Histopathological, morphological, and molecular characterization of fish-borne zoonotic parasite Eustrongylides Excisus infecting Northern pike (Esox lucius) in Iran

Abstract

Eustrongylides excisus is a fish-borne zoonotic parasite known to infect various fish species, including Northern pike (Esox Lucius). This nematode, belonging to the family Dioctophymatidae, has a complex life cycle involving multiple hosts. This study aimed to investigate the occurrence of Eustrongylides nematodes in Northern pike (E. Lucius) collected from Mijran Dam (Ramsar, Iran). Between June and October 2023, an investigation was conducted on Northern pike from Mijran Dam in Ramsar, Iran, following reports of reddish parasites in their muscle tissues. Sixty fish were examined at the University of Tehran, revealing live parasites in the muscles, which were then analyzed microscopically and preserved for a multidisciplinary study. The skeletal muscle tissues of 85% (51/60) of fish specimens were infected by grossly visible larvae which were microscopically identified as Eustrongylides spp. In histopathological examination, the lesion was composed of encapsulated parasitic granulomatous myositis. Microscopically, the cystic parasitic granulomas compressed the adjacent muscle fibers, leading to their atrophy and Zenker's necrosis. Moreover, epithelioid macrophages, giant cells and mononuclear inflammatory cells were present around the larvae and between the muscle fibers. Finally, a molecular analysis by examining the ITS gene region, revealed that they belong to the species E. excisus. Eustrongylidiasis in northern Iran necessitates further research into the biology, epidemiology, and control of Eustrongylides nematodes, focusing on various hosts. This study is the first to comprehensively characterize E. excisus in Northern pike in Ramsar, Iran, raising concerns about possible zoonotic transmission.

Keywords: Eustrongylides excisus; Freshwater ecosystem; Granulomatous myositis; Iran; Northern pike.

Fig. 1

(a-d) Macroscopic aspect of Northern pike (E. lucius) invasion with E. excisus. (a) Nematodes are found within the supra-axial muscles and in close proximity to the vertebral column (arrows). (b) Tissue reaction to the presence of parasites in the muscle tissues (arrowheads) and the nematode in motion (arrow). (c) Nematodes are found in supra-axial muscles (arrows), and dark red alive nematodes are removed from the fish (arrowheads). (d) Significant discoloration of multiple live nematodes 9 days after isolation from the fish (white arrowheads) compared to the nematode 2 days after isolation from the fish (black arrowhead)

Fig. 2

(a-d) Microscopic aspect of Eustrongyloides excisus isolated from Northern pike (E. lucius). (a) Dark-field and DIC images of the anterior region of a live nematode. Two rows of labial papillae (both inner (white arrows) and outer (black arrows)) are observed (b) A light microscope image of the anterior extremity of an Azocarmine-stained nematode displaying neural rings (white arrow) and inner papillae (arrowhead). Two cuticle layers are observed (black arrows). (c) Dark-field and DIC images of the posterior region of a live female nematode. (d) A light microscope image of the posterior extremity of a live male nematode and an Azocarmine-stained nematode

Fig. 3

(a) The scanned image depicts a section of muscular tissue, revealing presence of parasites between the epaxial muscle fibers (EM). The nematodes have replaced large portions of the epaxial skeletal muscle. (b) Longitudinal (arrowhead) and transverse (arrow) sections of the parasite. (c-d) The cuticle (arrowhead), muscle fiber (arrow), esophagus (white asterisk), and intestine (black asterisks) in the transverse section of the parasite are seen

Fig. 4

(a) Atrophy of muscle fibers (arrowheads) is seen. (b) Segmental necrosis (arrowheads). (c) Inflammatory cell infiltrations into the inter- (asterisk) and intramuscular (arrowheads) fibers are seen. (d) Note the presence of epithelioid macrophages (arrowheads)

Fig. 5

(a) A binucleated cell (arrowhead) is seen between muscle fibers. (b) Vacuolated cells (arrows) and eosinophilic material (arrowhead) in the lumen of the cyst are present. Two cuticle layers are observed (white arrowheads). (c-d) Masson’s trichrome is positive for collagenous fibers (arrowheads) surrounding the parasite. Muscle fibers (asterisk). External cuticle layer in red color and internal cuticle layer in blue color are seen (arrows)

Fig. 6

Molecular phylogenetic analysis based on the nucleotide sequences of the ITS rDNA gene was performed using the maximum likelihood method and the general time reversible model. The tree was generated for comparison of two isolates in the present study and some previously submitted to NCBI GeneBank