key: cord-009865-c5xj9asg
authors: Ortega, Cesar; Cañas‐Lopez, Leticia; Irgang, Rute; Fajardo, Raúl; Poblete‐Morales, Matías; Valladares-Carranza, Benjamin; Tapia‐Cammas, Diana; Avendaño‐Herrera, Ruben
title: First detection of spring viraemia of carp virus in common carp (Cyprinus carpio L.) affected by a septicaemic disease in Mexico
date: 2019-03-15
journal: J Fish Dis
DOI: 10.1111/jfd.12969
sha: 
doc_id: 9865
cord_uid: c5xj9asg

Spring viraemia of carp (SVC) is an infectious disease responsible for severe economic losses for various cyprinid species, particularly common carp (Cyprinus carpio carpio). The causative agent is the SVC virus (SVCV), a member of the Sprivivirus genus, Rhabdoviridae family, and a List 1 pathogen notifiable by the World Organization for Animal Health. This study describes the diagnosis of an SVCV pathogen isolated in October 2015 from wild common carp inhabiting a natural lagoon in central Mexico. While neither an epidemic nor fish mortalities were reported, the collected killed specimens exhibited clinical signs of disease (e.g., exopthalmia, moderate abdominal distension and haemorrhaging, as well as internal haemorrhages and adhesions). Histological results of injuries were consistent with the pathology caused by SVCV. This finding was supported by the isolation of a virus in EPC and BF‐2 cells and subsequent RT‐PCR confirmation of SVCV. The phylogenetic analyses of partial SVCV glycoprotein gene sequences classified the isolates into the Ia genogroup. These findings make this the first report of SVCV detection in Mexico, extending the southern geographical range of SVCV within North America. However, since this pathogen was detected in fish inhabiting a natural body of water without tributaries or effluents, it is difficult to estimate the risk of SVCV for other wild/feral cohabitating cyprinid species in the lagoon. The status of this virus is also unknown for other bodies of water within this region.

The common carp (Cyprinus carpio L) is a freshwater and brackish fish originated in Asia and has been farmed in China since at least 475 BC. Due to its ability to tolerate eutrophic waters and adverse environmental conditions, carp are farmed across a wide range of geographical and climatic areas worldwide (FAO, 2018) . The common carp was introduced to Mexico from Asia in 1889 (Wakida-Kusunoki & Amador-del-Ángel, 2011) and is now distributed across the country, with the purpose of providing animal protein to rural populations and additional work opportunities to the community (Wakida-Kusunoki & Amador-del-Ángel, 2011) . In 2017, carp production reached 53,421 tons, reflecting an annual estimated growth of 7.43% (CONAPESCA, 2017) .

Despite resilience to a wide range of environmental, climatic and geographical conditions, carp are susceptible to a variety of infectious diseases. Primary among these diseases is the spring viraemia of carp virus (SVCV), a notifiable pathogen according to the World Organization for Animal Health (OIE, 2017a) and a virus responsible for mortalities associated with acute haemorrhaging. To date, SVCV has been reported in common carp and other freshwater cyprinids in Asia and Europe, while detection on the American continent has been reported in the United States, Canada (Ahne et al., 2002; Ashraf et al., 2016; Garver et al., 2007; Goodwin, 2011) and

Brazil (Alexandrino, Ranzani-Paiva, & Romano, 1998; OIE, 2017a) .

Nevertheless, SVCV has not yet been detected in Mexico.

SVCV, the type species of the Sprivivirus genus and member of the Rhabdoviridae family (Afonso et al., 2016; ICTV, 2017; OIE, 2017a) , exhibits a genome structure that includes five genes encoded in the order 3-N-P-M-G-L-5, representing the nucleocapsid, phosphoprotein, matrix protein, glycoprotein and polymerase protein genes, respectively. SVCV infects different carp species, as well as other cyprinids and ictalurids (Ashraf et al., 2016) , and poses sanitary concerns in the global aquaculture industry. Other aquatic rhabdoviruses are also a threat to wild and farmed fish species. These include the viral haemorrhagic septicaemia virus (VHSV) and the infectious haematopoietic necrosis virus (IHNV) of the Novirhabdovirus genus, both of which are also listed as notifiable aquatic animal diseases by the OIE (2017a).

