Integrated epidemiology and multi-assay evidence of anthelmintic resistance in ovine gastrointestinal nematodes of Northern India

Abstract

Gastrointestinal parasitic (GIP) infections are a major constraint to sustainable small ruminant production worldwide due to increasing anthelmintic resistance caused by extensive reliance on chemical anthelmintics. In this study, a total of 2308 faecal samples were collected and examined from naturally infected sheep across northern India, encompassing 15 districts of Punjab and 07 of Himachal Pradesh. An overall prevalence of 96.4% for GIP infections was recorded with strongyles being predominant followed by coccidia. Quantitative faecal examination revealed 62.6% light, 10.2% moderate and 27.2% severe nematode infection with Haemonchus (60.0%) being the most prevalent genera as per coproculture examination. The logistic regression analysis revealed season and deworming status to be the significantly associated risk factors with prevalence of GIPs. The efficacy of fenbendazole, levamisole and ivermectin was evaluated at 14 sheep farms across the study region by in-vivo (faecal egg count reduction test, FECRT) and in-vitro assays (egg hatch test, larval development assay). The findings of FECRT revealed multi-anthelmintic resistance status (resistance against more than one class of anthelmintic) across all farms with an overall percent efficacy (mean ± SE) for fenbendazole, levamisole and ivermectin as 61.6 ± 5.0%, 57.4 ± 6.4% and 57.4 ± 5.2%, respectively. Results of in-vitro assays revealed 78.6%, 78.6% and 100% sheep farms as resistant against fenbendazole, levamisole and ivermectin, respectively. The high prevalence of GINs can be linked to its resistant status observed against commonly used anthelmintics, highlighting the need to investigate novel chemical and non-chemical approaches for effective worm management in the region.

Introduction

Gastrointestinal nematodes (GIN) such as Haemonchus contortus, Trichostrongylus spp., and Teladorsagia spp. are major parasitic threats to global sheep production, causing reduced growth, wool and meat yield, poor reproductive performance and increased mortality, particularly in pasture-based systems¹. The economic impact of GIN in India is equally striking with losses ranging from ₹1,194 to ₹1,674 per animal depending on faecal egg count intensity². Due to its hematophagous nature, high fecundity, short life cycle and ability to survive under diverse environmental conditions, H. contortus is the principal contributor to economic losses and is particularly difficult to control because of rapid pasture contamination with eggs and larvae during favourable conditions³. For decades, GIN control has relied heavily on broad-spectrum anthelmintic groups such as benzimidazoles, imidazothiazoles and macrocyclic lactones⁴. While these drugs initially revolutionized parasite control and improved productivity, their repeated and often indiscriminate use has led to the widespread anthelmintic resistance (AR) in sheep-producing regions⁵. Multi-anthelmintic-resistant (resistance status against more than one class of anthelmintic) H. contortus populations are now well documented globally^6–9.

India, home to approximately 74.26 million sheep as per the 20^th Livestock Census¹⁰, ranks among the world’s leading small ruminant producers. Sheep rearing contributes substantially to the livelihoods of small and marginal farmers. In northern India, traditional migratory grazing systems, communal pastures and limited access to veterinary services shape the epidemiology and transmission patterns of GINs with high prevalence and predominance of H. contortus^3,11. Transmission peaks during and after the monsoon season, when humidity and temperature favour the free-living larval stages survival. Host factors such as age and nutritional status along with management practices like communal grazing and frequent prophylactic deworming, create conditions highly conducive to heavy infection burdens and AR selection¹².

Although, AR has been recognized in India for decades, with early reports of benzimidazole¹³ and levamisole resistance¹⁴, most available studies remain geographically fragmented. Recent findings show declining efficacy of fenbendazole and levamisole, and emerging indications of ivermectin resistance in northern India^3,15. These investigations have relied on in-vivo (faecal egg count reduction test, FECRT) assay, often based on single-farm data and lacking complementary in-vitro data, limiting their broader interpretive value. The absence of a national framework for AR surveillance, early-warning systems and harmonized control guidelines further hampers effective worm management. Although, World Association for the Advancement of Veterinary Parasitology (WAAVP) provides internationally accepted standards for FECRT design and interpretation¹⁶, their consistent implementation across India remains limited. Similarly, integrated parasite management strategies such as targeted selective treatment, refugia maintenance and breeding for host resistance are seldom practiced¹⁷.

Given this background, comprehensive, regionally representative studies that combine epidemiological assessment with multi-assay AR evaluation are urgently needed. Northern India, in particular, represents a critical region where high infection pressure and frequent anthelmintic use create ideal conditions for AR emergence and spread. The present study aims to address this gap by qualitative and quantitative faecal examination, coproculture‑based species identification, FECRT, egg hatch test (EHT) and larval development assay (LDA) for fenbendazole, levamisole and ivermectin resistance in sheep populations. This study aims to establish regionally relevant benchmarks for infection burden, drug efficacy and generate evidence to support sustainable parasite management strategies under northern Indian sheep production systems.

