Morphological and molecular identification of common Eimeria species in sheep and goats of Bajestan area, Khorasan Razavi Province

Document Type : Research Article

Authors

Mohammad Abdollahi ¹

Asma Keyvanlou ¹

Gholamreza Razmi ²

¹ Department of Pathobiology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran

² Ferdowsi University of Mashad

10.22067/ijvst.2026.94932.1590

Abstract

Coccidiosis is a gastrointestinal disease caused by Eimeria spp in small ruminants. Limited information is available regarding the identified Eimeria species in sheep flocks in Khorasan Razavi Province. The present study aimed to identify the common Eimeria species and detect the pathogenic species in sheep and goats flocks in the Bajestan area. Between September 2021 to June 2022, a total of 400 fecal samples were collected from the rectum of sheep and goats. Samples initially were examined using the Willis method for the presence of Eimeria infestation, and then the Oocyst per gram (OPG) OPG level was determined using the Clayton Lane method. Samples with high OPG values were cultured in a 2.5% potassium dichromate solution. After the sporulation of the cultured sample, the Eimeria species was identified by the morphological characteristics. Additionally, the DNA of sporulated Eimeria oocysts from sheep and goats was extracted and tested using the PCR method. Two positive samples of each of these animals were selected and submitted for sequencing. In the present study, the prevalence of Eimeria spp infection was 69.5% (139/200) in sheep and 69% (138/200) in goats. In morphological studies, Eimeria faurei and Eimeria ovinoidalis were identified in sheep, and Eimeria alijevi, Eimeria hirci, and Eimeria christenseni were identified in goats. PCR amplification confirmed the presence of Eimeria spp in positive samples. The findings indicate a high prevalence of mixed Eimeria spp. infection in sheep and goats in the Bajestan area. In addition, two Eimeria species in sheep and three species in goats were morphologically identified.

Keywords

Prevalence, Identification, Eimeria

goat, sheep

Subjects

• Parasitology

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Abbreviations

OPG: Oocyst per gram

PCR : polymerase chain reaction

Introduction

Coccidiosis is one of the most common worldwide gastrointestinal disease affecting ruminants and poultry [ 1 ]. Coccidiosis caused by Eimeria spp. is a high-morbidity parasitic disease affecting various animal species, including sheep and goats [ 2 ]. Eimeria spp. Infections may result in either acute or chronic intestinal disorders in small ruminants and are responsible for significant economic losses worldwide [ 3 ].

Eimeria spp. is host-specific, which means an Eimeria species that infects goats does not infect sheep, and vice versa [ 4 ]. Many species of Eimeria have been identified in sheep and goats.

Among these species, three species of E. ovinoidalis, E. ahsata, and E. crandalis are known as pathogenic species in sheep, and four species of E. caprina, E. ninakohlyakimovi, E. christenseni, and Eimeria arloingi are known as pathogenic species in goats [ 5 ]. Among the known pathogenic species of Eimeria in goats are E. arloingi and E. ninakohlyakimovi that are known as the dominant pathogenic species in Iran as well as many other parts of the world [ 6 ].

Environmental and climatic factors are crucial in development, survival, and transmission of coccidiosis. Eimeria oocysts can survive in the environment for extended periods, ranging from a few weeks to several months, under favorable conditions moderate temperature and adequate humidity [ 7 ].

Coccidiosis is primarily spread through the ingestion of sporulated oocysts of Eimeria spp [ 8 ]. The life cycle of Eimeria consists of three main stages: asexual reproduction (schizogony) and sexual reproduction (gametogony) within the host's body, and sporogony outside the host in the external environment [ 9 ]. The location of these stages, whether in the small or large intestine, depends on the Eimeria species involved [ 4 ].

Coccidiosis in small ruminants occurs in both clinical and subclinical forms [ 10 ]. The clinical form is associated with diarrhea and death, while the subclinical form shows weight loss and reduction in animal production both in sheep and goats [ 7 , 11 ]. Studies indicate that most domestic ruminants become infected with coccidiosis at some point during their lifetime [ 7 , 12 ], with lambs between 1 and 6 months of age being the most susceptible [ 13 ]. Adult animals generally exhibit milder clinical signs due to high immunity from repeated infections [ 4 , 14 ].

