Epidemiology and genetic analysis of Oestrus ovis from slaughtered sheep in Sulaymaniyah province, Iraq

Oestrosis is a condition of nasal myiasis caused by infestation from the larvae of flies in the Oestrus genus (Diptera: Oestridae). It is considered a serious parasitic disease in goats and sheep as well as other animal species at times (20). According to Bosly (10), the most frequent clinical signs in animals are sneezing and nasal discharge. Parasitic larvae significantly impact the host’s health and result in decreased production of milk, meat and wool (4). The sheep nose botfly (Oestrus ovis L.) induces serious cavitary myiasis in regions globally where goats and sheep are raised (14). Oestrus ovis can be identified by examining the morphological characteristics of the larvae, such as the slits on the posterior spiracular plates, by noting the clinical signs, and sometimes by finding adult flies (30).

A few years ago, molecular methods were widely utilised to gain understanding of the classification and categorisation of different insects. In particular, certain regions of ribosomal and mitochondrial DNA have been observed to be strong genetic indicators for addressing taxonomic identification challenges in certain myiasiscausing flies from the Oestridae family, as discussed in studies by Otranto et al. (31, 33) and Otranto and Traversa (32). The mtCOX1 gene was selected to identify the larval relationships within the Oestridae family. Because of its large size and the presence of both highly conserved and variable regions with varying rates of mutation, it has been shown to be essential for different molecular phylogenetic purposes (25). The COX1 658 base pair (bp) area is a common and widely accepted genetic marker for animal taxonomy (19). Over the past decade, DNA barcoding has proven to be an effective method for examining genetic relationships and distinguishing various insect species in the Oestridae family using COX1 sequences (24, 28).

Several research studies have been carried out on O. ovis in the northern cities of Iraq (21, 39), as well as in specific Arabic countries (1, 26, 29). Iran (40), Türkiye (5, 34), and other neighbouring countries have also been taken into account. Infestation with O. ovis in sheep is found in varying percentages across Europe, from 21.9% to 93.7% (8). Dorchies et al. (13) reported a rate of 28.4% in France, while Alcaide et al. (2) stated that in Spain, the rate was 71.1%.

The existing research on O. ovis occurrence and molecular characteristics in the Kurdistan region of Iraq, particularly in Sulaymaniyah province, is limited. The goals of this research were to evaluate the monthly and seasonal presence of O. ovis and associated risk factors in slaughtered sheep at a Sulaymaniyah abattoir, and to investigate the genetic relationships of O. ovis larvae using PCR and partial sequencing of the mtCOXI gene.

In this study, the occurrence of O. ovis in sheep was found to be 22.25%, mirroring rates reported by Metwally et al. (26) in Saudi Arabia (22.3%), by Karatepe et al. (22) in Türkiye (22.52%) and by Shareef (39) in the Sulaymaniyah province of Iraq (20.96%). This discovery shows a lower rate compared to previous studies in different countries: 49.7% in Iran by Shoorijeh et al. (40), 38.71% in Türkiye by Özdal et al. (34), 58.03% in Jordan by Abo-Shehada et al. (1), 42.33% in Libya by Negm-Eldin et al. (29), 43.2% in Greece by Papadopoulos et al. (35), 71.1% in Spain by Alcaide et al. (2) and 91% in Italy by Caracappa et al. (12). On the other hand, certain authors documented lower occurrence rates: 8.67% in Egypt (3), 13.7% in Brazil (42), 17.2% in the Nineveh province of Iraq (21) and 24.8% in France (13). The diversity in prevalence rates might be due to variations in fly population, host immunity, animal types, weather patterns, farming techniques, medication usage and geographical location.

The average infestation intensity was 3.36 larvae per host, surpassing Shareef's (39) findings of 1.98 larvae per sheep in Iraq, but lower than Özdal et al.’s (34) results of 4.02 larvae per sheep in Türkiye. The variability in climate, age of animals and differences in host resistance could account for this (34).

The study revealed a greater proportion of L3 larvae (54%) as compared to L2 (30%) and L1 (15%). Yilma and Dorchies (47), Özdal et al. (34) and Mohammed et al. (27) reported a higher proportion of L3 compared to L2 and L1 in France, Türkiye and Iraq, respectively. Similarly, Alcaide et al. (2) found a higher percentage of L1 compared to L2 and L3 in south-western Spain, where the climate and altitude are similar. Larvae in the L1 stage may have been missed because of their small size and possible concealment in places like turbinates and ethmoid bones, or may have been destroyed in the nasal cavities during the hypobiotic phase (7).

The sheep had the highest infestation rates in summer and spring and the lowest in winter. In summer, the adult O. ovis fly becomes more active, as reported by Dorchies et al. (13), whereas in winter, the larvae experience periods of diapause and a reduction in their growth rate, as indicated by Yilma and Dorchies (47). This study showed that the rate of infestation in sheep was greatly impacted by the varying seasons. This is consistent with the investigation carried out by Özdal et al. (34) and differs from the research by Metwally et al. (26) that showed no significant effect of seasons on nasal botfly infestation levels. A study conducted in Egypt by Amin et al. (3) showed that infestation rates were highest during autumn at 17.91%, while winter had the lowest rate at 7.85%. Concordantly, researchers in Iraq found that the highest percentage was in autumn at 76.92%, while the lowest was in winter at 30.76% (27). Changes in environmental conditions could be the cause of these variations.