Results

Prevalence of gastrointestinal parasites

Out of 2308 animals examined, 96.4% (n = 2226) were overall positive for GIPs and 78.7% (n = 1752) had mixed infections. Strongyles were the most prevalent GIP constituting 97.0% (n = 2159) of positive samples, followed by coccidia (73.9%; n = 1644), Strongyloides spp. (15.4%; n = 344), Moniezia spp. (11.9%; n = 265), Trichuris spp. (7.6%; n = 170) and least were Fasciola spp. (1.1%, n = 24) and amphistomes (1.0%; n = 22). The quantitative assessment of strongyle-positive faecal samples showed egg per gram (EPG) count (mean ± SE) of 1118.9 ± 42.5 with 62.6% (n = 1351) having low, 10.2% (n = 221) moderate and 27.2% (n = 587) severe levels of infection. Seasonal comparison indicated that EPG levels were highest during the monsoon (1371.6 ± 92.8), followed by summer (1089.0 ± 62.5) and lowest during winters (897.1 ± 56.2).

Identification of third stage strongyle larvae (L₃) after copro-culture revealed seven distinct genera viz. Haemonchus (60.0 ± 2.3%), Trichostrongylus (23.4 ± 1.7%), Cooperia (0.9 ± 0.3%), Oesophagostomum (6.8 ± 1.0%), Bunostomum (0.1 ± 0.1%), Ostertagia (7.9 ± 1.5%) and Chabertia (0.9 ± 0.3%). A significantly (P < 0.05) higher abundance of Haemonchus was recorded during the monsoon (68.9 ± 2.8%), whereas, Trichostrongylus (27.6 ± 3.4%) and Ostertagia were higher during the winters (13.5 ± 3.4%) (Table 1).

Table 1.

Seasonal distribution (%) of gastrointestinal nematodes (Mean ± SE) in sheep of northern India.

Season	Haemonchus spp.	Trichostrongylus spp.	Cooperia spp.	Oesophagostomum spp.	Bunostomum spp.	Ostertagia spp.	Chabertia spp.
Summer	61.5 ± 3.5ab	24.0 ± 2.3ab	1.1 ± 0.4	8.2 ± 2.4	0.0	4.5 ± 1.3a	0.7 ± 0.4
Monsoon	68.9 ± 2.8b	18.2 ± 2.1a	1.1 ± 0.6	6.3 ± 1.5	0.0	5.0 ± 1.3a	0.5 ± 0.3
Winter	50.6 ± 3.9a	27.6 ± 3.4b	0.6 ± 0.3	5.9 ± 1.2	0.4 ± 0.3	13.5 ± 3.4b	1.4 ± 0.8
Overall	60.0 ± 2.3	23.4 ± 1.7	0.9 ± 0.3	6.8 ± 1.0	0.1 ± 0.1	7.9 ± 1.5	0.9 ± 0.3

Values followed by same letters in same column does not differ statistically at a significance level of 5%.

Analysis of risk factors associated with prevalence of gastrointestinal parasites

The geographical location had a significant (P < 0.01) association with prevalence of GIPs as Strongyloides spp. and Moniezia spp. were higher in Himachal Pradesh (HP), whereas, strongyles, coccidia and Trichuris spp. were higher in Punjab. Significant seasonal variations were recorded for distribution of strongyles (P < 0.01), Strongyloides spp. (P < 0.01), Moniezia spp. (P < 0.05) and Trichuris spp. (P < 0.05) with strongyle and Trichuris spp. exhibiting highest prevalence in monsoon and Moniezia spp. peaking in summer. Deworming status of the host was significantly associated with distribution of strongyle (P < 0.01), Strongyloides spp. (P < 0.01), coccidia (P < 0.01) and Trichuris spp. (P < 0.05), with higher prevalence consistently being recorded in hosts with no history of deworming in last three-month period. Host age also demonstrated significant association with prevalence of Strongyloides spp. (P < 0.05), Moniezia spp. (P < 0.01) and Trichuris spp. (P < 0.05) (details in Fig. 1).

Fig. 1.

Prevalence of gastrointestinal parasites with respect to associated risk factors in sheep of northern India. *P<0.05; **P<0.01.

The logistic regression analysis showed that season and deworming were significant predictors of GIP infections of sheep populations of northern Indian states. Animals examined during monsoon (P = 0.005; OR = 2.06; 95% CI = 1.23–3.45) and summer (P = 0.001; OR = 2.53; 95% CI = 1.41–4.53) had significantly higher odds of infection compared with those in winter season. The odds were higher in summer due to maturation of hypobiotic larvae and spring rise phenomenon. Deworming status of host showed the strongest association, with non-dewormed sheep (P < 0.001; OR = 2.54; 95% CI = 1.59–4.07) exhibiting a markedly higher risk of infection (Table 2).

Table 2.

Logistic regression analysis of various risk factors associated with gastrointestinal parasites in sheep of northern India.

