked footbaths. Daily cleaning of feeding and watering equipment was performed by only 25% of respondents, while the majority (75%) cleaned them sporadically or not at all. About 70% of animals were housed on bare concrete or dirty soil.

Health issues were prevalent: 65% of respondents frequently observed diarrhea and coughing, 40% reported pruritus, and 35% observed alopecia. Only 20% demonstrated some knowledge of disease etiology. While 30% of respondents reported occasional access to veterinary services, 25% relied on traditional remedies primarily Mitragyna inermis, Combretum glutinosum, and Bridelia ferruginea commonly used to treat bleeding and gastrointestinal parasites. Additionally, 70% of interviewees administered dewormers themselves to manage both internal and external parasites in their herds. Additional details are presented in the table provided as a Supplementary File.

Fecal sample collection

In each WALL sheep farm, individual fecal samples (minimum 20 g) were collected directly from the rectum of 40 randomly selected lambs, aged on average 4.6 ± 0.4 months (range: 4.0–5.5 months), using clean disposable gloves. Each sample was sealed in a plastic rectal sleeve by tying a knot midway and stored at 4 °C. All samples were processed and analyzed on the same day to ensure sample integrity.

Parasitological methods

All fecal samples were analyzed within 24 h of collection using two diagnostic techniques: the McMaster method and the Mini-FLOTAC method.

The modified McMaster technique

The modified McMaster technique [15] was performed following standard operating procedures. Three grams of fresh feces were placed into a container, and 42 mL of saturated sodium chloride solution (NaCl; specific gravity = 1.2) was added, yielding a dilution ratio of 1:15. The mixture was thoroughly homogenized and filtered three times through a 250 μm wire mesh to remove coarse debris. The resulting suspension was transferred between two bowls ten times to ensure uniform mixing. A 0.5 mL aliquot of the suspension was then loaded into each chamber of a standard McMaster slide. After 10 min of flotation, gastrointestinal helminth parasite eggs and coccidia oocysts were counted using a light microscope at 100× that refers to the total magnification, within the two grids corresponding to a total counting volume of 0.3 mL. Fecal egg count (FEC) values, expressed as eggs/oocysts per gram of feces (EPG/OPG, respectively), were calculated by multiplying the total counts by 50 (the multiplication factor).

The Mini-FLOTAC technique

The protocol described by Cringoli et al. [12] was followed to perform the Mini-FLOTAC technique. Two (02) grams of fresh feces were placed into the Fill-FLOTAC container, and 18 mL of saturated sodium chloride solution (NaCl; specific gravity = 1.2) was added, resulting in a dilution ratio of 1:10. The mixture was thoroughly homogenized using the Fill-FLOTAC’s integrated stirring stick, then filtered directly through the device. The resulting suspension was used to fill the two chambers of the Mini-FLOTAC apparatus. After a flotation period of 10 min, the reading disc was rotated, and helminth eggs and coccidia oocysts were examined under a light microscope at 100× that refers to the total magnification. Fecal egg count (FEC) values, expressed as egg/oocyst per gram of feces (EPG/OPG), were calculated by multiplying the total count by 5.

To ensure the quality and reliability of the parasitological analyses for both techniques, we followed quality control procedures as described by Rinaldi et al. [16]. Fecal samples were analyzed within an average of 7 h after collection. The specific gravity of the flotation solution was verified using a hydrometer, and the precision of fecal weight measurements was ensured through scale calibration. Each fecal sample was examined in triplicate for both techniques, resulting in a total of six readings per sample (three for McMaster and three for Mini-FLOTAC). All microscopic examinations were conducted by at least one trained expert in parasitological diagnostics, using morphological criteria described in standard manuals, including the WHO’s Bench Aids for the Diagnosis of Intestinal Parasites, second edition [17].

Statistical analysis

Data from each farm and parasite taxon were arranged in 2 × 2 contingency tables based on the detection outcomes (positive or negative) of gastrointestinal parasites by the McMaster and Mini-FLOTAC techniques. The agreement between the two diagnostic methods was assessed using Cohen’s Kappa coefficient (κ). This statistic evaluates the level of concordance between two categorical datasets beyond what is expected by chance. Kappa values were interpreted according to the scale proposed by Landis and Koch [18]: κ < 0 indicates no agreement; 0.00–0.20, poor agreement; 0.21–0.40, fair agreement; 0.41–0.60, moderate agreement; 0.61–0.80, good agreement; and 0.81–1.00, almost perfect agreement. The 95% confidence intervals (CI) for each κ value were calculated to assess the precision of the agreement estimates. The misclassification rate was calculated as the proportion of animals classified as uninfected by the McMaster technique but found to be positive using the Mini-FLOTAC method.

