Bull 431 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Makawi, Z. A. Bull. Iraq nat. Hist. Mus. (2024) 18 (2): 431- 445. https://doi.org/10.26842/binhm.7.2024.18.2.0431 ORIGINAL ARTICLE ISOLATION AND IDENTIFICATION OF EIMERIA SCHNEIDER, 1875 SPECIES (APICOMPLEXA, EIMERIIDAE) FROM GOATS IN WASIT PROVINCE, IRAQ Zainab A. Makawi Iraq Natural History Research Center and Museum, University of Baghdad, Baghdad, Iraq. E-mail: zainab@nhm.uobaghdad.edu.iq Recived: 10 July 2024, Revised: 1 Nov. 2024, Accepted: 7 Nov. 2024, Published: 20 December 2024 This work is licensed under a Creative Commons Attribution 4.0 International License ABSTRACT Eimeriosis is a major problem affecting ruminants worldwide. The disease is primarily caused by Eimeria species, which are specialized for each host and grow in the small and large intestine of animals. The losses due to subclinical infections (especially weight loss) and clinical disease (diarrhea) make the species of this genus a very significant economic concern. Therefore, this study was conducted in some areas of Wasit Province. A total of 180 fecal samples from goats, of both sexes and covering different age groups and months, were collected. All fecal samples were examined microscopically, and 75 positive fecal samples were taken for molecular examination and further analyzed using conventional PCR, sequencing and phylogenetic analysis. Microscopic results showed that the overall infection rate was 41.6%. The incidence of Eimeria species ranged from 5.55% to 22.22% across three different species of the genus Emeria Schneider, 1875, namely E. arloingi (Marotel, 1905), Martin, 1909 (22.22%), E. christenseni Levine, Ivens & Fritz, 1962 (13.88%), and E. hirci Chevalier, 1966 (5.55%). Regarding the PCR reaction, results from the 18S rRNA, COI gene and genetic sequencing, Confirmed that the fecal samples were positive for Eimeria Schneider, 1875 species. Keywords: COI gene, Eimeriosis, Eimeria species, Goat, 18S rRNA, Wasit Province. INTRODUCTION Intestinal infection of goats has been associated with a species of protozoan that belongs to the genus Eimeria Schneider, 1875 (Apicomplexa: Eimeriidae) (Mohamaden et al., 2018). Any stage of an animal's life may experience mixed infection with pathogenic and/or nonpathogenic species, with younger animals being more vulnerable (Keeton and Navarre, 2018; Smith et al., 2019). According to Chartier and Paraud (2012), Eimerosis, one of the most significant parasite diseases affecting goats, has spread around the globe. Due to high mortality and morbidity rates, indigent development and costly treatments, this condition results in substantial economic losses (Temizel et al., 2011). This parasite is transmitted via mature oocysts, BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Iraq Natural History Research Center & Museum, University of Baghdad https://jnhm.uobaghdad.edu.iq/index.php/BINHM/Home Copyright © Bulletin of the Iraq Natural History Museum Online ISSN: 2311-9799-Print ISSN: 1017-8678 https://doi.org/10.26842/binhm.7.2024.18.2.0431 https://orcid.org/0000-0003-1136-9121 mailto:zainab@nhm.uobaghdad.edu.iq https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ https://jnhm.uobaghdad.edu.iq/index.php/BINHM/Home 432 Bull. Iraq nat. Hist. Mus. 18 (2): 431-445. Isolation and identification of Eimeria which require specific environmental conditions, such as humidity and temperature, to develop and transform into sporozoite- containing structures (Malla and Saida, 2022). According to Rehman et al. (2011) and Cavalcante et al. (2012), the cause of eimeriosis is an intestinal protozoan parasite belonging to the genus Eimeria. Seventeen species of Eimeria have been identified in goats worldwide. Among these, E. christenseni Levine, Ivens & Fritz, 1962, E. arloingi (Marotel, 1905) Martin, 1909, E. caprina, and E. ninakohlyakimovae Yakimoff & Rastegaieff, 1930, are considered significant infections (Silva and Lima, 1998; Rajakaruna et al., 2012). Young animals under the age of four as well as animals of any age maintained in overcrowded conditions with added stressors are more severely affected. Examples of such stressors include weaning, nutritional adjustments, travel, temperature extremes, and so forth (Gul, 2007). Infected animals excrete oocysts in their faeces (Temizel et al., 2011). By ingesting sporulated oocysts, goats become infected. As sporulated oocysts enter intestinal epithelial cells in the small intestine, they release sporozoites causing electrolyte and nutritional malabsorption (Temizel et al., 2011; Zachary and McGavin, 2014). Eimeria species was diagnosed using a variety of techniques, including molecular, serological, and fecal testing (Woods et al., 2000). Molecular methods are particularly sensitive and specific for fecal analyses (Sweeny et al., 2011). Eimeria has been diagnosed by PCR, which amplifies DNA, in a various hosts, providing reliable results across a range of samples (Kawahara et al., 2010). The aim of the study was to identify the species of Eimeria infecting goats in Wasit Province using genetic sequencing. MATERIALS AND METHODS Sampling: In Wasit province, 180 fecal samples were collected from goats (83♂♂ and 97♀♀) aged between 5 months and over 6 years, from three sites: Al-Suwaira, Al-Aziziyah, and Al- Shaihemiyh Districts, with 60 fecal samples were collected from each site. From November 2022 to April 2023, fecal fecal samples weighing between 10-20 g were randomly collected from various farms. Immediately, while donning gloves, the samples were collected directly from the rectum and preserved at 4 °C until analysis (Ekawasti et al., 2021). Microscopic examination: After collection, the fecal samples were examined at the vertebrate laboratory of the Iraq Natural History Research Center and Museum, University of Baghdad, for further testing. These include direct smear, flotation in saturated salt solution, and culture with 2.5% Potassium Dichromate (wt./vol.)] and put in screw-cupped plastic containers. A microscope was used to identify the different species of Eimeria by using 40X power (Eckert et al., 1995). Molecular study DNA extraction: DNA was extracted from 75 fecal samples which were collected and stored previously at -20ºC using DNA extraction kit /Korea. 433 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Makawi, Z. A. Primers: The primers used for PCR amplification were designed according to Albanese et al. (2019) based on 18S rRNA with Primer sequence F (5'- CGCGCAAATTACCCAATGAA- 3'), R (5'- ATGCCCCCAACTGTCCCTAT- 3’) gene at 454 bp and Bawn et al. (2020) on COI (cytochrome oxidase subunit I) gene sequences with Primer sequence F (5′-AGGTGTTGCTAATGGAGCCAA-3′), R (5′ ACAGCTGAGAAGTATGCTCTGG-3′) at 405 bp. PCR reaction preparation: PCR reaction was performed using Master mix (Promega/USA) Component, including Master Mix or GoTaq® Green Master Mix (12.5µl), Forward primer (10 picomols/µl (1 µl), Reverse primer (10 picomols/µl (1 µl), DNA (1.5µl) and Distill water (9µl). PCR thermocycler conditions: The thermocycler conditions for PCR were conducted usinga conventional PCR thermocycler system. For the 18S rRNA gene, the conditions included: Initial Denaturation (Temperature 95 ᵒC for 5 min with 1 cycle), Denaturation 2 (Temperature 95 ᵒC for 45 sec), Annealing (Temperature 56 ᵒC for 45 sec), Extension 1 (Temperature 72 ᵒC for 1 min) with 35 cycle and Extension 2 (Temperature 72 ᵒC for 5 min with 1 cycle). While COI gene was conducted using conventional PCR thermocycler system which included Initial Denaturation (Temperature 94 ᵒC for 3 min with 1 cycle), Denaturation 2 (Temperature 94 ᵒC for 45 sec), Annealing (Temperature 58 ᵒC for 30 sec), Extension 1 (Temperature 72 ᵒC for 1 min) with 35 cycle and Extension 2 (Temperature 72 ᵒC for 7 min with 1 cycle). Sequencing of DNA and phylogenetic tree: Genetic sequencing was performed on seven positive samples in order to identify Eimeria species at the molecular level. The UPGMA (unweighted pair group method with arithmetic mean) approach was used to infer the evolutionary history. To obtain the bioinformatics for local goat Eimeria and to assign accession numbers for local Eimeria species in Iraq, a phylogenetic tree and the Genbank database were used. The 18s rRNA PCR product and the COI gene were shipped from the Korean company Macrogen. Once the sequences of the Eimeria species were acquired, they were submitted to the NCBI-GenBank to obtain Genbank accession numbers. Molecular Evolutionary Genetics Analysis (MEGA6) and multiple sequence alignment based on Clustal W alignment analysis were used for the DNA sequencing study. This includes phylogenetic tree analysis and multiple sequence alignment which were conducted using Clustal W alignment analysis and molecular evolutionary genetics analysis (MEGA6). Known sequences from NCBI-Blast were used as references for comparison was to identify Eimeria species through phylogenetic analysis. RESULTS This research was carried out in Wasit Province with no significant difference among the areas studied, a total of 180 fecal samples 