Skin Lesion-Associated Pathogens from Octopus Vulgaris: First Detection of Photobacterium Swingsii, Lactococcus Garvieae and Betanodavirus
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Vol. 115: 147–156, 2015 DISEASES OF AQUATIC ORGANISMS Published July 23 doi: 10.3354/dao02877 Dis Aquat Org Skin lesion-associated pathogens from Octopus vulgaris: first detection of Photobacterium swingsii, Lactococcus garvieae and betanodavirus G. Fichi1,*, G. Cardeti2, S. Perrucci3, A. Vanni3, A. Cersini2, C. Lenzi3, T. De Wolf4, B. Fronte3, M. Guarducci1, F. Susini1 1Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, S.S. dell’Abetone e del Brennero 4, 56123 Pisa, Italy 2Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, Via Appia Nuova 1411, 00178 Roma, Italy 3Dipartimento di Scienze Veterinarie - University of Pisa, Viale delle Piagge 2, 56124 Pisa, Italy 4Maricoltura di Rosignano Solvay, Via P. Gigli snc, 57016 Rosignano Solvay, Italy ABSTRACT: The common octopus Octopus vulgaris Cuvier, 1798 is extremely important in fish- eries and is a useful protein source in most Mediterranean countries. Here we investigated patho- gens associated with skin lesions in 9 naturally deceased specimens that included both cultured and wild common octopus. Within 30 min after death, each octopus was stored at 4°C and microbi- ologically examined within 24 h. Bacterial colonies, cultured from swabs taken from the lesions, were examined using taxonomical and biochemical analyses. Vibrio alginolyticus and V. para- haemolyticus were only isolated from cultured animals. A conventional PCR targeting the 16S ribosomal RNA (rRNA) gene and sequencing were performed on 2 bacterial isolates that re- mained unidentified after taxonomical and biochemical analysis. The sequence results indicated that the bacteria had a 99% identity with Lactococcus garvieae and Photobacterium swingsii. L. garvieae was confirmed using a specific PCR based on the 16S-23S rRNA internal transcribed spacer region, while P. swingsii was confirmed by phylogenetic analyses. Although all animals ex- amined were found to be infected by the protozoan species Aggregata octopiana localised in the intestines, it was also present in skin lesions of 2 of the animals. Betanodavirus was detected in both cultured and wild individuals by cell culture, PCR and electron microscopy. These findings are the first report of L. garvieae and betanodavirus from skin lesions of common octopus and the first identification of P. swingsii both in octopus skin lesions and in marine invertebrates in Italy. KEY WORDS: Common octopus · Vibrionaceae · Streptococcaceae · Nodaviridae · Aggregata octopiana Resale or republication not permitted without written consent of the publisher INTRODUCTION market value (Castellanos-Martínez & Gestal 2013). Octopus on-growing is currently being developed on The common octopus Octopus vulgaris Cuvier, an industrial scale in Galicia (Spain) (Castellanos- 1798 is a cephalopod mollusc that is extremely im- Martínez & Gestal 2013), while experimental studies portant in fisheries and represents an important pro- in Italy have examined reproduction and larvae on- tein source in most Mediterranean countries (Estévez growing (De Wolf et al. 2011). One of the disadvan- et al. 1996). Since the decline of traditional fisheries, tages of octopus aquaculture is the increase in the O. vulgaris has been considered a candidate species incidence of diseases caused by bacteria, viruses and in European aquaculture because of its easy acclima- parasites that could be a serious risk to production tization to farming conditions, ra pid growth and high (Castellanos-Martínez & Gestal 2013). Despite the *Corresponding author: [email protected] © Inter-Research 2015 · www.int-res.com 148 Dis Aquat Org 115: 147–156, 2015 importance of O. vulgaris, little information is avail- lesions were examined. Within 30 min after death, able on diseases occurring in wild populations of this each octopus was stored at 4°C and microbiologically species (Farto et al. 2003). Viruses, bacteria and par- examined within 24 h. asites have been found associated with skin lesions Six sample animals were cultured specimens (3 in cephalopods (Scimeca 2012). fe males and 3 males) obtained from the research Bacteria in skin lesions of different Octopus species, centre for marine species ‘Maricoltura di Rosig- including O. vulgaris, O. joubini and O. bria reus, have nano Solvay’ (MRS) in Livorno, Italy; these been reported mainly from cultured subjects, but also included 4 adults (2 males and 2 females), which from wild individuals (Romalde & Barja 2010). The were in the final stage of their reproductive cycle, main signs observed in diseased octopus are skin ul- and 2 sub-adults (1 male and 1 female) at the cers, which can result in deep wounds in the head, beginning of the production