Prevalence, Molecular Identification, and Phylogenetic Analysis of Strongylus equinus in Horses in Al-Muthanna Province, Iraq

Introduction
Horses are domestic animals that are important to resource-poor populations in both rural and urban areas, providing transportation services in difficult terrain or for military ceremonies, and playing a role in land tillage and cultivation (Andarge et al., 2017; Idoko et al., 2021; Alaba et al., 2022). The global horse population, particularly in Iraq, has declined significantly over the past few decades. This reduction can be attributed to the persistent reliance on traditional methods of horse care and management, such as the use of traditional farms or individual stables for each horse, as well as to inadequate veterinary attention, coupled with the diminishing availability of grasslands and forests (Devkota et al., 2021; Ememe et al., 2024).
Equines exhibit elevated susceptibility to a diverse array of pathogenic gastrointestinal helminths, particularly large strongyles such as Strongylus vulgaris, Strongylus equinus, and Strongylus edentatus, which are the primary causative agents of strongylosis, one of the most important parasitic diseases that affects more than 90% of the equine population (Alhaitami, 2023). Equine large strongyle infections are associated with a range of clinical manifestations, such as reduced productivity, colic, diarrhea, growth impairment, and reproductive problems, and they can lead to pathological changes that include damage to visceral organs, verminous arteritis, and thrombosis, leading to death (Kaur et al., 2019; Kompi et al., 2021). These changes arise not only from the presence of adult parasites within the intestinal tract but also from larval migration through intestinal tissues and systemic organs (Asefa & Dulo, 2017; Berihun et al., 2024). Horses become infected by ingesting feed contaminated with feces containing third-stage larvae (L3). The larvae migrate through the bloodstream to organs such as the liver, spleen, and pancreas, before settling and maturing in the large intestine (Kaur et al., 2019; Ola-Fadunsin et al., 2019; Alaba et al., 2022)
The historical diagnosis of gastrointestinal nematode infections in equines has primarily relied on microscopic identification of nematode eggs or larvae in fecal samples, using flotation techniques and or larval cultures (Bizuayehu & Bedada, 2018). However, these techniques are constrained by difficulties in distinguishing species solely based on egg or larval morphology (Lichtenfels et al., 2008; Jasim & Al-Amery, 2023). Since conventional diagnostic methods have limitations, there is a critical need to adopt molecular approaches, especially polymerase chain reaction (PCR) and sequencing, to accurately identify and distinguish S. equinus infection (Bohórquez et al., 2015; Diekmann et al., 2024). Also, this study contributes to the one health approach to equine parasitology by combining molecular surveillance with epidemiological risk-factor analysis, which is crucial for preventing zoonotic spillover and improving livestock health in developing regions. 
In Iraq, there is very little information about the prevalence of Strongylus spp. despite the significant health risks it poses to horses. This study aimed to evaluate the prevalence of S. equinus and the impact of sex, age, and seasonal factors on its incidence in the study area, which had not been previously studied. Besides, this study is the first in Iraq and the region to use molecular diagnostics and phylogenetic analysis to detect S. equinus in horses, thereby establishing critical baseline data for future epidemiological studies and control strategies. 

Materials and Methods 
Study area and sampling 
Between January and the end of October 2023, a cross-sectional study was conducted to determine the prevalence of Strongylus spp. and to characterize S. equinus molecularly. A total of 118 horses (48 females and 70 males) of different ages were randomly selected from stables in the Alkider, Rumathia, and Samawah of Al-Muthanna Governorate, southern Iraq (Figure 1).

Sample size (n=118) was calculated based on an expected prevalence of 50% from previous studies, a 95% confidence interval, and a precision ±9%. Five-gram fecal samples were obtained from each horse, either during rectal examination by a plastic glove or from fresh fecal deposits. Each sample was individually stored in a container, clearly labeled with age, sex, and date of collection, and transported to the laboratory in insulated containers with ice packs for direct examination using the flotation method. Then, samples that tested positive by microscopic examination were kept at 4 °C for later DNA extraction. Horses were divided into three age categories, namely under four years, four to seven years, and over seven years, based on owner-provided information and dental formulas, as per the methods outlined by Mezgebu et al. (2013).