Risk Factors	Category	No. of sample		OR	P-value	95% CI
		Examined	Positive (%)
Location	Punjab	1620	1570 (96.9)	1.53	0.06	0.97–2.41
	HP	688	656 (95.4)	Reference
Season	Monsoon	829	806 (97.2)	2.06	0.005	1.23–3.45
	Winter	775	732 (94.5)	Reference
	Summer	704	688 (97.8)	2.53	0.001	1.41–4.53
Age	< 6 M	107	103 (96.3)	1.17	0.78	0.39–3.45
	6 M to 1Y	508	486 (95.7)	Reference
	> 1Y	1693	1637 (96.7)	1.32	0.27	0.80–2.19
Sex	Male	273	266 (97.4)	1.45	0.35	0.66–3.19
	Female	2035	1960 (96.3)	Reference
Deworming	Present	405	377 (93.1)	Reference
	Absent	1903	1849 (97.2)	2.54	< 0.001	1.59–4.07

HP: Himachal Pradesh; OR: Odds Ratio; CI: Confidence Interval.

Anthelmintic efficacy test

Faecal egg count reduction test

The faecal egg count reduction percentage (FECR%), 90% confidence intervals (CI) and efficacy status for fenbendazole, levamisole and ivermectin across 14 selected sheep farms are summarised in Table 3. The overall efficacy as mean ± SE% (range) for fenbendazole, levamisole and ivermectin was 61.6 ± 5.0 (18.8–82.3%), 57.4 ± 6.4 (11.1–85.2%) and 57.4 ± 5.2 (26.5–79.7%), respectively, revealing multi-resistance status against these drugs in GINs of selected sheep farms. Further, L₃ obtained from culturing pooled faecal samples of the control and treatment groups at 14 days post treatment (DPT) revealed variations in distribution of GIN genera (%) among the groups. Haemonchus remained the predominant genus across all groups, with elevated proportions in fenbendazole (78.5 ± 4.5%), ivermectin (75.8 ± 2.8%) and levamisole (79.6 ± 2.3%) treated groups when compared to control (68.6 ± 3.3%). In contrast, Trichostrongylus decreased in all treatment groups (Table 4).

Table 3.

The efficacy (%) of fenbendazole, levamisole and ivermectin at selected sheep farms of northern India.

Farm	Fenbendazole		Levamisole		Ivermectin
	Efficacy (%) (90% CI)	Status	Efficacy (%) (90% CI)	Status	Efficacy (%) (90% CI)	Status
BNN	60.7 (30.7–77.8)	R	46.0 (22.1–62.6)	R	34.5 (-57.9-72.8)	R
CHMB	82.3 (62.2–91.7)	R	85.2 (71.0-92.5)	R	79.7 (26.0-89.8)	R
FDK	55.5 (13.4–77.1)	R	50.7 (-12-78.3)	R	29.2 (-117-76.9)	R
GSP	46.1 (-76.6-83.5)	R	37.7 (-53.7-74.7)	R	82.7 (57.6–92.9)	R
HAMR	76.4 (-9.3-94.9)	R	84.5 (58.8–94.2)	R	72.6 (26.0-89.8)	R
KGRA	81.6 (59.4–91.7)	R	78.4 (48.6–90.9)	R	72.3 (39.6–87.3)	R
KUU	82.2 (61.2–91.9)	R	72.6 (27.2–89.6)	R	70.5 (24.7–88.4)	R
LDH	58.7 (12.3–80.5)	R	11.1 (-63.4-51.6)	R	26.5 (-93.6-71.3)	R
MDI	73.7 (17.1–91.6)	R	72.4 (26.7–89.6)	R	69.1 (15.7–88.7)	R
MET	18.8 (-115.6-69.4)	R	59.1 (5.8–82.2)	R	49.6 (-27.6-80.1)	R
MOGA	47.9 (-51.2-82.1)	R	72.0 (40.8–86.8)	R	62.4 (-62.1-91.3)	R
NSS	36.1 (-31.5-69.0)	R	31.4 (-44.6-67.4)	R	28.5 (-29.4-60.4)	R
SAG	81.1 (63.9–90.0)	R	82.3 (65.6–90.9)	R	54.7 (9.4–77.3)	R
TTO	61.1 (16.9–81.8)	R	20.9 (-129-72.7)	R	71.3 (12.4–90.6)	R

R: Resistant; CI: Confidence Interval.

Table 4.

Distribution (%) of various nematode genera (Mean ± SE) on 14th day post treatment of faecal egg count reduction test.

