Introduction

Chicken coccidiosis is a gastrointestinal illness that hinders development and weakens the immune system, causing elevated fatalities [1]. The presence of coccidiosis is widespread in the majority of tropical and subtropical areas, where farming practices frequently involve the utilization of deep bedding. Such practices create an environment that is conducive to the year-round spreading of Eimeria species [2]. Coccidiosis is caused by more than 1000 species of Eimeria apicomplexan parasitic protozoa and has been reported to infect a variety of host animals including chickens, turkeys, ducks, sheep, rabbits, cows, and domestic cats and dogs [3]. In chickens, nine species of Eimeria can cause intestinal lesions that comprise E. tenella, E. acervulina, E. maxima, E. brunetti, E. necatrix, E. mitis, E. praecox, E. mivati, and E. hagani[4]. Among them, E. acervulina, E. tenella, and E. maxima are considered the most crucial in terms of economics [5]. In coccidiosis, multiple species of Eimeria are often infected simultaneously, which not only contributes to the virulence of the disease, but also leads to incorrect diagnosis [6, 7]. The severity of gut lesions caused by these species varies depending on their level of pathogenicity in different gut areas [8].

The use of poultry rearing and keeping systems in crowded conditions that allow them to come in close contact, increases the chance of disease persistence. According to 2016 prices, the estimated worldwide cost of controlling coccidiosis is £10.36 billion [2, 9] and, therefore, the diagnosis, prevention, and treatment of this disease cannot be ignored in the poultry industry[10].

The significance of coccidiosis in the financial losses of poultry in Iran is yet to be fully acknowledged [11], but in this country, the broiler industry has developed rapidly in recent years and broilers are mainly raised in deep litter systems and the issue of coccidiosis remains a significant concern despite the ongoing implementation of anticoccidials as food supplements[12].

It is crucial to identify the Eimeria species that are currently circulating and infecting various types of poultry farming, including both backyard and commercial operations, as it provides the bedrock for effective control measure [13]. The morphological method based on microscopic examination of oocysts and several parasitological parameters was the first-generation method used to identify Eimeria species [14]. Nevertheless, the effectiveness of this method has been diminished due to various constraints, including intricacy, proficiency, and perplexing distinctions across different species [15]. This has given room for molecular approach [16], and thus, recognition and genomic analysis of various types of Eimeria are crucial for the prevention, monitoring, and management of coccidiosis [17].

The different effective molecular techniques including polymerase chain reaction (PCR) were provided for the identification of all the seven species of Eimeria in chickens [18]. PCR-dependent tests have the capability to precisely identify the Eimeria species that affect livestock, even if they have mixed infections with a low occurrence rate of 0.05% (two oocysts per PCR/4000)[7]. The target regions that were used in these techniques consist of small subunit rRNA [19], 5S rRNA [20], first and second internal transcribed spacers(ITS-1; ITS-2) of nuclear ribosomal DNA [21,22,23], and sequence-characterized amplified region (SCAR) derived from random amplified polymorphic DNA (RAPD) profiles [24].

The objective of the present study is to provide an up-to-date status of coccidiosis in Iran by investigating the occurrence of Eimeria infection in poultry farms, and we tried to give details about the molecular existence of Eimeria species in chicken farms from various regions of Iran.

Material and Methods

Research Period and Specimen Collection

From July 2019 to December 2022, the complete intestinal tracts including upper intestine, mid intestine, cecum, and rectum from clinically confirmed field samples were collected from chickens of commercial farms(broiler and layer flocks) from some regions of Iran. The samples were placed in ice packs and immediately transferred to the Laboratory of Protozoa, Department of Parasitology, Razi Vaccine and Serum Research Institute, Iran. In the Protozoology Laboratory, all intestinal tracts were examined for the presence of the oocysts of Eimeria spp. by microscopic analysis and morphological methods that were confirmed with molecular techniques.

