Morphological and Molecular Detection of Dicrocoelium dendriticum Infection in Ruminants

Introduction
Dicrocoelium dendriticum, called lancet liver fluke, is a common parasitic liver and gall bladder disease in wild and domestic animals. It is also observed in horses, camels, rabbits, pigs, and humans (Scala et al., 2019). In addition, the human infection rate has increased in recent years (Jafari Rad et al., 2022; Abdollahpour et al., 2023). D. dendriticum is an important species in economic industries and veterinary medicine (Arbabi et al., 2018). Dicrocoelium has a complicated life cycle (Iyaji et al., 2018; Hosseini et al ., 2022). Its first intermediate hosts are various species of snails, and its second intermediate hosts are various species of ants. The disease can cause diarrhea, weight loss, anemia, digestive disorders, edema, growth delay, reduced mild production, biliary obstruction cholangitis, acute urticarial, and cirrhosis (Ofori et al., 2015). Dicrocoeliosis, a food-borne zoonotic disease, is caused by three species of Dicrocoelium, including Dicrocoelium hospes and Dicrocoelium chinensis (Khan et al., 2023). D. hospes is an endemic disease in West Africa, D. chinenisis in Europe and eastern Asia, and D. dendriticum endemic in Asia, Europe, North Africa, and America (Chougar et al., 2019; Lianou et al., 2023). This study aimed to characterize the morphology and molecular characteristics of the parasite D. dendriticum in cattle and sheep and to detect its various species in these animals.

Materials and Methods

Sample collection
A total of 122 liver samples were taken from 64 sheep and 58 cattle slaughtered in the different areas in Mosul City for both sexes. The found worms were placed in 70% ethanol alcohol and stored at -20°C for the polymerase chain reaction technique (Mahdee et al., 2022). 

Morphological examination 
Fifteen adult worms were selected randomly from the livers of sheep and cattle. The worm was placed between two glass slides, treated with formalin acetic acid alcohol solution, stained with hematoxylin, and mounted in Canada balsam (Ebrahim Pour et al., 2020). Morphometric measurements of parasites were performed under the light stereo microscope according to the study by Jwher et al. (2021). The macroscopic examination of the liver involved its color, consistency, and size (Makawi et al., 2023; Kadhum et al., 2022). 

DNA extraction
According to the manufacturer’s instructions, the DNA of 20 worms was extracted using a pure column DNA extraction kit, Bioron. Restriction fragment length polymorphism (RFLP)-PCR was performed to detect the DNA of D. dendriticum. The amplification of DNA was done using a specific primer to D. dendriticum, NADH dehydrogenase subunit gene (nad1), the primer Forward (5′-TTAAAACACACCCACCAGAA-3′), and primer reverse (5′-GTGTTTTGGTTTAATGTTTT-3′) (Gorjipoor et al., 2015). The reaction solution of 25 µL contained 5 µL of DNA extracted. Then, 1 µL of each primer, 12 µL of mater mix (IDAA TM–kit), and 6 µL of distilled water were mixed. The amplification program includes keeping at 95ºC for 5 minutes with 30 cycles at 95ºC for 2 minutes, at 58.4ºC for 1 minute, and 72ºC for 2 minutes. Final amplification products were diagnosed using 1% agarose gel electrophoresis (900 bp), after which 2 µL of Thermo Scientific™ FastDigest Green Buffer (10 x) (Thermo Fisher Scientific Inc.) was added to 10 μL of PCR mix with 18 µL of distilled water. Afterward, the mixture was incubated for 40 minutes at 37°C. The reaction products (206 and 694 bp) were analyzed by 1% agarose gel electrophoresis (Shokouhi et al., 2018).

Statistical analysis
Statistical analysis was done using the chi-square test in SPSS software, version 17 (SPSS Inc., Chicago, USA). 

Results
The infection rate of D. dendriticum was 23%, and the infection rates in sheep and cattle were 34.4% and 10.7%, respectively. The results show a significant difference in infection in both sheep and cattle. The results are not significant at P<0.05 between males and females for both sheep and cattle (Table 1).

Morphometric measurements of D. dendriticum in sheep and cattle are presented in Table 2.

It also shows that the mean of the internal-external diameter of the oral and ventral suckers, in addition to diameters of testes, vitelline glands (length, width), and the testes orientation in the D. dendriticum (Tandem testes) (Figures 1 and 2).

The current study clarifies that D. dendriticum is the only species isolated from sheep and cattle, according to morphometric and molecular studies. Macroscopic diagnosis of the infected livers shows discoloration, swelling, wrinkled spots, hyaline and biliary fibrosis, and lack of sharpness of liver margins (Figure 3).

In addition, many flukes are detected in the liver’s bile ducts. As for the results of molecular analysis, the amplification of DNA of worms by using PCR-RFLP used a specific primer of D. dendriticum nad1 fragment (900 bp), and then digestion to produce 206 and 694 bp fragments. All samples were positive for D. dendriticum (Figure 4).

Discussion
Dicrocoeliosis is one of the important parasitic diseases in veterinary medicine that infects the livers of domestic and wild animals (Kleiman et al., 2007). According to the current study, the percentages of infected sheep and cattle slaughtered were 34.4% and 10.7%, respectively, with significant differences. These results agree with the previous study from another country by Ebrahim Pour et al., (2020), which declared that the infection percentages in sheep and cattle were 36.7% and 6.09%, respectively. Ahmadi and Mahdavi (2010) reported that the infection percentages in sheep and cattle were 22.4% and 4.5%, respectively. The differences in the percentage of D. dendriticum in Iraq are probably due to the grazing behavior, environmental conditions, and management system. In addition, the current study evaluated the influence of sex for the host on D. dendriticum and found no statistically significant difference for the sex in the percentages of this infection (Maurelli et al., 2007).
All specimens collected from the sheep and cattle were morphologically identified as D. dendriticum based on the measurement and morphological keys, such as body length, maximum body width, testes orientation, and other morphometric measurements, and this result agreed with Sandoval et al. (2013) study. In addition, other studies in Austria, Italy, and Germany showed that the location of testes in D. dendriticum determined the species of Dicrocoelium. The testicles of D. dendriticum are tandem but bilateral in D. chinensis (Javanmard et al., 2022). This finding is in agreement with our current study. The macroscopic observation of infected livers aligned with the results described by Liu et al. (2014). Manga Gonzalez et al. (2010) showed discoloration, swelling, hyaline, change of liver margins, and biliary fibrosis. The results of the molecular techniques of this current study agree with the results described by Gorjipoor et al., (2015). However, according to morphometric measurements and molecular techniques, D. dendriticum exclusively causes diploidosis in sheep and cattle in Mosul, Iraq. 

Conclusion 
This study is the first to identify D. dendriticum in Mosul City, Iraq. The information gathered from the current research will reveal the rate of D. dendriticum infection in Iraq. Moreover, these parasites must be molecularly identified to be controlled and prevented.

Ethical Considerations

Compliance with ethical guidelines
Ethical approval was issued by the Institutional Animal Care and Use Committee of the College of Veterinary Medicine, University of Mosul on September 6, 2022 (Code: UM.VET.2022.16).

Funding
This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors. 

Authors' contributions
All authors equally contributed to preparing this article.

Conflict of interest
The authors declare no conflict of interest.

Acknowledgments
All authors thanks of College of Veterinary Medicine, University of Mosul.

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