Group	Haemonchus spp.	Trichostrongylus spp.	Cooperia spp.	Oesophagostomum spp.	Ostertagia spp.	Chabertia spp.
Control	68.6 ± 3.3	22.1 ± 3.8	1.8 ± 1.0	4.3 ± 1.6	3.0 ± 1.3	0.2 ± 0.2
Fenbendazole	78.5 ± 4.5	17.2 ± 4.2	0.0	2.4 ± 1.3	0.8 ± 0.6	0.0
Ivermectin	75.8 ± 2.8	21.7 ± 2.9	0.0	1.9 ± 0.8	0.4 ± 0.4	0.2 ± 0.2
Levamisole	79.6 ± 2.3	17.9 ± 2.9	0.0	0.9 ± 0.5	1.6 ± 0.9	0.0

Egg hatch test

The results of EHT conducted on eggs obtained from the pooled faecal samples at 0 DPT showed 78.6% (11/14) sheep farms exhibiting benzimidazole resistant GIN populations based on their effective concentration at 50% inhibition of egg hatching (EC₅₀) values. Three sheep farms, CHMB (0.049 µg/mL), KGRA (0.018 µg/mL) and KUU (0.069 µg/mL) were found susceptible with EC₅₀ as > 0.1 µg/mL. The EC₅₀ values in resistant farms (n = 11) ranged from 0.183 to 0.786 µg/mL (details in Table 5). The regression plots illustrating inhibition of egg hatching in relation to log concentrations of thiabendazole for different farms are presented in Fig. 2.

Table 5.

Results of in-vitro tests at various selected sheep farms of northern India.

Farm	Thiabendazole (EHT)			Levamisole (LDA)				Ivermectin (LDA)
	Slope ± SE	aEC50 (95% CI)	Status	Slope ± SE	aEC50 (95% CI)	bRR (95% CI)	Status	Slope ± SE	aEC50 (95% CI)	cRR (95% CI)	Status
BNN	2.47 ± 0.09	0.419 (0.38–0.46)	R	0.71 ± 0.04	0.430 (0.30–0.58)	4.94 (3.45–6.67)	R	0.55 ± 0.04	1.150 (0.79–1.61)	28.75 (1.61–19.75)	R
CHMB	1.79 ± 0.13	0.049 (0.04–0.06)	S	0.96 ± 0.06	0.638 (0.42–0.91)	7.33 (4.83–10.46)	R	0.71 ± 0.05	0.705 (0.39–1.14)	17.62 (9.75–28.50)	R
FDK	2.48 ± 0.09	0.786 (0.68–0.91)	R	1.05 ± 0.06	1.897 (1.26–2.73)	21.80 (14.48–31.38)	R	0.718 ± 0.05	10.434 (6.38–17.82)	260.85 (159.50-445.50)	R
GSP	2.65 ± 0.11	0.237 (0.20–0.27)	R	0.95 ± 0.04	0.913 (0.73–1.12)	10.49 (8.39–12.87)	R	0.70 ± 0.04	0.604 (0.40–0.86)	15.10 (10.00-21.50)	R
HAMR	2.25 ± 0.09	0.307 (0.25–0.37)	R	0.53 ± 0.05	0.015 (0.005–0.03)	0.17 (0.06–0.34)	S	0.67 ± 0.05	0.086 (0.03–0.17)	2.15 (0.75–4.25)	R
KGRA	1.63 ± 0.21	0.018 (0.01–0.03)	S	0.49 ± 0.04	0.123 (0.05–0.23)	1.41 (0.57–2.64)	S	0.57 ± 0.05	0.126 (0.06–0.22)	3.15 (1.50–5.50)	R
KUU	1.03 ± 0.06	0.069 (0.03–0.12)	S	0.87 ± 0.05	1.079 (0.71–1.55)	12.40 (8.16–17.82)	R	1.18 ± 0.07	4.176 (2.93–5.78)	104.40 (73.25–144.50)	R
LDH	2.55 ± 0.09	0.675 (0.62–0.74)	R	1.31 ± 0.06	4.185 (2.76–6.24)	48.10 (31.72–71.72)	R	0.79 ± 0.04	0.692 (0.47–0.97)	17.30 (11.75–24.25)	R
MDI	2.32 ± 0.10	0.641 (0.57–0.72)	R	1.34 ± 0.07	1.286 (0.93–1.72)	14.78 (10.69–19.77)	R	0.90 ± 0.05	2.466 (1.50–3.91)	61.65 (37.50-97.75)	R
MET	2.42 ± 0.10	0.784 (0.73–0.84)	R	1.33 ± 0.06	2.862 (1.69–4.59)	32.90 (19.43–52.76)	R	0.81 ± 0.05	0.321 (0.19–0.49)	8.03 (4.75–12.25)	R
MOGA	1.81 ± 0.07	0.580 (0.50–0.67)	R	0.84 ± 0.04	1.033 (0.63–1.59)	11.87 (7.24–18.28)	R	1.02 ± 0.05	0.360 (0.25–0.49)	9.00 (6.25–12.25)	R
NSS	1.93 ± 0.09	0.198 (0.18–0.22)	R	0.69 ± 0.04	0.498 (0.34–0.69)	5.72 (3.91–7.93)	R	1.19 ± 0.07	8.620 (4.53–15.75)	215.5 (113.25-393.75)	R
SAG	2.45 ± 0.12	0.244 (0.18–0.31)	R	0.52 ± 0.04	0.058 (0.01–0.15)	0.67 (0.11–1.72)	S	0.86 ± 0.05	0.961 (0.61–1.42)	24.03 (15.25–35.50)	R
TTO	1.64 ± 0.07	0.183 (0.16–0.20)	R	1.33 ± 0.07	2.071 (1.69–2.51)	23.80 (19.43–28.85)	R	0.69 ± 0.04	2.472 (1.44–4.09)	61.80 (36.00-102.25)	R

R: Resistant; S: Susceptible; CI: confidence interval; ^aEC₅₀ values in µg/mL.