Intensity of gastrointestinal infection was assessed by calculating the arithmetic means of EPG of feces for strongylids-type nematodes and OPG of feces for Eimeria spp., using corrected multiplication factors specific to each technique: 50 for the McMaster method and 5 for the Mini-FLOTAC method.

Prior to analysis, data were tested for normality using the Shapiro–Wilk test, which confirmed that EPG and OPG values followed a normal distribution. Consequently, differences in mean EPG/OPG values between diagnostic methods (McMaster vs. Mini-FLOTAC) and parasite groups were assessed using a two-way Analysis of Variance (ANOVA) to account for the effects of both diagnostic method and parasite type.

Where significant interactions or main effects were detected (p < 0.05), Tukey–Kramer Honest Significant Difference (HSD) post hoc tests were used to perform pairwise comparisons. All statistical analyses were conducted in R software [19], and results are presented as means ± standard error of the mean (SEM).

The reference values for infection intensity defined as light infection when FEC < 200 EPG/OPG, moderate infection when 200 ≤ FEC ≤ 500 EPG/OPG, and severe infection when FEC > 500 EPG/OPG were adopted from Alowanou et al. [20]. These thresholds were used in the present study to categorize the intensity of gastrointestinal parasitic infections among WALL sheep. This stratification enabled a clearer assessment of the epidemiological profile within and across farms, supporting more informed decision-making regarding selective anthelmintic treatment, herd health prioritization, and sustainable parasite control planning.

Prevalence was defined as the proportion of positive samples out of the total samples examined. For each parasite species, differences in prevalence between techniques were evaluated using the two-proportion z-test, performed in R using the prop.test() function [19].

Precision of the diagnostic techniques was assessed by calculating the coefficient of variation (CV) for each set of triplicate counts per technique. Mean CVs with corresponding 95% confidence intervals were then computed. Precision was expressed as a percentage by subtracting the CV from 100. These precision estimates were calculated for both the raw egg/oocyst counts and the final EPG/OPG values, which included the respective multiplication factors for each technique. Statistical significance was set at p < 0.05 for all analyses.

Results

Parasites species identified using Mini-FLOTAC and McMaster techniques

The degree of agreement between the McMaster and Mini-FLOTAC techniques in detecting gastrointestinal parasites in fecal samples from West African Long-legged sheep across farms in southern Benin is summarized in Table 1.

Table 1.

Agreement of the results obtained by parasitological techniques for the detection of Gastrointestinal parasites in fecal samples from crossbred sheep in Southern Benin

Farms		McMaster		Mini-FLOTAC		Kappa (CI 95%)
Farms	Parasites	Negative	Positive	Negative	Positive
Toffo	strongylids	08	32	00	40	1.00 (1.00–1.00)
	Strongyloides sp.	12	28	05	35	0.52 (0.28–0.76)
	Nematodirus spp.	38	02	32	08	0.31 (0.01–0.61)
	Marshallagia spp.	40	00	21	19	0.00 (0.00–0.00)
	Moniezia spp.	29	11	14	26	0.44 (0.19–0.69)
	Eimeria spp.	00	40	00	40	1.00 (1.00–1.00)
Allada	strongylids	02	38	00	40	1.00 (1.00–1.00)
	Strongyloides sp.	9	31	00	40	0.42 (0.17–0.67)
	Nematodirus spp.	37	03	21	19	0.25 (0.05–0.55)
	Marshallagia spp.	37	03	29	11	0.25 (0.05–0.55)
	Moniezia spp.	40	00	28	12	0.00 (0.00–0.00)
	Eimeria spp.	00	40	00	40	1.00 (1.00–1.00)
Tori-Bossito	strongylids	05	35	01	39	0.76 (0.53–0.99)
	Strongyloides spp.	02	38	00	40	1.00 (1.00–1.00)
	Nematodirus spp.	28	12	15	25	0.48 (0.23–0.73)
	Marshallagia spp.	40	00	11	29	0.00 (0.00–0.00)
	Moniezia spp.	37	03	15	25	0.39 (0.14–0.64)
	Eimeria spp.	00	40	00	40	1.00 (1.00–1.00)
Zê	strongylids	01	39	00	40	0.80 (0.56–1.00)
	Strongyloides spp	05	35	02	38	0.76 (0.52–1.00)
	Nematodirus spp.	27	13	12	28	0.49 (0.23–0.75)
	Marshallagia spp.	40	00	38	02	0.05 (0.15–0.05)
	Moniezia spp.	40	00	35	5	0.06 (0.16–0.04)
	Eimeria spp.	00	40	00	40	1.00 (1.00–1.00)
Sô-Ava	strongylids	07	33	00	40	0.53 (0.28–0.78)
	Strongyloides spp.	08	32	05	35	0.52 (0.27–0.77)
	Nematodirus spp.	40	00	18	22	0.00 (0.00–0.00)
	Marshallagia spp.	33	7	12	28	0.24 (0.04–0.52)
	Moniezia spp.	39	01	18	22	0.25 (0.03–0.53)
	Eimeria spp.	00	40	00	40	1.00 (1.00–1.00)