Coprological examination
To identify Strongylus spp. eggs, fecal samples collected from all horses were processed fresh daily via the flotation technique with sheather’s sugar solution (prepared with 454 g of sugar, 355 mL of distilled water, and 6 mL of phenol as a preservative; specific gravity 1.27), and the samples were examined under a light microscope at 10x (Alhaitami, 2023). The eggs were identified based on their morphological characteristics, as previously described (Aldelami & Albadrani, 2009).

DNA extraction and PCR 
Using the Qiagen DNA stool mini kit (Germany), genomic DNA was extracted from 50 fecal samples of horses that tested positive for S. equinus infection based on the flotation technique. Afterward, each eluted DNA sample was measured using a NanoDrop spectrophotometer (Thermo, USA) to determine its concentration and purity by assessing the absorbance ratio at 260/280 nm. Moreover, the integrity of the extracted DNA was assessed via 1% agarose gel electrophoresis supplemented with 0.05% ethidium bromide (Dalimi & Jaffarian, 2024). Partial S. equinus sequences from GenBank (Accession No. AJ228250.1) were used to design primers targeting a 220 bp internal transcribed spacer (ITS-1) rDNA fragment for detecting infection in horses, which were ordered from Macrogen (Korea) (Table 1).

The PCR amplification mixture for the detection of the ITS-1 region of rDNA included 25 µL of GoTaq® DNA polymerase 2X Master Mix (Promega, USِِA, and Cat. No. M7122) that contains (Taq polymerase, dNTPs, MgCl₂, and buffer), 5 µL DNA template (normalized to 150 ng), 1.5 μL of a 10 μM working stock of each primer (StrongF, StrongR), resulting in a final concentration of 0.3 µM for each primer, and completed the volume to 50 µL with nuclease-free water. The PCR amplification protocol consisted of an initial denaturation at 94 °C for 5 minutes, followed by 35 cycles of denaturation at 94 °C for 25 seconds, annealing at 56 °C (optimized based on primer Tm calculations and gradient PCR) for 25 seconds, and extension at 72 °C for 40 seconds. A final extension step at 72 °C for 5 minutes was performed. The specificity of the 220 bp product was confirmed by DNA sequencing. After amplification, 5 µL of each PCR product was subjected to electrophoresis on a 1.5% agarose gel and visualized under UV light using a transilluminator (Abdullah & Ali, 2021).

Sequencing and phylogenetic analysis
Three high-quality PCR products with strong band intensity and purity (37, 54, 83) were randomly selected and prepared for bidirectional Sanger sequencing at Macrogen (Korea). The three collected sequences were blasted using online NCBI (BLASTn) software to confirm and collect the aligned sequences. Phylogenetic analysis was performed in MEGA-X v10.0.5 using the Kimura 2-parameter method with 1000 bootstrap replications to ensure tree reliability, with three threads (Hade, 2020). The results of the three local S. equinus sequences were submitted to the NCBI-GenBank based on sequence analysis of the ITS-1 region of rDNA with the following accession numbers (PQ900954.1, PQ900955.1, and PQ900956.1).

Statistical analysis
Statistical analyses were performed using SPSS software, version 26.0 (IBM, USA). Prevalence rates were expressed with 95% confidence intervals (CI). The chi-square tests were used to assess associations between infection status and risk factors (age, sex, and season). Variables with P<0.2 were further analyzed using multivariate logistic regression to estimate adjusted odds ratios (aOR) with 95% CI. A P<0.05 was considered significant.

Results 
A parasitological examination of equine fecal samples confirmed the presence of Strongylus spp. eggs (Figure 2).

Epidemiologically, out of the 118 horses examined from January to the end of October 2023, 50 tested positive for Strongylus spp., with a prevalence rate of 42.3% based on the flotation technique. Concerning risk factors, infection rates were considerably higher in horses under 4 years of age (57.1%); however, this disparity was not statistically significant (P>0.05) (Table 2).

Also, the analysis demonstrated a statistically significant difference in infection rates based on sex, with female horses showing notably higher prevalence (56.2%) compared to male horses (32.8%) (P≤0.05) (Table 2). Moreover, the study identified significant seasonal variations in infection prevalence, with peak rates recorded in March (83.3%) and April (72.2%); these variations were statistically highly significant (P≤0.05) (Table 3).