^bResistance ratio=EC₅₀ of field/EC₅₀ of susceptible (Susceptible isolate EC₅₀ value of 0.36 µM or 0.087 µg/mL as per Ruffell et al.⁶⁰

^cResistance ratio calculated by using EC₅₀ value of susceptible isolate (0.04 µg/mL) as per Kaur et al.³²

Fig. 2.

Regression plots of egg hatching/larval development inhibition against log concentrations of anthelmintics for various sheep farms.

Larval development assay

Based on LDA results, 78.6% (11/14) and 100% (14/14) selected farms showed resistance against levamisole and ivermectin, respectively (Table 5). The effective concentration at 50% inhibition of larval development (EC₅₀) values in levamisole-resistant farms ranged from 0.430 to 4.185 µg/mL with resistance ratios (RR) in the range of 4.94 to 48.10 indicating moderate to severe resistance levels, whereas, the susceptible farms exhibited much lower EC₅₀ values (0.058 to 0.123 µg/mL). The EC₅₀ values for ivermectin-resistant farms ranged from 0.086 to 10.434 µg/mL with variable RR (2.15–260.85) and extremely high resistance levels in FDK and NSS farms. Regression plots depicting larval development inhibition against log concentrations for both levamisole and ivermectin are presented in the Fig. 2.

The correlation coefficient (r) for resistance status against fenbendazole, levamisole and ivermectin revealed by in-vivo test (FECRT) and in-vitro tests (EHT and LDA) was estimated as -0.539, -0.631 and − 0.517, respectively, revealing a strong inverse correlation between drug efficacy (FECRT) and resistance status (EHT and LDA).

Discussion

This study presents a comprehensive assessment of the epidemiology of GIP infections and AR status in sheep populations across northern India covering states of Punjab and HP. The findings of the present study indicate a very high burden of GIP infections; with 96.4% of the examined sheep found positive. The prevalence recorded in the present study is comparable to earlier reports from Punjab¹⁸ and HP¹⁹, while being markedly higher than that documented from the neighboring regions of Jammu²⁰ and Kashmir Valley²¹. Further, across India, it is higher than those reported from Maharashtra²², Karnataka²³ and Andhra Pradesh²⁴, but are comparable to Haryana²⁵. Our finding is also closer to reports from Nepal²⁶ and Africa²⁷ but higher than that reported from Ethiopia²⁸ and Bangladesh²⁹. Variations in the findings reported by other studies might be due to differences in agro-ecological conditions, methodological approaches, sample size, study design, management and husbandry practices, deworming strategies and intensity of the parasite exposure across various regions.

Our findings showed that strongyles were the most prevalent GIPs in sheep (97.0%), which agrees with many earlier reports^{19,20,22–27,29}. This can be attributed to the high biotic potential of strongyles and the semi-intensive grazing system that promotes continual exposure to the infective stages under favourable environmental conditions.

Based on the quantitative analysis, the present study observed an average EPG count (mean ± SE) of 1118.9 ± 42.5, which is lower than earlier reports from Punjab (1441.9 ± 77.7)¹⁸ but agrees to that from HP (1017.5 ± 205.6)¹⁹, possibly reflecting the influence of the larger sample size evaluated in this investigation. Also, higher EPG counts were observed during the monsoon period, while comparatively lower counts were recorded in the winter season likely due to environmental constraints that limit the survival and development of free-living pre-parasitic stages of the worms³⁰.

Coproculture and identification of L₃ showed the presence of several nematode genera, with Haemonchus being predominant which can be attributed to higher susceptibility of sheep to Haemonchus as compared to other genera³⁰. Further, our results agree with those reported by earlier workers^25,28. A significantly higher abundance of Haemonchus was noted during the monsoon, whereas Trichostrongylus and Ostertagia were more prominent in the winter season, indicating seasonal shifts in the dominance of strongyle genera.

The findings of the present study indicate that season and deworming status of host emerged as the most important risk factors significantly influencing the occurrence of GIP infections. Consistently higher infection rates in non-dewormed animals can be attributed to uninterrupted parasite life cycles and accumulated pasture contamination^24,27,28. A non-significant higher prevalence was detected in female host which may be due to greater susceptibility of females to the parasitic infections as physiological conditions such as pregnancy and lactation can reduce their immune responses³¹. Host age also showed a significant influence and higher infection rates were recorded in younger animals (< 6 months) and may be linked with weaker immune systems in younger individuals compared with older ones³².