The occurrence of oestrosis varied from 4% in January to 45.16% in June. This aligns with findings from Özdal et al. (34), but contradicts Karatepe et al. (22) who found the peak infestation rate to be in October. This research demonstrated that all larval phases were observed consistently during the whole period, except for the absence of first-stage larvae in January, April, November, and December. Shareef (39) observed that Iraq only had second and third instar larvae present. Even though the overall presence of first stage larvae was low in the study, there was a higher prevalence of L1 larvae from June to October, possibly because of new larvae from adult flies. Larvae of O. ovis stop growing during hypobiosis, whether it be in winter in temperate regions or during the hot and dry season in Sahelian countries (47).

We examined how risk factors like gender, age and breed impacted the results. We discovered that the breed of sheep had a notable impact on the rate of infestation. Infection rates were notably more elevated in imported breeds compared to local breeds (26.50% vs 15.65%, P-value < 0.05). Possible explanations for this could involve animals’ adaptation to the surroundings, genetic differences, previous exposures and stress factors. A significantly higher occurrence of oestrosis was observed in female animals in comparison to male animals (27.05% vs 17.08%, P-value < 0.05). The findings align with the previous studies conducted by Uslu and Dik (46) and Shoorijeh et al. (40). On the other hand, Metwally et al. (26), Mohammed et al. (27) and Negm-Eldin et al. (29) observed a higher incidence of the condition in males as opposed to females. The higher number of female animals in the groups could lead to a higher susceptibility to O. ovis larva infestation, which is also influenced by differences in physiology and behaviours between males and females (29). Oestrosis is found in a higher percentage of adult sheep (26.88%) compared to sheep (16.19%) that are 1 year old or younger. This discovery is consistent with those of several research studies (1, 29, 35, 39, 46), but contrasts with those of other investigations (5, 22). One potential explanation for the higher prevalence in mature animals may be their heightened appeal to female flies and their larger nasal openings in comparison to juvenile animals. Furthermore, mature animals exhibit decreased respiratory rates compared to juvenile animals, potentially assisting the female fly in egg laying and larvae entering nasal sinuses. Moreover, a juvenile animal may possess antibodies against oestrosis inherited from its mother, as suggested by Silva et al. (41).

According to Ogo et al. (30), the physical attributes and morphological identification of the larvae should not be relied upon. Molecular identification of fly species causing myiasis can be considered a dependable substitute for morphological identification because of the challenges in identifying larvae at the genus level (18, 30). Bosly (11) discovered O. ovis larvae at a molecular level, demonstrating its potential as a dependable substitute for conventional morphological identification techniques. This study focused on investigating the genetic diversity of O. ovis by analysing mtCOX1 gene sequences extracted from the nasal passages of sheep in Iraq at a molecular level. The mitochondrial DNA enables a more accurate identification of larvae species, with closely related species having unique developmental traits (17, 36).

In our study, the novel mutations resulted in new haplotypes that were previously not reported from any country. However, the sequences from our study had the highest similarity, ranging from 97.40 to 99.83%, with haplotypes isolated from Iraq (accession Nos MW555828–MW555829), Iran (accession Nos MW316742, KX268655), Turkey (accession No. MT124626), Croatia (accession No. MN845130), Bosnia and Herzegovina (accession No. MG755264), India (accession No. ON000070), Mexico (accession No. OR440699), Brazil (accession No. KR820703), and USA (accession No. NC_059851). These findings show that genetic differences may exist among samples collected from various regions. Hence, variations in O. ovis isolates might be due to differences in animal breeds, geographic locations, and climates (10, 29).

Phylogenetic analysis showed that the sheep sequences in this study had intraspecific variations and were highly similar to mtCOX1 gene sequences of O. ovis isolates from sheep in various countries including Iraq, Iran, Türkiye, Croatia, Bosnia and Herzegovina, India, Mexico, Brazil and the USA. On the other hand, Rajabi et al. (38) found that there was no variation within isolated sequences from Iranian sheep. There is a higher level of diversity within the genes encoding proteins in mtDNA when compared to ribosomal genes, as observed by Tan et al. (44). The research suggests that similarities among Iraqi isolates, Iran and Türkiye may be due to common geographical conditions and increased trade, as indicated by molecular alignment and phylogenetic tree analysis (6). Moreover, all O. ovis samples analysed in this study, as well as those retrieved from GenBank, were clustered together in a monophyletic group, consistent with earlier research (10, 26, 29, 38).

The current research indicates that oestrosis is a significant issue for sheep in Sulaymaniyah province in Iraq, and it is anticipated to impact animal production and welfare. The findings showed that the infestation rate was 22.25% in total and was notably higher in imported breeds compared to local breeds, adults compared to young sheep, females compared to males, and during the summer season compared to other seasons. The study area contained five unique haplotypes; two had been identified in Iraq and Iran before, while three were specific to our region. Five new mutations were discovered within regional haplotypes. This study provides valuable molecular information on O. ovis larvae in sheep in Iraq through the analysis of COX1 sequence barcodes. The results indicated a strong correlation among O. ovis strains from various countries, despite the dataset including only a few sequences. The study findings demonstrate that COX1 is an effective molecular marker for identifying and characterising botfly species.