Genomic DNA was successfully extracted and purified from 50 fecal samples of horses infected with Strongylus spp. using the QIAGEN DNA stool mini kit (Germany). DNA concentrations, measured using a Thermo Scientific Nanodrop spectrophotometer (USA), ranged from 52.1 to 211.4 ng/μL, with a purity of 1.8-1.9. Besides, agarose gel electrophoresis confirmed the integrity and purity of the extracted DNA, showing a distinct genomic band without degradation. Furthermore, PCR was performed using specific primers (StrongF, StrongR) to diagnose S. equinus infections in horses. The results indicated that 22(44%) out of 50 horses tested positive for S. equinus using PCR (Figure 3).

Finally, the result of ITS-1 in local S. equinus based on phylogenetic analyses were recorded outstanding 100% and 97% identity matching between the current study isolates (PQ900954.1, PQ900955.1., and PQ900956.1) and an Australian strain isolated from horses (AJ228250.1, AJ228248.1), and 94% with Chinese isolate (KP693438.1) (Figure 4).

Discussion
Gastrointestinal parasites, particularly Strongylus spp., represent a critical challenge to equine health and productivity in developing nations. These helminths significantly compromise livestock output through impaired digestive efficiency, reduced reproductive performance, and elevated costs associated with therapeutic and prophylactic interventions (Sori et al., 2017; Devkota et al., 2021). Epidemiological analysis revealed that 50 of the 118 horses were positive for Strongylus spp. infection with a prevalence rate of 42.3%. This finding is consistent with several prior studies (Hasson, 2014; Oli & Subedi, 2018; Negash et al., 2021; Berihun et al., 2024) but contrasts with others, which documented equine infection rates of 26.5%, 100%, and 71.8%, respectively (Studzińska et al., 2012; Sheferaw & Alemu, 2015; Alhaitami, 2023). The observed variations in Strongylus spp. prevalence rates attributed to multiple factors, including differences in sample size, diagnostic methodologies, selection criteria, environmental conditions, pasture management practices, and inconsistent anthelmintic administration (Sheferaw & Alemu, 2015; Alhaitami, 2023; Berihun, 2024).
Furthermore, this study shows that sex possessed a significant impact on the prevalence of infections caused by Strongylus spp., which is consistent with previous findings (Hasson, 2014; Alhaitami, 2023; Kebede et al., 2024), and contradicts findings with (Mathewos et al., 2021; Ilić et al., 2023; Berihun et al., 2024), who failed to establish a statistically significant correlation between sex and infection rate. This difference may be attributed to lactation and pregnancy, which can both be immunosuppressive due to stress from cyclical hormonal changes (Alaba et al., 2022; Jasim et al., 2024). Additionally, the epidemiological findings indicated that horses < 4 years of age exhibited the highest prevalence of Strongylus spp. infection (57.1%). However, the difference in infection rates among age groups was not statistically significant. These findings are consistent with those of Egbe-Nwiyi et al. (2019) but conflict with those of Alemayehu et al. (2022) and Kebede et al. (2024). The increased incidence of infection in young animals may be attributed to waning maternal immunity, greater exposure to the infective stages of the parasite, and irregular anthelmintic treatment regimens (Singh et al., 2016; Jürgenschellert et al., 2022).
Besides, the study indicated a pronounced seasonal correlation in Strongylus spp. infection prevalence, with the highest rate observed in March (83.3%) and the lowest in August (18.8%). These findings correspond with prior studies (Faraj et al., 2007; Hasson, 2014; Singh et al., 2016) and contrast with those of Diekmann et al. (2024), who noted an elevated prevalence of Strongylus spp. in July and December. The seasonal fluctuations in Strongylus spp. infection rates can be attributed to various interconnected causes, such as the increase in incidence in March, which aligns with the spring birthing season in Iraq, a period characterized by physiological stress and immunosuppression in equines, which increases vulnerability to infection. Simultaneously, ideal climatic conditions, including moderate spring temperatures and humidity, increase embryonation and larval growth, facilitating transmission (Faraj et al., 2007; Singh et al., 2016; Jasim et al., 2024). Additionally, the extended prepatent period of Strongylus spp. spanning several months may delay egg excretion in infected equinus until the subsequent grazing season, coinciding with infection peaks with favorable environmental conditions (Studzińska et al., 2012). 
This study improved upon earlier methods by combining traditional microscopy with PCR-based molecular diagnostics, demonstrating greater sensitivity compared to. Using PCR to target the ITS-1 region, a 220-bp fragment was amplified, confirming S. equinus in 22(44%) of horse samples. This finding aligns with recent work by Diekmann et al. (2024), which highlighted molecular diagnostics’ superior specificity compared to traditional methods, confirming only 1 of 12 morphologically positive samples with PCR. The discrepancies may be due to the inherent limitations of factors in the sensitivity and specificity of morphological diagnostics, such as the developmental stage of the parasite, sample fixation protocols, transportation and preservation conditions, sample size adequacy, and the examiner’s expertise in parasitological identification or the morphological overlap among Strongylus species (Bowman, 2020; Ghafar et al., 2023).
In addition, the use of ITS-1 as a molecular marker for precise species differentiation of S. equinus represents an innovative approach in Iraq. Its reliability compared to traditional diagnostic methods is likely due to the ITS-1 region’s significant evolutionary conservation, which renders it an effective molecular marker for taxonomic and phylogenetic research (Halvarsson & Tydén, 2023; Al-Biatee et al., 2024; Ahn et al., 2024). Also, the ITS-1 partial sequence in local S. equinus, based on phylogenetic analyses, showed 97%–100% nucleotide sequence identity between the current study isolates (PQ900954.1, PQ900955.1, and PQ900956.1) and an Australian strain isolated from horses, and 94% with a Chinese isolate. The high nucleotide similarity between Iraqi isolates and strains from Australia and China may be due to commercial animal movement—particularly through intermediary countries, rather than direct introduction from those regions or shared ancestral parasite lineages, offering new insights into global transmission patterns of equine parasites (Beltran-Alcrudo et al., 2019). This work is the first study in Iraq to combine molecular diagnostics and phylogenetic analysis for S. equinus. These findings provide baseline data for parasite control programs, inform veterinary health policies, and contribute to the global understanding of Strongylus spp. genetic diversity.

