Detection of lymphocystis disease virus (LCDV) from yellowbar angelfish (Pomacanthus maculosus Forsskål, 1775) in Iran: Histopathological and phylogenetic analysis

Highlights

• Whitish clusters of nodules most often on the fins and skin were seen. In addition, wet smears of skin nodules revealed hypertrophic LCDV-infected host dermal cells.

• The hypertrophic lymphocystis cells containing thick smooth hyaline capsules (HC) were revealed in the histopathologic micrographs of the fins and skin whitish nodules and spleen sections.

• Many inclusion bodies which were strongly stained by hematoxylin and eosin (H&E), were observed peripherally near the membrane of hyaline capsule.

• Specific amplicons with an expected size of 609 bp were detected in the homogenate of internal organs and skin lymphocystic lesion samples collected from the moribund fish.

• The phylogenetic tree based on 170 the MCP gene nucleotide sequences revealed that IR_LCDV_RGZIS isolates clustered into the genotype V group.

Abstract

Lymphocystis disease (LCD) is a benign viral disease characterized by the appearance of hypertrophic fibroblastic cells in the connective tissues of fish. In December 2021, a group of yellowbar angelfish (Pomacanthus maculosus Forsskål, 1775) which had significant mortalities (10%) and showed whitish nodules on the fins and skin, were packed in water-filled plastic bags, supplied with oxygen, and transported to the Ornamental Fish Clinic, Faculty of Veterinary Medicine, University of Tehran (Tehran, Iran). Following the clinical inspection and observation of whitish clusters of nodules mostly on the fins and skin by stereomicroscopy, wet smears of skin and fins revealed numerous hypertrophic lymphocystis cells. Histopathological study of the whitish nodules in skin and fins revealed many hypertrophic cells with thick hyaline capsules, irregular nucleus, and multiple intracytoplasmic inclusion bodies. In addition to the extension of hypertrophic lymphocystis cells' growth until the superficial muscle layer, infiltration of inflammatory cells and hemorrhage between hypertrophic cells are also observed. The hyaline capsule of hypertrophic fibroblasts stained with the PAS. Molecular characterization (PCR) confirmed the presence of Lymphocystis disease virus (LCDV) in the external nodules and the internal organs homogenate of the infected fish. Sequence analysis of the MCP gene of the detected virus (IR_LCDV_RGZIS) assigned it to genogroup 5. This is the first detection and characterization of LCDV in yellowbar angelfish (Pomacanthus maculosus) in Iran.

Introduction

Ornamental fish culturing has become one of the most vital and profitable industries in numerous countries all over the world (Monticini, 2010; Mousavi et al., 2020, Mousavi et al., 2021; Rahmati-Holasoo et al., 2022b). Cultivation and propagation of ornamental fish have been increasing in the last decades in Iran (Rahmati-Holasoo et al., 2010b; Shokrpoor et al., 2022). The yellowbar angelfish (Pomacanthus maculosus Forsskål, 1775) is a marine perciform ornamental fish which has been classified into the Pomacanthidae family (Froese and Pauly, 2019; Li et al., 2016; Soeth et al., 2018). This fish species originating from the tropical reef bottoms (Allen et al., 1998), presents an attractive body coloration and is easy to maintain in captivity as a commercial marine ornamental fish (Fenner, 1996; Stratton, 1994). The omnivorous yellowbar angelfish (P. maculosus) is distributed in the western Indian Ocean, Red Sea, the coast of Mozambique, Gulf of Oman, and the Persian Gulf, mostly in areas around the rocky and coral reefs but rarely in the sandy areas (Allen et al., 1998; Froese and Pauly, 2019; Pyle et al., 2010). International Union for Conservation of Nature (IUCN) has mentioned the yellowbar angelfish (P. maculosus) as the least concerned in the list of threatened species (IUCN, 2009).

Iridoviruses are large, complex, and double-stranded DNA viruses (Jancovich et al., 2012; Williams et al., 2005) which are mentioned as the emergent pathogenic agents (Hick et al., 2016) with a host range limited to insects and ectothermic vertebrates such as fish, amphibians, and reptiles (Williams et al., 2005). Lymphocystis disease virus (LCDV), as the causative agent of lymphocystis disease (LCD), has been classified into the genus Lymphocystivirus of the family Iridoviridae, and has an icosahedral symmetry with a capsid of approximately 200 ± 50 nm in diameter (Kitamura et al., 2006). Lymphocystis, a benign viral disease (Wolf, 1988), has a global distribution and would affect more than 140 marine and freshwater fish species from 34 families worldwide (Anders, 1989; Stoskopf, 2015). Currently, complete genome sequences of three LCDV strains, Lymphocystis disease virus 1 (LCDV-1) from European flounder Platichtys flesus (L.) in Europe and tentative species including Lymphocystis disease virus 2 (LCDV-2), and Lymphocystis disease virus China (LCDV-C) from Japanese flounder Paralichthys olivaceus in China were determined (Tidona and Darai, 1997; Williams et al., 2005; Zhang et al., 2004). In addition, another recently-discovered lymphocystivirus has been isolated from white mouth croaker (Micropogonias furnieri) and suggested as LCDV-4, although not yet accepted by the International Committee on Taxonomy of Viruses (ICTV) (Doszpoly et al., 2020).

Lymphocystis disease has been reported in several fish species, such as flower horn fish (hybrid cichlid) (Rahmati-Holasoo et al., 2010a), false clownfish (Amphiprion ocellaris) (Pirarat et al., 2011), black rockfish (Sebastes schlegelii) (Zheng et al., 2016), true clownfish (Amphiprion percula) (Lam et al., 2020), whitemouth croaker (Micropogonias furnieri) (Doszpoly et al., 2020; Perretta et al., 2020), gilthead sea bream (Sparus aurata) (Cherif et al., 2020), grass carp (Ctenopharyngodon idella) (Shahi et al., 2020), and Indian glassy fish (Parambassis ranga) (Rahmati-Holasoo et al., 2022a).

Viral diseases of ornamental fish have rarely been reported in Iran. Koi herpesvirus (KHV) and carp pox from koi carp (Cyprinus carpio L.), and lymphocystis disease from a flower horn fish have been reported (Rahmati-Holasoo et al., 2010a, Rahmati-Holasoo et al., 2016, Rahmati-Holasoo et al., 2020). This study provides the first detection of lymphocystis disease (LCD) based on the clinical, histopathological and molecular characterization of yellowbar angelfish (P. maculosus) in Iran.