Morphological and Molecular Identification of Eimeria spp. Infecting Broiler Chicken Farms in Iran

1. Introduction
Coccidiosis is an infectious parasitic disease that impacts a diverse range of birds, such as chickens, turkeys, and other domestic fowl [1]. The disease is caused by protozoa belonging to the genus Eimeria, which invade the birds’ intestinal lining [2]. Coccidiosis can quickly spread throughout a group of birds via contaminated feed, water, or bedding. Preventive measures, such as maintaining proper sanitation, administering vaccine, and following biosecurity protocols, are crucial for controlling the disease and ensuring the well-being and productivity of the birds [3]. Identifying and studying these parasites require the use of morphological and molecular techniques. Morphological identification involves examining physical traits of Eimeria, including the size, shape, and structure of their oocysts [4]. Furthermore,sequencing and molecular identification techniques can analyze the genetic material of Eimeria [5]. Combining these methods provides more reliable identification of Eimeria parasites [6]. The pathogenicity of various Eimeria species ranges from moderate to severe, underscoring the importance of determining species composition for effective control and prevention measures [7]. In Iran, where poultry farming is crucial for the economy and food security, Eimeria infections are a significant worry for poultry breeders [8]. Despite the widespread presence of these parasites, there is limited available information on the morphological and molecular characteristics of Eimeria species infecting domestic poultry in Iran.

2. Materials and Methods

2.1. Design and collection of study animals
Fresh broiler chicken feces (18–45 days old) were collected from a total of 149 poultry farms in Zanjan County (50 farms), Abhar County (35 farms), Gheidar County (33 farms), and Khoram Dareh County (31 farms) between June and December 2023. Each farm had a breeding stock of 5000 to 15,000 chickens. The feces were collected from various points within each broiler chicken house, including the four corners and center, using plastic bags. The oocyst samples were stored at to 4 °C, at which they sporulate poorly (or were kept at 4 °C to inhibit premature sporulation)

2.2. Morphological identification
Two grams of each sample were added to 60 milliliters of saturated saline solution. After the mixture was filtered through gauze and centrifuging at 1500 rpm for 10 minutes, a small drop of the sample was used to create a liquid film for observation under a light microscope. Samples that tested positive for oocysts were subjected to a flotation techniqueto collect oocysts for incubation in 2.5% potassium dichromate. They were then allowed to sporulate at 27 ºC for 5 days [9]. The oocysts that had formed spores were subsequently stored at 4 °C for further molecular examination. Eimeria identification was conducted by analyzing the oocysts and sporocysts, taking into account factors such as their size, shape, wall composition, and color [10].

2.3. Genomic extraction
The sporulated oocysts were rinsed in TE buffer and then broken down using 0.5 mm glass. DNA was extracted using a Genomic DNA Kit (Qiagen in Hilden, Germany), according to the manufacturer's protocol. 

2.4. Molecular methods
The molecular identification of Eimeria was carried out using polymerase chain reaction (PCR), as previously described in a previous study [9] (Table 1). 

The PCR reaction mix was comprised of 12.5 µL of PCR master mix, 20 µM of each forward and reverse primers, 1 µL of DNA template, and nuclease-free water to make a total of 25 µL. The amplification process began with an initial denaturation step at 94 °C for 10 min, followed by 35 cycles involving 94 °C for 30 sec, 52.5-65 °C for 30 sec, and 72 °C for 1 min. A final extension step at 72 °C for 5 min concluded the process. The PCR products were then analyzed on agarose 1.5% agarose gel, were stained with ethidium bromide, and visualized under UV light. 
Data collected from the study were analyzed using SPSS software, version 20. Values of P<0.05 were considered significant. The chi-square test (χ2) for association was used to measure statistical significance.

Statistical analysis 
Data collected from the study were analyzed using SPSS software, version 20. Values of P<0.05 were considered significant. Chi-square test (χ2) for association was used to measure statistical significance.

3. Results

3.1. Morphological identification
Out of the 149 poultry farms that were examined, 59.7% tested positive for Eimeria spp. Gheidar County showed the highest infection rate among the samples collected, standing at 81.8%. In contrast, the prevalence was relatively lower in Khoram Dareh at 38.7%. The prevalence rates in Zanjan and Ahar were recorded at 56% and 62.8%, respectively. There was a significant difference between the prevalence of Eimeria sp. and different geographical areas (P<0.05). The morphological characteristics of the isolated Eimeria species indicated the presence of five distinct species in the samples analyzed, namely E. tenella, E. necatrix, E. mitis, E. maxima, and E. acervulina.

3.2. Molecular identification
The molecular data confirmed the morphological studies. The study found that all positive samples showed multiple infections by two to four different species of Eimeria. A molecular examination performed in poultry farms in Zanjan revealed the presence of E. tenella, E. necatrix, E. acervulina, E. mitis, and E. maxima (Figure 1). 