75 (41.6%) of the analyzed were positive for Eimeria oocysts. A significant association (p=0.013) was observed between November 2022 and April 2023. Both gender (83♂♂ and 97♀♀) showed infection rates that varied from 22.22 to 5.55% across various locations, with males having a higher infection rate than 434 Bull. Iraq nat. Hist. Mus. 18 (2): 431-445. Isolation and identification of Eimeria females (48.1% and 36%) respectively, though the difference was not statistically significant. Goat fecal matter was used to identify three distinct Eimeria species based on their morphological characteristics in addition to the measurements (length and width) of oocyst, using a microscope ruler (ocular stage). The species were identified based on oocyst size, appearance, the presence or absence of micropyle, and the structure of the oocyst wall. The identified species included E. arloingi (22.22%), E. christenseni (13.88%) and E. hirci (5.55%) with the differences being highly statistically significant (Pls. 1-3; Tabs. 1-4). Table (1): Microscopic examination with infection rate Eimeria species in goats concerning the area. Area Number of Samples Analyzed. Number of positive (%) P value Al-Suwaira 60 20 33.3 0.18 Al-Aziziyah 60 25 41.6 Al- Shaihemiyh 60 30 50.0 Total 180 75 41.6 [The results of chi square analysis revealed that there was no significant association (p=0.18) between the area]. Table (2): The infection rate with Eimeria species according to months of study. Months/2022 and 2023 Number of Samples Analyzed. Number of positive (%) P value November 30 10 33.3 December 30 13 43.3 January 30 15 50.0 February 30 20 66.6 0. 013 March 30 10 33.3 April 30 7 23.3 Total 180 75 41.6 [The results showed a statistically significant association (p=0.013) between the month of infection or sample collection and the rate of infection with February being the month of higher percentage of infection compared to others (66.6 % positive cases)]. Table (3): The infection rate with Eimeria species according to goat's gender. Gender Number of samples analyzed. Number of positive P value Male 83 40 (48.1) 0.10 Female 97 35 (36) Total 180 75 (41.6) [The results of chi square analysis revealed that there was no significant association (p=0.10) between gender and the rate of infection]. 435 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Makawi, Z. A. Table (4): Total infection rate with morphological characteristics of different Eimeria species in goats. [The results showed a very highly statistically significant association (p ≤0.001) among the species of the parasite and the infection rate with E. arloingi constituting the high percentage of infection (22.22 %) of all]. Plate (1): Eimeria arloingi (left sporulated and right unsporulated). (Oocysts are elliptic in form, have thick walls, and are coloured yellowish brown. They also have polar and micropyle caps). Length × Width (µm) Observed (Min- max) Oocysts morphology P value % Number of positive Number of samples analyzed Species 33 * 15 Micropyle presents with a prominent cap ≤0.001 ≤0.00 22.22 40 180 E. arloingi 41*23 Micropyle presents with a prominent cap 13.88 25 E. Christensen 22*18 Micropyle present with cap 5.55 10 E. hirci 436 Bull. Iraq nat. Hist. Mus. 18 (2): 431-445. Isolation and identification of Eimeria Plate (2): Eimeria hirci (with a micropyle and micropyle cap, oocysts are light brown to brownish yellow in morphology and ellipsoidal to subspherical in shape). Plate (3): Eimeria christensen (Pear-shaped oocyst with a thick wall, micropyle cap, and polar cap; yellowish brown in tone). Molecular results Results obtained by PCR analysis: Seventy five fecal samples, which were examined traditionally in the lab, where further subjected to molecular examination. The samples tested positive result for Eimeria species using the 18S rRNA and COI gene (amplicons size H 454 bp and 405 bp) through the conventional PCR technique. DNA sequencing results: Seven Eimeria positive samples were selected from the 75 positive samples for sequencing and phylogenic analysis. Sequenced samples were analysed using MegaX software and Multiple Alignments were created. The positive isolates were compared with NCBI isolates and the percentage of similarity was obtained. The sequences were then submitted to the NCBI-GenBank to acquire accession number codes for local Eimeria species in goat. The results revealed that two species were diagnosed by targeting 437 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Makawi, Z. A. the 18S rRNA gene as E. arloingi (accession no. : PP862861.1, : PP862862.1, : PP862863.1, : PP862864.1) and Eimeria christenseni (accession no. PP862865.1, PP862866.1) (Tab.5), while the results revealed that three species were diagnosed by targeting the COI gene as Eimeria hirci (accession no. PP873185.1), E. arloingi (accession no. PP873186.1, PP873187.1, PP873188.1, PP873189.1) and Eimeria christenseni (accession no. PP873190.1, PP873191.1) (Tab. 6). Table (5): The NCBI-BLAST homology sequence identity (%) between local Eimeria isolates from goat’s faeces that were deposited in 18S rRNA gene bank and NCBI-BLAST deposited strains. No. Accession No Identical to Genbank accession No. Country Identity % 1 ID: PP862861.1 E. arloingi ID: ON259586.1 China:Shaan xi 100% 2 ID: PP862862.1 E. arloingi ID: ON259586.1 China:Shaan xi 99% 3 ID: PP862863.1 E. arloingi ID: ON259586.1 China:Shaan xi 99% 4 ID: PP862864.1 E. arloingi ID: ON259586.1 China:Shaan xi 99% 5 ID: PP862865.1 E. christenseni ID: ON259585.1 China:Shaan xi 99% 6 ID: PP862866.1 E. christenseni ID: ON259585.1 China:Shaan xi 100% Table (6): The NCBI-BLAST homology sequence identity (%) between local Eimeria isolates from goat’s faeces that were deposited in COI gene bank and NCBI-BLAST deposited strains. No Accession No Identical to Genbank accession No. Country Identity % 1 ID: PP873185.1 Eimeria hirci ID: LC508122.1 Japan 100 2 ID: PP873186.1 E. arloingi ID: LC508123.1 Japan 99 3 ID: PP873187.1 E. arloingi ID: LC508123.1 Japan 100 4 ID: PP873188.1 E. arloingi ID: LC508123.1 Japan 99 5 ID: PP873189.1 E. arloingi ID: LC508123.1 Japan 100 6 ID: PP873190.1 E. christenseni ID: LC508121.1 Japan 100 7 ID: PP873191.1 E. christenseni ID: LC508121.1 Japan 100 https://www.ncbi.nlm.nih.gov/nucleotide/ON259586.1?report=genbank&log$=nuclalign&blast_rank=1&RID=61JR0FU0016 https://www.ncbi.nlm.nih.gov/nucleotide/ON259586.1?report=genbank&log$=nuclalign&blast_rank=1&RID=61JR0FU0016 https://www.ncbi.nlm.nih.gov/nucleotide/ON259586.1?report=genbank&log$=nuclalign&blast_rank=1&RID=61JR0FU0016 https://www.ncbi.nlm.nih.gov/nucleotide/ON259586.1?report=genbank&log$=nuclalign&blast_rank=1&RID=61JR0FU0016 https://www.ncbi.nlm.nih.gov/nucleotide/ON259585.1?report=genbank&log$=nuclalign&blast_rank=1&RID=61K7W898016 https://www.ncbi.nlm.nih.gov/nucleotide/ON259585.1?report=genbank&log$=nuclalign&blast_rank=1&RID=61K7W898016 https://www.ncbi.nlm.nih.gov/nucleotide/LC508122.1?report=genbank&log$=nuclalign&blast_rank=1&RID=6C9UZDV6016 https://www.ncbi.nlm.nih.gov/nucleotide/LC508123.1?report=genbank&log$=nuclalign&blast_rank=1&RID=6CAY6RH5013 https://www.ncbi.nlm.nih.gov/nucleotide/LC508123.1?report=genbank&log$=nuclalign&blast_rank=1&RID=6CAY6RH5013 https://www.ncbi.nlm.nih.gov/nucleotide/LC508123.1?report=genbank&log$=nuclalign&blast_rank=1&RID=6CAY6RH5013 https://www.ncbi.nlm.nih.gov/nucleotide/LC508123.1?report=genbank&log$=nuclalign&blast_rank=1&RID=6CAY6RH5013 https://www.ncbi.nlm.nih.gov/nucleotide/LC508121.1?report=genbank&log$=nuclalign&blast_rank=1&RID=6CB7YH7Y013 https://www.ncbi.nlm.nih.gov/nucleotide/LC508121.1?report=genbank&log$=nuclalign&blast_rank=1&RID=6CB7YH7Y013 438 Bull. Iraq nat. Hist. Mus. 18 (2): 431-445. Isolation and identification of Eimeria Phylogenetic analysis Phylogenetic tree analysis of Eimeria species based on the currently identified sequences (18S ribosomal RNA and COI gene) is represented with coloured triangle and their corresponding accession numbers. As it can be seen, the Iraqi strains mainly appeared closely related to each other. However, when targetingthe18S ribosomal RNA diagnosed E. arloingi and Eimeria christenseni were found to be closely related to an isolates from Shaanxi, China (Diags. 1, 2). Meanwhile, targeting the COI gene diagnosed Eimeria hirci, E. arloingi, and Eimeria christenseni as being similar to an isolate from Japan (Diags. 3, 4). Diagram (1): Gel electrophoresis image (1% agarose) shows the positive samples of Eimeria spp. (amplicons size H – 454 bp) by targeting 18S rRNA gene. Diagram (2): Phylogenetic tree analysis for Eimeria species by targeting 18S rRNA gene 439 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Makawi, Z. A. Diagram (3): Gel electrophoresis image (1% agarose) shows the positive samples of Eimeria species (amplicons size H – 405 bp) by targeting COI gene. Diagram (4): Phylogenetic tree analysis for Eimeria spp. by targeting COI gene. DISCUSSION According to the findings, a variety of Eimeria species, including single, double, and multiple infections, parasitize goats residing in this area. The overall