Eating colostrum in lambs on the first day of birth creates immunity against the disease for several weeks after birth [ 15 , 16 ]. Nevertheless, when animals ingest large number of sporulated oocysts, the invasive stages of Eimeria spp. can cause enteritis and diarrhea [ 4 , 17 ]. This invasion cause destruction of intestinal mucosal cells, and cell rupture and may lead to diarrhea and dehydration. Also, it may disrupt the absorption of nutrients, albumin and electrolytes, and, in severe cases anemia, hypoalbuminemia and electrolyte imbalance [ 4 ].

Coccidiosis primarily affects young and immature ruminants, and its clinical manifestation includes symptoms such as diarrhea, anorexia, poor growth, and weight loss [ 18 ]. Although hemorrhagic diarrhea due to coccidiosis is less common in lambs and kids, it is more prevalent in calves [ 4 , 19 ]. Necropsy findings in affected small ruminants may include catarrhal enteritis, bleeding, and inflammation of the mucous membranes of the small and large intestines [ 20 ]. Coccidiosis is often a self-limiting disease and sometimes improves without specific treatment [ 21 ]. Numerous drugs are prescribed to treat and prevent coccidiosis, including amprolium, sulfonamides, lasalocid, and monensin. Environmental hygiene should be observed to avoid the disease, and rotation of grazing animals is effective; drugs can also be used for prevention [ 22 , 23 ].

According to studies, Eimeria parasites' prevalence in different parts of Iran varies between 16% and 89.91 %, and infection with more than one Eimeria species has also been reported between 56% and 100% of positive samples in different herds [ 24 , 25 , 26 , 27 , 28 , 29 ]. Despite the importance of sheep and goat breeding in Khorasan province, limited studies have investigated the species composition of Eimeria species in this region. Therefore, the present study aimed to identify common morphological species of Eimeria and molecular confirmation of pathogenic species in sheep and goats in the Bajestan area.

Result

Out of 200 fecal samples collected from sheep in the study area , 139 samples were positive for Eimeria oocysts, representing a prevalence rate of 69.5%. Similarly, among the 200 fecal samples obtained from goats, 138 samples were positive, indicating a prevalence rate of 69%. The Clayton lane method was used to determine the OPG values in samples that tested positive for Eimeria oocysts. The mean OPG values (± standard deviation) recorded for sheep and goats during different months are presented in Table 1.

Table 1.OPG level of fecal samples in sheep and goats in different months

Results of OPG Month	Sheep	Goat
	Mean ± SD	Mean ± SD
November	51.45±104	85.46±40
December	71.07±57	38.83±47
January	56.65±39	162.45±61
February	64.23±37	338±304.
March	13.51±12	131.24±81

Using the morphological identification keys available for Eimeria species in goats, E. christenseni (5.5%), E. elijoi (13.63%), and E. hirci (0.5%) were identified. However, due to the similarity in morphology between the three species of E.caprina, E. caprovina, and E. sefronika (46.96%), and the similarity between the two species of E.arloingi and E.jolchijovi (16.16%), it was not possible to distinguish these species from each other. Accurate identification of these species required molecular testing, as shown in Figure 1.

Figure 1. A: E.elijoi, B: E. arloingi or E. jolchijovi, C: E. caprina or E. sefronika, D: E.christenseni in goats.

Also, based on the observations made, two species of E.faurei (74.74%) and E. ovinoidalis (8.08%) were identified in sheep. However, due to the similarity in morphology between the species of E. ahsata, E. bacquensis, and E. granulosa (5.05%), it is not possible to definitively distinguish these species from each other through morphological methods, and accurate identification requires molecular testing as shown in (Figure 2).

Figure 2. A: E. ahsata or E.bacquensis, B: E. faurei, C: E.ovinoidalis in sheep.

