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Characterization of Neoparamoeba Pemaquidensis Strains: PCR-RFLP of the Internal Transcribed Spacer Region from the Amoeba and Endosymbiont

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Characterization of Neoparamoeba Pemaquidensis Strains: PCR-RFLP of the Internal Transcribed Spacer Region from the Amoeba and Endosymbiont DISEASES OF AQUATIC ORGANISMS Vol. 76: 141–149, 2007 Published June 29 Dis Aquat Org Characterization of Neoparamoeba pemaquidensis strains: PCR-RFLP of the internal transcribed spacer region from the amoeba and endosymbiont Charles G. B. Caraguel1, 2, Nathanaëlle Donay1, 2, Salvatore Frasca Jr.3, Charles J. O’Kelly 4, Richard J. Cawthorn1, 2 Spencer J. Greenwood1, 2,* 1AVC Lobster Science Centre and 2Department of Pathology & Microbiology, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward Island C1A 4P3, Canada 3Department of Pathobiology & Veterinary Science, University of Connecticut, 61 North Eagleville Road, Storrs, Connecticut 06269-3089, USA 4Bigelow Laboratory for Ocean Sciences, PO Box 475, 180 McKown Point Road, West Boothbay Harbor, Maine 04575, USA ABSTRACT: Neoparamoeba pemaquidensis continues to be an ongoing problem for commercial finfish aquaculture and has also sporadically been associated with mass mortalities of commercially relevant marine invertebrates. Despite the ubiquity and importance of this amphizoic amoeba, our understanding of the biology as it applies to host range, pathogenicity, tissue tropism, and geographic distribution is severely lacking. This may stem from the inability of current diagnostic tests based on morphology, immunology, and molecular biology to differentiate strains at the subspecies level. In the present study, we developed a polymerase chain reaction-restriction fragment length polymor- phism (PCR-RFLP) method based on the internal transcribed spacer (ITS) region that can accurately differentiate amoeba strains of N. pemaquidensis. The investigation focused on the complications of the amoeba ITS microheterogeneity in the development of a subspecies marker and the use of the endosymbiont, Ichthyobodo necator related organism (IRO), ITS region as an alternative marker. The combination of host amoeba and endosymbiont ITS PCR-RFLP analyses was successfully used to cor- rectly identify and characterize an N. pemaquidensis isolate from an outbreak of amoebic gill disease in Atlantic salmon Salmo salar from the west coast of North America (Washington State, USA). KEY WORDS: Internal transcribed spacer · Strain marker · PCR-RFLP · Microheterogeneity · Ichthyo- bodo necator related organism · Parasome Resale or republication not permitted without written consent of the publisher INTRODUCTION droebachiensis (as Paramoeba invadens Jones, 1985; Mullen et al. 2005). Neoparamoeba pemaquidensis Page, 1987 is a The genus Neoparamoeba is identified, in part, by ubiquitous marine amoeba present in free-living and the presence of one or more membrane-bound inclu- parasitic forms. As a pathogen, N. pemaquidensis is sions (‘paranuclear organelle’ or ‘parasome’) near the recognized worldwide as the causative agent of amoe- nucleus of the amoeba. Recent molecular evidence bic gill disease (AGD) in sea-farmed salmonids places members of the genus Neoparamoeba in a tight (Munday et al. 2001) and non-salmonid fish hosts sublineage within the Gymnamoebia (Fiala & Dyková (Dyková et al. 1995, 1998, 1999, 2000, Fiala & Dyková 2003, Peglar et al. 2003). Molecular phylogenetic analy- 2003). Disease outbreaks in marine invertebrates have sis has also revealed that the parasome is an endosym- also been attributed to N. pemaquidensis in both biont closely related to the prokinetoplastid Ichthy- American lobster Homarus americanus (Mullen et al. obodo necator (Dyková et al. 2003, Moreira et al. 2004). 2004, 2005) and green sea urchins Strongylocentrotus This molecular association forced reconsideration of the *Corresponding author. Email: [email protected] © Inter-Research 2007 · www.int-res.com 142 Dis Aquat Org 76: 141–149, 2007 proper name of this endosymbiont, and the more ap- The ability to further characterize pathogens at a propriate name of Ichthyobodo necator related organ- subspecies or strain level using genetic markers with ism (IRO) has been proposed (Caraguel et al. in press). sufficient variability has revealed previously unrecog- Several diagnostic methods have been developed for nized differences in parasite biology and has provided the identification of Neoparamoeba pemaquidensis. a clearer picture of host-pathogen interactions. In both Isolation in culture of amoebae and histopathological Giardia duodenalis and Cryptosporidium parvum, identification represent primary techniques based on genotyping and subtyping of isolates from different morphological features of the amoeba; however, both hosts have led to the discovery of new species, as well methods are inconsistent and lack specificity (Munday as the distinction of both host-adapted and cosmo- et al. 1993, Dyková & Novoa 2001, Dyková et al. 2005). politan species (Caccio et al. 2005). Thus, the charac- More specific and sensitive immunological methods terization of Neoparamoeba pemaquidensis at the incorporating polyclonal antibodies have been de- subspecies level may provide new information and signed for screening biological material, including an also allow us to reconsider our current understanding indirect fluorescent antibody test (IFAT; Howard & of the geographic distribution, host range, and tissue Carson 1993) and an immuno-dot blot test (Douglas- tropism of this amphizoic amoeba. Firstly, N. pema- Helders et al. 2001). All current immunological quidensis has been isolated from a variety of marine techniques, however, have limitations in the specific coastal environments from around the world (Fiala & identification of N. pemaquidensis, with reported Dyková 2003) as well as from a diverse assemblage of cross-reactivity of the polyclonal antisera with N. aes- marine organisms (Cann & Page 1982). However, it is tuarina and Pseudoparamoeba pagei (Douglas- unknown whether N. pemaquidensis detected in tis- Helders et al. 2001). sue or recovered from finfish have the potential to Because of the difficulties in characterizing Neop- infect invertebrates in geographically nearby locales. aramoeba species, there has been increased use of Secondly, N. pemaquidensis have been characterized molecular tools for identification and phylogenetic as ‘pathogenic’ when isolated from infected fish (e.g. studies. The 18S ribosomal RNA (rRNA) gene was ATCC 50172) and as ‘environmental’ when found in appropriately the first genetic marker used for molecu- the marine ecosystem (e.g. CCAP 1560/5). Although lar characterization of the genus Neoparamoeba (Elliot pathogenic strains can lose virulence during in vitro et al. 2001, Fiala & Dyková 2003, Peglar et al. 2003, culture (Morrison et al. 2005), it is currently unknown Wong et al. 2004, Dyková et al. 2005, Mullen et al. whether environmental strains can become patho- 2005). As a useful specific marker for Neoparamoeba genic. Thirdly, N. pemaquidensis is an external para- species (Dyková et al. 2005), the 18S rRNA sequences site during finfish gill infections (Adams et al. 2004) but have high levels of similarity (98.1 to 99%) among an internal parasite in lobsters and sea urchins (Mullen sequences from different isolates of N. pemaquidensis et al. 2004, 2005). Therefore, it has yet to be deter- (Wong et al. 2004). Two nested PCR methods for the mined if particular strains of N. pemaquidensis have detection of N. pemaquidensis based on the 18S rRNA specific host or tissue tropisms. Clearly, subspecies gene have been developed to detect and identify the identification of N. pemaquidensis by using a broadly pathogen. The first is specific for N. pemaquidensis applicable molecular marker could have direct appli- and is relatively efficient when used after culture- cations in disease monitoring, surveillance, and epi- enrichment of environmental and host-derived sam- demiological studies during outbreaks. ples (Elliot et al. 2001, Wong et al. 2004). The second The internal transcribed spacer (ITS) region, located nested PCR generates a 165 base pair product from between the 18S and 28S rRNA genes, was targeted to Paramoebidae/Vexilliferidae amoebae but does not explore the level of intra-specific and intra-genomic amplify templates from Pseudoparamoeba pagei or variability (Brown et al. 2004, Ruggiero & Procaccini Korotnevella hemistylolepis and more importantly 2004, Beszteri et al. 2005). The Neoparamoeba does not amplify templates from N. pemaquidensis pemaquidensis ITS region showed both qualitative strain ATCC 50172 (Mullen et al. 2005). These PCR and quantitative inter-strain variability that was protocols clearly illustrate useful features as well as mainly localized to the more variable regions of the ITS some of the current limitations of molecular character- 1 and ITS 2 (Caraguel et al. in press). A detailed inves- izations. As with all initial studies, the benefits and tigation of intra-strain variability revealed the exis- applicability will be assessed as more sequence data tence of very high levels of microheterogeneity in become available; until then, they should only be used these ITS regions. The existence of this intragenomic with caution in large-scale diagnostic applications. variability potentially precluded the use of the N. Moreover, both methods are unable to discriminate pemaquidensis ITS region as a good genetic marker. isolates at the subspecies level due to the low degree of Phylogenetic analysis of the N. pemaquidensis ITS intraspecific variability of the 18S rRNA marker. region revealed that microheterogeneity did not Caraguel et al.: Molecular characterization of N. pemaquidensis strains 143 obscure the monophyletic
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