Kappa < 0 there wasn´t agreement, 0 to 0.20 poor agreement, 0.21 to 0.40 fair agreement, 0.41 to 0.60 moderate agreement, 0.61 to 0.80 good agreement and 0.81 to 1 almost perfect agreement (Landis & Koch, 1977 [18] )

CI Confidence Interval, k Kappa

For strongylids eggs, a perfect agreement (κ = 1.00; 95% CI: 1.00–1.00) was observed in Toffo and Allada, and an almost perfect agreement was found in Zê (κ = 0.80; 95% CI: 0.56–1.00) and Tori-Bossito (κ = 0.76; 95% CI: 0.53–0.99). However, only a moderate agreement was observed in Sô-Ava (κ = 0.53; 95% CI: 0.28–0.78). For Strongyloides spp., agreement ranged from moderate to perfect. Perfect agreement (κ = 1.00; 95% CI: 1.00–1.00) was recorded in Tori-Bossito, while good agreement was found in Zê (κ = 0.76; 95% CI: 0.52–1.00). Moderate agreement was observed in Toffo (κ = 0.52), Sô-Ava (κ = 0.52), and Allada (κ = 0.42). Agreement for Nematodirus spp. detection was lower overall. Fair agreement was noted in Toffo (κ = 0.31; 95% CI: 0.01–0.61), Allada (κ = 0.25), and Zê (κ = 0.49), while no agreement was observed in Sô-Ava (κ = 0.00) due to complete discordance in detection between techniques.

For Marshallagia spp., the agreement was generally poor to nonexistent. The kappa values were κ = 0.00 in Toffo, Tori-Bossito, and Sô-Ava, κ = 0.25 in Allada, and κ = 0.05 in Zê, indicating either no agreement or only slight agreement between the techniques. Moniezia spp. detection showed fair agreement in Toffo (κ = 0.44), while Allada and Zê had poor agreement (κ = 0.00 and κ = 0.06, respectively). Tori-Bossito and Sô-Ava also showed fair agreement (κ = 0.39 and κ = 0.25, respectively). For Eimeria spp., a perfect agreement (κ = 1.00; 95% CI: 1.00–1.00) was consistently observed across all farms, as both techniques identified all samples as positive.

The misclassification rates of parasite identification

The misclassification rates of parasite identification by McMaster relative to Mini-FLOTAC are illustrated in Fig. 2. This metric reflects the proportion of animals whose infections by Nematodirus spp., Marshallagia spp., or Moniezia spp. were undetected by the McMaster technique but correctly identified by Mini-FLOTAC.

Fig. 2.

The rate of misclassification obtained by the Mini-FLOTAC when compared to McMaster technique. Each bar of the chart represents the proportion of animals infected par Nematodirus spp., Marshallagia spp. and Moniezia spp. undetected by the McMaster technique but declared by the Mini-FLOTAC technique. Error bars indicate the standard error of the mean (SEM)

Across all farms, the Mini-FLOTAC technique consistently revealed higher diagnostic sensitivity, with misclassification rates for McMaster ranging from approximately 2.5% to 12.5%, depending on the parasite species and location. The highest overall misclassification rate was observed in Sô-Ava, where Marshallagia spp. had a misclassification rate of 12.5%, indicating a significant underestimation of prevalence by McMaster in this location (p < 0.05). Similarly, Zê exhibited misclassification rates of 8.5% for Marshallagia spp. and around 2.8% for both Nematodirus spp. and Moniezia spp., underscoring McMaster’s limited ability to detect these parasites in this context. In Tori-Bossito, the misclassification rate for Marshallagia spp. reached 7.5%, while Allada showed lower but still notable discrepancies (~ 3.5%) for all three parasite taxa. The lowest misclassification rate was seen in Toffo, with only ~ 2.5% of Marshallagia spp. cases missed by McMaster.