Sample Processing and Oocyst Preparation

All the intestinal tracts were examined macroscopically for clinical lesions associated with coccidial infections, and the superficial mucous layer and contents of all parts of the intestinal tract were microscopically examined for the presence of Eimeria oocysts. The superficial mucous layer and intestinal contents were flushed from the intestinal tract and suspended in 2.5% potassium dichromate solution to enhance sporulation process of immature oocysts to a total volume of 50 ml. First, all samples were examined by direct wet mount preparation. Then, the above intestinal contents of each intestinal tract were analyzed using a modified standard fecal flotation technique [25]. Briefly, 5 ml from each sample suspension was pelleted by centrifugation at1500 × g for 5 min. The resulting pellet was resuspended in 10 ml phosphate-buffered saline solution (PBS) and passed through a 1-mm mesh size sieve to remove coarse fecal debris. The resulting filtrate was pelleted again by centrifugation at 1500 × g for 5 min and the resulting pellet was resuspended in 10 ml saturated sucrose solution, and this suspension was used in a standard gravity via fecal flotation using 22 mm × 22 mm coverslips. Following flotation, by centrifugation at 500 × g for 5 min, the coverslip was affixed to slides and thoroughly inspected for the existence of Eimeria oocysts. The slides were microscopically screened at 100 × , 400 × , and 1000 × magnifications, and the detected oocysts were identified by their morphometric characteristics as mentioned in references [26, 27]. Morphometric analysis was performed using a computer-aided image analysis system BA-300 (Motic) at 400 × magnification. The variables including the width and the lengths of the parasites were analyzed. The intestinal tract contents of the positive oocyst samples were incubated at 26 °C for 7 days to permit sporulation to be completed. The sporulated oocysts were washed with PBS to remove potassium dichromate, concentrated by flotation, resuspended in PBS, and then stored at 4 °C and  – 20 °C.

DNA Extraction

All positive samples were employed for DNA extraction by the modified phenol–chloroform extraction procedure. Initially, 250 μL of concentrated oocysts was centrifuged at 10,000 g for 5 min and the supernatant was discarded. A volume of 250 μL of PBS was added to each tube and mixed thoroughly. Then, it was centrifuged at 10,000 g for 5 min and the supernatant was discarded. A volume of 250 μL of PBS was added and completely mixed with the sediment. The suspension was subjected to five cycles of freezing and thawing in liquid nitrogen for 5 min each, followed by a 60 °C water bath for 5 min. The specimens were diluted with 500μL of lysis buffer (50 mM Tris–HCL, pH8.0; 25 mM EDTA, and 400 mM NaCl) and then vortexed until entirely homogenized. After adding 50 μL of 10% SDS (resulting in a final concentration of 1%) and proteinase K (100 μg/ml), the microtubes were incubated overnight in a water bath at 60 °C. The next day, ribonuclease A (100 μg/ml) was added to the sample to remove RNA and incubated at 37 °C for 30 min. In the next step, undissolved proteins and debris were precipitated by adding 150 μL 5 M NaCl to the suspension and kept at 4 °C for 15 min. After that, the samples were mixed with 500 μL phenol–chloroform–isoamyl alcohol (24:24:1) mixture and the microtubes were turned up and down slowly for 10 min. It was then centrifuged at 13,000 g for 15 min, and the aqueous supernatant phase was transferred to a new 2-mL microfuge tube. Chloroform was added as the same amount of supernatant, gently rotated, and centrifuged at 13,000 g for 10 min, and this step was repeated one more time. DNA precipitation was performed by adding an equal volume of cold isopropanol and 1/10 volume of sodium acetate solution (3 M, pH 5.2), kept at  – 20 °C overnight, followed by centrifugation at 13,000 g for 30 min to induce DNA separation. Ultimately, the sediment was rinsed two times with 70% chilled ethanol, dehydrated, and reconstituted in 50 μL of distilled water. The isolated DNA was preserved at  – 20 °C until PCR examination [28].

