Open Veterinary Journal, (2023), Vol. 13(9): 1099–1105

Original Research

10.5455/OVJ.2023.v13.i9.5

Molecular identification and phylogenetic confirmation of Sarcocystis species in slaughtered camels in Al-Najif province, Iraq

Ola A. Aggar* and Mohammed T. S. Al-Zubaidi

Department of Parasitology, College of Veterinary Medicine, University of Baghdad, Baghdad, Iraq

*Corresponding Author: Ola A. Aggar. Department of Parasitology, College of Veterinary Medicine, University of Baghdad, Baghdad, Iraq. Email: Ula.Abd1105a [at] covm.uobaghdad.edu.iq

Submitted: 10/06/2023 Accepted: 06/08/2023 Published: 30/09/2023

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Abstract

Background: Sarcocystis is an intracellular parasite of particular importance as it infects many domestic animals as camels that play the role of intermediate host for the parasite.

Aim: This study aimed to identify Sarcocystis species in camels by molecular assay with confirmation of local isolates by phylogenetic analysis.

Methods: A total of 200 slaughtered camels (Camelus dromedarius) that were slaughtered in Al-Najaf province (Iraq) abattoirs from October (2021) to July (2022) were subjected to collect the fresh tissues from four organs (esophagus, diaphragm, skeletal muscle, and heart), to be tested later by the conventional polymerase chain reaction (PCR). Then, a total of 20 positive genomic DNA samples were sequenced, named, got specific access numbers (OP785703.1 to OP785722.1), and compared with the NCBI-GenBank isolates.

Results: Targeting Cox1 gene, 80% of collected tissues were found positive by the conventional PCR assay. Phylogenetic tree analysis revealed that the local Sarcocystis isolates were identical to Indian S. cameli isolates at 99.70%–99.90%. Significantly, an increase in Sarcocystis infection was seen in the esophagus compared to the diaphragm, skeletal muscle, and heart; older (>4 years) than younger (≤4 years) camels, and in females more than males.

Conclusion: To the best of our knowledge, this represents the first molecular study in Iraq that identifies Sarcocystis cameli in camels. However, additional epidemiological and molecular studies in camel populations as well as in other domestic and wild animals appeared to be necessary.

Keywords: Sarcocystosis, S. cameli, Camelus dromedarius, PCR, Sequencing.

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Introduction

Sarcocystis, first reported in 1843, is a genus of intracellular protozoan parasite which belongs to Sarcocystidae family, Eucoccidiorina order of Apicomplexa phylum. Worldwide, considerable production losses and health illness in animals have been recognized due to the presence of >200 species in the genus of Sarcocystis that infects a wide array of domestic and wild animals (Dubey et al., 2015a; Abbas et al., 2023). The life cycle of a parasite involves two hosts; definitive (predator) and intermediate (prey) hosts. However, Sarcocystis spp. having greater specificity to intermediate than definitive hosts (Prakas et al., 2023). In the intermediate host, the development of asexual stages of the parasite occurs usually in skeletal, cardiac, esophageal, and diaphragmatic muscles and to a lesser extent in CNS (Dubey et al., 2015a; Gajadhar et al., 2015; Prakas et al., 2021). The definitive host can be infected during ingestion of contaminated tissues with mature Sarcocystis that undergoes sexual reproduction to excrete the oocysts/sporocysts to the environment by feces (Lindsay and Dubey, 2020; Razooqi et al., 2022).

One-humped or dromedary camels (Camelus dromedarius) reared widely in arid regions of many world countries (Burger et al., 2019; Zhu et al., 2019) has been used mainly for the production of meat that is characterized by the low percentages of fats and cholesterol when comparison to meats of other animal species (Faye, 2016; Kadim et al., 2018). However, the existence of parasite in camel meats might potentially downgrade their quality to be consumed by humans (Hamidinejat et al., 2013; Mohammadpour et al., 2020).