In Mexico, sanitary control measures associated with diseases in farmed carp are scarce, especially in relation to viral agents. López-Jiménez (1987) reported on the occurrence of non-published cases of infectious ascites attributed to bacterial infections of Aeromonas spp. and Pseudomonas spp., whereas the occurrence of viral cases was largely ruled out, excepting one case in which coronavirus-like viral particles were microscopically identified. In turn, Vélez-Hernández, Constantino-Casas, García-Márquez, and Osorio-Sarabia (1998) All 10 specimens were collected from Tecocomulco, a natural freshwater lagoon in the state of Hidalgo that is approximately 1,769 hectares in size and without tributaries or effluents (Rico-Sánchez, Rodríguez-Romero, López-López, & Sedeño-Díaz, 2014) . The lagoon is formed by run-off and springs from the surrounding mountains, has an average length of 10.5 km, is between 1.0 and 3.5 km wide and has a depth, depending on periods of drought, between 70 cm and 2 m (Novelo, Montejano, Cantoral, & Tavera, 2005) . No disease outbreaks and/or disease-related mortalities have been reported for the lagoon for any of the different fish populations. Common carp (≈250 g) were captured using a net that traps the fish through the gills. Many of the collected fish presented signs of septicaemic disease, characterized by moderate exophthalmia, abdominal distension and haemorrhaging in the ventral and lateral areas, as well as at the base of the fins.

All fish were killed via anaesthetic overdose (240 mg/L tricaine methanesulfonate 222; Sigma) and immediately subjected to postmortem examination. Initial external observations were conducted, and the lesions of each specimen were recorded. Examinations were then conducted for the presence of external parasites, and scrapings were obtained from gills and skin for microscopic observation at 10× and 40× magnifications. In addition, the microscope slides were Gram-stained.

For histological analyses, internal samples were taken from each fish, fixed in vials containing 10% buffered formalin, dehydrated and embedded in paraffin wax following standard procedures. Each tissue was sectioned at 5 μm and stained with haematoxylin and eosin to describe histopathological alterations, as in Ortega et al. (2016) . Sections were observed at different magnifications under an Olympus BH-2 light microscope.

The aspect, colour and contents of the body cavity were also reviewed. Samples for bacterial isolation were aseptically taken from external skin lesions, the kidney, spleen, liver and brain of each specimen and streaked onto tryptone soy agar (TSA; Oxoid) and Columbia sheep blood agar (AES Laboratoire, France). All plates were aerobically incubated at 28°C and examined every 24 hr for 7 days. If no colony growth was observed after a week, the cultures were considered negative.

The clinical signs presented by the collected carp led to the presumption that an agent similar to SVCV could be responsible. To blind-passed at least once before being classified as negative.

Electron microscopy was carried out on EPC monolayers grown in 6-well tissue culture plates. The cells were inoculated with supernatants from CPE-positive cultures of Carp-1 and Carp-2. At 48 hr post-inoculation, the cell culture medium was removed. The monolayers were harvested by scraping, centrifuged for 1 hr at 500 g, fixed in glutaraldehyde (2.5% in 0.1 M sodium cacodylate buffer, pH 7.4) and post-fixed in 2% osmium tetroxide for 1 hr.

Cells were dehydrated with ascending concentrations of acetone and then embedded in epoxy resin (Epon 812; Electron Microscopy Sciences) and acetone. Semi-fine slices (150 μm) were obtained using an ultramicrotome and were mounted on slides contrasted with toluidine blue (Electron Microscopy Sciences).

Once the area of interest was identified, fine slices (80 μm) were obtained and mounted on copper grids contrasted with uranyl acetate and lead citrate (Electron Microscopy Sciences). Finally, samples were observed using a Jeol 1010 electron transmission microscope at 60 kV.

Cell cultures were collected for virus identification when CPE ap- The amplified product was then assessed through semi-nested PCR designed to amplify a 606-bp product (primers: SVCV-F1 and SVCV-R4). The amplification procedure for both protocols adhered to those described by Stone et al. (2003) . All PCR amplifications were carried out in a G-storm Thermal Cycler T Gradients and were separated by electrophoresis on 1.5% (w/v) agarose gel, visualized with 1/10,000

GelRed Nucleic Acid Gel Staining (Biotium) and photographed under UV light. A GeneRuler 100 bp Plus DNA Ladder (Thermo Scientific) was employed as a molecular mass marker. A sample was considered positive if the expected sizes of the primary and secondary PCR products were 714 and 606 bp, respectively. 

This report describes an SVCV rhabdovirus as the cause of an infective outbreak among wild common carp in Mexico. While SVCV can replicate in fish, bird and mammalian cell cultures, optimal cell systems are those derived from cyprinid fish, such as EPC (Fijan et al., 1983) , which grows at 20-22°C. The CPE of SVCV is characterized by a margination of nuclear chromatin followed by a rounding up, detachment and lysis of cells. The presently obtained results are consistent with these descriptions. However, given that SVCV is closely related to the pike fry rhabdovirus, an SVCV-specific RT-PCR was used to obtain a more precise identification (Stone et al., 2003) . The second round of the semi-nested PCR protocol amplified a prominent 606-bp product corresponding to nucleotides 373-978 of the SVCV glycoprotein. Genetic clustering and geographical origin are closely linked for SVCV strains, which can be divided into the Asian and European clades (Miller et al., 2007; Stone et al., 2003) . Phylogenetic analysis of the partial glycoprotein genes obtained for the SVCV isolates (Carp-1 and Carp-2) for mortalities in adult rainbow trout. Likewise, tilapia, zebrafish and even trout could act as carriers of this viral agent (Emmenegger et al., 2016; OIE, 2017b) .