Intensity of parasitism in WALL sheep

Intensity of infection by strongylids

The comparison of the McMaster and Mini-FLOTAC techniques in estimating the intensity of strongylids infections revealed significant differences in EPG values across most of the five farms (Table 2). Overall, the Mini-FLOTAC technique consistently detected higher mean EPGs than the McMaster method.

Table 2.

Mean of EPG (± SEM) of strongylids in 40 fecal samples per sheep farm using McMaster and Mini-FLOTAC techniques

Sheep farms	Mean FEC of strongylids		Mean of EPG of strongylids
	McMaster	Mini-FLOTAC	McMaster	Mini-FLOTAC	p-value
Tori	42.31 ± 1.42	465.71 ± 9.49	2115.5a ± 31.61	2328.55b ± 33.20	0.045
Allada	39.69 ± 1.34	410.54 ± 8.70	1984.5a ± 30.03	2052.71ab ± 31.62	0.051
Zê	17.00 ± 1.11	275.71 ± 7.91	850.00a ± 28.46	1378.55b ± 30.83	0.033
Toffo	10.43 ± 0.79	88.63 ± 6.32	521.5a ± 25.28	443.15a ± 23.73	0.420
Sô-Ava	19.43 ± 0.95	160.29 ± 7.12	971.5a ± 26.86	801.45a ± 25.32	0.380

Values are presented as means ± standard error of the mean (SEM), based on 40 fecal samples per farm. Different lowercase letters indicate a significant difference between the means of EPG values at p < 0.05. P-values refer to differences between techniques (paired tests). The average strongylids egg count was used exclusively as the basis for calculating EPG values

For example, in Tori, the mean EPG obtained using Mini-FLOTAC was 2,328.55 ± 33.20, which was significantly higher than that of McMaster (2,115.5 ± 31.61; p = 0.045), with statistically distinct groupings. In Allada and Zê, similar trends were observed, with significantly higher EPGs recorded using Mini-FLOTAC compared to McMaster (p < 0.05). In Zê, Mini-FLOTAC yielded an EPG of 1,378.55 ± 30.83 versus 850.00 ± 28.46 for McMaster (p = 0.033). However, in Toffo and Sô-Ava, although Mini-FLOTAC tended to yield higher EPG values, the differences were not statistically significant (p = 0.420 and p = 0.380, respectively).

Intensity of parasitism by coccidia

Table 3 presents the comparison of OPG values for coccidia between the two diagnostic techniques across the same farms. Overall, Mini-FLOTAC detected significantly higher OPGs than McMaster in nearly all locations (p < 0.001), with the exception of Toffo, where no statistically significant difference was observed (p = 0.485).

Table 3.

Mean of coccidia OPG (± SEM) of coccidia in 40 fecal samples per sheep farm using McMaster and Mini-FLOTAC techniques

Sheep farms	Mean oocysts of coccidia		Mean of OPG of coccidia
	McMaster	Mini-FLOTAC	McMaster	Mini-FLOTAC	p-value
Tori	261.31 ± 15.81	3,893.29 ± 23.72	13,065.5a ± 110.68	19,466.45b ± 129.65	0.0002
Allada	241.26 ± 15.02	3,632.51 ± 22.14	12,063.0a ± 107.52	18,162.55b ± 126.49	0.0005
Zê	104.86 ± 12.65	1,977.14 ± 18.97	5,243.0a ± 98.03	9,885.7b ± 118.59	0.0003
Toffo	308.20 ± 16.60	3,053.77 ± 20.55	15,410.0a ± 113.84	15,268.85a ± 128.07	0.4850
Sô-Ava	421.34 ± 17.39	4,473.91 ± 24.51	21,067.0a ± 118.59	22,369.55b ± 137.56	0.0002

Values are presented as means ± standard error of the mean (SEM), based on 40 fecal samples per farm. Different lowercase letters indicate a significant difference between the means of coccidia OPG values at p< 0.05. P-values refer to differences between techniques (paired tests). The average coccidia oocyst count served exclusively as the basis for calculating OPG values

For instance, in Tori, the mean OPG value obtained with Mini-FLOTAC was 19,466.45 ± 129.65, significantly higher than that of McMaster (13,065.50 ± 110.68; p = 0.0002). Similar trends were observed in Allada and Sô-Ava, where Mini-FLOTAC consistently yielded significantly greater OPGs (p < 0.001). Even in Zê, where the baseline infection levels were lower, Mini-FLOTAC recorded higher OPGs than McMaster (p = 0.0002). However, in Toffo, both techniques produced comparable OPG values (15,268.85 ± 128.07 for Mini-FLOTAC vs. 15,410.00 ± 113.84 for McMaster; p = 0.485).

