Indian Journal of Animal Research

  • Chief EditorK.M.L. Pathak

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Indian Journal of Animal Research, volume 57 issue 9 (september 2023) : 1202-1208

Metagenomic Studies for the Detection of Parelaphostrongylus odocoilei, Ostertagia leptospicularis and Eimeria ahsata in Northern Cyprus Sheep for the First Time

J.T. Olaifa1,*, E. Ozgor2
1Department of Bioengineering, Cyprus International University, Haspolat, Lefkosa, North Cyprus.
2Department of Molecular Biology and Genetics, Cyprus International University, Haspolat, Lefkosa, North Cyprus.
Cite article:- Olaifa J.T., Ozgor E. (2023). Metagenomic Studies for the Detection of Parelaphostrongylus odocoilei, Ostertagia leptospicularis and Eimeria ahsata in Northern Cyprus Sheep for the First Time . Indian Journal of Animal Research. 57(9): 1202-1208. doi: 10.18805/IJAR.BF-1560.
Background: Gastrointestinal parasitism is considered one of the major health problems of ruminants in developing and undeveloped countries in the world. Despite the universal knowledge of these parasites, little is known about their prevalence and distribution in Northern Cyprus. This study is aimed at the microscopic and metagenomic study of gastrointestinal parasites across Cyprus sheep farms.

Methods: A total of 200 sheep faecal samples were randomly collected across sheep farms in suburban regions of Northern Cyprus during 2021-22. Samples were analyzed using the centrifugal floatation technique. Data were subjected to chi-square test analysis on SPSS to determine the relationship between age, sex of animals and prevalence of gastrointestinal nematodes. Samples were randomly selected to extract DNA and subjected to metagenomic analysis after which DNA has been extracted from the samples.