infection frequency was found to be 75 (41.6%) across three regions of the Waist province including, Al- Suwaira, Al-Aziziyah, and Al- Shaihemiyh, and there was no significant difference observed among areas. This study's high infection rate reflects unsanitary living Conditions, which are likely a contributing cause to the spread of eimeriosis, and grazing in exposed, contaminated places. According to the infection rate of Eimeria parasite, this study disagrees with that of Macedo et al. (2019) who reported infection rates of Eimeria spp. at 73.91% and 60% in goats. While Hasan and Mahmood (2021) recorded a subclinical eimeriosis rate of 69.05% in goats. Senbeto and Chekol (2022), who found that infection with Eimeria species was 50.5% in goats. In addition, several studies have documented high infection rates (57.5%, 68.4%, 58%, 82.03% ) in small ruminants (El-Alfy et al., 2020; Ayana et al., 2022; Carneiro et al., 2022), in Iraq, Egypt, Ethiopia and Brazil respectively. Higher prevalence rates were recorded in Vietnam 86.85%, India 90.96%, and Myanmar 60.0% (Bawn et al., 2020; Singh et al., 440 Bull. Iraq nat. Hist. Mus. 18 (2): 431-445. Isolation and identification of Eimeria 2020; Nguyen-Ho-Bao et al., 2022). The present study's prevalence rates of Eimeria spp. oocysts in goats are lower than those previously reported. Eser et al. (2020) recorded a much lower infection rate as being 20.33% in Turkey. In the current study, the highest infection rates were recorded in February 66.6% and January 50, 66.6, and 50%, with a significant association (p=0.013) between infection rate and month. This finding aligns with the observation of Ibrahim (2012) who reported that the infection rate was higher during the rainy season (60.22%) compared to the dry season (45.81%). It is also consistent with Minnat (2014), who recorded a hight infection rate in February with a rate (100%). The increase in rainfall, humidity, and fall in temperature during February and January may attributive the higher infection rates of Eimeria sp. (Saleh, 2011). Regarding gender, no significant difference was observed in the current study. Similarly, Ibrahim (2012) observed a slightly higher infection rate in males (55.19%) compared to females (51.63%). Additionally, Verma et al. (2018) also discovered that the rates of infection in males were greater than that in females, 71.55% and 71.39% respectively. Male animals may be more susceptible to infections due to the immunosuppressive effects of high androgen levels, particularly testosterone, in their plasma during the reproductive season (Souza et al., 2015). The mentioned species were diagnosed based on the measurements described by Taylor et al. (2007) and Verma et al. (2018). Numerous publications have been conducted on this particular subject. In Iraq, Al-Bakray and Daoud (2005) recorded E. Christensen (36.5%) and E. hirce (18.5%) in goats at the Mosul abattoir. This aligns with findings by Diao et al. (2022), who discovered Eimeria species in goats in China, including E. arloingi (49.7%), E. Christensen (41.2%) and E. hirce (37.8%). Goz et al. (2006) identified E. hirce (18%) and E. Christensen (6%), were found in research on eimeriosis in goats done in Brazil by Cavalcante et al. (2012). This study is further comparable to molecular study by Bawn et al. (2020), who verified the COI sequences of E. christenseni, E. hirci and E. arloingi using COI and 18S r DNA sequences reporting sequence similarities of 98.9%, 98.4%, and 98.5% in Nay Pyi Taw area. Abdel Khader and Jarad (2022) in Al-Diwaniyah Province showed 87(87%) were positive for 18S rRNA gene of Eimeria spp. Seasonal differences the number of animals included in different researches, regional and agro-climatic diversity, local control approaches, and differences in the predominance of Eimeria species may all play a role in this variation. CONCLUSIONS The current study identified three species of Eimeria through morphological and molecular analyses by targeting the18S rRNA and COI genes (amplicons size H 454 bp and 405 bp). The identified species include: Eimeria arloingi, E. hirci, and E. christenseni. Hence, it is imperative to conduct a thorough research to mitigate the infections caused by Eimeria in goats. This necessitates the development of a comprehensive database to effectively reduce the prevalence of these parasites. Additionally, the use of anticoccidial medications and maintaining sanitary conditions are vital control measures that must be implemented. The current study identified three