Case Report: First occurrence of Lymphocystis disease virus 3 (LCDV-Sa) in Wild Marine Fish in Tunisia

Lymphocystis Disease (LD) is a rarely fatal, chronic and slowly developing disease, that affects over 150 different marine and fresh water fi sh species [1-8]. These include species that are of a particular importance for fi sh farming, such as Sparus aurata (gilthead sea bream). The typical sign of lymphocystis disease is the presence of small pearl-like nodules on the skin and fi ns of affected fi sh, that may occur singly or more generally grouped in raspberry-like clusters of tumorous appearance [9]. The external clinical signs of affected fi sh make them unmarketable [10] and more susceptible to other bacterial, viral or parasitic infections; increasing mortality rates and important economic loss. The etiological agent of LCD is the Lymphocystis Disease Virus (LCDV), a member of the genus Lymphocystivirus, family Iridoviridae. The Major Capsid Protein (MCP) gene represents an important molecular marker for the LCDV genotyping [11,12]. Data obtained on the basis of MCP sequences currently support the existence of nine genotypes in the genus Lymphocystis virus [13]. The genotype VII represents isolates obtained from sea bream and senegalese sole Solea senegalensis [14].


Introduction
Lymphocystis Disease (LD) is a rarely fatal, chronic and slowly developing disease, that affects over 150 different marine and fresh water fi sh species [1][2][3][4][5][6][7][8]. These include species that are of a particular importance for fi sh farming, such as Sparus aurata (gilthead sea bream). The typical sign of lymphocystis disease is the presence of small pearl-like nodules on the skin and fi ns of affected fi sh, that may occur singly or more generally grouped in raspberry-like clusters of tumorous appearance [9]. The external clinical signs of affected fi sh make them unmarketable [10] and more susceptible to other bacterial, viral or parasitic infections; increasing mortality rates and important economic loss. The etiological agent of LCD is the Lymphocystis Disease Virus (LCDV), a member of the genus Lymphocystivirus, family Iridoviridae. The Major Capsid Protein (MCP) gene represents an important molecular marker for the LCDV genotyping [11,12]. Data obtained on the basis of MCP sequences currently support the existence of nine genotypes in the genus Lymphocystis virus [13]. The genotype VII represents isolates obtained from sea bream and senegalese sole Solea senegalensis [14].
Tunisia imports more than 70% of the juvenile fi sh needed for marine cage farming. Currently, the aquatic bio-security and fi sh health management protocols used are minimal. Existing measures for disease prevention rely on the use of general prophylactic practices, such as good husbandry practices, reduced stocking density and enforcing the virological control of specimen to be introduced in the farming sites in order to detect carrier fi sh. This manuscript describes the detection of lymphocystis disease virus (LCDV) in gilthead sea bream, from both asymptomatic and diseased fi sh, collected at several Tunisian farms. It also describes the detection of LCDV in a number of asymptomatic specimens belonging to different wild fi sh species collected around farming facilities.

Materials and methods
During the routine auto-control for viral diseases by Tunisian fi sh farmers during 2015-2016, gilthead sea bream

Abstract
The results of the present study describe the molecular identifi cation of Lymphocystis Disease Viral partial genome (LCDV-Sa) and histopathology in skin nodules and internal organs of Tunisian gilthead sea bream (Sparus aurata). The report supports as well as the existence of multiple reservoirs of LCDV within wild fi sh species caught near the cage facility; delivering a list of possible vector species susceptible to transmit the disease to farmed sea bream. Histology sections revealed irregular nucleus with basophilic cytoplasmic inclusions. In addition, to the best of our knowledge, molecular results added new viral reservoirs to the list of susceptible fi sh species described worlwide, including Sardinella aurita, Sardina pilchardus, Trachurus trachurus, Sarpa salpa, Diplodus vulgaris, Diplodus puntazzo, Liza aurata, Sparus aurata, Diplodus anularis, and Spicara maena. The virus detection was not correlated with neither the fi sh species nor the sampling temperature which varied between 15°C and 25°C. Partial sequence analysis of the MCP gene indicated that the newly identifi ed LCDV strains were clustered within genotype VII and shared 96-100% of sequence identity with previously identifi ed Tunisian LCDV sequences from farmed sea bream.
fi sh were randomly collected from four production facilities located at the Sahel region (Central-East coast), approximately 8 to10 kilometers far from each other. A total of 37 pooled samples were obtained from clinically ill and healthy Sparus aurata specimens, belonging to different growth stages were analyzed by molecular tools in order to detect LCDV. Samples included newly imported juveniles from different South European hatcheries. In addition, 96 wild fi sh specimens were caught around or inside S. auarta cages and processed in order to screen new viral reservoirs. Wild specimen species were identifi ed based on Food and Agriculture Organization fi sh identifi cation sheets [15].
Internal organs (liver, kidneys, and spleen), nervous tissues The phylogenetic tree was constructed using the MEGA6 with UPGMA method and the fi nal phylogenetic tree was drawn with the Coral DRAWX6 program. Because the sequences from GenBank were heterogeneous in length, the longer sequences were cut and adapted to the shortest. The reliability of the tree was inferred using the bootstrap method with 1000 replicates [18]. The partial nucleotide sequences of the LCDV strains screened in the present study were not deposited in Gen Bank because they are less than 200 pb.