Result: Our investigations in the Northern Cyprus sheep farms allowed us to identify three species of gastrointestinal parasites namely Parelaphostrongylus odocoilei, Ostertagia leptospicularis and Eimeria ahsata which are new to this region. The GINs recovered are Haemonchus, Moniezia and Toxocara among others. A high prevalence (85%) of gastrointestinal parasites was recorded. Results showed a considerable significant relationship (P<0.05) between the age of animals and parasite prevalence.
The cost of managing diseases caused by gastrointestinal nematodes in ruminant animal production is a major pain for farmers globally (Francis and Šlapeta, 2022). The losses incurred are due to disease manifestation, low productivity and a higher susceptibility to other infections (Kamal et al., 2021; Khan et al., 2021). GIT parasitism can alter milk’s nourishing characteristics, such as protein, lessened fat and lactose composition. Livestock animals can become diseased with diverse parasites and disseminate them to other animals around them (Khan et al., 2021). Many protozoans are usually found in ruminant animals such as sheep and goats (Isah, 2019). Though microscopy has been a conventional technique of choice for parasitological studies, it permits the identification of the parasites typically to genus level, since the eggs of interrelated species are repeatedly indistinguishable (Chessa et al., 2020), immunological, hybridization and molecular methods were established and utilized for a better taxonomic identification (Jaeger and Iñiguez, 2014; (Chessa et al., 2020)).  
Molecular parasitological examinations are primarily centred on PCR amplification and Sanger sequencing of short barcoding loci as 18S rDNA, primarily making use of primers for a precise parasite taxon. The latest established next-generation sequencing (NGS) permits the identification of many taxonomic groups simultaneously by direct shotgun sequencing of DNA hauled out of the samples (metagenomics) or by PCR-centred metabarcoding of target genes (Srivathsanet al., 2016), this technique has been applied on human samples i (Güler and Süer, 2021; Chihi et al., 2022) and  swine samples i (Wylezich et al., 2019), but has never been applied to parasitology on any ruminants in Northern Cyprus.  The infection of gastrointestinal parasites is influenced by many factors such as the climatic condition, the host nature, farming operations, the functional state of the host and the epidemiology of the parasite of interest. Owing to the significant challenges with drug resistance, there is an apparent necessity and substantial global interest in the advance of enhanced methods of controlling GIT parasites for which epidemiology of parasites is a prerequisite.          
Hence, the present study is aimed to investigate the prevalence of GIT parasites in 200 faecal samples collected across the sheep farms of Northern Cyprus utilizing microscopy and metagenomic analysis. Moreover, three gastrointestinal parasites of economic importance were discovered through metagenomic analysis for the first time in Northern Cyprus.
Ethical approval for the present study was obtained from the Ethics Committee of Cyprus International University (020-7952). All samples were collected across Northern Cyprus sheep farms between September 2021 and February 2022. The farms raise sheep and goats largely for milk and meat production. Microscopic studies were carried out in the microbiology laboratory of Cyprus International University.
A total of 200 faecal samples were collected from 200 sheep considering age and sex of animals. The samples were collected and kept in airtight containers. Upon arrival at the laboratory, each sample was prepared using the centrifugal floatation technique (Zajac, 2002) The samples were recovered and kept at 4oC until they were checked for the presence or absence of nematodes and Eimeria oocysts.
Processed samples were identified up to genus level based on morphological description (Shorb, 1940; Christie and Jackson 1982).
Further, QIAamp PowerFecal Pro DNA Kit (Qiagen, Hilden, Germany) was used for the extraction and purification of the genomic DNA from positive samples. The primer pair used for the construction of amplicon libraries targeted a region of nearly 1400 bp covering the V1-V9 region of the 18SrRNA gene. Oxford Nanopore Technologies Nanopore barcode DNA sequences of the generated library were added to the 5' end of the target-specific primer pairs. The target-specific primer-connector sequences specific to 18SrRNA are TTTCTGTTGGTGCTGATATTGC-GRGTTTGA TYHTGGCTCAG -3' for the forward primer and 5'-ACTTG CCTGTCGCTCTCTATCTTC-TACCTTG TTAYGACTT-3' for the reverse primer. The taxonomic annotations were performed by comparing the created OTUs according to the RDP 18SrRNA database and results were obtained by correlating the OTUs detected as the same genus. Pie charts were created with various statistical analyzes using Minitab and R programs according to the organisms to which the OTUs matched, their quantitative values and the metadata data of the samples.
Statistical analysis for prevalence was carried out employing SPSS software for Windows version 22.0 (SPSS Inc., Chicago, USA). Chi-square test was carried out to determine the relationship between variables and the relationship between variables was assumed to be real when the probability of finding the observed difference by chance was less than 5% (P<0.05). The complete prevalence was estimated as a percentage of the number of sheep infested in the entire number of sheep investigated.
Prevalence of parasite
Out of the 200 samples analysed for the presence of gastrointestinal parasites from the farms, 75 are male and 125 are females. At least one parasite was present in 160 of the analyzed samples amounting to 80% parasite prevalence. Thus, eggs of gastrointestinal nematodes (GIN) recovered from the samples as eggs in the genus of Moniezia, Cooperia, Oesophagostomum, Parelaphostrong ylus, Trichurus, Heamonchus, Nematodirus, Ostertagia, Toxocara and other strongyles (Fig 1). The differentiation of srongyle eggs were done morphologically with the aid of microscopic using aforementioned keys. Eimeria oocysts were spotted in 65 of the total number of samples analyzed. It was also observed that eggs of GIN in the genera of Heamonchus were the most encountered (80) accounting for 40% prevalence (Table 1). Our results of high prevalence are similar to those of Kulišić et al. (2013) and Mphahlele et al., (2021) and gastrointestinal parasite infection has a direct effect on the health and productivity of animals, thereby causing a reduction in output and, consequently, a decrease in the owner’s and community’s income (Mathewoset al., 2022). The predominance of Haemonchus over other GIT parasites has also been reported by Tariq et al. (2008) and Wani et al. (2011) in Kashmir Valley. The major reason for this prevalence can be connected to the fact that Haemonchus females are principally fertile egg layers (laying about 10,000 eggs a day) (Tramboo et al., 2015). The high prevalence of GIT parasites observed in the current study could be as a result of the accessibility of appropriate climatic conditions such as fairly high temperatures and rainfall that support the lengthy survival and development of an infective larval stage of most nematodes (Shearer and Ezenwa, 2020; Mphahlele et al., 2021).