The AR in GIN of small ruminants has been detected using in-vivo and in-vitro approaches. The principal in-vivo methods include FECRT and controlled efficacy test, whereas in-vitro assays encompass EHT, LDA, larval paralysis, migration and motility tests³³. While the controlled efficacy tests were used extensively in the past, their application has declined due to high cost, procedural complexity and animal welfare concerns³⁴. The present investigation utilized a combination of FECRT, EHT and LDA to assess the AR status in GINs of sheep. The FECRT results revealed widespread multi-resistance status, with low efficacy of fenbendazole, levamisole and ivermectin across the studied sheep farms, reflecting reduced therapeutic effectiveness of these commonly used anthelmintics. These results corroborate several earlier reports from various regions of India documenting reduced efficacy of fenbendazole^15,35–41, levamisole^15,35,39 and ivermectin^15,36,39,41 against GINs of sheep. Similarly, reports from different regions of world have consistently demonstrated the widespread occurrence of multiple AR in GINs of sheep^42–44. In contrast, the findings of in-vitro assays provided further resolution of AR pattern with EHT detecting benzimidazole resistance in 78.6% of sheep farms. The present findings are comparable to reports from Karnataka⁴⁵ but higher than that reported from Uttarakhand³⁸ and Tamil Nadu⁴⁶ states of India.

The LDA detected levamisole resistance in 78.6% and ivermectin resistance in 100% of sheep farms. This widespread resistance particularly against ivermectin poses a serious risk to sustainable sheep production and smallholder livelihood in the region. Three sheep farms (KUU, KGRA and CHMB) classified as resistant against fenbendazole by FECRT were susceptible for benzimidazoles by EHT. This discrepancy between resistance detection methods can be attributed to the use of different benzimidazole analogues, with thiabendazole employed in the EHT and fenbendazole used in the FECRT, as thiabendazole is considered as most reliable compound for EHT for defining benzimidazole resistance^32,38. The EHT and FECRT are well validated to reliably detect BZ-resistant nematodes, however, the former provides more quantitative estimate of resistance levels. Further, the discrepancies in results of these tests recorded in present study may be associated with the fact that the tests fail to equally detect the resistance if the frequency of R genes in a population is below 25%⁴⁷.

Further, the in-vivo and in-vitro assay results were mostly concordant, however exceptions were observed for CHMB with levamisole and GSD with ivermectin, where comparatively higher in-vivo efficacy was recorded despite resistance being detected by the in-vitro assays. There have been few reports documenting susceptibility of levamisole and ivermectin^15,36,48,49 from India. Although limited data are available from India on the application of LDA for detecting levamisole and ivermectin resistance, evidence from other regions of the world indicates its utility in GINs of sheep^50,51. On these lines, our findings are comparable for ivermectin while indicating higher resistance to levamisole.

The reduced efficacy and emergence of resistance to fenbendazole, levamisole and ivermectin observed in the present study may be linked to prevailing farm management practices in the region, particularly indiscriminate and repeated use of the same anthelmintic over extended periods and the blanket deworming of the entire flocks without considering individual infection status. In addition, failure to accurately weigh the animals before treatment results in widespread underdosing, particularly in large flocks, as doses are commonly based on estimated average flock weight rather than the heaviest animals. The limited resources to permit test and treat the animals only after faecal examination along with lack of awareness about the use of FAMACHA for screening of animals at farm levels add to the indiscriminate use of anthelmintics in the region. Also, the selection of anthelmintic in majority of farms is through self-medication rather than veterinarian’s prescription. These practices markedly increase the risk of AR development⁵². Further, Haemonchus emerged as the predominant genera among treated sheep relative to other GINs, indicating a greater tolerance to anthelmintic drugs, which may be linked to its high fecundity and substantial genetic variability compared with other nematode species^30,53.

Key molecular mechanisms involve mutations in the β-tubulin gene associated with benzimidazole resistance, alterations in glutamate- and GABA-gated chloride channels linked to macrocyclic lactone (ivermectin) resistance and changes in nicotinic acetylcholine receptors responsible for levamisole resistance⁴. Although, these molecular mechanisms have been extensively reported in different parts of the world, their epidemiological significance in the region remains insufficiently explored. Therefore, it is imperative to investigate the role of associated genetic markers to assess the distribution frequency and quantification of resistance alleles in regional field populations of GINs. In this regard, techniques such as PCR-based assays and their variants along with sequencing as diagnostics will provide valuable insights towards frequency and spread of resistance. Furthermore, correlating these findings with phenotypic resistance data obtained through in-vivo and in-vitro assays will enhance our understanding of genotype–phenotype relationship in AR development. Such integrated approaches prove crucial for developing evidence-based control strategies against GINs of sheep and for mitigating further spread of AR in the region.