Prevalence of gastrointestinal parasites detected by McMaster and Mini-FLOTAC techniques

The comparative analysis of the prevalence of gastrointestinal parasites across five sheep farms using McMaster and Mini-FLOTAC techniques is presented in Table 4. While both techniques detected the same types of helminths and protozoa, Mini-FLOTAC consistently showed higher detection rates, with several statistically significant differences across parasite taxa and farms (p < 0.05).

Table 4.

Prevalence (%) [95% CI] og Gastrointestinal parasites detected by each of the two techniques in each of the five sheep farms

Farms	Technique	strongylids	coccidia	Strongyloides spp.	Nematodirus spp.	Moniezia spp.	Marshallagia spp.
Tori	McMaster	71.43ᵃ [54.9–84.4]	85.71ᵃ [70.6–94.3]	62.86ᵃ [46.0–77.3]	48.57ᵃ [32.0–65.5]	11.43ᵃ [3.2–27.0]	62.86ᵃ [46.0–77.3]
	Mini-FLOTAC	77.14ᵃ [60.9–88.6]	85.71ᵃ [70.6–94.3]	74.29ᵃ [57.9–86.9]	62.86ᵃ [46.0–77.3]	25.71ᵇ [13.1–42.6]	74.29ᵃ [57.9–86.9]
Allada	McMaster	80.00ᵃ [64.1–90.9]	68.57ᵃ [51.8–82.1]	28.57ᵃ [15.2–45.9]	37.14ᵃ [22.4–54.2]	–	28.57ᵃ [15.2–45.9]
	Mini-FLOTAC	88.57ᵃ [74.6–96.0]	85.71ᵇ [70.6–94.3]	48.57ᵇ [32.0–65.5]	51.43ᵇ [34.5–68.0]	18.50 [7.8–34.9]	48.57ᵇ [32.0–65.5]
Zê	McMaster	80.00ᵃ [64.1–90.9]	45.71ᵃ [29.2–63.1]	5.31ᵃ [1.1–14.6]	62.86ᵃ [46.0–77.3]	–	–
	Mini-FLOTAC	100.00ᵇ [90.0–100.0]	65.71ᵇ [48.7–80.4]	45.71ᵇ [29.2–63.1]	74.29ᵃ [57.9–86.9]	21.16 [9.4–37.3]	25.12 [12.4–41.5]
Toffo	McMaster	80.00ᵃ [64.1–90.9]	20.00ᵃ [8.6–36.9]	11.43ᵃ [3.2–27.0]	48.12ᵃ [31.6–64.9]	27.25ᵃ [14.3–44.2]	11.43ᵃ [3.2–27.0]
	Mini-FLOTAC	100.00ᵇ [90.0–100.0]	37.14ᵇ [22.4–54.2]	25.71ᵇ [13.1–42.6]	65.47ᵇ [48.9–79.8]	42.56ᵇ [26.4–59.7]	25.71ᵇ [13.1–42.6]
Sô-Ava	McMaster	65.71ᵃ [48.7–80.4]	17.14ᵃ [6.9–33.6]	2.86ᵃ [0.1–14.9]	–	32.5ᵃ [17.5–50.9]	14.52ᵃ [5.1–30.2]
	Mini-FLOTAC	80.00ᵇ [64.1–90.9]	14.29ᵃ [5.7–29.3]	8.57ᵃ [2.4–21.6]	24.71 [12.1–41.9]	66.8ᵇ [49.8–80.9]	57.14ᵇ [40.1–72.7]

The comparison of proportion values (%) of parasite species in each sheep farm, obtained by both techniques, was done through the two-proportion z-test in R with the function prop.test using the two-proportion z-test. Different lowercase letters indicate a significant difference between values at p < 0.05

For strongylids nematodes, prevalence values ranged from 65.71% to 100%. Mini-FLOTAC yielded significantly higher prevalence than McMaster in Zê (100.00% vs. 80.00%, p < 0.05), Toffo (100.00% vs. 80.00%), and Sô-Ava (80.00% vs. 65.71%). In Tori and Allada, although Mini-FLOTAC recorded numerically higher values (77.14% and 88.57%, respectively), these were not significantly different from McMaster (71.43% and 80.00%). For coccidia (Eimeria spp.), Mini-FLOTAC also outperformed McMaster in terms of detection. Significant differences were observed in Allada (85.71% vs. 68.57%, p < 0.05), Zê (65.71% vs. 45.71%), and Toffo (37.14% vs. 20.00%). However, no statistically significant differences were found in Tori and Sô-Ava, where prevalence values were similar.