Fig 1: Images of parasite genera found in the microscopy study (a) Monieza (b) Eimeria oocysts (c) Trichurus (d) Oesophagostomum (e) Ostertagia (f) Nematodirus (g) Heamonchus (h) Parelaphostrongylus (i) Monieza.

Table 1: Prevalence of GIT nematodes and Eimeria in Northern Cyprus sheep farms.

Effect of age on gastrointestinal parasites prevalence
The results showed that younger animals are more susceptible to gastrointestinal parasites infection than the older animals (Table 2), animals under the age of 2 years generally gave a result of above 90% prevalence in terms of infection and this shows that there is a statistically significant relationship between the age of animals and prevalence (P<0.05). The higher prevalence in younger sheep is in agreement with the results of Abebe et al., (2018) who reported significantly higher GIT parasites prevalence in younger animals in Ethiopia but disagree with that of Tramboo et al., (2015) as they established an overall higher prevalence in older animals than younger ones. The high prevalence of parasites in younger animals indicates a high degree of susceptibility, unlike the adults which acquire immunity to some of these GIT parasites.

Table 2: Age wise prevalence of gastrointestinal parasites among Northern Cyprus sheep.

Poly-parasitism of gastrointestinal parasites
Out of the 200 examined sheep, 95 (47.5%) were cross infected with two parasites, 10 of the analyzed samples (5%) were infected with three gastrointestinal parasites and 60 (30%) were infected with one gastrointestinal parasite while 40 (20%) were negative for the presence of GI parasites. The present study has indicated the presence of mixed infection with two or more GIT parasite genera in sheep and this is in agreement with the findings of other researchers (Asif et al., 2008; Samuel et al., 2016). Polyparasitism has been proposed to be a vital source of morbidity and loss of production in sheep (Samuel et al., 2016). Additionally, the incidence of interaction and compromisation of the immune system of the host by polyparasitism has been detailed to intensify their vulnerability to other diseases or parasites (Wang et al., 2006). Therefore, polyparasitism is a significant issue in sheep production in the present study area.
Effect of animal sex on prevalence
In terms of animal sex, observations revealed that all the parasites encountered were found in the faecal samples of  the male animals except for Oesophagosuturom  while three of the 12 encountered parasites namely Ostertagia, Trichurus and Oesophagostomum were not found in any of the female animals across the farms. Though, the interpretations were not substantial. The effect of sex on the vulnerability of sheep to infections could be ascribed to innate tendency and distinctive proneness due to hormonal regulation. The total number of samples collected from male animals tested during the study was 75 and 58 were positive for at least one endoparasite while 102 out of 125 samples taken from the female animals yielded a positive result accounting for 81.6% prevalence therefore a higher prevalence of major gastrointestinal parasite infection was observed in female animals though the p-value (0.33) suggests that the relationship between the sex and prevalence is not statistically significant (P>0.05) (Table 3). This is a result of the same exposure of both sexes and they are as well from the same agroecology. The influence of sex on the susceptibility of sheep to infections could be ascribed to innate tendency and distinctive proneness as a result of hormonal regulation. The existing gender-wise remarks are in agreement with the finding of Tariqet al. (2008) and Gauly et al., (2006) who described the occurrence of gastrointestinal parasite infection in sheep as higher but not statistically significant in females than in their male counterparts.

Table 3: Prevalence of GIT parasites according to the animal sex.