Notably, the prevalence of these species varied, with E. acervulina being the most common (55.7%) in Zanjan Province, followed by E. maxima (48.3%), E. mitis (20.1%), E. tenella (20.1%), and E. necatrix (13.4%), as shown in Table 2.

Mixed infections with two or more Eimeria species were found in 64 out of 103(62.1%) positive samples (Table 3). The most prevalent combination was E. acervulina, E. maxima, which were present in 23 out of 101 (22.3%) positive samples.

4. Discussion
Eimeria spp, a type of protozoan parasite, can result in notable financial losses within poultry facilities due to decreased productivity, increased mortality, and additional expenses related to disease management [11-13]. In Iran, Eimeria infections are frequently observed in domestic poultry, highlighting the necessity for a thorough characterization of the various species affecting these birds. The main goal of this study was to evaluate the occurrence of Eimeria species in broiler chickens in Zanjan Province, Iran. The current study aims to investigate the prevalence of coccidiosis and the diversity of Eimeria species in local poultry in Zanjan Region, where such infections had not been previously examined.
In this study, fecal specimens were collected from 149 domestic poultry facilities situated in four distinct urban areas within Zanjan Province, across various seasons. The findings revealed that out of the 149 processed samples, 89 tested positive for Eimeria oocysts, indicating a prevalence rate of 59.7%. Notably, the prevalence of these species varied, with E. acervulina being the most common (55.7%) in Zanjan Province. However,in a previous study, and unlike our study, the highest prevalence was related to E. Tenella [13, 14].
In a study on poultry farms of Sistan in 2018, five species of Eimeria, including E. tenella, E. maxima, E. acervulina, E. necatrix, and E. mitis, have been reported and the prevalence of poultry infection to Eimeria species were reported to be 20.96% [8]. Analysis of Eimeria species present in poultry across Zanjan Region identified the presence of five distinct species: E. acervulina, E. maxima, E. mitis, E. necatrix, and E. tenella. E. acervulina had the highest infection rate (55.7%), while E. tenella had the lowest rate (13.4%) across all regions in Zanjan provinces. A study conducted previously in Iran also reported the presence of five Eimeria species in poultry farms, E. tenella, E. maxima, E. acervulina, E. necatrix, and E. mitis, with an overall prevalence of 55.96% [15]. Another study in the same area found the prevalence of Eimeria infection in poultry farms to be 21.53% [16]. Additionally, in previous study in Iran [17], broiler chickens showed a high infection rate of coccidiosis (75%), which was much higher than that of the current study. The current study in Zanjan revealed a prevalence of 59.7%, indicating a need to increase coccidiosis control measures in the province.
Moreover, a study in Brazil identified eight Eimeria species in free-range chickens, with E. necatrix having the highest prevalence at 25%, followed by E. mitis (18.3%), E. mivati (17.3%) E. tenella (12.4%), E. brunetti (9.9%), E. acervulina (9.1%), E. praecox (4.8%) and E. maxima (1). Highly skilled individuals are necessary for accurately identifying Eimeria species, as there is a notable overlap in characteristics across the various species [2, 13, 18]. Alongside morphological analysis, molecular methods like PCR and sequencing have become essential in accurately identifying Eimeria species. By targeting specific genetic markers based on conserved ITS1 regions of rDNA [19, 13], the chicken coccidian species can be identify more accurately [20]. 

5. Conclusion 
In the present study, the results of molecular methods were coinciding with morphological descriptions, which agreed with previous findings reported. The present study presented findings on the occurrence of Eimeria species in poultry through ITS1-PCR in Zanjan, Iran. It confirmed the existence of E. tenella, E. acervulina, E. mitis, E. necatrix, and E. maxima in poultry farm in Zanjan, Iran, relying on morphological features and validated by molecular PCR. F103.

Acknowledgements
We thank the Principal and staff members of the Department of Microbiology of Birjand University of Medical Sciences for providing the necessary facilities to carry out the research work.

Compliance with ethical guidelines
This study was approved by the Institutional Animal Ethics and Research Committee of the Department of Veterinary Parasitology, Abhar Branch, Islamic Azad University, Abhar, Iran. 

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

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

Authors' contributions
Conceptualization, study design, experiments, data interpretation, review and editing: All authors; Data acquisition: Mojtaba khoini and Rahmat Solgi; Writing the original draft: Rahmat Solgi.

Conflict of interest
The authors declared no conflict of interest.