Conclusion
According to survey data, strongylosis is a major veterinary disease affecting local horses, with a prevalence of 42.3%. It occurs year-round, peaking in spring, and is more common in horses under 4 years of age and in females. Besides, the phylogenetic analysis revealed close genetic similarity among Iraqi, Australian, and Chinese strains, highlighting the evolutionary conservation of the ITS1 region of rDNA as a robust molecular marker for taxonomic and phylogenetic studies. To advance understanding of the parasite’s genetic diversity and support One Health goals, the study recommends future initiatives, including whole-genome sequencing of S. equinus, expanded surveillance in Iraq, the application of molecular diagnostics to larger equine populations, and an evaluation of anthelmintic resistance.

Ethical Considerations
Compliance with ethical guidelines
Sample collection was performed in compliance with the World Organisation for Animal Health (OIE) animal welfare guidelines and the ARRIVE guidelines. Before collecting the samples, we obtained verbal consent from the horse owners.

Funding
This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors.

Authors' contributions
Study design, super vision, review and editing: Hussein Jabar Jasim; Experiments and data collection: Naer Abdulbari Madlool Alkaabawi; Data interpretation, statistical analysis, and writing the original draft: Zahraa Abd Alhammza Abbass and Mohammed Mijbas Mohammed alomari; Final approval: All authors.

Conflict of interest
The authors declared no conflict of interest.

Acknowledgments
The authors extend their gratitude to the veterinary clinicians and support staff at Al-Muthanna Provincial Veterinary Hospital, as well as the equine breeders, for their invaluable contributions to fieldwork and sample acquisition. 