Conclusion

The findings of this study revealed a high prevalence of GIP in sheep, with strong evidence of widespread multi-resistance across the studied farms of northern India to the widely used broad spectrum anthelmintics, as confirmed by both in-vivo and in-vitro assays. The combined use of these assays proved effective in detecting both the magnitude and complexity of AR in sheep. This reflects intense and sustained selection pressure likely driven by frequent, indiscriminate and prophylactic use of the same anthelmintics without resistance monitoring. Collectively, these findings highlight the necessity for region-specific and evidence-driven parasite control programmes, emphasizing targeted selective treatment, routine resistance surveillance, improved grazing management and incorporation of alternative control measures.

Materials and methods

Study region

The study was conducted in the north-western region of India with latitude of 29.30° N to 33.12° N and longitude of 73.55° E to 79.04° E covering two states of Punjab and Himachal Pradesh with elevations ranging from 180 m to 6975 m above mean sea level. According to Koppen-Geiger climatic classification, the study region falls under humid subtropical (Cfa), warm semi-arid (Bsh) and sub-temperate (Cwb) climates⁵⁴.

Sampling methodology

Faecal samples were collected from indigenous sheep of all ages and sexes across 15 districts of Punjab and 07 of HP (Fig. 3). For each sample, information on the animal’s age, sex, management system and deworming history for the previous three months was documented. The prevalence of GIPs was analysed using a repeated cross-sectional design with a random sampling method during different seasons (summer, winter and monsoon). All samples were collected with owners’ consent following standard parasitological procedure to maintain uniformity and accuracy.

Fig. 3.

Area selected for the epidemiological survey and location of sheep farms for faecal egg count reduction test.

A total of 2308 faecal samples were collected, comprising 1620 samples from Punjab and 688 from HP during the year 2024-25. Approximately 5–10 g faecal sample was collected directly from the rectum following proper restraint, placed in a suitably labelled sample collection vial, carried to the Postgraduate Laboratory, Department of Veterinary Parasitology in insulated ice boxes and stored at 4 °C until processing.

Coproscopic examination

Each sample was subjected to both qualitative and quantitative faecal examination. For qualitative coprological examination, simple sedimentation and floatation (using saturated salt solution) techniques were used to identify the parasitic ova/oocysts/cysts based on their morphological characteristics³⁰. The quantitative assessment was performed using the McMaster egg counting technique⁵⁵ to determine the EPG of faeces. Infection intensity was grouped into three categories as low (EPG < 800), moderate (EPG 800–1200) and severe (EPG > 1200)⁵³. Further, to identify the genera of GINs, standard faecal culturing technique was used followed by the Baermann’s technique to recover the L₃ stages⁵³. The L₃ were killed using Lugol’s iodine solution on a clean glass slide and distinguished based on key morphological characteristics including the appearance of anterior and posterior ends, intestinal cells’ number, cranial retractile body and length of the oesophagus⁵⁶.

Faecal egg count reduction test

The FECRT was conducted at 14 selected sheep farms across northern India to assess the efficacy status of commonly used and widely preferred dewormers belonging to the benzimidazole (fenbendazole), imidazothiazole (levamisole) and macrocyclic lactone (ivermectin) classes (Fig. 3). Sheep reared under semi-intensive management system mainly maintained on grazing with additional feeding of concentrates were selected for the study. The owners were informed about the details of study to prevent self-medication and disposal of selected animals during the study period. The selection criteria of naturally infected animals followed the guidelines issued by the WAAVP for determining efficacy with EPG ≥ 150 and ensuring no anthelmintic treatment to animals in past 12 weeks^57,58.

The anthelmintics evaluated included fenbendazole 2.5% suspension (MSD, India) administered @ 5.0 mg/kg, ivermectin 0.08% oral solution (Virbac, India) @ 0.2 mg/kg and levamisole 150 mg tablets (KLAB, Uttarakhand, India) @ 7.5 mg/kg body weight. Animals with EPG ≥ 150 were randomly allocated into four groups: three treatment groups (fenbendazole, levamisole and ivermectin) and one control group, with each group comprising of 10 ± 2 animals. Each group received its respective anthelmintic dose based on individual body weight and animals were marked by using different coloured nylon ropes around their necks. Faecal samples from each animal were collected at day 0 and 14 DPT, strongyle eggs were enumerated using McMaster egg counting technique to determine the FECR%⁵⁷. Further, pooled faecal samples from each group collected 14 DPT were cultured and L₃ were identified, to assess the effect of the anthelmintic drug on the GIN genera⁵⁶.

Egg hatch assay

The EHT was performed to detect benzimidazole resistance as per the procedure of Coles et al.⁵⁹. Briefly, strongyle eggs were extracted from the pooled faecal samples collected at D0, fresh egg suspension was prepared and dispensed into 48-well culture plate (VWR, Radnor, PA, USA) @ 150–200 eggs per well in 190 µL of distilled water. The stock solution (Solution A, 1 mg/mL) of thiabendazole (Sigma-Aldrich, St. Louis, MO, USA) was prepared by dissolving 2 mg of the drug in 2 mL of dimethyl sulfoxide (DMSO) (SRL, India). From this stock, 80 µL was taken and mixed with 920 µL of 2% DMSO to obtain Solution B (80 µg/mL), which was then used in treatment wells at concentrations of 0.06, 0.125, 0.25, 0.5, 1.0, 2.0 and 4.0 µg/mL in 10 µL volumes with 2% DMSO in the control well. Each concentration was tested in triplicate after incubating the sealed plate at 28 °C for 48 h in a BOD incubator. After incubation, one drop of Lugol’s iodine was added per well and a minimum of 100 dead/embryonated eggs or larvae were enumerated under a compound microscope (CX-21 Olympus, Japan).

