Detect of the eggs of P. equorum in the feces of horses by traditional method and molecular techniques in Baghdad, Iraq | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Iraqi Journal of Veterinary Sciences | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Article 1, Volume 38, Issue 2, April 2024, Pages 245-250 PDF (732.79 K) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Document Type: Research Paper | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
DOI: 10.33899/ijvs.2023.138252.2779 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Authors | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Suha T. Al-Biatee* 1; Balkes F. Hade* 2; Haider M. Al-Rubaie* 1 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1Department of Parasitology, College of Veterinary Medicine, University of Baghdad, Baghdad, Iraq | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
2Department of Microbiology, College of Veterinary Medicine, University of Baghdad, Baghdad, Iraq | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Abstract | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
The risk of P. equorum infection in horses remains critical. Little studies were conducted to investigate prevalence and molecular analysis of P. equorum in Baghdad city, Iraq. In this study traditional detection followed by molecular technique depending on ITS-2 region were used as an attempt to evaluate this nuclear region as a genetic marker to diagnose the parasitic infection. One hundred and thirty-eight fecal samples of horses were collected and examined by direct wet mount smears, floatation method by NaCl. Extraction of genomic material, nested PCR was done followed by phylogenic analysis depending on ITS-2 region performed for the first time in Iraq and genetic substitutions to analyze Iraqi horses. Nested PCR were done after determining the total infection rate 6.52% by conventional technique, including 3.84% in males and 10% in females with significant difference. Highly infection rate 11.42% was recorded in the age group under 2 years and the lower infection rate 4% was found in the age group between 2-4 years with significant difference. The Iraqi isolates were recorded in the Gen Bank under the accession numbers MZ400507.1, MZ400508.1, MZ400509.1, and MZ4005010.1; while, phylogenetic analysis recorded an identity range between 97-100% with China, Australia and USA isolates. P. equorum is more distributed in younger horses than elderly in Baghdad city and ITS-2 region is a certain molecular marker for detection P. equorum isolates in Iraqi horses. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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equorum; Nested PCR; Internal transcribed spacer-2 region; Phylogenetic tree; Iraq | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Introduction
Materials and methods
Ethical approval Samples were collected and transferred after obtaining approval from the regulations of Iraqi Ministry of Agriculture. The research protocols for the study were approved by the College of Veterinary Medicine, University of Baghdad, and were included in laboratory standards after animal utilization protocol certification No. P. G. 1904 in 2/10/2022.
Samples collection One hundred and thirty-eight horse fecal samples were collected from different areas in Baghdad city during the period from the beginning of December till the end of October 2022. The fecal samples of 25 g were divided into two parts, the first one 10 g for fecal analysis by using direct wet mount smears and flotation method by using NaCl (13) and the other part 15 g for the molecular study.
Statistical Analysis The Chi-square was used for assess the significant differences among factors at P≤0.01 (14).
DNA extraction DNA extraction from P. equorum using G- spin DNA extraction kit (Intron, Korea) was done according to the manufacturer´s recommendation. Estimation of Genomic DNA was detected by Nanodrop spectrophotometer to estimate the concentration and its purity at absorbance 260-280 nm.
Primers Two sets of primers were designed targeting the ITS-2 region; the first PCR run with ITS-2 first run PCR primer; PE1F5'-GCTGCGTTCTTCATC GAT-3' and PE1R5'-TTAGTTTCTTTT CT CCG CT- 3’ at 400 bp PCR product depend on (15), as well as the second PCR run with ITS-2 second PCR run primer; PE2F5'-AAT GCC ATA TAT GAA ATA TAT ACG-3' and PE2R5'-TTA GTT TCT TTT CCT CCG CT-3’ at 290 bp PCR product depend on (16).
PCR mix and amplification profile The DNA was amplified by using nested PCR (first and second) reaction and for gene diagnosis a mixture of the specific interaction components volume include Master mix-Maxime Pre-Mix Kit (i-Taq), template DNA 1.5µl, 1µl forward primer (10pmo); 1µl reverse primer (10pmo) and 16.5µl Free nuclease water to maintain total PCR reaction volume 20 µl. The optimum thermocycler condition for gene detection was run under the condition (Table 1). Agarose gel Electrophoresis 1.5% had been done to determine DNA pieces and to detect the result of PCR interaction and compare it with marked standard DNA to distinguish the bundles size (17).
Table 1: Thermocycler conditions targeting ITS-2 region for 1st and 2nd PCR reaction
Sequencing and phylogenic tree analysis Approximately 290 bp Amplicons from four worm samples were sequenced using Sanger sequencing and the resulting sequences were submitted on NCBI and analyzed using MEGA6.0 (18).