species of Eimeria according to morphological and molecular study by targeting 18S rRNA and COI gene (amplicons size H 454 bp and 405 bp) including: Eimeria arloingi, E. hirci, and E. christenseni. Hence, it is 441 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Makawi, Z. A. imperative to conduct thorough research to mitigate the infection caused by Eimeria in goats; this necessitates the development of a comprehensive database to effectively reduce the prevalence of these parasites. Additionally, the utilization of anticoccidial medications and the maintenance of sanitary conditions are vital control measures that must be implemented. Furthermore, it is essential to regulate open habitat grazing and implement updated preventative measures, while also conducting further research on eimeriosis, especially in Waist Province, giving the limited studies on goats in this region and Iraq in general. CONFLICT OF INTEREST STATEMENT " Regarding the publishing of this article, the authors declare that they have no conflicts of" interest. LITERATURE CITED Abdel Khader, W. H. and Jarad, N. I. 2022. Detection of Eimeria species in domestic goats with both microscopic and molecular methods in Al-Diwaniyah Province. International Journal of Health Sciences, 6(S8): 682-698. [CrossRef] Al-Bakray, H. S. and Daoud, M. S. 2005. Occurrences of Eimeria spp. in goats in Mosul abattoir Iraq. Iraqi Journal of Veterinary Sciences, 19(2): 97-103. (In Arabic, with English abstract) [Click here] Albanese, G. A., Lee, D. H., Mueller, A. E. and Jordan, B. J. 2019. Identification of a Short DNA Bar Code in the 18S rDNA for improved differentiation of common Eimeria species infecting chickens. Journal of Parasitology, 105(5): 816-820. [CrossRef] Ayana, D., Temesgen, K., Kumsa, B. and Alkadir, G. 2022. Dry season Eimeria infection in dairy cattle and sheep in and around Adama and Bishoftu Towns, Oromia, Ethiopia. Veterinary Medicine: Research and Reports, 13: 235-245. [CrossRef] Bawn, S., Win, T. Z. B., Win, S. Y., Htun, L. L., Nakao, R. and Katakura, K. 2020. First detection of Eimeria species in Myanmar domestic goats with both microscopic and molecular methods. Parasite, 27 (38): 1-7. [CrossRef] Carneiro, P. G., Sasse, J. P., Silva, A. C. S., Seixas, M., Paschoal, A. T. P., Minutti, A. F., Martins, T. A., Cardim, S.T., Rodrigues, F. S. and Barros, L. D. 2022. Prevalence and risk factors of Eimeria spp. natural infection in sheep from northern Paraná, Brazil. Revista Brasileira de Parasitologia Veterinária, 31(1): e017421. [CrossRef] Chartier, C. and Paraud, C. 2012. Coccidiosis due to Eimeria in sheep and goats: a review. Small Ruminant Research, 103(1): 84-92. [CrossRef] https://doi.org/10.53730/ijhs.v6nS8.9757 https://www.iasj.net/iasj/download/04def86a6e3a1b37 http://dx.doi.org/10.1645/19-34 https://doi.org/10.2147%2FVMRR.S377017 https://doi.org/10.1051/parasite/2020037 https://doi.org/10.1590/S1984-29612022004 https://doi.org/10.1016/j.smallrumres.2011.10.022 442 Bull. Iraq nat. Hist. Mus. 18 (2): 431-445. Isolation and identification of Eimeria Cavalcante, A. C. R., Teixeira, M., Monteiro, J. P. and Lopes, C. W. G. 2012. Eimeria species in dairy goats in Brazil. Veterinary Parasitology, 183(3-4): 356-358. [CrossRef] Diao, N., Zhao, B., Chen,Y., Wang ,Q., Chen , Z., Yang, Y., Sun ,Y. Shi, J., Li, J., Shi, K., Gong, Q. and Du, R. 2022. Prevalence of Eimeria spp. among goats in China: A Systematic Review and Meta-Analysis. Frontiers in Cellular and Infection Microbiology, 12(806085): 1-11. [CrossRef] Eckert, J., Braun, R., Shirley, M. and Coudert, P. 1995. Guidelines on techniques in coccidiosis research, Luxembourg: office for official publications of the European Communities, Published by the European Commission Directorate-General XII Science, Research and Development Agriculture Biotechnology L-2920, Luxembourg 306 pp. [Click here] El-Alfy, E., Abbas, I., Al-Kappany, Y., Al-Araby, M., Abu-Elwafa, S. and Dubey, J. P. 2020. Prevalence of Eimeria species in sheep (Ovis aries) from Dakahlia governorate, Egypt. Journal of Parasitic Diseases, 44(3): 559-573. [CrossRef] Ekawasti, F., Nurcahyo, R. W., Firdausy, L.W., Wardhana, A. H., Sawitri, D. H., Prastowo, J. and Priyowidodo, D. 2021. Prevalence and risk factors associated with Eimeria species infection in cattle of different geographical regions of Indonesia, Veterinary World, 14(9): 2339-2345. [CrossRef] Eser, M., Guksu, A., Cicek, H., Erez, M. S. and Cicek, H. 2020. Prevalence of Eimeria species in sheep Eskisher province. Eurasian Journal of Veterinary Sciences, 36(4): 261- 266. [CrossRef] Gul, A. 2007. The prevalence of Eimeria species in goats in Igdir. Turkish Journal of Veterinary and Animal Sciences, 31(6): 411-414. [Click here] Goz, Y., Aydin, A., Yuksek, N. and Deger, S. 2006. Frequency of coccidian species in goats in Van province of Turkey. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 12(2): 163-165. [Click here] Hasan, K. A. and Mahmood, O. I. 2021. Prevalence of Eimeria Species in sheep and goat in Tikrit City, Iraq. Indian Journal of Forensic Medicine and Toxicology, 15(2): 2032-2036. [CrossRef] Ibrahim, M. M. 2012. Prevalence of Eimeria species of the domestic goats Capra hircus Linnaeus, 1758 in Al-Baha area, Saudi Arabia. Egyptian Academic Journal of Biological Sciences, 4(1): 165-172. [Click here] https://doi.org/10.1016/j.vetpar.2011.07.043 https://doi.org/10.3389/fcimb.2022.806085 ../../../USER/Downloads/Biotechnologyguidelinesontechniquesincoccidiosisresearch.pdf https://doi.org/10.1007%2Fs12639-020-01229-1 https://doi.org/10.14202/vetworld.2021.2339-2345 http://dx.doi.org/10.15312/EurasianJVetSci.2020.307 https://journals.tubitak.gov.tr/cgi/viewcontent.cgi?article=2508&context=veterinary https://vetdergikafkas.org/uploads/pdf/pdf_KVFD_355.pdf http://dx.doi.org/10.37506/ijfmt.v15i2.14662 https://eajbsz.journals.ekb.eg/article_14297_992b3e3a68b127cf7445a69ccad973cc.pdf 443 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Makawi, Z. A. Kawahara, F., Zhang, G., Mingala, C. N., Tamura, Y., Koiwa, M., Onuma, M., Nunoya, T., 2010. Genetic analysis and development of species-specific PCR assays based on ITS-1 region of rRNA in bovine Eimeria parasites. Veterinary Parasitology, 174(1- 2): 49-57. [CrossRef] Keeton, S. T. N. and Navarre, C. B. 2018. Coccidiosis in large and small ruminants. Veterinary Clinics of North America - Food Animal Practice, 34(1): 201-208 . [CrossRef] Rajakaruna, R. S., Bandara, N. W. and Rajapakse, R. P. 2012. Coccidia infections in goats in Kandy and Nuwara Eliya districts in Sri Lanka. The Sri Lanka Veterinary Journal, 59(A): 1-6. [ResearchGate] Malla, T. K. and Saida, L. A. 2022. A survey of ecto and endo parasites of house mouse Mus musculus Linnaeus, 1758 of Erbil City, Kurdistan Region, Iraq. Bulletin of the Iraq National History Museum, 17 (2): 267-290. [CrossRef] Macedo, L. O., de Santos, M. A. B., Silva, N. M. M., da Barros, G. M. M., do, R., Alves, L. C., Giannelli, A., Ramos, R. A. N. and Carvalho, G. A. 2019. Morphological and epidemiological data on Eimeria species infecting small ruminants in Brazil. Small Ruminant Research, 171:37-41. [CrossRef] Minnat, T. R. 2014. Detection of gastrointestinal parasite infection of sheep and goats in Diyala Province-Iraq. AL-Qadisiya Journal of Veterenarr Medicine Science, 13(2): 118-123. [Click here] Mohamaden, W. I., Sallam, N. H. and Abouelhassan, E. M. 2018. Prevalence of Eimeria species among sheep and goats in Suez Governorate. Egypt International Journal Veterinary Sciences Medicine, 6(1): 65-72. [CrossRef] Nguyen-Ho-Bao, T., Lu, T. A. and Nguyen, H. H. 2022. Studies on Molecular Characteristics of Eimeria arloingi in Goats (Capra hircus) in Vietnam. Journal of Science, Engineering and Technology, 10(1): 19-28. [Click here] Rehman, T. U., Khan, M. N., Khan, I. A. and Ahmad, M. 2011. Epidemiology and economic benefits of treating goat coccidiosis. Pakistan Veterinary Journal, 31(3): 227-230. [Click here] Silva, A. C and Lima, J. D. 1998. Eimeria minasensis. (Apicomplexa: Eimeriidae) in the domestic goat Capra hircus, from Brazil. Memórias do Instituto Oswaldo Cruz Rio de Janeiro, 93(6): 741-744. [CrossRef] Saleh, B. A. H. 2011. Epidemiological study of sheep coccidiosis in Thi-Qar Province. M.Sc. Thesis in Department of Animal Production, College of Agriculture, Basra University, Iraq, 320 pp.[Click here] https://doi.org/10.1016/j.vetpar.2010.08.001 https://doi.org/10.1016/j.cvfa.2017.10.009 https://www.researchgate.net/publication/237091830%20Coccidia_infections_in_goats_in_Kandy https://doi.org/10.26842/binhm.7.2022.17.2.0267 https://doi.org/10.1016/j.smallrumres.2018.12.006 https://vetmed.uodiyala.edu.iq/uploads/2019/drtariq/Detection%20of%20gastrointestinal%20parasite%20infection%20in%20sheep%20and%20goat%20of%20Diyala%20province.pdf http://dx.doi.org/10.1016/j.ijvsm.2018.02.004 https://journals.southernleytestateu.edu.ph/index.php/jset/article/view/3 ../../../USER/Downloads/Documents/227-230.pdf https://doi.org/10.1590/s0074-02761998000600009 https://iqdr.iq/search?view=4098ed21f984c2 444 Bull. Iraq nat. Hist. Mus. 18 (2): 431-445. Isolation and identification of Eimeria Senbeto, Y. A. and Chekol, A. B. 2022. Prevalence of small ruminant coccidiosis in Pawe District, Metekel Zone, Benishangul Gumuz Regional State, Ethiopia International Journal Advance Resource Biology Sciences, 9(7): 262-272.