Detect of the eggs of P. equorum in the feces of horses by traditional method and molecular techniques in Baghdad, Iraq

1. equorum is more distributed in younger horse than elderly in Baghdad city.
2. Semi nested PCR an accurate diagnosis method for detection equorum.
3. ITS-2 region was good marker for detection equorum isolates in Iraqi horse by molecular technique.
4. ITS-2 region partial sequence recorded low genetic diversity (0.0035) and closely to Australia, China and USA isolates for the first time to Iraqi isolates.

Introduction

1. equorum is a member in the phylum Nematoda, a roundworm which predominantly affecting the health of foals and yearling horses and decreased in elderly with a wide geographical distribution, this parasite starts its life cycle in small intestine of the host during meet and mate between adult worms, then female adult worms produce huge number of eggs which laid in host small intestine then exit with feces or hatches to larval stage which migrate to liver and lung (1-6). Worm burdens can achieve high numbers (7). The infection is manifested clinically as nasal discharge, ill thrift (8), ceased growth, inappetence, rough hair coat, lethargy (9). In severe cases, other signs detected like pneumonia respiratory, bronchial hemorrhage, intestinal impaction, colic or even liver and lungs damage were diagnosis during migration larval phase (1). Many authors detected P. equorum as main species of Parascaris spp. eggs in feces of horse (10,11). Other study detected P. univalens mitochondrial genome with high sequences similarity with P. equorum and identical genetic maps (12). Due to scarce of data and the importance of P. equorum in horses, this study was designed to estimate the prevalence and molecular detection and some risk factors (sex and age) on the infection rate.

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 1^st and 2^nd 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 1^st 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 2^nd. 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

98. equorum is a nematode roundworm of young horses with wide geographical range (19). This study describes the results of detection of the infection in horses in Baghdad city by using microscopic examination; direct wet mount smears and flotation methods of fecal samples and molecular confirmative diagnosis by Nested PCR. The results of the present study show spherical to sub spherical shape of P. equorum eggs and their measurements between 10⁴´98.2 μm, length and width respectively. This agreed with Zajac and Conboy (20) who found that eggs of the parasite spherical in shape with diameter 90-100 μm. The total infection rate of P. equorum was 6.52%. This result disagreed with previous studies in some countries such as Lind and Christensson (6) who found 48% positive eggs samples for P. equorum. Also, Romero et al. (21) analyzed fecal samples by coproparasitological concentration-flotation technique of 218 horses found 11.93% were infected with Parascaris spp.

 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 (3^rd 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

1. equorum occurs in a high percentage in horses at Baghdad city. ITS-2 region is a good molecular marker for diagnosis the infection of P. equorum. Molecular detection with nested PCR flowed by phylogenic tree analysis were very useful to detection and diagnosis parasite P. equorum in horses.

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.