PCR Analyses

All samples that were determined to be positive for the microscopic existence of Eimeria species oocysts were examined for further confirmation of the presence of the internal transcribed spacer-1 (ITS-1) gene by single PCR assay using species-specific primers (Table 1) as previously described [18, 21, 22, 29] by the following procedures:

Table 1 Primers designed for the ITS-1 region specific to seven Eimeria species capable of infecting chickens
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All PCRs were done in a total 25 μL PCR mixture containing 12.5 μL of 2 × PCR master mixes (Sinaclon, Iran), 25 pmol of species-specific forward and reverse primers (2 μL), 2 μL of DNA template, and 8.5 μL of distilled water. For each PCR reaction, positive controls (DNA extracted from commercial vaccine) and negative controls (double-distilled water) were included. Each amplification was performed with the following cycling parameters: 1 cycle of 95 °C for 5 min, 35 cycles of 95 °C for 30 s, 58 °C or 65 °C for 30 s, and 72 °C for 60 s, and a final cycle of 72 °C for 10 min. After the PCR amplification, the resultant DNA fragment was detected by agarose gel (1.5% (w/v)) electrophoresis of 5μL of the PCR products.

Phylogenetic Investigation of ITS1gene

The amplified ITS1 gene’s affirmative PCR outcomes were obtained from the gel through the utilization of the gel purification kit (Vivantis, Malaysia) according to the manufacturer’s instructions. The PCR results were directly sequenced both in the forward and reverse directions by Pishgam Co. (Tehran, Iran). The results of sequences were aligned with partial sequences of ITS1gene of the other Eimeria species from different studies and compared with sequences available in GenBank by BioEdit Sequence Alignment Editor program [30]. The maximum-likelihood method with MEGA10 software was utilized to perform the evolutionary analyses and construct the phylogenetic tree. The bootstrap outcomes were calculated for 1000 replicates [31].

Results

Sample Collection and Microscopic Analysis

A total of 64 fully confirmed clinically intestinal tracts were collected from different parts of Iran. From these 64 intestinal tracts, 82 samples were prepared from the different sites involved in the digestive tract. In morphological assessment, 23 samples could not be isolated and its information was not evaluated. The information of the collected samples that were photographed and analyzed with a special microscopic camera using Motic software is mentioned in Table 2. As mentioned in the table, different images were taken from each sample under different microscopic fields, and the average length and width of the oocysts of each slide were calculated, and a total average of all the slides of each sample was obtained, and finally, the morphological species was determined. Some of the captured images are presented as examples in Fig. 1.

Table 2 The epidemiological, morphometrical, and molecular information of the collected and analyzed samples
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Fig. 1
figure 1

As an example of microscopic analysis, different images were taken from sample E-5-1 under different microscopic fields and the total average of all the slides indicates the morphological species of E. tenella

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Using morphological methods, the following seven species of Eimeria were identified: E. acervulina (15/59; 25.42%), E. tenella (30/59; 50.84%), E. maxima (12/59; 20.33%), E. praecox (1/59; 1.69%), E. necatrix (2/59; 3.38%), E. mitis (5/59; 8.47%), and E. mivati (2/59; 3.38%). Mixed infections were found in eight (13.55%) samples.

Molecular Analyses

By using PCR method and specific primers, fragments with a length of 145 to 330 bases were amplified. In molecular assessment, 31 samples could not be isolated and its information was not evaluated. Totally, the following five species were identified using molecular methods: E. acervulina (35/51; 68.62%), E. tenella (33/51; 64.70%), E. maxima (6/51; 11.76%), E. brunetti (5/51; 9.80%), and E. necatrix (2/51; 3.92%). Mixed infections were found in 23 (45.09%) samples. The comparison of the results of the morphometric and molecular methods is presented in Table 3.