Regarding Strongyloides spp., Mini-FLOTAC detected significantly higher prevalence in Allada (48.57% vs. 28.57%, p < 0.05), Zê (45.71% vs. 5.31%), and Toffo (25.71% vs. 11.43%). In Tori and Sô-Ava, the differences were not statistically significant despite Mini-FLOTAC reporting numerically higher values. The detection of Nematodirus spp. varied across farms. Mini-FLOTAC significantly outperformed McMaster in Allada (51.43% vs. 37.14%) and Toffo (65.47% vs. 48.12%). No significant difference was observed in Tori (62.86% vs. 48.57%) or Zê (74.29% vs. 62.86%). Notably, McMaster failed to detect any case of Nematodirus spp. in Sô-Ava, while Mini-FLOTAC recorded a prevalence of 24.71%, though the difference could not be statistically tested due to the absence of McMaster-positive cases. For Moniezia spp., the prevalence was significantly higher with Mini-FLOTAC in Tori (25.71% vs. 11.43%), Toffo (42.56% vs. 27.25%), and Sô-Ava (66.8% vs. 32.5%). In Allada and Zê, McMaster detected no cases, while Mini-FLOTAC identified 18.50% and 21.16% prevalence, respectively. Finally, for Marshallagia spp., Mini-FLOTAC significantly increased detection in Allada (48.57% vs. 28.57%), Toffo (25.71% vs. 11.43%), and Sô-Ava (57.14% vs. 14.52%). In Zê, Mini-FLOTAC identified a 25.12% prevalence while McMaster detected none, highlighting a possible underestimation by the latter.

Diagnostic precision of the McMaster and the Mini-FLOTAC techniques

The comparison of analytical precision between the McMaster and Mini-FLOTAC techniques is presented in Table 5. In Tori, Mini-FLOTAC yielded a CV of 17.05% (95% CI: 12.81–25.08) with a precision of 82.94% (95% CI: 80.05–89.68), compared to a CV of 42.81% (95% CI: 38.15–41.55) and precision of 57.18% (95% CI: 43.22–76.75) for McMaster. A similar trend was observed in Allada, where Mini-FLOTAC showed a CV of 17.05% and a precision of 82.94%, while McMaster recorded a CV of 42.69% and a precision of only 57.30%. Zê demonstrated the most substantial difference in favor of Mini-FLOTAC, with a remarkably low CV of 12.37% (95% CI: 8.05–18.89) and a high precision estimate of 87.62% (95% CI: 83.05–95.78). In contrast, McMaster’s CV reached 42.71%, with a precision of 57.28%. In Toffo, the CV of Mini-FLOTAC remained low at 18.94%, associated with a precision of 81.05%, whereas McMaster showed a higher CV of 33.23% and a lower precision (66.76%). Similarly, in Sô-Ava, Mini-FLOTAC achieved a CV of 16.90% (precision 83.09%), compared to McMaster’s CV of 33.19% and precision of 66.80%.

Table 5.

Coefficients of variation (CV), standard deviations (SD), and precision estimates for the two egg-counting techniques evaluated in each of the five sheep farms

Sheep farms	Techniques	CV	SD		Precision (%)
			Eggs* Counted	EPG**
Tori	McMaster	42.81 (38.15–41.55)	5.78	289	57.18 (43.22–76.75)
	Mini-FLOTAC	17.05 (12.81–25.08)	17.94	89.7	82.94 (80.05–89.68)
Allada	McMaster	42.69 (38.28–41.09)	3.33	166.5	57.30 (43.50–76.95.50.95)
	Mini-FLOTAC	17.05 (12.94–25.25)	11.85	59.25	82.94 (79.55–90.08)
Ze	McMaster	42.71 (37.28–40.63)	3.93	196.5	57.28 (43.75–75.95)
	Mini-FLOTAC	12.37 (8.05–18.89)	10.58	52.9	87.62 (83.05–95.78)
Toffo	McMaster	33.23 (33.90–37.73.90.73)	3.49	174.5	66.76 (43.08–82.16)
	Mini-FLOTAC	18.94 (12.38–25.91)	10.59	52.95	81.05 (77.32–90.45)
Sô-Ava	McMaster	33.19 (33.61–37.92)	6.29	314.5	66.80 (45.68–84.25)
	Mini-FLOTAC	16.90 (10.16–25.01)	19.05	95.25	83.09 (81.85–94.28)

95% confidence intervals presented in parenthesis

EPG Eggs per gram of feces

*Eggs counted represents the raw count before multiplying with the multiplication factor for each technique and **EPG is the fecal egg count multiplied by 50

Discussion

This study represents the first comprehensive field-based evaluation of the Mini-FLOTAC and McMaster techniques for diagnosing gastrointestinal (GI) parasitic infections in WALL sheep within the agroecological context of southern Benin. The results provide compelling evidence that Mini-FLOTAC outperforms the modified McMaster technique in multiple diagnostic dimensions: sensitivity, spectrum of detection, quantitative accuracy, and precision. These findings have significant implications for parasite surveillance and control programs, particularly in resource-constrained settings.