Larval development assay

The LDA was carried with eggs obtained from pooled samples to assess resistance status against levamisole and ivermectin⁶⁰. The fresh egg suspension containing 150–200 eggs was transferred to each well of a 48-well culture plate (VWR, USA) and the final volume of 440 µL was adjusted using distilled water. Then 50 µL of LDA media along with 10 µL of amphotericin-B (Sigma-Aldrich, USA) were added to each well. Following this, plates were placed in a BOD incubator at 28 °C for 24 h to allow hatching of eggs, after which the wells were examined for the presence of larvae. The stock solutions (Solution A, 1 mg/mL) of levamisole and ivermectin (Sigma-Aldrich, USA) were prepared by dissolving 1 mg of each drug in 1 mL of DMSO. From Solution A of levamisole and ivermectin, 320 µL each was withdrawn and mixed with 1680 µL of 2% DMSO to obtain Solution B (160 µg/mL) of levamisole and ivermectin. This solution was used in the treatment wells at final concentrations of 0.31, 1.25, 5.0, 20.0 and 80.0 µg/mL in 500 µL volumes while control wells received only 2% DMSO. After sealing, plates were incubated again at 28 °C in a BOD incubator for 6–7 days to allow development of L₃. Thereafter, one drop of Lugol’s iodine was added to each well, and the contents were transferred into 1.5 mL microcentrifuge tubes. The samples were then centrifuged in a MiniSpin Plus (Eppendorf, Germany) at 3000 rpm for 4 min and the supernatant was discarded. Approximately, 200 µL of the remaining sediment was gently mixed and placed onto a clean glass slide. The counts of unhatched eggs, larval stages consisting of L₁, L₂ and L₃ for each well were then recorded using a compound microscope.

Data analysis

Epidemiological data were analysed using RStudio 4.3.3 and IBM SPSS Statistics 20 software. The logistic regression and Chi-Square tests (model goodness-of-fit test) were applied to determine the association between risk factors and prevalence of GIP infections, with odds ratios and P-values at a 95% CI used to assess the significance. For FECRT, arithmetic mean egg counts before and after treatment, along with 90% CI for percentage reduction, were calculated using the open-source software available at www.fecrt.com⁶¹ and resistance status was interpreted accordingly. Tukey’s HSD post-hoc test was applied to compare GIN L₃ prevalence between pre- and post-treatment groups and across seasons. The EC₅₀ values with 95% CI for both EHT and LDA were estimated through Probit analysis using Polo-Plus software. Isolates showing EC₅₀ values greater than 0.1 µg/mL of thiabendazole in the EHT were classified as resistant⁵⁹. For LDA, the RR for selected farms against levamisole and ivermectin were calculated as the ratio between EC₅₀ of field isolates and that of susceptible reference isolates. For levamisole, the EC₅₀ of susceptible isolate (0.36 µM or 0.087 µg/mL) reported by Ruffell et al.⁶⁰ was used. For RR calculation against ivermectin, the EC₅₀ of the susceptible ASR isolate (0.04 µg/mL) reported by Kaur et al.³² from the region was used. To evaluate the relationship between in-vivo and in-vitro resistance detection assays, the correlation between FECRT drug efficacy (%) and the EC₅₀ values (µg/mL) obtained from the EHT/LDA for each anthelmintic was calculated, and the r value was determined.

Author contributions

K.C., A.K., J., M., D.S. and H.S. Sampling, conducting the experiment, data analysis, preparation of figures and manuscript writing N.K.S. Conceptualization, funding acquisition, experiment designing, manuscript writing and project administration. All the authors reviewed and approved the final version of the manuscript.

Funding

The study was supported under the National Livestock Mission (NLM) by the Department of Animal Husbandry & Dairying, Ministry of Fisheries, Animal Husbandry & Dairying, Government of India, New Delhi (R-30012/1/2022-DADF-Dept-Part (2) (E-24308) dated 27/02/2023).

Data availability

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

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

Ethical approval for collection of faecal samples and administration of anthelmintic drugs to the naturally infected sheep was obtained from the Institute Animal Ethics Committee (IAEC), Guru Angad Dev Veterinary & Animal Sciences University, Ludhiana vide IAEC/2023/134 − 55 dated 22.05.2023 (GADVASU/2023/IAEC/69/19). The sheep owners were sensitized about the purpose of study and all the protocols were conducted in accordance with relevant guidelines and regulations.