Table 3 The comparison of the morphometrical and molecular information of the analyzed samples
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Phylogenetic Analysis

After sequencing of 13 selected nucleotide sequences of the ITS1 gene of positive samples, they were submitted to the GenBank database with the following accession numbers: Eimeria tenella (ON869221.1, ON869066.1, ON869065.1, ON869064.1), Eimeria acervulina (ON869063.1, ON869062.1, ON869061.1, ON853616.1), Eimeria maxima (ON869223.1, ON869219.1), Eimeria necatrix (ON869220.1, ON869218.1), and Eimeria brunetti (ON869222.1). To identify the most similar sites, existing sequences were subjected to the Basic Local Alignment Search Tool (BLAST) and after alignment with BioEdit, the results showed that our sequences matched with each other and with other positive cases of Eimeria spp. ITS1gene that were deposited in Gen Bank (Supplementary Table S1). The phylogeny of the sequenced Eimeria spp. was investigated by computerized analysis of the ITS1 gene sequences of 48 Eimeria spp. isolates which included 13 samples obtained in the present work and samples of Eimeria spp. and other apicomplexan parasites from 35 other studies (Fig. 2). Phylogenetic trees using maximum likelihood (Fig. 2) showed similarity among Eimeria spp. isolates in this study and with some other Eimeria spp. in animals from different parts of the world that were deposited in GenBank.

Fig. 2
figure 2

Phylogenetic connections were acquired from 13 affirmative sequences in this investigation along with sequences from other investigations utilizing the maximum likelihood technique based on their ITS1gene sequences reinforced by 1000 bootstrap replicates. The red figures symbolize the sequences obtained in this investigation

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Discussion

Parasites are important because of their profound impact on their hosts’ lives, which sometimes causes serious diseases. In the meantime, when the parasite host plays a main role in human life for economics and nutrition, this importance is doubled.

The avian farming sector is rapidly expanding within the field of agriculture, playing a significant role in promoting worldwide nourishment [32]), and consequently, a crucial component of the economy’s growth. Chicken, a major poultry bird, contributes greatly to agricultural production through the supply of meat and eggs [33]. However, chickens also are host to many lethal illnesses which bog down productiveness and compromise welfare, ensuing in excessive mortality in a few cases. They are exposed to many different pathogens, of which the most famous and important are coccidian parasites [34]. Among numerous ailments that impact chickens on a worldwide scale, coccidiosis is a commonly known condition linked with a significant rate of death in the poultry sector [1]. Although there has been significant progress in the development of avian coccidiosis vaccines in recent decades, controlling avian coccidiosis is still a major challenge for the global poultry industry [10].

In any way, the prevention and control of this disease is very important, and the epidemiological studies on the prevalence of Eimeria species are useful tools for this aim [18]. According to these different studies, coccidiosis is widely regarded as a parasitic ailment in the poultry sector, as evidenced by the prevalence of Eimeria spp. infection in different parts of the world [35]. For example, researchers reported an infection frequency of 92.8% in Colombia [36]; 90% in Argentina [37]; 92% in Romania [18]; 79.4% in North India [38]; 65.8% in East China [39]; 66.8% in Karbala and Babylon provinces, Iraq [40]; and 78.7% in South Korea [41]. Recently, in a research from Serbia, DNA of Eimeria spp. (E. acervulina (37%), E. maxima (17%), E. mitis (25%), and E. tenella (48%)) were found in 59% of investigated samples [42]. In a study performed in Ethiopia, the overall Eimeria spp. infection prevalence was 27.1% [43], and in another research conducted in Riyadh City, Saudi Arabia, 120 domestic poultry farms were examined and 30 were found to be infected with oocysts of Eimeria spp. (25%) [34].