The Mini-FLOTAC technique detected a broader range of GI parasites than the McMaster method, including Nematodirus spp., Marshallagia spp., and Moniezia spp., which were entirely missed by McMaster. This expanded diagnostic reach is critical in endemic zones where polyparasitism is common and where certain parasite species, although less prevalent, may cause substantial morbidity, particularly in young animals. This result aligns with previous findings by Alowanou et al. [20] and Mohammedsalih et al. [21], who demonstrated the superior detection capability of Mini-FLOTAC in small ruminants, camels, and rabbits under various environmental and management conditions.

The higher sensitivity observed with Mini-FLOTAC can be attributed to multiple technical advantages. First, the dilution ratio used in Mini-FLOTAC (1:10) reduces fecal debris and improves clarity under microscopic examination, enhancing the visibility of eggs. Second, the total examined volume is greater (2 mL across two chambers) than that of McMaster (0.3 mL), increasing the likelihood of detecting eggs, particularly when infection intensities are low. These advantages have been validated across various host species [12, 22].

The estimated prevalence of GI parasites was significantly higher using Mini-FLOTAC (78.92%) compared to McMaster (54.62%), revealing the potential underestimation of parasite burden by the latter. This underestimation is not trivial, as it can lead to false assumptions about herd health status and inappropriate deworming decisions. Misclassification rates observed in our study ranged from 11.42% to 17.14%, representing infected animals that would have been missed if only McMaster had been used. These findings mirror the report by Dias de Castro et al. [23], who demonstrated that Mini-FLOTAC has a much lower detection threshold (as low as 5 EPG) compared to McMaster (50 EPG). As emphasized by Bosco et al. [24], this increased sensitivity is particularly important for monitoring low-level infections and detecting early-stage infections before clinical symptoms or production losses occur.

An important finding of this study is that Mini-FLOTAC consistently provided higher fecal egg counts than McMaster, which indicates its greater sensitivity in estimating parasite burden. Underestimation of egg counts, as frequently observed with McMaster, may lead to misclassification of infection intensity and to inaccurate assessments of herd-level transmission risk. In addition, Mini-FLOTAC revealed a broader composition of gastrointestinal nematodes, including Nematodirus spp., Marshallagia spp., and Moniezia spp., which were less consistently detected by McMaster. These results emphasize that Mini-FLOTAC offers a more comprehensive description of parasite burden and species composition in small ruminants, which is essential for epidemiological monitoring and control strategies.

The Mini-FLOTAC method consistently yielded higher FECs across all study farms, a finding consistent with other studies in cattle, pigs, camels, and equines [21, 25, 26]. For example, mean strongylid EPGs were systematically higher in sheep using Mini-FLOTAC compared to McMaster, reaffirming its greater quantitative sensitivity. This increase in FECs has diagnostic and epidemiological relevance. Higher egg recovery reflects more accurate quantification of parasite burden, which is essential not only for identifying heavily infected individuals but also for evaluating anthelmintic efficacy and transmission risk. Notably, the farms with higher mean FECs also exhibited higher standard deviations but lower coefficients of variation, indicating consistent recovery despite fluctuating absolute values—an observation echoed by Mohammedsalih et al. [21] in camels.

Among all GI helminths detected, strongylids dominated the parasitic landscape, consistent with previous reports from Benin and other parts of West Africa [27, 28]. The predominance of Haemonchus contortus and Trichostrongylus colubriformis, both prolific egg producers, contributes to this pattern. Under optimal conditions warm temperatures, high humidity, and continuous grazing H. contortus can produce up to 5,000 eggs/day [3], rapidly contaminating pastures and sustaining transmission cycles.

The high prevalence of strongylids observed in this study is further exacerbated by suboptimal herd management practices. All farms surveyed practiced continuous grazing without rotational schedules, likely due to urban encroachment and land scarcity, as detailed in the preliminary survey. These conditions are conducive to persistent pasture contamination, and as [3] pointed out, the lack of grazing rest periods significantly increases nematode survival and infection risk.