[CrossRef] Smith, B. P., Van Metre, D and Pusterla, N. 2019. Large animals internal Medicine. 6th Edition. Elsevier. Veterinary science and veterinary medicine Large Animal Internal Medicine, 1949pp. [Click here] Singh, A. K., Shanker, D., Rout, K. P., kumar, A. and Kumar, P. 2020. Studies on Eimeria species in goats of Mathura region, Uttar Pradesh, India. Haryana Veterinary, 59(1): 131-132. [Click here] Souza, L. E. B., Cruz, J. F., Nato, M. R. T., Albuquerque, G. R., Melo, A. D. B. and Tapia, D. M. T. 2015. Epidemiology of Eimeria infections in sheep raised extensively in a semiarid region of Brazil. Revista Brasileira de Parasitologia Veterinária, 24(4): 410-415. [CrossRef] Sweeny, J. P. A., Ryan, U. M., Robertson, I. D., Yang, R., Bell, K. and Jacobson, C. 2011. Longitudinal investigation of protozoan parasites in meat lamb farms in southern western Australia. Preventive Veterinary Medicine, 101(3-4): 192-203. [CrossRef] Temizel, E.M., Demir, G., Selcuk, O., Catık, S., Senlik, B. and Senturk, S. 2011. Effect of treatment with clindamycin in an outbreak of coccidiosis in goat kids in Turkey. Journal of Biological and Environmental Sciences, 5(13): 37-40. [Click here] Taylor, M. A., Coop, R. L. and Wall, R. L. 2007. Veterinary parasitology. Blackwell Publishing Ltd. 3rd Edition, Blackwell Publishing, Oxford, 600 pp. [Click here] Verma, R., Sharma, D. K., Paul, S., Gururaj, K., Dige, M., Saxena, V. K., Rout, P. K., Bhusan, S. and Banerjee, P. S. 2018. Epidemiology of common gastrointestinal parasitic infections in goats reared in Semi- Arid Region of India. Journal of Animal Research, 8 (1): 39-45. [CrossRef] Woods, W. G., Whithear, K. G., Richards, D. G. Anderson, G. R., Jorgensen, W. K. and Gasser, R. B. 2000. Single-strand restriction fragment length polymorphism analysis of the second internal transcribed spacer (ribosomal DNA) for six species of Eimeria from chickens in Australia. International Journal for Parasitology, 30(9): 1019- 1023. [CrossRef] Zachary, J. F. and McGavin, M. D. 2014. Pathologic basis of veterinary disease, 5th edition. The Canadian Veterinary Journal, 55(4):372. [Click here] http://dx.doi.org/10.22192/ijarbs.2022.09.07.026 https://shop.elsevier.com/books/large-animal-internal-medicine/ https://www.cabidigitallibrary.org/doi/pdf/10.5555/20203435702 https://doi.org/10.1590/s1984-29612015070 https://doi.org/10.1016/j.prevetmed.2011.05.016 https://dergipark.org.tr/en/pub/jbes/issue/38011/438913 https://dergipark.org.tr/en/pub/jbes/issue/38011/438913 https://books.google.iq/books/about/Veterinary_Parasitology.html?id=tPpEofdsZ5gC&redir_esc=y http://dx.doi.org/10.30954/2277-940X.2018.00150.07 https://doi.org/10.1016/S0020-7519(00)00084-9 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3953940/ 445 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Makawi, Z. A. Bull. Iraq nat. Hist. Mus. (2024) 18 (2): 431- 445. Eimeria Schneider, 1875عزل و تشخيص انواع جنس (Apicomplexa, Eimeriidae) من املاعز في محافظة واسط، العراق زينب علوان مكاوي مركز بحوث ومتحف التاريخ الطبيعي, جامعة بغداد, بغداد, العراق. 20/12/2024، النشر: 7/11/2024القبول: ،1/11/2024املراجعة: ،10/7/2024االستالم: الخالصة يعد مرض األيميريا من املشاكل الرئيسية التي تواجه املجترات في جميع أنحاء العالم. ويحدث هذا املرض في الغالب بسبب أنواع األيميريا املتخصصة لكل مضيف والتي تنمو في األمعاء الدقيقة والغليظة للحيوانات. إن الخسائر الناجمة من العدوى رية )فقدان الوزن على وجه الخصوص( واألمراض السريرية )اإلسهال( دون السري تجعل أنواع هذا الجنس مصدر قلق اقتصادي كبير للغاية. لذلك, أجريت هذه عينة من براز املاعز, من 180الدراسة في بعض مناطق محافظة واسط. حيث تم جمع عينات البراز كال الجنسين, شملت فئات عمرية وأشهر مختلفة, وتم فحص جميع عينة براز موجبة للفحص الجزيئي وتحليلها بشكل أكبر باستخدام 75باملجهر وتم أخذ تفاعل البوليميراز املتسلسل التقليدي والتسلسل والتحليل النشوئي. وأظهرت النتائج ٪. حيث تراوحت معدل اإلصابة بأنواع 41.6املجهرية أن املعدل اإلجمالي لإلصابة كان Emeria ٪ لتحديد ثالثة أنواع مختلفة لجنس22.22٪ إلى 5.55من األيميريا Schneider, 1875, وشملت االنواع االتية , : E. arloingi Martin, 1909 (Marotel, 1905) (22.22 )٪ ،E. christenseni Levine, Ivens & Fritz, 1962 (13,88و ,)٪ Chevalier, 1966 E. hirci (5,55 فيما ي )٪ تعلق البراز أن عينات والتسلسل الجيني S rRNA , COI 18 أظهرت نتائج جين، PCR بتفاعل . Eimeria Schneider, 1875النواع الجنس كانت إيجابية