In this study, two DNA samples extracted from Eimeria oocysts collected from goats and sheep were tested by PCR along with positive and negative controls. Both sheep and goat samples showed a band slightly larger than 300 bp (Figure 3).

Figure 3. Electrophoresis results of PCR with special primers; M: Marker 100 bp, 1, 2: positive sample(goat), 3: positive sample(sheep), 4: negative control, 5: positive control.

Discussion

The present study revealed a prevalence of coccidiosis of 69.5% in sheep and 69% in goats in the Bajestan area w of Khorasan Razavi Province. The prevalence of coccidiosis has been reported in sheep and goats in different studies conducted in various countries and regions. The prevalence rates vary from 54% to 90.09%, depending on the location, climate, and diagnostic methods. The highest prevalence (90.09%) has been reported in China, while the lowest prevalence was recorded in Ordon [ 34 , 35 , 36 ].

In Iran, Yakhchali et al. reported a 16.7% prevalence of coccidiosis in sheep in Tabriz [ 37 ]. Nourollahifar et al. reported a 63% prevalence of coccidiosis in sheep in Rudsar [ 38 ]. Many Eimeria species have been identified in sheep and goats, some of which can cause disease. According to studies, E.ovina has the highest pathogenicity in sheep; E. ahhsata and E. crandalis are next in line, and E. faurei, has low pathogenicity [ 39 ]. In goats, E. ninakohlyakimoi and E.caprina have the highest pathogenicity, and E. arloingi has low pathogenicity [ 40 ]. In the present study, morphological examination identified E.faurei and E. ovina in sheep. However, due to the morphological similarities among E.ahhsata, E.granulosa, and E. bukidnonensis, a conclusive identification solely based on morphological characteristics was not possible.

Additionally, in goats, species such as E. elijoyi, E. christenseni, and E. hirci were identified using available morphological keys. However, the close morphological resemblance between E. arloingi and E. jolchijovi, along with similarities among E. caprina, E. caprovina, and E.espinosa, prevented an accurate diagnosis from being made using the morphological approach[ 31 ].

In research carried out by Yakhchali et al. involving sheep fecal samples from Malayer region, E. faurei was found to be the most common species among lambs younger than six months of age, and the second most common species overall after E. intracata, [ 25 ].

Vasilkova et al. identified E.elijoyi and E. arloingi as dominant species in goats following examination of various Eimeria species in sheep and goats [ 41 ]. In this study, the highest prevalence in sheep was related to E. faurei, and in goats was related to one of E. caprina, E.caprovina, or E. espheronica.

Interestingly, E. ovinoidea frequently reported as a dominant species in sheep in many studies, was reported with low prevalence in this study. In goats, E. ninakohlyakimoi was reported with high prevalence in various studies, but was not identified in this study. The OPG level recorded in this study did not follow a specific pattern during different months and fluctuated during different months. According to studies, the OPG level observed in sheep and goats in Bajestan were lower compared to those reported in other regions of Iran and the worldwide. Standard methods for identifying different Eimeria species, including morphological and histological approaches, are time-consuming and often lack sufficient sensitivity. Overlapping morphological characteristics between Eimeria species further complicate accurate identification. Therefore, the development and evolution of molecular diagnostics for identifying and determining the characteristics of parasites have been very helpful [ 42 ].

The 18S rRNA gene has been used as a molecular target to distinguish closely related Eimeria species and for conducting phylogenetic analyses. Amplification of fragments ranging between 384 and 546 bp has been effectively amplified using primer sets targeting the 3' end of the 18S rRNA gene and the 5' end of the 5.8S rRNA gene within the ITS-1 regions of the Eimaria genus [ 32 ]. In the present study, PCR results confirmed the presence of Eimeria spp in fecal samples in sheep and goats. however, sequencing of the PCR samples yielded low quality results, which prevented the identification of the dominant species of sheep and goats in the Bajestan area. Consequently, it is essential to gather a larger number of representative samples of various Eimeria species from sheep and goats; sequencing these samples can facilitate phylogenetic analysis and precise identification of morphologically similar Eimeria species.