Mini-FLOTAC demonstrated significantly better precision than McMaster, with CVs often below 20% compared to values exceeding 30% for McMaster. Precision is a critical metric for laboratory diagnostics, as it indicates consistency across replicates and sampling times. Higher precision in Mini-FLOTAC stems from its more rigorous sample homogenization (using 2–5 g of feces) and the stability of its counting chambers, which are designed to minimize inter-chamber variability. The findings are congruent with Rinaldi et al. [16] and Kida et al. [29], who showed that Mini-FLOTAC offers better reproducibility across repeated measures, especially when used with high-specific-gravity solutions (e.g., zinc sulfate at SG = 1.32). Although we used saturated NaCl (SG = 1.20) in this study, the performance remained superior, suggesting that even under conservative flotation conditions, Mini-FLOTAC maintains diagnostic integrity.

In addition to its diagnostic advantages, Mini-FLOTAC presents logistical benefits. Unlike the original FLOTAC, it does not require centrifugation or electricity, making it well-suited for field conditions and veterinary outreach programs in rural areas. Its compatibility with portable kits such as Fill-FLOTAC further enhances its ease of use and standardization, as recommended by Cringoli et al. [12].

Given the increasing threat of anthelmintic resistance and the need for targeted selective treatment, Mini-FLOTAC offers an effective solution for evidence-based parasitic control. Its ability to detect low parasite burdens and track FEC trends over time enables practitioners to fine-tune deworming protocols and reduce unnecessary drug use. However, deworming decisions should not rely solely on FEC values but should also consider clinical indicators (e.g., FAMACHA© scores, packed cell volume) and productive parameters, which together provide a more accurate basis for treatment.

Conclusions

This study confirms that the Mini-FLOTAC technique significantly outperform the traditional McMaster method in diagnosing gastrointestinal parasitism in sheep under field conditions in Sub-Saharan Africa. By offering enhanced sensitivity, broader parasite detection, higher fecal egg and oocyst counts, and superior diagnostic precision, Mini-FLOTAC represents a robust and scalable solution for parasite surveillance in small ruminants. Its capacity to detect infections commonly overlooked by McMaster, coupled with its reproducibility and ease of use, suggests its suitability for integrated parasite control strategies in low-resource settings. The adoption of Mini-FLOTAC can improve the accuracy of epidemiological assessments and support evidence-based interventions to reduce parasitic burdens and mitigate the emergence of anthelmintic resistance in smallholder farming systems.

Abbreviations

GI

Gastrointestinal

FEC

Fecal Egg Count

EPG

Eggs per grams of feces OPG-Oocysts per grams of feces

CV

Coefficient of Variation

CI

Confidence Interval

SEM

Standard Error of the Mean

WALL

West African Long-Legged (sheep)

WAD

West African Dwarf (sheep)

TST

Targeted Selective Treatment

NaCl

Sodium Chloride

WAAVP

World Association for the Advancement of Veterinary Parasitology

MAFF

Ministry of Agriculture, Fisheries and Food (UK)

UAC

Université d’Abomey-Calavi

FSA

Faculté des Sciences Agronomiques

LESA

Laboratoire d’Ethnopharmacologie et de Santé Animale

IFS

International Foundation for Sciences

Authors’ contributions

Study conception, study design, data analysis and drafting of the original manuscript: G.G.A. Study conception, study design and drafting of the original manuscript: G.A., C.H., J.D., H.A.K. and N.O.K. Review of the manuscript: C.B.Z. Review of the manuscript and supervision: P.A.O., A.B.D. and S.H.A. All the authors read and approved the manuscript for publication.

Funding

The first author was supported by the International Foundation for Sciences, IFS (grant number: 3-B/5792-2).

Data availability

The data that support the findings of this study are available on request from the corresponding author.

Declarations

Ethics approval and consent to participate

All experimental protocols were approved by the Ethics Committee for Animal Research of the Agricultural Techniques and Sciences Department at the National University of Sciences, Technologies, Engineering and Mathematics (UNSTIM), under the approval number UNSTIM/ENSET/STA-2023-095. All procedures involving animals were conducted in strict accordance with relevant national and institutional guidelines and regulations. Furthermore, the study and its reporting are fully compliant with the ARRIVE guidelines (https://arriveguidelines.org). Informed consent was obtained from all participants, including the owners of the sheep from whom fecal samples were collected for analysis.

Consent for publication

All the authors (corresponding and associate authors) agree to the publication of the results of this study.

Competing interests

The authors declare no competing interests.