Although Sarcocystis spp. can induce significant pathology or even mortality, most diseased camels generally exhibit subclinical illness (Metwally et al., 2020). Thus, the diagnosis of Sarcocystis spp. in camels using traditional tools represents a challenge for the researchers. Hence, molecular identification of Sarcocystis spp. in Iraqi camels with confirmation of local isolates by phylogeny was aimed in this study.

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Materials and Methods

Study animals and sampling

Totally, 200 slaughtered camels (C. dromedarius) of different ages (<2 to ≤5 years) and sexes (107 males and 93 females) were selected to the present study from the abattoir of Al-Najaf province (Iraq) during October (2021)–July (2022). Approximately, 100 g of fresh meat were obtained from the esophagus, diaphragm, skeletal muscle, and heart of each slaughtered camel, kept in individual plastic labeled containers, and saved frozen at −20C for later molecular examination.

Molecular examination

Conventional polymerase chain reaction (PCR)

In accordance with the manufacturer instruction of the gSYAN DNA Extraction Kit (iNtRON Biotechnology, South Korea), the DNAs were extracted from collected samples and examined directly by the Nanodrop System (Thermo Scientific, UK) to estimate the purity and concentration of these DNAs samples (Gharban et al., 2023). According to the manufacturer instructions of GoTaqTM Green PCR MasterMix Kit (Bioneer, Korea), the Mastermix tubes were prepared at a modified final volume of 25 μl (5 μl of DNA template, 2 μl of forward primer, 2 μl of reverse primer, 4 μl of Green PCR Mastermix, and 12 l of nuclease-free water) using one set of primers [(F: 5′-ATG GCG TAC AAC AAT CAT AAA GAA-3′) and (R: 5-ATA TCC ATA CCG CCA TTG CCC AT-3′)] targeting Cox1 gene at a product size of 1,058 bp (Metwally et al., 2020). The PCR thermocycler was carried as follows: one cycle for initial denaturation (5 minutes/95°C); 35 cycles for denaturation (30 seconds/95°C), annealing (30 seconds/54°C) and extension (1 minute/72°C); and one cycle for final extension (5 minutes/72C). Agarose gel stained with 3 μl of Ethidium Bromide and Ladder Marker (100–2,000 bp) were analyzed by electrophoresis at 80 Volt, 80 Am for 90 minutes. PCR products of previously diagnosed and confirmed Sarcocystis spp. isolate was used as a positive control; while nuclease-free water was used as a negative control.

Finally, the DNA fragments were visualized under the UV-Transilluminator (ATTA/Korea).

Phylogenetic analysis

Sequencing of some positive DNAs was performed to confirm the species of Sarcocystis, and study the genetic relationship between the local and NCBI-BLAST Sarcocystis isolates. Targeting the Cox1 gene, a total of 20 positive DNA samples in addition to forward primer were sent for the Macrogen Company (South Korea) to be sequenced by the AB DNA Sequencing System. First, the local isolates of Sarcocystis species were recorded in the National Center For Biotechnology Information (NCBI)-GenBank to obtain their access numbers; and then, the Phylogenetic tree analysis was performed by the MEGA 6.0 Software.

Statistical analysis

GraphPad Prism (GraphPad Software Inc., USA) Software was served to evaluate significant variation between results of this study using the one way-analysis of variance. Values were represented as percentages (%), and significant differences were p ˂ 0.05 (Gharban, 2023).

Ethical approval

The current study was licensed by the Scientific Committee of the Department of Parasitology in the College of Veterinary Medicine, University of Baghdad, Baghdad, Iraq.

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Results

Targeting Cox1 gene, 80% (160/200) of meat samples were positive by the conventional PCR assay at a product size of 1,058 bp (Fig. 1).

The genomic DNAs of 20 positive samples were named respectively as Sarcocystis spp. IQN.1 to IQN.20. Comparative analysis of local isolates with the NCBI-GenBank Sarcocystis isolates showed that there were nucleotide alignment similarities (*) and substitution mutations in Cox1 gene.

Comparative homology sequence identity (%) to detect genetic variations between the local Sarcocystis spp. IQN-isolates (total no. 20) and the NCBI-BLAST Sarcocystis cameli Indian (MW651858.1) isolate of cox1 gene. This identity showed that there was 99% of similarity and 0.150%–0.50% of total genetic mutations/changes (Table 1, Fig. 2).