Results
Morphology of parasite eggs The morphological appearance of twenty-five eggs of the parasite from different samples revealed a spherical to sub spherical shape and their measurements were 95-120μm length and 90-110 μm width (Figure 1).
Figure 1: P. equorum egg by direct wet mount smears (X10).
Total infection rate The total infection rate of P. equorum in horses was 6.52% (9/138) with a significant difference between males 3.84% and females 10% (Table 2). The rate of infection was the highest 11.42% in age group under 2 years, followed by the age group4 years old 5.66% and the lower infection rate 4% occurred in the age group between 2-4 years with significant difference (Table 3).
Table 2: Distribution of P. equorum infection according to sex of study animals
Table 3: Prevalence of P. equorum infection according to age of horses
Molecular analysis Molecular findings detected that DNA purity and concentration were 1.8 and 300 ng/ µl respectively. Nested PCR technique was done for 10 randomly collected samples, using two primers for ITS-2 region. Results of amplification of 5 samples with 400 bp in first PCR run and 290 bp in second run were showed by electrophoresis of 1.5% agarose (Figures 2 and 3). Four positive local isolates were sequencing and submitted in NCBI with accession numbers MZ400507.1; MZ400508.1; MZ400509.1 and MZ4005010.1 then phylogenic analysis indicated that Iraqi isolates were closely to Australia, China and USA isolates with very low genetic diversity at 0.0035 (Figure 4). Genetic substitutions for Iraqi isolate compared with China isolate ID: MK209647.1. Results indicated transition in location 742 A/G with isolate MZ400507.1. Transition and trans-version with isolate MZ4005010 in the locations 495 C/T,501G/T,709 T/C and 719 T/C (Transition) and 550 A/C,692 G/C and 707A/T (Trans-version) while other isolates showed no genetic substitution (Table 4).
Figure 2: Nested 1st PCR reaction targeting the ITS-2 region at 400 bp. Lane (M): Ladder marker (1500-100 bp); Lanes (1-5): Positive PCR products.
Figure 3: Nested 2nd. PCR reaction targeting the ITS-2 region at 290 bp. Lane (M): Ladder marker (1500-100 bp); Lanes (1-5): Positive PCR products.
Figure 4: Phylogenetic tree analysis of local and global P. equorum isolates.
Table 4: P. equorum ITS-2 region substitutions Iraqi isolates
Discussion
According to age, P. equorum infections occur commonly in foals, yearlings and seldom harbored in horses older than 4 years (6). Regarding gender, Nielsen et al. (13) reported that the equine ascarid occurred predominantly in foals and this is in agreement with the results of the present study. Due to resistant of eggs to the environmental conditions, this leads to increasing the infection rate of P. equorum and this agreed with Reinemeyer (9) who indicated that the survival of larvae eggs up to 10 years and the highly development of infective stage (3rd larvae) within 10 days potentiate the survival of the parasite and increased the infection in the field (22). Breed with place of origin were significantly associated with helminth infection (21). Nielsen et al. (13) stated that no studies in naturally infected horses have detected occurrence of the two Parascaris species. The molecular results of the present study indicated that ITS-2 region was good genetic marker for detecting different nematode parasites, P. equorum for the first time in Iraq, that agreement with other studies which detected parasites infection and manifested dependent of genetic marker on ITS region as good molecular marker (23-30), due to difficulty of diagnosis of parasite by traditional methods (30). The four sequences were fell into same cluster and aligned with other 10 sequences of ITS-2 region isolate from different countries depend on final aligned sequence which closely related taxa and only one Iraqi isolate was located outside main cluster as a highly supported sister cluster rooted in a basal position to the main group. Phylogenetic tree-maximum-likelihood was generated from final alignment sequences and showed the main four Iraqi isolate of P. equorum and eight isolates of P. equorum identified in China and one from each Australia and USA. Most isolates of Iraq connected with main clades with (97-100%) identity, coinciding with other studies (16,23,31). Many studies used phylogenic tree comparisons with molecular parasites genome revealed high genetic similarities among local isolates and the globally registered sequences (31-39). Molecular detection and Phylogeny one of identification methods used in many studies (40-43). Comparing local ITS-2 region sequences of P. equorum with accession MK209647.1, results showed many genetic substitutions (transition and trans-version) which indicated that Nested-PCR technique followed by phylogenic and substitution analysis to ribosomal rDNA ITS-2 region was suggested for estimation of genetic diversity which considered the main genetic line study to detect genetic homology (23,24).
Conclusion
Acknowledgments The authors would like to thanks College of Veterinary Medicine, University of Baghdad, Iraq/ for providing the necessary facilities and administrative support.
Conflict of interests
The author has no conflict of interest. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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