Also, in some studies in Iran, different prevalence rates of poultry coccidiosis were reported. An investigation to show the prevalence of coccidiosis in broiler farms in Hamadan province, western Iran, indicated that the overall rate of coccidiosis was 31.8%; the individual rates were as follows: E. acervulina (75.7%), E. tenella (54.3%), E. necatrix (28.6%), and E. maxima (20%). Mixed infections were observed in all of the positive farms[11]. A cross-sectional study conducted in Marand city, Iran, revealed 35.2% of broiler farms were positive for coccidian oocysts [44]. A study conducted to examine the occurrence of various Eimeria species in the raised broiler chickens of Khoy city, located in the northwest region of Iran, West Azerbaijan province, revealed that the bedding materials were contaminated with five distinct Eimeria species: E. maxima (32.67%) in six farms (23.07%), E. mitis (24%) in six farms (23.07%), E. acervulina (18%) in five farms (19.23%), E. tenella (14.67%) in four farms (15.38%), and E. necatrix (10.67%) in three farms (11.58%) [45]. Also, several studies have been conducted in the city of Mashad (38%) in North East Iran [12] and Golestan province (36%) in North Iran [46]. The prevalence of infection was significantly greater in Tabriz (55.96%), located in the northwest of Iran [47] and Hamadan (75%), situated in the west of Iran [48]. A research recorded four species of Eimeria, namely E. maxima, E. tenella, E. acervulina, and E. necatrix, from the poultry farms of Hamadan. Meanwhile, the first three species have also been detected in the bedding of the poultry farms situated in various parts of Iran [12, 46]. In a study, 50 field samples from different parts of Iran were examined for the presence of Eimeria oocysts and the prevalence was estimated to be 30% [49]. Another study was conducted to determine the occurrence of Eimeria species in the bedding material of broiler and layer farms located in Tehran and Alborz provinces. The findings revealed the presence of three Eimeria species, including E. tenella, E. maxima and E. acervulina in broiler with amount of 25.55%, 31.39% and 14.77%, respectively, and four Eimeria species, including E.tenella, E. maxima, E. acervulina and E. necatrix in pullet with amount of 21.51%, 20.64%, 8.33% and 1.03% respectively [50].

Various studies mentioned above have shown the presence of different Eimeria species in different parts of the world and different regions of Iran, which shows the high prevalence of parasites all over the world. Of course, it should be pointed out that most of the studies conducted in Iran have been performed using microscopic methods, and to our knowledge, this is the initial research carried out in Iran to identify the sequence of a particular gene in poultry Eimerian parasite and document it in databases.

The present research showed that seven species of Eimeria parasite were reported using morphological method and five species using molecular method. In the meantime, E. brunetti was only identified using molecular methods, and E. praecox, E. mitis, and E. mivati were identified only using morphological methods, which shows the inaccuracy of morphometric methods and the necessity of using molecular methods in the accurate identification of species. Accurate identification of circulating species is very important to determine the appropriate strategy to deal with the disease and to determine the appropriate vaccination and drug treatment program. Our study shows that using the molecular method, the highest prevalence was related to E. acervulina, but using morphological methods, the highest prevalence was observed related to the species of E. tenella. In any way, these two species as the main disease-causing species should be considered in control programs and in treatment and vaccination strategies.

A study claimed that one of the main preconditions for reducing the risk of coccidiosis is improvement of biosecurity measures on farms. Continuous education of farm employees, change of coccidiostats in feed, inclusion of phytobiotics, and good farm hygiene should be the main goals of all broiler farms in this region[42].

Conclusions

The present study is an update on the situation of poultry coccidiosis in Iran and confirms the presence of different species of this parasite among some of the commercial chicken farms in this area. This study provides information on the identification of the most frequently identified coccidial species and their distribution, in the region. Further investigation is required, although this marks a significant initial stride toward enhancing comprehension of the present condition of coccidial challenge in Iran. To our knowledge, this is the first data on the molecular detection, identification, and characterization of Eimeria spp. in poultry population of different regions of Iran. The consequent epidemiological information of this research can be employed to develop guidelines for controlling measures that are better suited to the present condition of this disease. The increased occurrence of E. acervulina and E. tenella as solitary or combined infections in broiler farms in Iran may be attributed to decreased susceptibility to anticoccidials agents and inadequate management practices, particularly in smaller and medium-sized farms. More research is necessary to assess the drug sensitivity of these strains of parasite and manage the use of coccidiostats.