Results
Histological results revealed that the lesions sampled from the skin and fi ns of infected S. aurata exhibited several characteristics that are known to be associated with LCD: ie,    Figure  2). The newly listed sequences were not deposited to a public access database as they were less than 200 nt.

Discussion
LCDV outbreaks are frequently observed in the Mediterranean gilthead sea bream aquaculture [19] even though, it is usually described as a self-limiting disease, there have been several reports on mortalities ranging up to 45 % in juvenile fi sh, which were related to secondary bacterial infections. Alternatively mortality may be linked to lymphocystis lesions which may severely impair fi sh respiration and/or feeding [20,21].
Very little proactive viral surveillance in North Africa there has been conducted, subsequently, few epidemiological data related to this virus in this region of the Mediteranean sea is available.
African Lymphocystis viral isolates have only been identifi ed in Cichlids; including species of Tilapia in Lakes Victoria (Nyanza) (Oreochromis variabilis and Haplochromis spp.), in Lake George (H. elegans) and in Lake Kitangiri (Tilapia amphimelas and O. esculentus) in East Africa [22]. In Tunisia, LCDV has only been identifi ed in farmed sea bream (S. aurata) specie [23].
The results of the present study support the existence of multiple reservoirs of LCDV at the farm facilities. To the best of our knowledge, this report describes the fi rst identifi cation of lymphocystis disease virus from wild Tunisian fi sh delivering a list of possible vector species susceptible to transmit the disease to farmed sea bream.
Genetic variations have been detected among LCDVs isolated from different hosts [10,24,25]. These affected species belong to evolutionarily advanced orders of teleosts fi shes, including these families: Cichlidae, Osphronemidae, Centrarchidae, Gobiidae, Chaetodontidae, Pomacentridae, Sciaenidae, Serranidae and Pleuronectidae. As far as the authors are aware, LCDV has not been previously reported in less-advanced fi sh orders, such as Siluriformes, Cyprinids and Salmonids.
The Iridoviridae family has been determined to have highly conserved regions within the MCP gene [11] making it an ideal target for the identifi cation of the virus. On the basis of LCDV Tunisian sequences that have been published to date, low genomic variability was observed among isolates from different fi sh farms spaced over 7 years. The survey indicates that the newly identifi ed LCDV isolates were clustered within genotype VII and shared 96-100% of sequence identity with other Tunisian LCDV isolates previously identifi ed in 2005 [13] and in 2011 [10]. Sequences were also closely related to strains isolated from other regions of south Europe [13]. This could be due to the active trade of fi sh among farms located in the Mediterranean Sea (Spain, Italy, France and Turkey), which make it diffi cult to draw any conclusion about the geographical distribution of the isolates. Full genome sequencing would allow a more thorough determination of the phylogenetic relationship between local and imported LCDV strains and

Acknowledgement
The present work was funded by the Tunisian ministry of Agriculture (IRESA, 03/0013). We generously thank Dr. Dolores Castro for her precious help to initiate and revise this work by supplying the LCDV-positive control and unpublished PCR primers and protocols to our lab.
The authors declared no potential confl icts of interest with respect to the research, authorship, and/or publication of this article.