References 
Abdullah, S. H., & Ali, S. A. (2021). Molecular study and phylogeny of Babesia spp. in native sheep from Sulaimani govenorate/ northern Iraq. Iraqi Journal of Agricultural Sciences, 52(5), 1077-1083.‏ [DOI:10.36103/ijas.v52i5.1445]
Ahn, S., Redman, E. M., Gavriliuc, S., Bellaw, J., Gilleard, J. S., & McLoughlin, P. D., et al. (2024). Mixed strongyle parasite infections vary across host age and space in a population of feral horses. Parasitology, 151(12), 1299–1316. [DOI:10.1017/S0031182024001185] [PMID] 
Alaba, B. A., Olajide, E. O., Omotosho, O. O., & Okemiri, D. C. (2022). Prevalence, severity and predisposing factors of gastrointestinal parasite infection in polo horses in Ibadan, Nigeria. Journal of Applied Veterinary Sciences, 7(3), 80-85.‏ [DOI:10.21608/javs.2022.140342.1150]
Al-Biatee, S. T., Hade, B. F., & Al-Rubaie, H. M. (2024). Detect of the eggs of P. equorum in the feces of horses by traditional method and molecular techniques in Baghdad, Iraq. Iraqi Journal of Veterinary Sciences, 38(2), 245-250.‏ [DOI:10.33899/ijvs.2023.138252.2779]
Aldelami, M. K., & Albadrani, B. A. (2009). Therapeutic efficacy of a mixture of Ivermectin and Closantel against gastrointestinal parasites in draft horses. Iraqi Journal of Veterinary Sciences, 23(1), 37- 42. [DOI:10.33899/ijvs.2009.5681]
Alemayehu, M. T., Abebe, B. K., & Haile, S. M. (2022). Investigation of strongyle prevalence and associated risk factors in horses in and around Alage District, Ethiopia. Journal of Parasitology Research, 2022, 3935008. [DOI:10.1155/2022/3935008] [PMID] 
Alhaitami, I. (2023). Diagnostic epidemiological differential study of the intestinal parasites infecting horses in Karbala province, Iraq. Journal of Kerbala for Agricultural Sciences, 10(3), 184-189. [DOI:10.59658/jkas.v10i3.1251]
Andarge, B., Muhammed, C., & Tibesso, G. (2017). Prevalence of major intestinal nematodes of equines in Jimma Town, South Western Ethiopia. International Journal of Veterinary Science and Research, 3(2), 69-73. [DOI:10.17352/ijvsr.000024]
Asefa, S., & Dulo, F. (2017). A prevalence of gastro-intestinal nematode parasitic infections in horses and donkeys in and around Bishoftu town, Ethiopia. Middle-East Journal of Applied Sciences, 3(3), 38-43. [Link] 
Beltran-Alcrudo, D., Falco, J. R., Raizman, E., & Dietze, K. (2019). Transboundary spread of pig diseases: The role of international trade and travel. BMC Veterinary Research, 15(1), 64. [DOI:10.1186/s12917-019-1800-5] [PMID] 
Berihun, A. M., Bizu, F., Maru, M., & Kassaw, S. (2024). Prevalence, associated risk factors, and identification of the genera of equine strongyles in horses and donkeys in and around Bishoftu, Ethiopia. Veterinary Medicine International, 2024, 3224113. [DOI:10.1155/vmi/3224113] [PMID] 
Bizuayehu, F., & Bedada, H. (2018). Epidemiology of horse GIT parasitism in Gondar Town and Wegera district. African Journal of Basic and Applied Sciences, 10 (3), 55-63. [Link]
Bohórquez, G. A., Luzón, M., Martín-Hernández, R., & Meana, A. (2015). New multiplex PCR method for the simultaneous diagnosis of the three known species of equine tapeworm. Veterinary Parasitology, 207(1-2), 56-63.‏ [DOI:10.1016/j.vetpar.2014.11.002] [PMID]
Bowman, D. D. (2020). The anatomy of the third-stage larva of Toxocara canis and Toxocara cati. Advances in Parasitology, 109, 39-61.‏ [DOI:10.1016/bs.apar.2020.03.002] [PMID]