leads to the perivasculitis, degeneration and, ultimately, necrosis of blood cells, which manifests as disseminated haemorrhages F I G U R E 2 Histological injuries in common carp affected by an SVCV pathogen. (a) Liver with perivascular haemorrhaging (asterisk), hepatic degeneration represented by karyolysis (arrow) and pyknosis (arrowhead); (b) lymphocytic pancreatitis (arrows), necrosis (arrowhead) and periacinar lymphocytic infiltration (inset); (c) kidney with haemorrhagic (asterisk) and interstitial mononuclear (arrow) nephritis, as well as tubular dilation with proteinuria (arrowhead); (d) splenic congestion (asterisk) and reticuloendothelial hyperplasia (arrowhead), as well as multifocal haemosiderosis (arrow); (e) interstitial enteritis (lamina propria; asterisk) and necrotic cryptitis (arrow) with epithelial necrosis; and (f) epithelial scaling and villous atrophy (arrow), as well as granulomatous ulcerative colitis (asterisk) [Colour figure can be viewed at wileyonlinelibrary.com] et al., 2002; Gaafar et al., 2011; Negele, 1977) .

Surprisingly, however, no signs of disease or fish mortalities have been previously described for the Tecocomulco Lagoon, a situation that is difficult to explain or justify. It is important to note that while this lagoon is rurally located, it is visited by carp fishermen and tourists or weekend visitors. The Tecocomulco Lagoon is also host to several other species, including fish, such as charal (Chirostoma sp.), carps (Cyprinus spp., Ctenopharyngodon idella, Hypophthalmichthys molitrix, Amblycephala megalobrema, Mylopharyngodon piceus) and

goldfish; amphibians, such as axolotl (Ambystoma velasci) and anurans like Rana moctezumae; and reptiles, such as the aquatic snake (Thamnophis eques).

Although Carp-1 and Carp-2 were isolated, the capacity and extent to which these isolates could cause mortality to carps and other fish species within the Tecocomulco Lagoon are unknown. (Novelo et al., 2005; Quiroz-Flores, Ramírez-García, & Lot-Helgueras, 2014) , macroinvertebrate populations (Rico-Sánchez et al., 2014) and pesticide presence/levels (Aguilar-Martínez, 2007) .

Therefore, the impact of the isolated SVCV pathogen on fish populations in the Tecocomulco Lagoon remains unclear, and information regarding mortalities associated with this or prior cases is unavailable. This information gap could be due to the large size of the lagoon (1,769 ha; Rico-Sánchez et al., 2014) or, perhaps, due to the asymptomatic nature of affected fish, which act as SVCV carriers (Ahne et al., 2002) . Furthermore, the lack of an active health-monitoring programme in Mexico (Ortega, 2012) and scarce knowledge of aquatic environments have conditioned local residents and fishermen to view low mortality rates as "normal," without concerns for identifying the cause. In the present case, the responsible laboratory only received samples and notified the health authority of suspected SVCV.

In fact, only five of the 10 sampled fish evidenced classical clinical signs consistent with an SVCV infection, which is a notable finding to consider for future health and research assessments. The obtained results are similar to those described by Garver et al. (2007) , who isolated SVCV from asymptomatic common carp in Lake Ontario & Macías-García, 2000) , which are species susceptible to or that act as carriers for SVCV (Ahne et al., 2002; Ashraf et al., 2016) .

In any case, the occurrence of this case should alert health authorities to be on guard for other cases. For example, after the first SVCV outbreak at a koi farm in North America in 2002, eight subsequent SVCV detections or outbreaks have been recorded (Emmenegger et al., 2016) . This exotic virus could, therefore, represent a potential threat to native and culture fish populations in Mexico. Further contributing to this potential threat is that one of the factors related to SVCV appearance is the temperature range in which fish live, with 10-17°C being optimum (Ahne et al., 2002) .

In Europe, the appearance of SVCV is related to temperature increases after winter. In the case of the Tecocomulco Lagoon, the water temperature fluctuates yearly between 13.1°C and 21.2°C (Delgadillo, 2012; Novelo et al., 2005; Quiroz-Flores et al., 2014) ;

during the sampling period, the temperature range was closer to 13.1-15.1°C (Delgadillo, 2012) . 

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