According to organ, significantly higher increases (p < 0.05) in the prevalence of sarcocysts were detected in the esophagus (91.43%), while the lower one (p < 0.05) was seen in the heart (62.5%). However, insignificant variation (p > 0.05) in the prevalence of Sarcocystis spp. between diaphragm (85.42%) and skeletal muscle (85.42%) was reported (Table 2).

According to the age of study camels, this study has seen that the parasite is more prevalent in camels aged >4 years (96%) than those aged ≤4 years (53.33%), (Table 3).

Regarding the sex of study camels, the infection rate of sarcocystosis was increased significantly (p < 0.05) in females (87.78%) in comparison with males (73.64%), (Table 4).

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Discussion

Sarcocystis infections have been demonstrated in different animal species such as cattle, buffalo, goats, and sheep (Dakhil et al., 2017; Al-Saadi et al., 2020; Swar and Shnawa, 2020; Abdullah, 2021). The molecular technique is one of the most useful diagnostic methods for the identification of many pathogens, but there is low molecular data about Sarcocystis infection in camels. In this study, the prevalence of Sarcocystis spp. in slaughtered camels was 80%. This agrees with the finding reported in camels (85.5%) in Egypt (Rabie et al., 2021).

In this study, the Cox1 gene revealed a high efficacy in the detection of Sarcocystis spp. in tissue samples of slaughtered camels. Different studies reported that DNA barcoding system using the Cox1 gene has a high efficacy in discrimination between the invertebrate and vertebrate species (Rodrigues et al., 2017; Mioduchowska et al., 2018; Ahmed et al., 2022). Gjerde et al. (2015) concluded that the Cox1 gene can work better with DNA samples derived from Sarcocystis, and sequences of the Cox1 gene can vigorously distinguish between different species of Sarcocystis. Rodrigues et al. (2017) demonstrated the suitability of Cox1 as a gene of parasitic reorganization from those of highly related. Other studies reported that Cox1 has a well phylogenetic character than other genes (Stöger et al., 2016; Wang et al., 2017). Furthermore, some authors claimed that the evolution of the Cox1 gene enables researchers to distinguish between closely related species and investigate intraspecific variation (Changbunjong et al., 2016; Viricel and Rosel, 2022). The Cox1 has been demonstrated as the potential barcode marker for joint analysis of nuclear and mitochondrial markers of various species (Rodrigues et al., 2017; Ahmed et al., 2022).

Fig. 1. Representative image shows the agarose-gel electrophoresis of PCR products targeting the Cox1 gene to detect the genus of Sarcocystis. Lane (M): Ladder marker (100–2,000 bp); Lane (1): positive control; Lane (2): negative control; Lanes (3, 7, 8): negative samples; Lanes (4–6 and 9–16): positive samples for Sarcocystis spp. at 1,058.

Table 1. NCBI-BLAST Homology sequence identity between local and GenBank isolates.

Fig. 2. Phylogenetic tree analysis based on Cox1 gene partial sequence in local IQN S. cameli isolates and NCBI-GenBank S. cameli isolates. Comparative identity for the genetic variations between the local isolates and NCBI-BLAST isolates showed that similarity was 98%; whereas, the total genetic mutations/changes were 0.2%–1.5%.

Despite the discovery of five different Sarcocystis species in camels, multiple studies have shown that only S. cameli and S. ippeni are considered to be the real species infecting dromedary camels, namely S. cameli, S. ippeni, S. camelicanis, S. camelocanis, and S. miescheri (Dubey et al., 2015b; Omer et al., 2017; Rabie et al., 2021).

Table 2. Total infection rate of sarcocystosis in camels according to organ.

Table 3. Total infection rate of sarcocystosis in camels according to age.

Table 4. Total infection rate of sarcocystosis in camels according to sex.