Dalimi, A., & Jaffarian, F. (2024). Molecular Characterization of Strongyloides Stercoralis in Mazandaran Province, North of Iran. Archives of Razi Institute, 79(3), 513-518.‏ [DOI:10.32592/ARI.2024.79.3.513] [PMID] 
Devkota, R. P., Subedi, J. R., & Wagley, K. (2021). Prevalence of gastrointestinal parasites in equines of Mustang district, Nepal. Biodiversitas Journal of Biological Diversity, 22(9), 3958-3963. [DOI:10.13057/biodiv/d220943]
Diekmann, I., Blazejak, K., Krücken, J., Strube, C., & von Samson‐Himmelstjerna, G. (2024). Comparison of morphological and molecular Strongylus spp. identification in equine larval cultures and first report of a patent Strongylus asini infection in a horse. Equine Veterinary Journal, 57(2), 522-529. [DOI:10.1111/evj.14134]
Egbe-Nwiyi, T. N., Paul, B. T., & Cornelius, A. C. (2019). Coprological detection of equine nematodes among slaughtered donkeys (Equus asinus) in Kaltungo, Nigeria. Veterinary World, 12(12), 1911-1915. [DOI:10.14202/vetworld.2019.1911-1915] [PMID] 
Ememe, M. U., Ukwueze, C. S., Onyeabor, A., & Isacc, K. K. (2024). Seasonal effects on the gastrointestinal parasites in horses at Port Harcourt Polo, Rivers State, Nigeria. Journal of Sustainable Veterinary and Allied Sciences, 6 (3), 204-210. [DOI:10.54328/covm.josvas.2024.198]
Faraj, A. A., Khadim, F. S., & Fadl, S.R. (2007). Distribution of Helminth Infections in gastro-intestinal tract of horses. The Iraqi Journal of Veterinary Medicine, 31(2), 82-92.‏ [Link]
Ghafar, A., Abbas, G., Beasley, A., Bauquier, J., Wilkes, E. J., & Jacobson, C., et al. (2023). Molecular diagnostics for gastrointestinal helminths in equids: Past, present and future. Veterinary Parasitology, 313, 109851.‏ [DOI:10.1016/j.vetpar.2022.109851] [PMID]
Hade, B. F. (2020). Molecular sequancing and phylogenic analysis to virulence nmuc-1 gene in visceral larvae migrans. Iraqi Journal of Agricultural Sciences, 51(3), 894-902. [DOI:10.36103/ijas.v51i3.1044]
Halvarsson, P., & Tydén, E. (2023). The complete ITS2 barcoding region for Strongylus vulgaris and Strongylus edentates. Veterinary Research Communications, 47(3), 1767-1771.‏ [DOI:10.1007/s11259-022-10067-w] [PMID] 
Hasson, R. H. (2014). A study in the prevalence of horse’s helminthes parasitic infection in Baquba city; Diyala province’. International Journal of Recent Scientific Research, 5(10), 1810-1813. [Link]
Idoko, I. S., Edeh, R. E., Adamu, A. M., Machunga-Mambula, S., Okubanjo, O. O., & Balogun, E. O., et al. (2021). Molecular and serological detection of piroplasms in horses from Nigeria. Pathogens, 10(5), 508. [DOI:10.3390/pathogens10050508] [PMID] 
Ilić, T., Bogunović, D., Nenadović, K., Gajić, B., Dimitrijević, S., & Popović, G., et al. (2023). Gastrointestinal helminths in horses in Serbia and various factors affecting the prevalence. Acta Parasitologica, 68(1), 56-69.‏ [DOI:10.1007/s11686-022-00636-z] [PMID]
Jasim, H. J., & Al-Amery, A. M. (2023). Biochemical and histopathological changes associated with Fasciola spp. in slaughtered buffaloes at Al-Muthanna Province, Iraq. Advances in Animal and Veterinary Sciences, 11(3), 446-452. ‏ [DOI:10.17582/journal.aavs/2023/11.3.446.452]
Jasim, H. J., Alkaabawi, N. A. M., & Kathem, A. N. (2024) Clinical, prevalence and molecular studies of Parascaris equorum in infected horses in Al-Muthanna Province. Journal of Animal Health and Production, 12(4), 466-472. [DOI:10.17582/journal.jahp/2024/12.4.466.472]