In conclusion, the present study identified E. faurei and E. ovinoidalis as the predominant species in sheep and E. elijoyi, E. christenseni, and E. hirci in goats based on morphological examination. Although, PCR amplification of ITS1 region was successfully performed for positive samples, sequencing samples had low quality and results were insufficient for phylogenetic analysis.

Declaration of Generative AI and AI-assisted technologies in the writing process

To reduce noise and preserve fine details in microscopic images, AI-based image enhancement methods were employed."

Sampling

This study was conducted from September 2021 to June 2022, and fecal samples of sheep and goats were collected to determine the frequency of Eimeria spp infection and identify Eimeria species in Bajestan, Khorasan Razavi province, Iran.

The sample size was calculated based on a previously reported prevalence rate of 20% in Zabol area [ 28 , 29 ]. Accordingly, 200 fecal samples per animal were collected. Each month, fecal samples of 40 sheep and 40 goats were collected directly from the rectum in special containers and transferred to the parasitology laboratory of the Faculty of Veterinary Medicine at Ferdowsi University of Mashhad.

Microscopic examination

The fecal samples were examined by the Willis method [ 30 ]. Samples that tested positive were separated and preserved for further testing. Oocysts per gram (OPG) were determined quantitatively by the Clayton Lane method [ 30 ]. After examination, oocysts were purified and transferred into 2.5% (w/v) potassium dichromate solution to allow sporulated at room temperature [ 30 ]. Species identification was performed based on oocyst size and morphology (shape, color, form index, presence or absence of micropyle and its cap, presence or absence of residual, polar, and Stieda bodies of the oocysts and sporocysts). One hundred sporulated oocysts were identified using the available morphological keys [ 30 , 31 ]. The size of Eimeria oocysts was determined using a digital microscope.

DNA extraction and PCR test

Positive Samples were isolated, examined under light microscope, and preserved for molecular detection. DNA was extracted from the fecal samples using Genomic DNA Purification kit (denazist Co, Mashhad) following the manufacturer's instructions. DNA samples were kept at −20 °C until processed for PCR assay.

The ITS-1 region of Eimeria spp. from each positive sample was amplified using PCR and the cycling conditions and primer sequences of the forward and reverse primers were as follows:

• Forward: 5'-GCAAAAGTCGTAACACGGTTTCC-3',
• Reverse: 5'CTGCAATTCACAATGCGTATCG-3

Cycling conditions were: initial denaturation at 94'C for 5 min followed by 35 cycles of 94°C for 45 s, 65°C for 1 min and 72°C for 1 min, this was followed by a final extension at 72°C for 7 min. The amplification products from ITS-1 rRNA were separated on a 1.8% agarose gel containing 1 µl/mL of green viewer for 40–60 min, and then imaged. From the PCR products, one positive sample from sheep, and one from goat were selected and sent for sequencing [ 32 , 33 ].

Sequencing

PCR positive samples using the primers were submitted for sequencing to a company in Iran (Topaz- Genekaush Co, Karaj, Iran). The submitted samples were sequenced using the MEGA-4 software, and after performing a blast, the sequenced samples from sheep and goats did not match any of the registered samples in the gene bank.

Statistical analysis

The prevalence of Eimeria spp infection in sheep and goats was calculated by SPSS software version 22(IBM company.USA)

Authors' Contributions

Conceptualization: G.R.R., Methodology: M.A., A.K, Investigation: M.A, A.K, G.R.R, Writing - original draft preparation: M.A., A.K., Writing - review and editing: G.R., Funding acquisition: G.R.R., Supervision: G.R.R.

Acknowledgements

The authors thank Mr Hamid Eshrati for his assistance with the laboratory examination. This study was supported by grant No 3/57226 from the Vice President of Research and Technology of Ferdowsi University of Mashhad, Iran.

Competing Interests

The authors declare that there is no conflict of interest.

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