season. The remaining mantle or arms (Scimeca 2012). Aeromonas cavieae, 3 specimens were commercial-caught wild speci- A. hydrophila, Pseudomonas stutzeri and various Vib- mens and included 2 adult males caught in Viareg- rio species (i.e. V. alginolyticus, V. anguillarum, V. gio (Lucca, Italy) and 1 adult male caught in harveyi and V. parahaemolyticus), have been associ- Livorno, Italy. ated with these lesions (Romalde & Barja 2010). In ad- dition, Farto et al. (2003) isolated V. len tus from gills, branchial-heart and skin lesions of wild common octo- Post-mortem examination pus in Spain, confirming its pathogenic potential. Aggregata spp. (Protozoa: Apicomplexa) coccidian Each octopus was examined for external clinical parasites are considered the dominant epizootiological abnormalities and post-mortem lesions. Samples for agents in wild and cultured octopus stocks in Europe parasitological in vestigation were taken and imme- (Gestal et al. 2002a, 2007). In severe forms, Aggregata diately analysed as fresh samples or fixed in 10% spp. infection spreads from the digestive tract to the buffered formalin for histological examination. Inoc- mantle and the gills, affecting the connective tissue ula for bacteriological investigation were taken and and epithelium of these organs and causing the promptly examined, while samples for virological appearance of white cysts associated with host cell investigation were stored at −80°C prior to examina- hypertrophy with nuclear displacement, inflamma- tion. tion, phagocytosis, ulceration, and destruction of the organ architecture (Gestal et al. 2002b, Castellanos- Martínez & Gestal 2013). A. octopiana is the only spe- Parasitological investigation cies of this genus reported in common octopus (Es- tévez et al. 1996, Castellanos-Martínez et al. 2013). Fresh smears and scrapes of skin, gill, intestine, Viruses infect most marine organisms, from bac - kidney and the mesenterium covering of the intestine teria to invertebrates and vertebrates (Pierce et al. and other internal organs were examined microscop- 2010). Cephalopods, including common octopus, ically for the presence of parasites. In addition, in have been found to contain virus-like particles, prob- order to investigate the presence of Aggregata spp. ably of the genus Iridovirus, associated with tumours in histological lesions, skin samples were fixed in in the muscle tissue of the arms and, in advanced 10% buffered formalin, dehydrated in a graded stages, on the funnel and the ventral surface of the ethanol series, embedded in paraplast (JB-4 Poly- mantle (Farley 1978, Hanlon & Forsythe 1990). sciences) and sectioned using a microtome. Sections The aim of the present study was to investigate the (3 µm) were stained with haematoxylin−eosin (Carlo pathogens associated with skin lesions in cultured Erba Reagents) following standard procedures and and wild common octopus. ob served under a light microscope. MATERIALS AND METHODS Bacteriological investigation Animals Inocula from skin mantle lesions, brain and bran - chial-heart were taken with a flame-sterilized loop Between May 2011 and December 2012, a total of 9 and plated onto 2% NaCl tryptone soya agar and naturally deceased specimens of wild and cultured blood agar plates. The cultures were incubated at adult and sub-adult common octopus exhibiting skin 25°C for 36 h. Developing colonies were examined Fichi et al.: P. swingsii, L. garvieae and betanodavirus in octopus 149 using taxonomical analyses according to Holt et al. Tissue homogenates were centrifuged at 3000 × g (1994), and biochemical reactions were performed (30 min), filtered through 0.45 µm membranes and using API test strips (Biomerieux™). incubated overnight at 4°C. Bluegill fry (BF-2), Strains that were not identified by taxonomical epithelioma papulosum cyprini (EPC) and striped and biochemical reactions were identified using snakehead (SSN-1) cell lines were routinely grown in 16S ribosomal RNA (rRNA) PCR assay. Bacterial 25 cm2 tissue culture flasks (BD FalconTM) in cell DNA was extracted using a QiaAmp Blood and culture EMEM supplemented with 10% FCS, 1% Tissue kit (Qiagen). A conventional PCR targeting L-glutamine and 1% antibiotic-antimycotic solution the 16S rRNA gene with the universal bacteria (Sig ma Aldrich) at 25°C. Sample extracts were inocu- primers 27F and 1525R (5’-GAG TTT GAT CCT lated at 2 ten-fold dilutions (1:10 and 1:100) onto 1 d GGC TCA G-3’ and 5’-AAG GAG GTG WTC CAR old BF-2, EPC and SSN-1 monolayers grown in CC-3’, respectively) was run as previously de - 24-well cell culture plates (BD FalconTM). BF-2 and scribed (Blackall et al. 2001). The resulting PCR ECP cell culture plates were incubated at 15°C for products were purified using a PCR purification kit the isolation of birnavirus and reovirus, and at 22°C (EuroGOLD Gel Extraction Kit, Euroclone) and