References

1. Partovi R, Seifi S, Samakkhah SA, Nikpay A. Appraisal of dietary prebiotic supplementation on meat properties and carcass characteristics of broiler chickens after experimental infection with Eimeria species. Iran J Vet Med. 2021; 15(3):346-57. [DOI:10.22059/ijvm.2020.303786.1005095]
2. Györke A, Pop L, Cozma V. Prevalence and distribution of Eimeria species in broiler chicken farms of different capacities. Parasite. 2013; 20:50. [DOI:10.1051/parasite/2013052] [PMID]
3. Shirley MW, Smith AL, Tomley FM. The biology of avian Eimeria with an emphasis on their control by vaccination. Adv Parasitol. 2005; 60:285-330. [DOI:10.1016/S0065-308X(05)60005-X] [PMID]
4. Haug A, Gjevre AG, Thebo P, Mattsson JG, Kaldhusdal M. Coccidial infections in commercial broilers: Epidemiological aspects and comparison of Eimeria species identification by morphometric and polymerase chain reaction techniques. Avian Pathol. 2008; 37(2):161-70.[DOI:10.1080/03079450801915130] [PMID]
5. Carvalho FS, Wenceslau AA, Teixeira M, Carneiro JA, Melo AD, Albuquerque GR. Diagnosis of Eimeria species using traditional and molecular methods in field studies. Vet Parasitol. 2011; 176(2-3):95-100. [DOI:10.1016/j.vetpar.2010.11.015] [PMID]
6. Matsubayashi M, Shibahara T, Matsuo T, Hatabu T, Yamagishi J, Sasai K, et al. Morphological and molecular identification of Eimeria spp. in breeding chicken farms of Japan. J Vet Med Sci. 2020; 82(5):516-9. [DOI:10.1292/jvms.19-0661][PMID]
7. Tewari AK, Maharana BR. Control of poultry coccidiosis: Changing trends. J Parasit Dis. 2011; 35(1):10-7. [DOI:10.1007/s12639-011-0034-7][PMID]
8. Shahraki F, Shariati F, Nabavi R, Jamshidian A. Coccidiosis in Sistan: The prevalence of Eimeria species in native chicken and its histopathological changes. Comp Clin Path. 2018; 27(4):1537-43. [DOI:10.1007/s00580-018-2770-x]
9. Huang Y, Ruan X, Li L, Zeng M. Prevalence of Eimeria species in domestic chickens in Anhui province, China. J Parasit Dis. 2017; 41(4):1014-9. [DOI:10.1007/s12639-017-0927-1][PMID]
10. Soulsby EJL. Helminths, arthropods and protozoa of domesticated animals. 1982. [Link]
11. Györke A, Kalmár Z, Pop LM, Şuteu OL. The economic impact of infection with Eimeria spp. in broiler farms from Romania. R Bras Zootec. 2016; 45(5):273-80. [DOI:10.1590/S1806-92902016000500010]
12. Fornace KM, Clark EL, Macdonald SE, Namangala B, Karimuribo E, Awuni JA, et al. Occurrence of Eimeria species parasites on small-scale commercial chicken farms in Africa and indication of economic profitability. PLoS One. 2013; 8(12):e84254. [DOI:10.1371/journal.pone.0084254][PMID]
13. Attia MM, Mahmoud MA, Abdelsalam M, Almutairi LA, Alqahtani MA, Areshi SM, et l. Molecular and histopathological characterization of Ascaridia galli and Eimeria tenella co-infection in Numida meleagris. Front Vet Sci. 2025; 12:1622170.
14. Nasiri V, Jameie F, Morovati Khamsi H. Detection, identification, and characterization of Eimeria spp. from commercial chicken farms in different parts of Iran by morphometrical and molecular techniques. Acta Parasitol. 2024; 69(1):854-64. [DOI:10.1007/s11686-024-00818-x] [PMID]
15. Nematollahi A, Moghaddam G, Pourabad RF. Prevalence of Eimeria species among broiler chicks in Tabriz (northwest of Iran). Mun Ent Zool. 2009; 4(1):53-8. [Link]
16. Yakhchali M, Fakhri M. [Prevalence of Eimeria species in free-range chickens of villages of Khoy suburbs, Iran (Persian)]. Vet Res Biol Prod. 2017; 30(1):206-11. [Link]
17. Shirzad MR, Seifi S, Gheisari HR, Hachesoo BA, Habibi H, Bujmehrani H. Prevalence and risk factors for subclinical coccidiosis in broiler chicken farms in Mazandaran Province, Iran. Trop Anim Health Prod. 2011; 43(8):1601-4. [Link]
18. Haug A, Thebo P, Mattsson JG. A simplified protocol for molecular identification of Eimeria species in field samples. Vet Parasitol. 2007; 146(1-2):35-45. [DOI:10.1016/j.vetpar.2006.12.015] [PMID]
19. Hamidinejat H, Shapouri MS, Mayahi M, Borujeni MP. Characterization of Eimeria species in commercial broilers by PCR based on ITS1 regions of rDNA. Iran J Parasitol. 2010; 5(4):48-54. [PMID]
20. Kumar S, Garg R, Moftah A, Clark EL, Macdonald SE, Chaudhry AS, et al. An optimised protocol for molecular identification of Eimeria from chickens. Vet Parasitol. 2014; 199(1-2):24-31. [DOI:10.1016/j.vetpar.2013.09.026][PMID]