Jürgenschellert, L., Krücken, J., Bousquet, E., Bartz, J., Heyer, N., & Nielsen, M. K., et al. (2022). Occurrence of strongylid nematode parasites on horse farms in Berlin and Brandenburg, Germany, with high seroprevalence of Strongylus vulgaris infection. Frontiers in Veterinary Science, 9, 892920. [DOI:10.3389/fvets.2022.892920] [PMID] 
Kaur, S., Singh, E., & Singla, L. (2019). Strongylosis in equine: An overview. Journal of Entomology and Zoology Studies, 7(5), 43-46.‏ [Link]
Kebede, I. A., Gebremeskel, H. F., Bandaw, T., & Ahmed, A. D. (2024). Prevalence and risk factors of parasitic gastrointestinal nematode infections of donkeys in Southern Ethiopia. Journal of Parasitology Research, 2024, 3073173.‏ [DOI:10.1155/2024/3073173] [PMID] 
Kompi, P.P., Molapo, S., & Ntsaoana, M.E. (2021). Prevalence and faecal egg load of gastrointestinal parasites in horses in Maseru district, Lesotho. Journal of Animal Health and Production, 9(1), 5-12. [DOI:10.17582/journal.aavs/2021/9.5.715.721]
Lichtenfels, J. R., Kharchenko, V. A., & Dvojnos, G. M. (2008). Illustrated identification keys to strongylid parasites (Strongylidae: nematoda) of horses, zebras and asses (Equidae). Veterinary Parasitology, 156(1-2), 4-161. [DOI:10.1016/j.vetpar.2008.04.026] [PMID]
Mathewos, M., Girma, D., Fesseha, H., Yirgalem, M., & Eshetu, E. (2021). Prevalence of gastrointestinal helminthiasis in horses and donkeys of Hawassa district, Southern Ethiopia. Veterinary Medicine International, 2021, 6686688.[DOI:10.1155/2021/6686688] [PMID] 
Mezgebu, T., Tafess, K., & Tamiru, F. (2013). Prevalence of gastrointestinal parasites of horses and donkeys in and around Gondar town, Ethiopia. Open Journal of Veterinary Medicine, 3(6), 267-272. [DOI:10.4236/ojvm.2013.36043]
Negash, W., Erdachew, Y., & Dubie, T. (2021). Prevalence of strongyle infection and associated risk factors in horses and donkeys in and around Mekelle City, Northern Part of Ethiopia. Veterinary Medicine International, 2021, 9430824.[DOI:10.1155/2021/9430824] [PMID] 
Ola-Fadunsin, S. D., Daodu, O. B., Hussain, K., Ganiyu, I. A., Rabiu, M., & Sanda, I. M., et al.(2019). Gastrointestinal parasites of horses (Equus caballus Linnaeus, 1758) and risk factors associated with equine coccidiosis in Kwara and Niger states, Nigeria. Sokoto Journal of Veterinary Sciences, 17(3), 35-43. [DOI:10.4314/sokjvs.v17i3.6]
Oli, N., & Subedi, J. R. (2018). Prevalence of gastro-intestinal parasites of horse (equus caballus linnaeus, 1758) in seven village development committee of Rukum district, Nepal. Journal of institute of science and technology, 22(2), 70-75. [Link]
Sheferaw, D., & Alemu, M. (2015). Epidemiological study of gastrointestinal helminthes of equinus in Damot-Gale district, Wolaita zone, Ethiopia. Journal of Parasitic Disease, 39(2), 315-320. [DOI:10.1007/s12639-013-0352-z] [PMID] 
Singh, G., Singh, N. K., Singh, H., & Rath, S. S. (2016). Assessment of risk factors associated with prevalence of strongyle infection in equines from Central Plain Zone, Punjab. Journal of Parasitic Diseases, 40(4), 1381–1385. [DOI:10.1007/s12639-015-0695-8] [PMID] 
Sori, G., Bekele, T., Geso, G, Ibrahim, H., Gobena, F., & Jarso, G., et al. (2017). Prevalence of equine strongyle infection and its associated risk factors in Jimma Town, Southwest Ethiopia. International Journal of Livestock Production, 8 (11), 187-191. [Link] 
Studzińska, M. B., Tomczuk, K., Demkowska-Kutrzepa, M., & Szczepaniak, K. (2012). The Strongylidae belonging to Strongylus genus in horses from Southeastern Poland. Parasitology Research, 111(4), 1417–1421. [DOI:10.1007/s00436-012-3087-3] [PMID]