Microbiota composition according to genus level
In this study, the gut microbiota community composition was analysed, Fig 2 shows the complete composition and abundance  of Eukaryota domain presents in the feacal samples. At the genus level, the top three predominant populations in the samples analysed were  Blastocystis,  Mucor, Ichthyobodo and Entemoeba. In terms of the composition, Blastocystsis which has the highest abundance with 33.5% followed by Mucor (14%), Ichthyobodo (13%), Entamoeba (8%), Parelaphostrongylus (4%), Spironucleus (3.5%) and Ripella (0.6%) with the least abundance among the families. Furthermore, it was observed that four species were present in the Blastocystis genus namely Blastocystis hominis, Blastocystis ratti, Blastocystis python and Blastocystis cycluri. Three species present in Mucor genus namely Mucor amphibiorum, Mucor mucedo and Mucor indicus while the other families were represented with just a species (Table 4).

Fig 2: Composition and abundance of domain Eukaryota in Cyprus sheep faecal sample.

Table 4: Taxonomy of species detected by metagenomic analysis.

Metagenomic analysis detects three species of GIT parasite
The results obtained from the present study of GIT parasites with a metagenomic tool detailed the identification of three parasites which are new to the study area. The GIT parasites detected from the analysis are Parelaphostrongylus odocoilei, Ostertagia leptospicularis and Eimeria ahsata. The krona graphs presented showed the percentage of each parasite species identified. Fig 3-6 revealed the species identified for the first time in the study area through the use of metagenomic analysis. Eggs in the genus of the three new species of parasite discovered were also identified in the microscopy analysis which shows some level of congruency between the two techniques. However, we could not identify species from other genera of parasite identified using microscopy analysis, this could be a result of the very low number of samples analysed in the metagenomic study as compared to microscopy analysis.

Fig 3: Composition and abundance of species found in Strongylida order.

Fig 4: Krona chart showing the only parasite discovered in the Parelaphostrongylus genus.

Fig 5: Krona chart showing the only parasite discovered in the Ostertagia genus.

Fig 6: Krona chart showing the only parasite discovered in the Eimeria genus.

The high prevalence of protostrongylid infections in Dall’s sheep in Subarctic and Arctic North America was first reported by Jenkins et al., (2006). The findings of Jenkins et al., (2005b) further suggest that adding to muscular and respiratory pathology, P. odocoilei also has the likelihood of causing neurological disease in thinhorn sheep (Jenkins et al., 2005b). Ostertagia leptospicularis which is not only found in sheep but also other ruminants such as cattle and goats is a gastrointestinal nematode of the genus Ostertagia, it develops in the abomasal glands of ruminants. The Ostertagiaspecieshave been connected to pathophy siological changes resulting from abomasal parasitism which include a reduced acidity of the abomasal contents, which is associated with a decreased peptic digestion and bacteriostatic activity (Hertzberg et al., 2000). The presence and prevalence of Eimeria ashata have been reported by Olmos et al., (2020) in Argentina, Souza et al., (2015) in Brazil and Reeg et al., (2005) in Germany. This is the first time the presence of Eimeria ashata in a ruminant animal is reported. Coccidiosis has been reported as a globally important clinical and subclinical parasitic infection in ruminant animals. Coccidiosis is caused by coccidian parasites of the genus Eimeria which add to enteric disease, particularly in young or stressed animals under poor farm management thereby leading to high mortality in young ruminant animals (Yusof and Isa, 2016). Furthermore, investigations in many countries such as  Brazil (Souza et al., 2015) and Egypt (Al-Alfyet al., 2020) have indicated that coccidiosis has a huge economic significance to the production of livestock owing to the clinical disease (diarrhoea) and sub-clinical disease (poor weight gain) caused by the parasite kept in large numbers under differs management methods.  
This study indicated that both GINs and Eimeria infections were leading in sheep farms in Cyprus. Younger animals were more susceptible to infection with GIT parasites. The combination of microscopic identification and metagenomic technique resulted in a more complete parasitological identification and could be recommended as a new approach in parasitological studies. The parasitic weight constituted a pronounced financial influence and limitation to the sheep venture in attaining improved and viable productions. Consequently, these results recommended a need for well-harmonized, healthy regulation of sheep farms by scientists and the spreading of awareness on GINs and Eimeria to breeders and farmers to lessen the incidence of infections.

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