In this study, the phylogenetic analysis discovered that the local study isolates were identical to the global NCBI-GenBank Indian S. cameli isolate. This is in agreement with the results of other studies as S. cameli is the more prevalent species of Sarcocystis in camels to yet (Valinezhad et al., 2008; Dubey et al., 2015b; Omer et al., 2017; Asopa et al., 2023). For the first molecular proof of S. cameli in camels in Iran (Motamedi et al., 2011), the bradyzoite DNA was amplified from the 18S rRNA gene fragment using traditional PCR.

Sarcocystis species can frequently infect the muscular tissue of the heart, tongue, esophagus, and diaphragm (Wahba et al., 2014; Ahmed et al., 2016). Our findings revealed that Sarcocystis spp. has existed in all examined organs but more commonly in the esophagus. Some research suggested that sarcocystosis is more frequent in the cremaster muscle (Bucca et al., 2011; Sağlam and Keleş, 2016). Shekarforoush et al. (2006) demonstrated that the heart is the most infected organ; while Al-Ani and Amr (2017) noted that camel’s diaphragm was the most frequently affected organ. According to Oryan et al. (2010), Sarcocystis appears to prefer the esophagus, tongue, and heart. In contrast, Gareh et al. (2020) reported that Sarcocystis can spread in various organs, mostly in the esophagus with a prevalence rate of 49%. Rabie et al. (2021) reported Sarcocystis in the esophageal, heart, and ocular muscles in addition to other organs, with a higher infection rate in the esophagus (85.5%). Asopa et al. (2023) showed that Sarcocystis spp. was prevalent in the tongue and esophagus of dromedary camels from Rajasthan’s Bikaner area. This variance in the distribution of S. cameli strains may be accounted for differences in the prevalence of Sarcocystis among camel organs (Hamidinejat et al., 2013).

Another substantial risk factor for infection was age. In this study, the infection rate was considerably higher in older camels (>4 years) than in younger camels (<4 years). Other reports in Iran (Hamidinejat et al., 2013), Saudi Arabia (Omer et al., 2017), and Egypt (El-Bahy et al., 2019) have revealed similar results. The slower development of detectable cysts may account for the low prevalence of Sarcocystis in young camels. Another explanation for increasing the rate of infection in older camels is that the older animals are slaughtered at a higher rate than younger ones. In addition, keeping young camels indoors may reduce their exposure to disease (Valinezhad, et al., 2008, Hamidinejat et al., 2013, Omer et al., 2017).

Sex was discovered as a crucial factor linked to infection. Our findings were consistent with numerous studies conducted in southern Ethiopia (Woldemeskel and Gumi, 2001), Iran (Valinezhad et al., 2008), and Egypt (Rabie et al., 2021) which found that male camels were more susceptible to infection than females. Omer et al. (2017) showed that the slaughtered female camels were usually over 4 years old, whereas male camels were under 2 years old. This disparity might be explained by the fact that most males are grazed outside for hard work, while most females are kept indoors for breeding under good and hygienic management. This might make males more susceptible to infection than females (Romero et al., 2017). An insignificant difference in the frequency of sarcocystosis between male and female camels was identified by Hamidinejat et al. (2013). A lack of relationship between sex and infection rates has been shown in similar studies on camels (Woldemeskel and Gumi, 2001; Shekarforoush et al., 2006; Valinezhad et al., 2008).

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Conclusion

To the best of our knowledge, this represents the first molecular study in Iraq that identifies Sarcocystis cameli in camels. However, additional epidemiological and molecular studies in camel populations as well as in other domestic and wild animals appeared to be necessary.

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Acknowledgments

The authors are thankful to the Colleges of Veterinary Medicine, University of Baghdad for providing the necessary facilities for the study. However, all materials used in this study were purchased by the authors and no external funds were received for this study.

Conflict of interest

The authors declare that there is no conflict of interest.

Author’s contribution

OAA: Collection of tissue samples, extraction of DNAs, and preparation of Mastermix tubes; MTSA: PCR analysis, sequencing, and statistical analysis of obtained results. All authors have written the manuscript and approved the final copy of it.

Funding

There is no external fund (private funding).

Data availability

All data supporting the findings of this study are available within the manuscript.

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